KR101535253B1 - 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 an induced pluripotent stem cell (HD-iPSC) derived from a Huntington's disease patient. Also, it was confirmed that Huntington biomarker was expressed when it was treated under specific conditions when it was subdivided into cells or when it was differentiated into neurons. The HD-iPSC can be useful for screening a therapeutic agent for Huntington's disease through the change of the biomarker, and the HD-iPSC can be effectively used as an autologous cell therapy agent of Huntington's disease if the genetic defect is removed have.

Description

[0001] The present invention relates to a method for screening a therapeutic agent for Huntington's disease using inducible pluripotent stem cells derived from Huntington's disease patients,

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

BACKGROUND ART It is known that neurodegenerative diseases are diseases caused by the brain as they age, caused by the death of neuronal cells, synapse formation between neurons, or functional problems. Degenerative neurological 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 symptom and the area of the affected brain.

Huntington's disease is a fatal genetic disorder that occurs at the rate of one person per 10,000 people. It is known that the symptoms usually occur in the early 30s to 50s and most of them die after 15 years. Typical symptoms include muscle spastic movements, mental disorders, memory loss, dyspnea, heart failure, and infection. The Huntington gene causes the production of a toxin protein called huntingtin in brain cell neurons, which eventually damages the nerves 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 the unstable trinucleotide repeat sequence CAG of this gene. Patients have 35 or more repeats in comparison to normal subjects, and the number of repeats can be accurately measured by PCR amplification. Because of the autosomal dominant inheritance, normal and alleles from the normal allele are detected in the patient. It is known that the timing and symptoms of onset are proportional to the number of repeats of the CAG repeat sequence.

Studies at the UT Southwestern Medical Center have reported that quinazoline-derived materials effectively block the transient receptor potential channel 1 (TRPC1) -mediated store-operated calcium entry pathway and delay the symptoms of Huntington's disease (Chemistry and Biology ).

Induced Pluripotent Stem Cells (iPSCs), on the other hand, are cells that have been induced to have pluripotency through artificial de-differentiation of already differentiated cells. Since iPSC can be variously differentiated into long-term cells such as brain and heart, many studies have been conducted as a therapeutic agent for treating various diseases using iPSC.

According to the status of patent application for degenerative neurological diseases, Alzheimer's is the most, 1/5 of Parkinson's disease, and Huntington's disease is less than 10%. appear.

Currently, there is no known way to treat Huntington's disease. In order to develop a treatment for Huntington 's disease, it is necessary to search for a drug candidate that increases or decreases the expression of the Huntington' s disease related gene.

It is an object of the present invention to provide a composition for treating Huntington's disease comprising Huntington's disease patient-derived induced pluripotent stem cells (HD-iPSC).

Induced pluripotent stem cells (HD-iPSC) derived from a Huntington ' s disease patient.

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 comprising Huntington's disease patient-derived induced pluripotent stem cells (HD-iPSC).

The term "induced pluripotent stem cells" or "iPSC" refers to cells that have become pluripotent by treating somatic or already differentiated cells. Herein, the method for treatment includes, but is not limited to, a compound, a method for genetic transformation or culturing under specific conditions, and the like.

The term "induced pluripotent stem cell derived from Huntington's disease patient" or "HD-iPSC" refers to an induced pluripotent stem cell produced from a subject having Huntington's disease or a cell derived from a subject suspected of genetically having Huntington's disease do. In particular, somatic cells obtained from patients having 72 CAG repeat sequences can be used.

In addition, the Huntington's disease-derived induced pluripotent stem cell (HD-iPSC) may be obtained by treating Oct4, Sox2, Klf4 and c-Myc genes in skin cells of a patient. At this time, treatment of Oct4, Sox2, Klf4 and c-Myc may be directly to the culture medium of the patient somatic cell and may be performed through episomal system or retroviral infection.

Another aspect is to provide a screening method for Huntington's disease candidate candidate substances. In one embodiment, the method comprises culturing a Huntington's disease-derived induced pluripotent stem cell (HD-iPSC) in an undifferentiated state; Administering the test compound or composition to the undifferentiated HD-iPSC; Measuring the Huntington's disease biomarker from HD-iPSC in which the test compound or composition has been treated with HD-iPSC and the test compound or composition; And selecting a test compound or composition in which the expression of the Huntington's disease biomarker is changed in the test group as compared with the control group.

