KR20130133598A - Screening method for candidate drugs for huntington's disease using induced pluripotent stem cells derived from huntington's disease patients - Google Patents

Abstract

The present invention relates to induced pluripotent stem cells derived from Huntington's disease patients (HD-iPSC). In addition, it was verified that when the induced pluripotent stem cells were treated under specific conditions, at a differentiation stage, or differentiated into neurocytes, a Huntington's biomarker is expressed. Through the change of the biomarker, the HD-iPSC is useful in the screening of candidate drugs for Huntington's disease. Further, when genetic defects are removed, the HD-iPSC is useful as an autologous cell therapeutic agent against Huntington's disease.

Description

Screening method for candidate drugs for Huntington's disease using induced pluripotent stem cells derived from Huntington's disease patients}

TECHNICAL FIELD The present invention relates to a method for screening a Huntington's disease therapeutic agent using induced pluripotent stem cells derived from Huntington's disease patients.

Degenerative neurological diseases (neurodegenarative diseases) is a disease that occurs in the brain with age, it is known that it is caused by the death of neuronal cells, synapse formation or functional problems between the neurons. Neurodegenerative diseases are classified into various diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease, and stroke depending on the main symptoms and the invading brain area.

Huntington's disease is a fatal genetic disease that occurs at a rate of 1 in 10,000 people. It is known to occur mainly in the early 30s to 50s, and most die within 15 years after the onset of symptoms. Representative symptoms include muscle spasms, mental disorders, memory loss, dyspnea, heart failure, and infection. The Huntington gene causes brain cells to produce a toxic protein called huntingtin in neurons, which eventually damages the nerve and causes a degenerative effect.

The Huntington gene (IT15 gene, located on chromosome 4) has already been reported, and most Huntington's disease patients have mutations in this gene's unstable trinucleic acid repeat CAG. Patients have 35 or more repetitions compared to normal patients, and can accurately measure repetition counts through PCR amplification. As an autosomal dominant gene, the patient detects both normal and abnormal allele bands. The onset and symptoms are known to be proportional to the size of the CAG repeat sequence.

A study by the UT Southwestern Medical Center reported that quinazoline-derived substances effectively block the Transient receptor potential channel 1 (TRPC1) -mediated store-operated calcium entry (SOC) pathway, which delays symptoms of Huntington's disease (Chemistry and Biology ).

Meanwhile, induced pluripotent stem cells (iPSCs) refer to cells induced to have pluripotency through artificial dedifferentiation of already differentiated cells. iPSC can be variously differentiated into organ cells such as brain, heart, etc., and many studies have been conducted as therapeutic agents for treating various diseases using iPSC.

According to the current state of patent applications for neurodegenerative diseases, Alzheimer's accounted for 1/3 of the cases, Parkinson's disease was about 1/5, and Huntington's disease was less than 10%. appear.

Currently, there is no known way to treat Huntington's disease. For the development of Huntington's disease therapies, it is necessary to search for drug candidates that increase or decrease the expression of Huntington's disease related genes.

An object of the present invention to provide a composition for treating Huntington's disease, including HPS-derived induced pluripotent stem cells (HD-iPSC).

Huntington's disease-derived induced pluripotent stem cells (HD-iPSC) is to provide a method for screening for Huntington's disease candidates.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In general, the nomenclature used herein is well known and commonly used in the art.

One aspect is to provide a composition for treating Huntington's disease, including HPS-derived induced pluripotent stem cells (HD-iPSC).

The term “induced pluripotent stem cells” or “iPSCs” refers to cells that have become pluripotent by treating somatic cells or already differentiated cells. The treatment method herein includes, but is not limited to, a compound, a genetic transformation, or a method of culturing under specific conditions.

The term "induced pluripotent stem cells derived from Huntington's disease patients" or "HD-iPSC" refers to induced pluripotent stem cells produced by dedifferentiation from cells extracted from individuals with Huntington's disease or those suspected of having genetically Huntington's disease. do. In particular, somatic cells obtained from patients with 72 CAG repeats can be used.

