KR102364890B1 - Expression cassette and vector with genes related alzheimer's disease and transgenic human pluripotent stem cell line made from it and alzheimer's diseae organoid and neuron model differentiated from this cell line - Google Patents

Abstract

The present invention relates to an expression cassette and a vector comprising Alzheimer's disease (AD)-related genes, a cell line transformed using same, and an organoid and a neuronal cell produced using the same, and more specifically, the expression cassette of the present invention comprises mutant genes of amyloid precursor protein (APP) and preseniline-1 (PS1), which are AD-related genes so as to be simultaneously expressed and maintained. Therefore, the present invention can provide an AD model having physiological characteristics of human cells/tissues by using human cells, and the disease model can be used as a platform for AD pathological mechanism research and drug screening. In addition, the present invention can provide a novel human disease model platform for AD molecular mechanism research. The present invention, reproducing pathological phenotypes of AD in two-dimensional and three-dimensional models, may contribute to the development of accurate human models for other diseases.

Description

Expression cassette and vector containing Alzheimer's disease-related mutant gene, human totipotent stem cell line transformed using the same, and Alzheimer's disease organoid and neuron model differentiated therefrom {EXPRESSION CASSETTE AND VECTOR WITH GENES RELATED ALZHEIMER'S DISEASE AND TRANSGENIC HUMAN PLURIPOTENT STEM CELL LINE MADE FROM IT AND ALZHEIMER'S DISEAE ORGANOID AND NEURON MODEL DIFFERENTIATED FROM THIS CELL LINE}

The present invention relates to an expression cassette comprising a gene related to Alzheimer's disease, a vector, a human pluripotent stem cell line transformed using the same, and brain organoid and neuronal Alzheimer's disease modeling using the same, and more specifically, the expression cassette of the present invention contains these genes so that APP (amyloid precursor protein) and PSEN1 (preseniline 1:Preseniline-1) mutant genes associated with Alzheimer's disease can be expressed simultaneously. The present invention also relates to a human totipotent stem cell line transformed using an expression cassette or vector containing the above genes, and further to a three-dimensional organoid and two-dimensional neuronal cell model derived from the cell line.

Alzheimer's disease is one of the degenerative brain diseases and accounts for 60% of dementias that cause cognitive loss due to the gradual degeneration of nerve cells. Depending on the cause, Alzheimer's disease is divided into familial Alzheimer's disease, which is caused by genetic factors, and sporadic Alzheimer's disease, which occurs in a large number of patients although the exact cause is unknown. When the brain tissue of a patient who died from Alzheimer's disease was examined, characteristic lesions such as neuritic plaque (or senile plaque) and neurofibrillary tangles were observed. General brain atrophy is seen.

Among them, neurites are formed by the accumulation of proteins and dead cells outside the cell, and their main component is a peptide called β-amyloid (Aβ) consisting of 42 or 43 amino acids. The cytoskeleton is abnormally agglomerated around the excessively phosphorylated Tau protein and looks like a bundle of threads.

It is known that APP and PSEN1, which are known as the representative causative genes of Alzheimer's disease, contribute to the overexpression of β-amyloid and the aggregation of Tau protein, which are the etiological mechanisms causing Alzheimer's disease.

β-Amyloid is produced from amyloid precursor protein (APP) through proteolysis. APP, a proprotein, is a protein having one transmembrane domain. It is expressed in several isotypes by alternate splicing and is known to pass through two metabolic pathways in the cell. One is a pathway in which p3 and sAPPα are produced by α-secretase and γ-secretase, and the other is a pathway in which β-amyloid and APPβ are produced by β-secretase and γ-secretase. Mutations in this APP protein are found in patients with familial Alzheimer's disease. Mutations discovered so far include APP670/671 (Swedish), APP672 (Flemish), APP716 (Florida), APP717 (London), and the like, and it has been found that the formation of β-amyloid is increased in these mutations.

Another gene that exhibits a mutation that causes familial Alzheimer's disease is presenilin 1 (PSEN1). PSEN1 is a protein with eight transmembrane domains that plays an important role in the development process and is known to act as a member of γ-secretase itself or a complex. More than 45 mutations that cause familial Alzheimer's disease have been reported throughout PSEN1 protein, and these mutations are also known to increase the amount of β-amyloid formation.

The onset of Alzheimer's disease due to the produced β-amyloid is accompanied by a process of nerve cell damage caused by hyperphosphorylation of Tau protein, and it is known that several kinases act on this hyperphosphorylation of Tau protein. In addition to Tau hyperphosphorylation, Tau tangle formation also plays a role in damaging nerve cells, and mutations in Tau that form tangles well were found.

In addition to aging of human brain cells, elucidating the mechanism of β-amyloid accumulation and aggregation and Tau protein tangle formation is expected to play an important role in the treatment of Alzheimer's disease. , and the need to establish a cell line or animal model in which the Tau gene mutation is expressed simultaneously.

