WO2020231089A1 - Induced pluripotent stem cells of alzheimer's disease patient with mutation (v715m) in amyloid precursor protein - Google Patents

Abstract

The present invention relates to: screening for Alzheimer's-related diseases by using induced pluripotent stem cells with a mutation in an amyloid precursor protein (val715met) derived from blood cells; a personalized therapeutic agent; and a preparation method therefor.

Description

Induced pluripotent stem cells of Alzheimer's disease patients with amyloid precursor protein mutation (V715M)

Using blood cells that are easier to collect than skin cells, it provides an induced pluripotent stem cell dedifferentiated from human PBMC containing an APP mutant gene having V715M substitution, which is a Korean specific amyloid precursor protein mutation.

Alzheimer's disease is the most common degenerative brain disease that causes dementia, and was first reported in 1907 by Dr. Alois Alzheimer, a German psychiatrist. Alzheimer's disease is characterized by a very slow onset and gradual progression. In the beginning, they mainly show problems with the memory of recent events, but as they progress, they are accompanied by other abnormalities in other cognitive functions such as language and judgment, and eventually all functions of daily life are lost.

Genetic factors have been reported to account for less than 10% of all Alzheimer's incidences, and people with immediate family members who have it have a higher risk of developing it than those who do not.

Alzheimer's disease is a family member of Alzheimer's disease if there is a mutation in the amyloid precursor protein gene (located on chromosome 21), prisenilin 1 gene (located on chromosome 14), and prisenilin 2 gene (located on chromosome 1). Although known, they are all only involved in the onset of premature (early) Alzheimer's disease, which occurs in the 40s to 50s, and is not related to the onset of most late-stage (old) Alzheimer's.

In the case of Alzheimer's disease, existing animal models have an inherent limitation in that it is difficult to reproduce the pathophysiological symptoms directly related to Alzheimer's disease, so the development of treatments or treatments using existing animal models is limited. That is, in most cases, the symptoms of Alzheimer's disease in humans do not appear even in a mouse model having the same gene mutation as those of Alzheimer's disease patients.

After inducing dedifferentiation of skin cells of Alzheimer's disease patients to make induced pluripotent stem cells (iPSCs), they were differentiated into neurons again to investigate their characteristics. As a result, the pathological markers of Alzheimer's disease of each patient can be measured. There has been a report of research results showing statistically significant differences when compared with normal subjects (Inoue et al., 2013; Israel et al., 2012). This iPSC-based research model is expected to be used as a good research model for verifying the efficacy of therapeutic agents that inhibit pathological biomarkers and searching for customized therapeutics tailored to each patient.

Therefore, research and development of induced pluripotent stem cells having characteristics that can be used for screening of new drugs, high biosimilarity, and pathological characteristics of Alzheimer's disease are required.

One aspect relates to induced pluripotent stem cells dedifferentiated from human PBMCs containing an APP mutant gene with a V715M substitution.

Another aspect relates to a nerve cell obtained by differentiating the stem cell into a nerve cell.

Another aspect is a stem cell line containing an APP mutant gene having a V715M substitution comprising the step of introducing a vector expressing a dedifferentiation factor into a PDMC containing an APP mutant gene having a V715M substitution isolated from the blood of an Alzheimer's patient. It relates to a method of manufacturing.

Another aspect is a method of screening for a drug effective for preventing or treating Alzheimer's disease, comprising the steps of: contacting the nerve cells with a test substance;

Measuring at least one of Aβ expression amount, phosphorylated tau protein expression amount, mitochondrial motility, and imbalance between mitochondrial division and fusion in the contacted neuron;

Comparing the measured results with the measured results from the nerve cells not in contact with the test substance; It relates to a drug screening method comprising a.

In the present specification, the “APP mutant gene” may be any mutant gene in which a normal gene and one or more bases are substituted for an amyloid precursor protein (amyloid (β)beta protein, aka Aβ) gene, and the mutant gene is 2 It may be a mutant gene in which at least two bases are continuously or discontinuously substituted, deleted, or added, and the 715th amino acid from the N-terminal of APP is substituted from Val to Met.