In addition, the HPS-derived induced pluripotent stem cells (HD-iPSC) may be obtained by treating Oct4, Sox2, Klf4 and c-Myc genes on skin cells of the patient. At this time, the treatment of Oct4, Sox2, Klf4 and c-Myc may be directly treated in the culture medium of the somatic cells of the patient, it may be performed through an episomal system (episomal system) or retrovirus infection.

Another aspect is to provide a method for screening a candidate drug for Huntington's disease. One embodiment comprises the steps of culturing the induced pluripotent stem cells (HD-iPSC) derived from Huntington's disease patients in an undifferentiated state; Administering a test compound or composition to said undifferentiated HD-iPSC; Measuring Huntington's disease biomarker from the HD-iPSC treated with the test compound or composition and the HD-iPSC untreated with the test compound or composition; And it provides a screening method of the Huntington's disease therapeutic agent candidate comprising the step of selecting a test compound or composition in which the expression of the Huntington's disease biomarker in the experimental group compared to the control group.

The screening method is as follows.

First, the induced pluripotent stem cells may include a step of maintaining a stable state in an undifferentiated state. The Huntington's disease-derived induced pluripotent stem cells are undifferentiated stem cells having pluripotency.

At this time, after differentiation into neuronal progenitor cells in the undifferentiated state can be differentiated into neurons. In addition, it can then differentiate into neurons from neuroprogenitor cells. At this time, the differentiation into neurons may be differentiated by treating brain-derived neurotrophic factor (BDNF), and in this case, it may be differentiated by treating brain-derived neurotrophic factor lacking bFGF.

Thereafter, administering the test compound or composition to the undifferentiated HD-iPSC or differentiated HD-iPSC.

The compound or composition may be organic and inorganic, and may be a single compound or a complex compound. In addition, it may be a protein, peptide, DNA, RNA, and the like, and any substance present in nature may be a target.

Thereafter, a step of measuring Huntington's disease biomarker from the HD-iPSC treated with the test compound or composition and the HD-iPSC untreated with the test compound or composition is provided.

In addition, the Huntington's disease biomarker may be EM48, BDNF protein, α-tubulin (α-tubulin). The biomarker can be used as a biomarker by measuring the increase or decrease of EM48, the increase or decrease of expression of BDNF protein, and the degree of acetylation of α-tubulin. In particular, the biomarker may be EM48.

Thereafter, the present invention provides a step of selecting a test compound or composition in which the expression of the Huntington's disease biomarker is changed in the experimental group compared to the control group.

In this case, the expression of Huntington's disease biomarker may be increased or decreased, but in the case of a therapeutic agent, the expression is preferably decreased in the case of EM48, and in the case of BDNF and α-tubulin acetylation, the expression is increased. It is preferable to be. The biomarker is preferably reduced or increased at a statistically significant level compared to the control.

Another embodiment comprises the steps of differentiating HPS disease-derived induced pluripotent stem cells (HD-iPSCs) from them into neurons; Administering a test compound or composition to said differentiated HD-iPSC; Measuring Huntington's disease biomarker from the HD-iPSC treated with the test compound or composition and the HD-iPSC untreated with the test compound or composition; And it provides a screening method of the Huntington's disease therapeutic agent candidate comprising the step of selecting a test compound or composition in which the expression of the Huntington's disease biomarker in the experimental group compared to the control group.

The screening method is as follows.

First, a step of differentiating HPS-derived induced pluripotent stem cells (HD-iPSCs) into neurons is provided. The step may further comprise maintaining the induced pluripotent stem cells in a stable state in an undifferentiated state. The Huntington's disease-derived induced pluripotent stem cells are undifferentiated stem cells having pluripotency.

At this time, after differentiation into neuronal progenitor cells in the undifferentiated state can be differentiated into neurons. In addition, it can then differentiate into neurons from neuroprogenitor cells. At this time, the differentiation into neurons may be differentiated by treating brain-derived neurotrophic factor (BDNF), and in this case, it may be differentiated by treating brain-derived neurotrophic factor lacking bFGF.

Thereafter, administering the test compound or composition to the undifferentiated HD-iPSC or differentiated HD-iPSC.