Several studies have attempted to establish transgenic mice for the study of the pathogenesis of Alzheimer's disease. The transgenes that have been the main targets of these trials are genes such as APP, PSEN1, Tau and ApoE4, which have been identified as causative genes in familial Alzheimer's disease. The transgenic mouse, which is mainly used in the present study, is a model that uses APP or PSEN1 gene mutations to increase the concentration of β-amyloid in the brain to form neurites. However, since it is difficult to know precisely the pathogenesis of Alzheimer's disease with β-amyloid alone, recent attempts have been made to simultaneously insert the Tau mutant gene. This is to create a model closer to human Alzheimer's disease by simultaneously expressing β-amyloid and Tau in the brain of transgenic mice.

In addition, when APP and PSEN1 are present in mutant forms at the same time, β-amyloid can be produced at an earlier stage. In TG2576, the first-generation transgenic mouse, the accumulation of β-amyloid in the brain starts after 12 months of age or more, whereas in the case of 5XFAD or APP/PSEN1 mice, β-amyloid accumulation begins within 6 months. Since the Duff group succeeded in developing a double transgenic mouse obtained by crossing APP and PSEN1 single transgenic mice in 1996, this transgenic mouse has been used in several studies to date. In fact, in the case of double transgenic mice, it can be observed that the period of spot formation is shortened from 3 months to 9 months due to the synergistic effect of the two genes. However, even in the development of various AD animal models, it is often difficult to apply the results derived from studies using animal models to humans due to genetic, physiological/pathological differences between animals and humans.

In order to improve this problem, the inventors of the present invention studied to produce an Alzheimer's disease model having human cell/tissue physiological characteristics using human cells, and as a result, the present invention was completed.

KR 10-2018-0130060

One object of the present invention is a) an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated, and b) amino acids 146 and 286 are To provide an Alzheimer's disease-related gene expression cassette comprising a PSEN1 mutant gene encoding a mutated presenilin 1 (PSEN1).

Another object of the present invention is to provide a recombinant expression vector comprising the expression cassette.

Another object of the present invention is to provide a cell line transformed using the recombinant expression vector.

Another object of the present invention is to provide nerve cells and organoids derived from the cell line.

One aspect of the present invention is a) an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated, and b) amino acids 146 and 286 are To provide an Alzheimer's disease-associated gene expression cassette comprising a PSEN1 mutant gene encoding a mutated presenilin 1 (PSEN1).

The APP mutant gene is an APP mutant gene encoding an amyloid precursor protein (APP) in which amino acids 176, 670, 671 and 717 are mutated. Specifically, the mutation of the 176 amino acid is I176V mutation (Florida, FL), the 670 amino acid mutation and the 671 amino acid mutation are K670/M67L (Swedish, Swe), respectively, and the 717 amino acid mutation is V717I (London) .Lon) mutation.

The PSEN1 mutant gene is a PSEN1 mutant gene encoding presenilin 1 (PSEN1) in which amino acids 146 and 286 are mutated. Specifically, the 146 amino acid mutation is M146L, and the 286 amino acid mutation is L286V.

As an embodiment of the present invention, the APP mutant gene may have the sequence of SEQ ID NO: 1. Specifically, the APP mutant gene is a mutation of amino acids 176, 670, 671 and 717 as described above compared to the normal APP gene of SEQ ID NO: 2.

As another embodiment of the present invention, the PSEN1 mutant gene may have the sequence of SEQ ID NO: 3. Specifically, the PSEN1 mutant gene is one in which amino acids 146 and 286 are mutated as compared to the normal PSEN1 gene of SEQ ID NO: 4.

As an embodiment of the present invention, the Alzheimer's disease-related gene expression cassette may include a promoter to further improve gene expression, and the promoter is a CMV promoter (eg, human or mouse CMV immediate -early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, SV40 promoter, adenovirus promoter (major late promoter), pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, tk promoter of HSV, SV40E1 promoter, respiratory syncytial virus (RSV) promoter, metallotionin promoter, β-actin promoter, ubiquitin C promoter, human interleukin-2 (IL-2) gene promoter, human lymphotoxin gene promoter, human granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter and CAG promoter selected from the group consisting of However, specifically, it may be a CAG promoter.

The CAG promoter is a kind of modified CMV promoter, cytomegalovirus immediate-early enhancer, chicken β-actin promoter, chimeric intron (chimeric intron), exon 1 (exon) 1) and a complex promoter including exon 2 of the rabbit β-globin gene (Hitoshi Niwa et al., Gene, 108:193-199, 1991, Monahan et al., Gene Therapy, 7:24-30, 2000). In the present invention, the CAG promoter allows expression to be maintained without disappearing for a long period of time even after differentiation induction after the expression cassette of the present invention is introduced into stem cells.