In the present specification, "vector" or "expression vector" refers to a plasmid, viral vector, or other medium known in the art capable of inserting a nucleic acid encoding a structural gene and expressing the nucleic acid in a host cell. Means. Preferably, it may be a viral vector. Examples of the viral vector include, but are not limited to, retroviral vectors, adenovirus vectors, Herpes virus vectors, abipox virus vectors, and lentiviral vectors. In particular, a method using Sendai virus is preferred. The Sendai virus vector is constructed so that all of the viral genes have been removed or altered so that a non-viral protein is produced in the infected cells by the viral vector. The main advantages of Sendai virus vectors for gene therapy are that they deliver large amounts of genes into cloned cells, accurately insert the transferred genes into cellular DNA, and do not cause continuous infection after gene transfection.

The “vector” or “expression vector” can be introduced into cells by a method known in the art. For example, but not limited thereto, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE dextran Transfection using (DEAE Dextran-mediated transfection), transfection using polybrene (polybrene-mediated transfection), electroporation, gene gun, and other known methods for introducing nucleic acids into cells Can be introduced into cells for production of transgenic animals.

The “nerve cell” or “neuron” refers to the structure and functional unit of the nervous system in the human body, and is preferably a neuron or a neuron in the brain.

Another aspect relates to a nerve cell obtained by differentiating the induced pluripotent stem cell into a nerve cell.

Another aspect is a stem cell line containing an APP mutant gene having a V715M substitution comprising the step of introducing a vector expressing a dedifferentiation factor into a PDMC containing an APP mutant gene having a V715M substitution isolated from the blood of an Alzheimer's patient. It relates to a method of manufacturing.

Another aspect is a method of screening for a drug effective for preventing or treating Alzheimer's disease, comprising the steps of: contacting the nerve cells with a test substance;

Measuring at least one of Aβ expression amount, phosphorylated tau protein expression amount, mitochondrial motility, and imbalance between mitochondrial division and fusion in the contacted neuron;

Comparing the measured results with the measured results from the nerve cells not in contact with the test substance; It relates to a drug screening method comprising a.

The time for floating culture of the PBMC may vary depending on the growth rate of cells, and it takes 4 hours in the present invention.

The term “treatment” refers to, or includes alleviation, inhibition of progression, or prevention of a disease, disorder or condition, or one or more symptoms thereof.

The terms “administering,” “applying”, “introducing” and “implanting” are used interchangeably and in a method or route that results in at least partial localization of a patch or composition according to one embodiment to a desired site. It may mean the placement of a patch or composition according to an embodiment into an individual by.

In the step of administering an Alzheimer's prophylactic and therapeutic candidate substance to the Alzheimer's model cell line, the prophylactic and therapeutic candidate substance is, for example, a test compound or a test composition, a small molecule compound, an antibody, or an antisense nucleotide. (antisense nucleotide), short interfering RNA (siRNA), short hairpin RNA (shRNA), nucleic acids, proteins, peptides, other extracts or natural products.

Raf-1, FAK1, MEK-1, RAD51, RAD52, GADD45 GAMMA, NF-kB p52, Rb2, Oct3, Klf4 in the markers for verifying whether the peripheral blood-derived mononuclear cells are dedifferentiated into induced pluripotent stem cells. SOX2, SSEA4 and TRA-1-81.