The compound or composition may be organic and inorganic, and may be a single compound or a complex compound. In addition, it may be a protein, peptide, DNA, RNA, and the like, and any substance present in nature may be a target.

Thereafter, a step of measuring Huntington's disease biomarker from the HD-iPSC treated with the test compound or composition and the HD-iPSC untreated with the test compound or composition is provided.

In addition, the Huntington's disease biomarker may be EM48, BDNF protein, α-tubulin (α-tubulin). The biomarker can be used as a biomarker by measuring the increase or decrease of EM48, the increase or decrease of expression of BDNF protein, and the degree of acetylation of α-tubulin. In particular, the biomarker may be EM48.

Thereafter, the present invention provides a step of selecting a test compound or composition in which the expression of the Huntington's disease biomarker is changed in the experimental group compared to the control group.

In this case, the expression of Huntington's disease biomarker may be increased or decreased, but in the case of a therapeutic agent, the expression is preferably decreased in the case of EM48, and in the case of BDNF and α-tubulin acetylation, the expression is increased. It is preferable to be. The biomarker is preferably reduced or increased at a statistically significant level compared to the control.

Using the present invention, an effective treatment for Huntington's disease can be developed. In addition, HD-iPSC can be used as a treatment for Huntington's disease. Therefore, HD-iPSC derived from Huntington's disease patients is considered to be industrially useful.

1 shows the properties of HD-iPSC-induced neural progenitor cells (HD-iPSC-NPC) and mature neurons (HD-iPSC-MN) in step 5.
(AD): Characterization of HD-iPSC-NPC at 5-NP. Immunohistochemical staining showed that NPC markers were expressed at high levels, (A): NESTIN, SOX2 and (B): Musashi expression and low levels of GFAP and (C): Tuj1 and MAP2. DAPI was used to counterstain each cell. Scale bar = 100 μm. (D): Semiquantitative RT-PCR analysis showing lack of expression of neural progenitor markers (ie Sox2, Nestin, Musashi) and expression of mature neurons (Map2) or astrocytes (GFAP). (EN): Characterization of HD-iPSC-induced MN (5-MN phase). (E): Immunocytochemical staining results indicating the expression level of markers mainly expressed in whole brain (OTX2) and dopaminergic neuronal (TH) regions (F): General neurons (Tuj1) and whole brain-specific neurons ( Marker expression for BF-1). (G): Expression of lateral ganglionic eminence (LGE) progenitor, GSH-1. (H): Expression of another LGE marker, DLX2, with marker for medium spiny projection neuron (MSN), DARPP-32. Neuronal double-positive responses to GSH-2 or DLX2 and DARPP-32 demonstrate that striatal MSN was formed. (I): Expression of calbindin, a marker of striatal neuron. (J): Expression of markers for mature neurons (MAP2) and synaptic vesicles (SVP32). (K): Expression of markers for mature neurons (NeuN) and GABA, a type of neurotransmitter. Scale bar = 100 μm. DAPI staining distinguished each cell presence. (L): FACS analysis showing gating strategy and immune response cells for NeuN and GABA. Note that 88% of NeuN-positive neurons were also immunoreactive with GABA. (M): Semiquantitative RT-PCR results showing expression of forebrain (BF-1 and GSH-2), striatum (calvindine), MSN (DARPP-32) and GABA-like (GAD67 and GABARα2) neurons (N) : Electrophysiological Measurement of HD-iPSC-MN. Note the presence of GABA-sensitive cells.
Figure 2 shows the behavioral analysis showing the functional recovery after transplantation of HD-iPSC-NPC in rats that induced HD lesions in only one hemisphere through lateral injection of quinolinic acid (QUIN). For comparison, the experimental group transplanted with H9- and F5-NPC was used as a control. (A): Stepping (walking) experiment showing similar trends as seen in F5 transplant group at week 6-12 for H9- and HD transplant group. There was no difference between the three groups treated. ** indicates that p <0.002. (B): Staircase experiment showing that the H9- and F5 transplant groups always showed significantly improved performance over the sham group. In the HD transplant group, behavioral recovery occurred from 4 to 12 weeks, indicating a slow recovery effect compared to other transplant groups. # Means p <0.04 and shows effect of H9- and F5 transplant group compared to sham group at week 2. (C): While the H9- and HD transplant groups generally induced significant behavioral recovery from 6 to 12 weeks compared to the sham group, the F5 transplant group tended to show a gradual recovery over time from the beginning of the transplant. Apomorphine-induced rotation experiments showing the presence of the # Means p <0.04 and shows effect of F5 group compared to HD- and sham group at 2 weeks.
Figure 3 shows immunohistochemical analysis showing the formation of neural tissue after transplantation of HD-iPSC-NPC into QUIN-lesioned mice. To investigate the fate of the transplanted cells, antibodies to either human-specific nuclei (AD, E, G) or human-specific mitochondria (F, H) were used. Implanted HD-iPSC-NPCs form (A): NESTIN, (B): MAP2, (C): DARPP-32, (D): GAD65 / 67, (E), GABA, (F): SVP38 Indicated. (G): Lack of PCNA-positive cells from transplanted cells. (H): Lack of EM48-positive cells from transplanted cells. Scale bar = 50 μm.
4 is an immunohistochemical analysis showing high sensitivity to HD aggregate formation for HD-iPSC. (A): When F5- and HD cells were treated with the proteasome formation inhibitor (MG133). HD cells formed aggregates from 5 μΜ, while F5 cells were unresponsive to MG132. (B): Expression analysis of EM48 at 4, 33, and 40 d post-intracery transplantation of H9-NPC and HD-iPSCNPC in brains of newborn mice. H9 cells did not form EM48-positive cells under any conditions, while HD cells formed EM48-positive cells at week 33. The same was true of the 40 weeks, and the results are not shown. Scale bar = 50 μm.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it is apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