The above screening method is as follows.

First, the induced pluripotent stem cells may be maintained in a stable state in an undifferentiated state. The Huntington's disease-derived induced pluripotent stem cells are pluripotent stem cells.

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

Thereafter, the step of administering the test compound or composition to undifferentiated HD-iPSC or differentiated HD-iPSC is provided.

The compound or composition may be an organic or inorganic substance and may be a single compound or a composite compound. In addition, it can be a protein, a peptide, DNA, RNA, etc., and all substances present in nature can be targeted.

Thereafter, a step of measuring the Huntington's disease biomarker from the HD-iPSC in which the test compound or composition has been treated with the HD-iPSC and the test compound or composition is not treated.

Also, the Huntington's disease biomarker may be EM48, BDNF protein, α-tubulin. The biomarker can be used as a biomarker by measuring changes in EM48, changes in the expression of BDNF protein, and changes in the degree of acetylation of alpha -tubulin. In particular, the biomarker may be EM48.

Thereafter, the test compound or composition in which the expression of the Huntington's disease biomarker is changed in the test group as compared with the control group is provided.

At this time, the expression of the Huntington's disease biomarker may be increased or decreased, but in the case of the therapeutic agent, the expression is preferably decreased in the case of EM48, and the expression is increased in the case of BDNF and a-tubulin acetylation . It is preferable that the biomarker is decreased or increased at a statistically significant level as compared with the control group.

Another embodiment is a method of treating a Huntington ' s disease-derived derived pluripotent stem cell (HD-iPSC) Administering the test compound or composition to the differentiated HD-iPSC; Measuring the Huntington's disease biomarker from HD-iPSC in which the test compound or composition has been treated with HD-iPSC and the test compound or composition; And selecting a test compound or composition in which the expression of the Huntington's disease biomarker is changed in the test group as compared with the control group.

The above screening method is as follows.

First, it provides a step of differentiating Huntington's disease-derived induced pluripotent stem cells (HD-iPSC) into neuronal cells. The step may further include the step of maintaining the induced pluripotent stem cells in a stable state in an undifferentiated state. The Huntington's disease-derived induced pluripotent stem cells are pluripotent stem cells.

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

Thereafter, the step of administering the test compound or composition to undifferentiated HD-iPSC or differentiated HD-iPSC is provided.

The compound or composition may be an organic or inorganic substance and may be a single compound or a composite compound. In addition, it can be a protein, a peptide, DNA, RNA, etc., and all substances present in nature can be targeted.

Thereafter, a step of measuring the Huntington's disease biomarker from the HD-iPSC in which the test compound or composition has been treated with the HD-iPSC and the test compound or composition is not treated.

Also, the Huntington's disease biomarker may be EM48, BDNF protein, α-tubulin. The biomarker can be used as a biomarker by measuring changes in EM48, changes in the expression of BDNF protein, and changes in the degree of acetylation of alpha -tubulin. In particular, the biomarker may be EM48.

Thereafter, the test compound or composition in which the expression of the Huntington's disease biomarker is changed in the test group as compared with the control group is provided.

At this time, the expression of the Huntington's disease biomarker may be increased or decreased, but in the case of the therapeutic agent, the expression is preferably decreased in the case of EM48, and the expression is increased in the case of BDNF and a-tubulin acetylation . It is preferable that the biomarker is decreased or increased at a statistically significant level as compared with the control group.

When the present invention is used, a therapeutic agent for an effective 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 highly likely to be used industrially.