As a specific example, the number and combination of markers selected for confirming the formation of dedifferentiated stem cells are not specifically limited, and various combinations may be selected and used. By measuring the level of expression of the biomarkers at the mRNA or protein level, it is possible to confirm whether or not dedifferentiated stem cells are formed. Since the nucleic acid sequence and protein sequence of the markers of the present invention are already known, detection of a marker for diagnosis can be usefully detected by using an agent capable of measuring mRNA or protein level based on the known sequence. . The detection of the marker protein is performed by contacting a sample with an antibody that specifically recognizes the protein and measuring its antigen-antibody complex formation. The amount of antigen-antibody complex formation can be quantitatively measured through the size of the signal of the detection label. The detection label may be selected from the group consisting of enzymes, fluorescent substances, ligands, luminescent substances, microparticles, redox molecules and radioactive isotopes, but is not limited thereto. Known assay methods for measuring protein levels include Western blot, ELISA, radioimmunoassay, radioimmunodiffusion method, octeroni immune diffusion method, rocket immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, Competitive or non-competitive analysis methods such as precipitin reaction, gel diffusion presipitin reaction, aggregation assay, fluorescence immunoassay, protein A immunoassay, FACS, protein chip, etc. may be used, but are not limited thereto. Can be done by In the above, ELISA is a direct sandwich ELISA using another labeled antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support, and reacts with another antibody that recognizes an antigen in a complex of an antibody and an antigen attached to a solid support. It includes a variety of ELISA methods such as indirect sandwich ELISA using a labeled secondary antibody that recognizes this antibody after being prepared.

By using blood cells that are easier to collect than skin cells, and using induced pluripotent stem cells of Alzheimer's patients with mutations in the amyloid precursor protein unique to Koreans, we provide customized treatments tailored to the characteristics of each patient's disease and a method of screening the treatment. do.

1, T1 is an MR image showing general cortical atrophy including the left temporal lobe, and FBB-PET is a result of increased amyloid absorption in the cerebral cortex and bilateral acinar.

Figure 2 is a family tree of the APP-V715M cell line. Patients indicated by arrows are the result of autosomal dominant genetic patterns.

3 is an expression result of undifferentiated markers such as OCT4, SOX2, Nanog, SSEA4, and Tra-1-81 in APP-V715M patients in which iPSC cell lines were established.

Figure 4 is an immunocytochemical analysis result showing the differentiation ability of iPSC cell lines forming three embryonic layers (triderm) including ectoderm (TUJ1, green), mesoderm (SMA, green), and endoderm (AFP, red). . Scale bar: 100 μm.

5 is a result showing genomic DNA sequence indicating the presence of a heterozygous V715M mutation (from GTG to ATG) in the APP gene of the APP-V715M iPSC cell line.

6 is a result of reverse transcription PCR analysis showing that there is no Sendai virus vector remaining in the cell.

7 shows the results of chromosomal karyotyping of the APP-V715M iPSC cell line.

Figure 8 is the results of in vivo teratoma analysis showing the formation of all three embryonic layers: Tuj1-positive neurons (ectoderm), cartilage (mesoderm, mesoderm) and intestinal epithelium (endoderm, endoderm).

9 is a result of detection of the expression levels of extracellular Aβ ₄₂ and Aβ ₄₀ cultured in iPSC-derived neurons by ELISA.

10 is an immunocytochemical analysis result showing the expression of Aβ precipitates anti-stained with Tuj1 (green) and DAPI (blue) using an antibody against Aβ ₄₂ (red) at 10 weeks of neuronal differentiation. Four figures (from left) are the z-stack image results of Aβ ₄₂ positive Aβ deposits (marked by arrows) in APP-V715M iPSC-derived neurons.

11 is a result of measuring the expression levels of Aβ ₄₂ and Aβ ₄₀ in TBS insoluble / SDS soluble cells in a total of 1 μg of protein using ELISA in differentiated neurons at 10 weeks.

12 is an immunocytochemical analysis result showing the expression of AT8 (phosphorylated tau protein) (red) and MAP2 (green) stained with DAPI (blue) in iPSC-derived neurons at 10 weeks of neuronal differentiation, AT8 (phosphorylated tau Protein) was expressed in soma (somatic cells) and neuritis.

13 is a result of quantification of immunocytochemical analysis normalized to MAP2-positive cells.

14 and 15 are Western blot analysis results showing an increase in AT8 and a ratio of AT8 / Tau5 in APP-V715M iPSC-derived neurons (scale bar: 10 μm).