Example  One : HD - iPSC Induction, culture and differentiation into neurons

<1-1> HD - iPSC Cultivation of

20% KO-SR (knockout serum replacement, Invitrogen), bFGF (10 ng / ml) and antibiotics (penicillin, streptomycin) according to the method described in Park IH et al. (Nat Protocol 2008; 3: 1180-1186) HD-iPSC was incubated and maintained in DMEM / F-12 medium. In addition, co-culture with PA6 stromal cells (Riken Cell Bank) (RCB1127) induced differentiation of HD-iPSCs into neurons. As a control, another HD-iPSC cell line (HD-iPSC2) derived simultaneously when human embryonic stem cell (ESC) (H9, WiCell), normal iPSC (F5) and HD-iPSC was established was used. HD-iPSCs with 72 CAG repeats are described in Park IH et al. (Cell 2008; 134: 877-886), which was originally developed for collaboration with our researchers. The cell line is currently available from Coriell (GM23225).

The colonies of the above-mentioned undifferentiated human embryonic stem cells and induced pluripotent stem cells were isolated manually. Thereafter, they were transferred onto freshly prepared PA6 cells in differentiation medium (DM-PA6) consisting of GMEM containing 10% KO-SR. After 4 days KO-SR in DM-PA6 was replaced with N2 supplement (Invitrogen). Then on days 11-13, a final neural rosette-like structure was formed, including neuronepithelial cells (NE cells), which were manually separated and adhered to non-sticky petri. Transferred to a dish and suspended in culture for 6 days to form neurospheres.

<1-2> nerve bulb ( NP A) cell generation

To obtain individual cells in the NP stage, neurospheres were treated with Accutase ^™ (Chemicon) and then separated and coated with poly L-ornithine (PLO; 15 μg / ml, Sigma) / fibronectin (FN; 1 μg / ml, Sigma) It was placed on a 60 mm ² tissue culture dish. NP cells were maintained in GMEM supplemented with 1% penicillin, 1% streptomycin, 1% non-essential amino acids, 0.1% β-mercaptoethanol, N2 supplement and 20 ng / ml bFGF (Invitrogen).

<1-3> Differentiation into Mature Neurons

To differentiate human embryonic stem cells and induced pluripotent stem cells into mature neurons, PLO / FN-coated dishes under DM medium added with 20 ng / ml brain-derived neurotrophic factor (BDNF, R & D Systems) without the addition of bFGF. Neurospheres were placed directly on the image.