Figure 1 shows the characteristics of HD-iPSC-induced neural progenitor cells (HD-iPSC-NPC) and mature neurons (HD-iPSC-MN)
(AD): Characterization of HD-iPSC-NPC in 5-NP. (A): expression of NESTIN, SOX2 and (B): Musashi and low-level expression of GFAP, and (C): expression of Tuj1 and MAP2 by immunocytochemical staining. DAPI was used to counterstain each cell. Scale bar = 100 μm. (D) Semi-quantitative RT-PCR analysis showing the expression of neural progenitor cell markers (ie, Sox2, Nestin, Musashi) and lack of mature neuronal cells (Map2) or astrocyte (GFAP) expression. (EN): Characterization of HD-iPSC-induced MN (5-MN stage). (E): Immunocytochemical staining results (F) showing the degree of expression of the markers mainly expressed in the whole brain (OTX2) and dopaminergic neuron (TH) regions: general neuron (Tuj1) and whole brain- Marker expression for BF-1). (G): Markers for lateral ganglionic eminence (LGE) progenitor cells, expression of GSH-1. (H): Expression of another LGE marker, DLX2, along with a marker for medium spiny projection neurons (MSN), DARPP-32. Neuronal double-positive responses to GSH-2 or DLX2 and DARPP-32 demonstrate the formation of striatal MSN (striatal MSN). (I): Expression of calbindin, a marker of striatal neurons. (J): Expression of markers for mature neurons (MAP2) and synaptic vesicles (SVP32). (K): Expression of markers for mature neurons (NeuN) and GABA, a neurotransmitter. Scale bar = 100 μm. DAPI staining was used to distinguish the presence of each cell. (L): Gating strategy for NeuN and GABA and FACS analysis showing immunoreactive cells. It is noteworthy that 88% of NeuN-positive neurons also showed immunoreactivity to GABA. (M): Semiquantitative RT-PCR results (N) showing the expression of the whole brain (BF-1 and GSH-2), striatum (calvidin), MSN (DARPP-32) and GABA (GAD67 and GABARα2) : Electrophysiological Measurements of HD-iPSC-MN. The presence of GABA-sensitive cells is noteworthy.
FIG. 2 shows the results of behavioral analysis showing the recovery of function of HD-iPSC-NPC after transplantation of quinolinic acid (QUIN) into HD lesion in one hemisphere. For comparison, the experimental group transplanted with H9- and F5-NPC was used as a control. (A): Stepping (gait) experiments showing similar trends as seen in the F5 transplant group from 6 to 12 weeks in the H9- and HD transplant groups. There was no difference between the three groups treated. ** indicates p < 0.002. (B): Staircase (staircase) experiments showing that the H9- and F5 grafts always show significantly improved performance over the sham group. HD transplantation group showed behavioral recovery from 4 to 12 weeks, indicating that the recovery effect was delayed compared with other transplantation groups. # Means p <0.04 and shows the effect of H9- and F5 transplantation group compared to sham group at 2 weeks. (C): While the H9- and HD transplantation groups induced a significant behavioral recovery from 6 to 12 weeks versus the sham group, the F5 transplantation group tended to show gradual recovery over time from the beginning of the transplantation Apomorphine-Induced Rotation Experiment. # Means p <0.04, and shows the 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 in QUIN-lesioned rats. To investigate the fate of transplanted cells, antibodies against either the human-specific nucleus (AD, E, G) or the human-specific mitochondria (F, H) The transplanted HD-iPSC-NPCs form (A): NESTIN, (B): MAP2, (C): DARPP-32, (D): GAD65 / 67, (E), GABA, Respectively. (G): Lack of PCNA-positive cells from transplanted cells. (H): lack of EM48-positive cells from transplanted cells. Scale bar = 50 μm.
Figure 4 is an immunohistochemical analysis showing high sensitivity to HD aggregate formation for HD-iPSC. (A): Treatment of F5- and HD cells with a proteasome formation inhibitor (MG132). F5 cells were unresponsive to MG132, whereas HD cells formed aggregates from 5 μM. (B): Analysis of expression of EM48 at 4 weeks, 33 weeks, and 40 weeks after transplantation of H9-NPC and HD-iPSCNPC into brain of newborn mice. H9 cells did not form EM48-positive cells under any conditions, whereas HD cells formed EM48-positive cells at 33 weeks of age. The results of 40 weeks were the same, and the results were not shown. Scale bar = 50 μm.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are merely illustrative of the present invention and that the scope of the present invention is not construed as being limited by these embodiments.