16 is a representative chymograph result for mitochondrial movement.

17 is an average result of an anterograde speed and a retrograde speed.

18 is a representative Western blot image result of four independent proteins.

19 is a result of quantification of proteins related to mitochondrial fusion (Mfn1 and Mfn2) and division (DRP1 and Fis1).

Example 1: Manufacturing process of induced pluripotent stem cells

Monocytes (MNCs) were freshly isolated from peripheral blood of APP-V715M patients using the Ficoll-Paque™ PLUS method (GE Healthcare, USA). The isolated peripheral-derived mononuclear cells (PBMCs) were suspended in monocyte culture for 4 days and then dedifferentiated using Sendai virus vector (SeVdp) expressing four reprogramming factors (OCT3/4, SOX2, cMYC, KLF4). Became. Three or more individual clones were picked for iPSC generation and the best growing clone was selected for further analysis. The selected clones are cultured for 4 weeks to form an induced pluripotent stem cell colony.

Example 2: teratoma formation and analysis of induced pluripotent stem cells

Genotyping of the APP-V715M single nucleotide mutation was performed by DNA sequencing (Cosmo Genetech, Korea). The APP gene was amplified by PCR using the following primers (forward primer: TTC AAG GTG TTC TTT GCA GA; reverse primer: CAT AGT CTT AAT TCC CAC TTG G). For teratoma formation, undifferentiated iPSCs were harvested and implanted subcutaneously in NOG mice. The teratoma was incised and separated at 12 weeks after injection, and then fixed with 4% paraformaldehyde (PFA). The tissue embedded in paraffin was cut and stained with hematoxylin / eosin to observe whether or not tridermal (ectoderm, endoderm, mesoderm) tissue was formed.

Experimental Example 1: MRI and pedigree characteristics of Alzheimer's disease (AD) patients with APP-V715M mutation

A 54-year-old male with an APP mutation (Exon17; c.2143G> A; p.V715M) was enrolled. The patient showed mobility and rigidity and did not understand verbal commands.

As shown in Fig. 2, the patient (indicated by an arrow) developed dementia in an autosomal dominant pattern.

As shown in FIG. 1, 　 taken MRI showed cortical atrophy, and 18F-Florbetaben amyloid PET (18FBB-PET) showed significant amyloid accumulation in both striatum and associated cortex.

Experimental Example 2: Confirmation of the validation marker of iPSC obtained in AD patients with APP-V715M mutation

The following experiment was conducted to find out whether iPSCs obtained in AD patients well represent pathological features of AD. In order to investigate the pathological characteristics of patients with the APP-V715M mutation, iPSC cell lines were constructed from AD patients with the APP-V715M mutation based on Examples 1 and 2. As shown in FIG. 3, the iPSC cell line with the APP-V715M mutation shows typical expression of induced pluripotent stem cell markers, and includes OCT4, SOX2, SSEA4 and TRA-1-81. The differentiation ability of the iPSC cell line was evaluated using in vitro embryoid body formation and in vitro teratoma test, and as shown in Figs. 4 and 8, the expression of trigerm-specific markers was shown.

As shown in Fig. 5, the genotype of the established iPSC cell line was confirmed by a conventional sequencing analysis method. As shown in FIG. 6, the SeVdp vector was not inserted into the established iPSC cell line, and as shown in FIG. 7, all of the karyotypes of chromosomes were normal.

Experimental Example 3: Whether APP-V715M iPSC-derived neurons increase Aβ and p-tau

In order to investigate the amount of amyloid expression in APP-V715M iPSC-derived neurons of patients with the APP-V715M mutation at the cellular level, the following experiment was conducted. The amount of extracellular amyloid-β expression was measured using the culture medium (CM) used, which was collected from neurons (1 × 10 ⁵ ) cultured 48 hours after the last medium was replaced at 10 weeks of differentiation. Intracellular amyloid was measured in a total of 1 μg protein of neurons differentiated for 10 weeks.