Example  2: HD-iPSC-derived neurons Character analysis  Confirm

<2-1> Identification using immunocytochemical method

To analyze marker expression of NP-stage and differentiated neurons, immunocytochemical analysis was performed using the following primary antibodies: human-specific nuclei (1: 200, Chemicon), human-specific Mitochondria (1: 200, Chemicon), human-specific Nestin (1: 200, Chemicon), SOX2 (1: 200, Chemicon), type III β-tubulin (Tuj1) (1: 500, Chemicon), Musashi (1 : 500, Chemicon), OTX2 (1: 500, Chemicon), BF-1 (1: 100, Santa Cruz), GSH-2 (1: 200, Santa Cruz), DLX2 (1: 250, Chemicon), MAP2 ( 1: 200, Chemicon), NeuN (1: 500, Chemicon), gammaaminobutyl acid (GABA) (1: 5000, Sigma), glutamic acid decarboxylase 65/67 (GAD65 / 67) (1: 200, Chemicon) , DARPP-32 (1: 100, Cell Signaling), Calbindin (1: 250, Chemicon), TH (1: 1000, Pel-Freez), SVP38 (1: 200, Sigma) and EM48 (1:50, Chemicon) .

To detect proliferating or undifferentiated cells in transplanted animals, proliferating cell nuclear antigen (PCNA) (1:50, Santa Cruz) and OCT4 (1: 250, Santa Cruz) were used. goat anti-mouse IgG-conjugated Alexa-555 (1: 200, Molecular Probes), goat anti-rabbit IgG-conjugated Alexa-488 (1: 200, Molecular Probes) and goat anti-mouse IgM-conjugated Alexa-555 (1 : 200, Molecular Probes) was used as the secondary antibody. Staining patterns were examined and photographed using a confocal laser-scanning microscope imaging system (LSM510, Carl Zeiss, Inc.).

<2-2> Cell Characterization Using Flow Cytometry

Fluorescence activated cell sorting (FACS) was performed using the FACS Calibur System (BD Bioscience) to quantify the percentage of GABA neurons formed in the neuronal population of HD-iPSC. Data analysis was performed according to the manufacturer's instructions (BD Bioscience).

<2-3> Cell Characterization by Electrophysiological Measurement

Cells attached to the glass cover slip were transferred to a tank mounted on the stage of an inverted microscope (IX-70, Olympus, Osaka, Japan). The bath (~ 0.15 ml) was filled to 5 ml / min and voltage-clamp experiments were performed at room temperature (22-25 ° C.). A patch pipette with about 2.5-3 MΩ free-tip resistance was connected to the head stage of a patch-clamp amplifier (Axopatch-1D, Axon Instruments, Foster City, CA). PCLAMP software v.9.2 and Digidata-1322A (Axon Instruments) were used to obtain data and apply command pulses. Each cell current was recorded at 10 kHz and a low pass filter was performed at 1 kHz. Current traces were saved and analyzed using Clampfit v.9.2 and Origin v.7.0 (Microcal, Northampton, MA). For comparison of the total cell current between cells, the magnitude of the current was measured in line with the membrane area measured by capacitance. The receptor agonist γ-aminobutyl acid (GABA) and the antagonist picrotoxin were measured on whole cell current.

Example  3: Through animal models HD - iPSC The effectiveness of treatment

<3-1> QUIN-Induced Experimental HD Animal Model Fabrication and Cell Transplantation

Animal experiments were performed in accordance with CHA University IACUC (Institutional Animal Care and Use Committee; IACUC090012). Adult male Sprague-Dawley rats (Orient, Seoul, Korea) weighing 250-280 g were used. Brain location coordinates of two areas (AP +0.7 mm, ML +2.5 mm, DV .4.5 mm, and AP +0.7 mm, ML +2.5 mm, DV -3.5 mm from the Bregma) Quinolinic acid (QUIN, Fluka; 0.3 mol / L x 1.5 μl) was injected. On day 7 after QUIN lesion induction, 2 μl of HD iPS-induced NP cells (100,000 cells / μl) were injected for 12 rats with the following coordinates: AP +0.2 mm, ML +2.2 mm and DV. 4.0 mm from the Bregma. For comparison, QUIN lesion mice were transplanted with 2 μl of medium (GMEM) containing the same number of H9-NPC (n = 8) or F5-NPC (n = 11). In the control group (n = 9), 2 μl of GMEM was injected at the same time. Transplanted animals received cyclosporine A intraperitoneally (10 mg / kg, Sigma) daily from one day before transplantation to 12 weeks after transplantation.