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 cultured and maintained in DMEM / F-12 medium containing DMEM / F-12. In addition, HD-iPSC was induced to differentiate into neurons by co-culturing with PA6 stromal cells (Riken Cell Bank) (RCB1127). As a control group, another HD-iPSC cell line (HD-iPSC2) was used which was derived from the co-culture when human embryonic stem cell (H9, WiCell), normal iPSC (F5) and HD-iPSC were established. HD-iPSC with 72 CAG repeats is described by Park IH et al. (Cell 2008; 134: 877-886) and was provided for collaborative research with this researcher. This cell line is now available from Coriell (GM23225).

The above-mentioned undifferentiated human embryonic stem cells and the colonies of the induced pluripotent stem cells were separated by hand. The cells were then 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). After that, a definitive neural rosette-like structure including neuroepithelial cells (NE cells) was formed on the 11th to 13th day of the car. This was manually separated, and the non- Transferred to a dish, and suspended in culture for 6 days to form neuropheres.

<1-2> Nerve Bulb NP ) Cell production

To obtain individual cells of the NP stage, the neurons 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) 60 mm ² tissue culture dish. NP cells were maintained in GMEM supplemented with 1% penicillin, 1% streptomycin, 1% nonessential amino acid, 0.1% beta-mercaptoethanol, N2 supplement and 20 ng / ml bFGF (Invitrogen).

<1-3> Differentiation into mature neurons

In order to differentiate human embryonic stem cells and inducible pluripotent stem cells into mature neuronal cells, a PLO / FN-coated dish was cultured under DM medium supplemented with 20 ng / ml brain-derived neurotrophic factor (BDNF, R & D Systems) The neurosphere was placed directly on the nerve.

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

<2-1> Confirmation by immunocytochemical method

To analyze the marker expression of the NP-phase and differentiated neurons, immunocytochemistry was performed using the following primary antibodies: human-specific nuclei (1: 200, Chemicon), human-specific (1: 200, Chemicon), human-specific Nestin (1: 200, Chemicon), SOX2 (1: 200, Chemicon), type III β-tubulin (1: 100, Santa Cruz), GSH-2 (1: 200, Santa Cruz), DLX2 (1: 250, Chemicon), MAP2 1: 200, Chemicon), NeuN (1: 500, Chemicon), gamma aminobutyric acid (GABA) (1: 5000, Sigma), glutamic acid decarboxylase 65/67 (GAD65 / 67) , DAPP-32 (1: 100, Cell Signaling), Calbindin (1: 250, Chemicon), TH (1: 1000, Pel-Freez), SVP38 .

Proliferating cell nuclear antigen (PCNA) (1:50, Santa Cruz) and OCT4 (1: 250, Santa Cruz) were used to detect proliferating or undifferentiated cells in transplanted animals. goat anti-rabbit IgG-conjugated Alexa-488 (1: 200, Molecular Probes) and goat anti-mouse IgM-conjugated Alexa-555 (1: 200, Molecular Probes) : 200, Molecular Probes) was used as the secondary antibody. The staining pattern was 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 a FACS Calibur System (BD Bioscience) to quantify the percentage of GABAergic neurons formed in the neuronal population of HD-iPSC. Data analysis was performed according to manufacturer's instructions (BD Bioscience).

<2-3> Investigation of cell characteristics using electrophysiological measurement

The cells attached to the glass cover slip were transferred into a bath placed on the stage of an inverted microscope (IX-70, Olympus, Osaka, Japan). The water tank (~ 0.15 ml) was filled at 5 ml / min and the voltage-clamp test was performed at room temperature (22 ~ 25 ° C). A patch pipette with about 2.5 to 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 acquire data and apply command pulses. All cell currents were recorded at 10 kHz and low pass filters at 1 kHz, respectively. Current traces were stored and analyzed using Clampfit v.9.2 and Origin v.7.0 (Microcal, Northampton, MA). For comparison of total cell currents between cells, the magnitude of the current was measured in accordance with the membrane area measured by the capacitance. The receptor agonist γ-aminobutyric acid (GABA) and the antagonist peakotoxin were measured on whole cell current.