Aβ ₄₀ and Aβ ₄₂ expression levels were measured extracellularly and intracellularly. As shown in FIG. 9, no significant difference in the Aβ ₄₀ expression was found, but neurons differentiated from APP-V715M iPSC showed a dramatic increase of more than 2 times in the Aβ ₄₂ expression level after 10 weeks of differentiation. In addition, the ratio of Aβ ₄₂ / Aβ ₄₀ was increased more than 2 times in the group differentiated from APP-V715M iPSC compared to the control group.

To detect Aβ precipitates, iPSC-derived neurons were stained with Aβ ₄₂ antibody at the neuron differentiation stage of 10 weeks. As shown in FIG. 10, the confocal microscopy image showed an increase in extracellular Aβ ₄₂ precipitate in neurons differentiated from APP-V715M iPSC compared to the control.

In addition, the TBS insoluble/SDS soluble fraction was extracted. The expression levels of Aβ ₄₂ and Aβ _{40 in} TBS insoluble/SDS soluble cells were measured using ELISA at 10 weeks of differentiation in a total of 1 μg of protein. Expression amount and the ratio of Aβ ₄₂ / Aβ ₄₀ in FIG as intracellular Aβ ₄₂ shown in 11 is significantly increased in APP-V715M iPSC-derived neurons.

The amount of expression of a specific protein is quantitatively measured using the Western blot method. The protein was separated on a polyacrylamide gel by electrophoresis, and transferred to a PVDF (polyvinylidene difluoride) filter. After that, after binding with the antibody (primary antibody) of the protein to be measured, a band is formed using a secondary antibody that reacts with this antibody. Based on the size marker for measuring the molecular weight of the protein, the band located at the molecular weight size of the protein to be viewed is quantified.

The amount of expression is measured by labeling a specific protein expressed with a fluorescent substance using an immunocytochemical analysis method. After binding the cells immobilized with 4% PFA with the primary antibody of the protein to be measured, the expression level of the protein to be viewed is observed using a secondary antibody to which a fluorescent substance reacting with this antibody is attached. It is possible to know whether a specific protein is present in the experiment group compared to the control group, and furthermore, it is possible to check whether the protein is present in a specific part of the cell.

It has been demonstrated in recent in vitro experiments that the increase and accumulation of Aβ peptides can induce tangle of nerve fibers composed of aggregated hyper-phosphorylated tau proteins. The Tau protein is usually present in a soluble form in the axon of mature neurons. However, when hyperphosphorylated, tau protein is abnormally accumulated in dendrite and cell bodies in AD. In order to investigate the expression level of phosphorylated tau protein in APP-V715M iPSC-derived neurons, immunocytochemical analysis was performed using an antibody against AT8 (phosphorylated at Ser202 / Thr205). As shown in FIG. 13, the 　APP-V715M iPSC-derived neurons showed a significant increase in AT8 expression in neurite and soma when compared with the control group.

In addition, as shown in Figs. 14 and 15, Western blot was performed to measure the amount of AT8 protein expression, indicating an increase in the amount of phosphorylated tau protein in APP-V715M iPSC-derived neurons. It was demonstrated that APP-V715M iPSC-derived neurons showed significant increases in both extracellular and intracellular Aβ expression levels and phosphorylated tau protein expression levels.

Experimental Example 4: Mitochondrial motility disorder and imbalance observation of mitochondrial fusion and division in neurons derived from APP-V715M iPSC

To investigate mitochondrial movement, imaging analysis of live cells was performed using a Mito-tracker (Ds-Red) at 10 weeks of continuous differentiation. Live cells were imaged using a Leica TCSSP5II confocal microscope. Neurons differentiated for 10 weeks were cultured with Mito-tracker red (Thermo-Fisher Cat.M7512) for 15 minutes to perform live cell imaging (LCI) analysis. The cells were maintained at 37 °C and 5% CO2 / 95% O ₂ (Live Cell Instrument, Seoul, Korea) was supplied during imaging. Slow image recording lasted up to 4 minutes 30 seconds at 2 second intervals. Mitochondrial chymographs were analyzed using KymographClear, a set of imageJ macro tools capable of generating chymographs from image sequences. Quantitative analysis of mitochondrial velocity was performed using KymographDirect.