<3-2> Behavioral Experiments in Animal Models

To determine if the lesions occurred extensively in the striatum after QUIN-lesion induction and at the same time to assess whether they recovered functionally after cell transplantation, a stepping test, a stepping test staircase test) and apomorphine-induced rotation experiments were performed.

<3-3> Perform statistical analysis

Statistical analysis of behavioral data was performed using the Statistical Analysis System (Enterprise 4.1; SAS Korea) owned by CHA. The measurements were analyzed using the PROC MIXED program, a linear mixed models procedure. The dependent variation was the median score obtained from each behavioral test and the independent variation was "Treatment" (H9-, HD-, F5- and sham groups) and "Week" (0w pre-transplantation, 2w, 4w, 6w, 8w, 10w) , And 12w) The results are expressed as mean ± standard error of the mean (SEM), p values less than 0.05 were considered significant.

<3-4> Immunohistochemical Analysis

For immunohistochemical analysis, mice were sacrificed at week 12 post-transplant and fixed by perfusion with 4% paraformaldehyde in their brains. A 40 μm frozen coronal section was prepared using cryostat (Microm, Germany). The antibodies used for immunohistochemical analysis were the same as those described for immunocytochemical analysis (see above). To identify lesions induced by QUIN injection, conventional DAB (3,3'-diaminobenzidine) with cresyl violet staining, Fluoro-Jade staining, and DARPP-32 antibody was performed simultaneously.

Example 4: HD-Therapeutic Screening with HD-iPSC

The undifferentiated HD-iPSC obtained in Example <1-2> or the HD-iPSC obtained in Example <1-3> is treated with various concentrations of test compounds of the HD therapeutic candidate group. The extent of acetylation of EM48, BDNF and α-tubulin in the experimental and untreated controls with HD treatment was measured. Huntington's disease therapeutic candidates are selected when EM48 is decreased, the expression of BDNF is increased, or the acetylation of α-tubulin is increased.

Claims (8)

Huntington's disease-derived HPS treatment composition comprising induced pluripotent stem cells (HD-iPSC). The composition of claim 1, wherein the HD-iPSC is derived from a patient having 72 CAG repeats. The composition of claim 1, wherein the induced pluripotent stem cells are obtained by treating Oct4, Sox2, Klf4, and c-Myc on somatic cells of a Huntington's disease patient. The composition of claim 3, wherein treating Oct4, Sox2, Klf4, and c-Myc is performed through retroviral infection. Culturing HPS-derived induced pluripotent stem cells (HD-iPSCs) in an undifferentiated state;
Administering a test compound or composition to said undifferentiated HD-iPSC;
Measuring Huntington's disease biomarker from the HD-iPSC treated with the test compound or composition and the HD-iPSC untreated with the test compound or composition; And
A screening method for a Huntington's disease therapeutic agent candidate comprising the step of selecting a test compound or composition in which the expression of the Huntington's disease biomarker in the experimental group compared to the control group. The method of claim 5, wherein the Huntington's disease therapeutic agent candidate is screened by increasing or decreasing the expression of the Huntington's disease biomarker. The method of claim 5, wherein the Huntington's disease biomarker is acetylation of EM48, BDNF, and α-tubulin. Differentiating HPS disease-derived induced pluripotent stem cells (HD-iPSCs) from them into neurons;
Administering a test compound or composition to said differentiated HD-iPSC;
Measuring Huntington's disease biomarker from the HD-iPSC treated with the test compound or composition and the HD-iPSC untreated with the test compound or composition; And
A screening method for a Huntington's disease therapeutic agent candidate comprising the step of selecting a test compound or composition in which the expression of the Huntington's disease biomarker in the experimental group compared to the control group.

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