Example  3: Through animal models HD - iPSC Of treatment effect

<3-1> QUIN-Induction Experiment HD animal model preparation and cell transplantation

Animal experiments were performed according to CHA University IACUC (Institutional Animal Care and Use Committee; IACUC090012). Adult male Sprague-Dawley rats (Orient, Seoul, Korea) weighing 250-280 g were used. (AP + 0.7 mm, ML + 2.5 mm, DV .4 mm, and AP +0.7 mm, ML + 2.5 mm, DV -3.5 mm from the Bregma) at one of the following locations Quinoline acid (QUIN, Fluka; 0.3 mol / L x 1.5 μl) was injected. On day 7 post-QUIN lesion induction, 2 μl HD iPS-induced NP cells (100,000 cells / μl) were injected into 12 mice in 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 implanted with 2 μl of medium (GMEM) containing the same number of H9-NPCs (n = 8) or F5-NPCs (n = 11). In the control group (n = 9), 2 μl of GMEM was injected simultaneously. The transplanted animals received cyclosporine A intraperitoneally (10 mg / kg, Sigma) every day from the day before transplantation to the 12th week after transplantation.

<3-2> Behavioral experiments in animal models

In order to determine whether extensive lesions were observed in the striatum after the induction of the lesion and to evaluate whether the lesion was functionally restored 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; The measured values were analyzed using PROC MIXED program, a linear mixed models procedure. Dependent variants were intermediate scores from each behavioral test, and the independent variables were "Treatment" (H9-, HD-, F5- and sham groups) and "Week" (0w pre-transplantation, 2w, 4w, 6w, , and was 12w). results are expressed as mean ± mean standard error (SEM). It was considered a small p value greater than 0.05 significantly.

<3-4> Immunohistochemical analysis

For immunohistochemical analysis, rats were sacrificed at 12 weeks post-transplantation and fixed by perfusion with 4% paraformaldehyde in their brains. A 40 μm frozen coronal section was prepared using cryostat (Microm, Germany). Antibodies used for immunohistochemical analysis were identical to those described in immunocytochemical assays (see above). To identify lesions induced by QUIN infusion, conventional DAB (3,3'-diaminobenzidine) with cresyl violet staining, Fluoro-Jade staining and DARPP-32 antibody was performed simultaneously.

Example 4 Screening of HD-Therapeutics Using HD-iPSC

The compound to be tested of the HD therapeutic agent group is treated at various concentrations in the undifferentiated HD-iPSC obtained in Example <1-2> or the HD-iPSC obtained in Example <1-3>. Measure the degree of acetylation of EM48, BDNF and a-tubulin in HD treated and treated and untreated controls. If EM48 is reduced, expression of BDNF is increased, or acetylation of α-tubulin is increased, it is selected as a candidate for the treatment of Huntington's disease.

Claims (9)

delete delete delete delete Culturing Huntington's disease patient-derived induced pluripotent stem cells (HD-iPSC) having 72 CAG repeat sequences in an undifferentiated state;
Adding the test compound or composition to the undifferentiated HD-iPSC;
Measuring the expression level of Huntington's disease biomarker from HD-iPSC in which the test compound or composition has been treated with HD-iPSC and the test compound or composition; And
Selecting a test compound or composition in which the expression of the Huntington's disease biomarker is changed in the test group as compared to the control group. The method according to claim 5, further comprising the step of adding a proteasome formation inhibitor to the undifferentiated HD-iPSC after the step of culturing the HD-iPSC in an undifferentiated state and before the step of adding the test compound or composition. Screening method of candidate substance. The method according to claim 5, wherein the Huntington's disease biomarker is acetylation of EM48, BDNF and a-tubulin. Dividing Huntington's disease patient-derived induced pluripotent stem cells (HD-iPSC) having 72 CAG repeat sequences into neuronal cells;
Implanting neurons differentiated from the HD-iPSC into the brain of a mouse;
Administering a test compound or composition to a mouse transplanted with said nerve cell;
Measuring the level of expression of a Huntington's disease biomarker of a neural cell of a mouse in which the test compound or the composition is administered and the neurocyte of the mouse to which the test compound or composition is administered; And
Selecting a test compound or composition in which the expression of the Huntington's disease biomarker is changed in the test group as compared to the control group. [Claim 9] The method according to claim 8, wherein the mouse to which the test compound or composition is administered is a mouse having 33 or more weeks of transplantation of a neural cell differentiated from HD-iPSC.

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