As shown in Fig. 17, the anterior velocity and the posterior velocity were significantly decreased in APP-V715M iPSC-derived neurons compared to the control group.

In addition, mitochondrial fusion and fission-related proteins, including mitochondria fusion-related proteins Mfn1 (membrane protein mitofusin 1), Mfn2 (membrane protein mitofusin 2), and mitochondria fission-related proteins DRP1 (dyinamine-related protein 1) and Fis1 (mitochondrial fission 1 protein). The expression pattern of was investigated. In particular, the amount of DRP1 expression was significantly increased in APP-V715M iPSC-induced neurons compared to the control group. This strongly suggests that high expression levels of Aβ and phosphorylated tau proteins can interfere with mitochondrial transport. This may be due to the impaired balance of mitochondrial fusion and division in APP-V715M iPSC-derived neurons. As a result of Western blot analysis as shown in FIGS. 18 and 19, it was found that the cleavage-related markers (DRP1 and Fis1) were increased, and the fusion-related markers (Mfn1 and Mfn2) were significantly decreased in the APP-V715M iPSC-derived neurons.

Claims (14)

1. Induced pluripotent stem cells dedifferentiated from human PBMCs containing an APP mutant gene with a V715M substitution.
2. The stem cell of claim 1, wherein the dedifferentiation comprises introducing a vector expressing a dedifferentiation factor into the PBMC.
3. The stem cell of claim 2, wherein the vector is a Sendai virus vector expressing OCT3/4, SOX2, cMYC and KLF4.
4. The stem cell of claim 1, wherein the stem cell expresses OCT4, SOX2, SSEA4 and TRA-1-81.
5. A nerve cell obtained by differentiating the stem cell of any one of claims 1 to 4 into a nerve cell.
6. The neuron according to claim 5, wherein the differentiation is made by a method comprising a neuroprecursor step.
7. The neuron according to claim 5, which expresses an APP mutant gene having a V715M substitution.
8. The neuron according to claim 5, which has a high expression amount of amyloid beta, a high expression amount of phosphorylated tau, mitochondrial dyskinesia, and an imbalance of mitochondrial division and fusion.
9. To prepare a stem cell line containing an APP mutant gene having a V715M substitution comprising the step of introducing a vector expressing a dedifferentiation factor into PDMC containing an APP mutant gene having a V715M substitution isolated from the blood of Alzheimer's patients. Way.
10. The method of claim 9, wherein the vector is a Sendai virus vector expressing OCT3/4, SOX2, cMYC, and KLF4.
11. The method according to claim 9, comprising the step of selecting an induced pluripotent stem cell by checking the expression of OCT4, SOX2, SSEA4, and TRA-1-81 in the peripheral-derived mononuclear cells in which the dedifferentiation is induced.
12. A method for producing a neuron expressing an APP mutant having a V715M substitution comprising the step of differentiating a neuron from the induced pluripotent stem cell of claim 9.
13. As a method of screening for drugs effective in preventing or treating Alzheimer's disease,

    Contacting the nerve cell of claim 5 with a test substance;

    Measuring at least one of Aβ expression amount, phosphorylated tau protein expression amount, mitochondrial motility, and imbalance between mitochondrial division and fusion in the contacted neuron;

    Comprising the step of comparing the measured result with the measured result from the nerve cell of claim 5 not in contact with the test substance.
14. The method of claim 13, wherein when one or more of the measurement results of a decrease in the Aβ expression level, a decrease in the phosphorylated tau protein expression level, an increase in mitochondrial motility, and a decrease in mitochondrial fusion and division imbalance appear, the test substance is used to prevent or treat Alzheimer's disease. The method further comprising the step of selecting as an effective drug.

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