In another embodiment of the present invention, the gene expression cassette may have the sequence of SEQ ID NO: 5. Specifically, the gene expression cassette may include a sequence for its expression together with the above-described APP mutant gene and PSEN1 mutant gene.

The Alzheimer's disease-related gene expression cassette may further include an enhancer to further improve gene expression, and the enhancer may include a known enhancer.

The gene expression cassette of the present invention is contained in a recombinant expression vector, a transgenic human pluripotent stem cell line, and a three-dimensional organoid and two-dimensional neuron produced using the same, which will be described later, so as to have human cell/tissue physiological characteristics Alzheimer's disease model can be provided.

Another aspect of the present invention is to provide a recombinant expression vector comprising the expression cassette.

As used herein, the term "recombinant expression vector" refers to a recombinant DNA molecule comprising a desired coding sequence and an appropriate nucleic acid sequence essential for expressing a coding sequence operably linked in a specific host organism. Promoters, enhancers, termination signals and polyadenylation signals available in eukaryotes are known.

As used herein, the term "operably linked" refers to a functional linkage between a gene expression control sequence and another nucleotide sequence. The gene expression control sequence may be at least one selected from the group consisting of an origin of replication, a promoter, and a terminator. The transcription termination sequence may be a polyadenylation sequence (pA), and the origin of replication may be an f1 origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, or a BBV origin of replication, but is not limited thereto. .

The recombinant expression vector according to an embodiment of the present invention is selected from the group consisting of viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, lentiviral vectors, retroviral vectors and adeno-associated viral vectors. it could be Vectors that can be used as recombinant expression vectors include plasmids used in the art (eg, pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1). , pHV14, pGEX series, pET series, pUC19, etc.), phage (eg, λgt4λB, λ-Charon, λΔz1, M13, etc.) or viral vectors (eg, adeno-associated virus (AAV) vectors, lentiviral vectors etc.) may be manufactured on the basis of, but is not limited thereto.

The recombinant expression vector of the present invention may further include one or more selectable markers. The marker is a nucleic acid sequence having characteristics that can be selected by conventional chemical methods, and includes all genes capable of distinguishing a transfected cell from a non-transfected cell. For example, herbicide resistance genes such as glyphosate, glufosinate ammonium or phosphinothricin, ampicillin, kanamycin, G418, Bleomycin ), hygromycin (hygromycin), may be an antibiotic resistance gene such as chloramphenicol (chloramphenicol), but is not limited thereto.

The production of the recombinant expression vector of the present invention can be prepared using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art. there is.

The recombinant expression vector comprises a restriction enzyme site, constructing a first vector from which the promoter and gene cluster are removed; preparing a recombinant second vector by inserting a promoter, a mutant APP gene, and a mutant PSEN 1 gene into the second vector; And inserting the second recombinant vector into the first vector: can be prepared through a recombinant expression vector manufacturing method comprising:.

The recombinant expression vector of the present invention can provide an Alzheimer's disease model with human cell/tissue physiological characteristics by being included in a recombinant cell line and three-dimensional organoid to be described later including the gene expression cassette described above.

Another aspect of the present invention is to provide a cell line transformed using the recombinant expression vector.

In order to prepare a transformed cell line into which the recombinant expression vector according to an embodiment of the present invention is introduced, a method known in the art for introducing a nucleic acid molecule into an organism, cell, tissue or organ can be used, As is known, it can be carried out by selecting an appropriate standard technique according to the host cell. Such methods include, for example, electroporation, calcium phosphate (CaPO4) precipitation, calcium chloride (CaCl2) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic liposome method , and lithium acetate-DMSO method and the like may be included, but are not limited thereto.

In one embodiment of the present invention, the cell line may be a human adult stem cell (hASC), a human embryonic stem cell (hESCs), or a human pluripotent stem cell (hiPSCs). And, specifically, it may be a human embryonic stem cell.

The transformed cell line may be prepared by a method for producing a transformed cell line comprising the step of transforming with the recombinant expression vector.

Another aspect of the present invention is to provide a nerve cell derived from the cell line.

The nerve cell is the cell line differentiated into a nerve cell, and may be prepared by a known method including a known material necessary for differentiation.

Another aspect of the present invention is to provide an organoid derived from the nerve cell.

As used in the present invention, "organoid" refers to a cell mass having a structure, cell components, and functions similar to those of living (human) organs. The organoid of the present invention may include a two-dimensional cell tissue cultured on a plate as well as a three-dimensional cell mass. Specifically, the organoid may be a tissue differentiated into a three-dimensional brain organoid or a two-dimensional nerve cell.

The organoid may be prepared through a method for preparing an organoid comprising the step of culturing the cell line.

The present invention can provide an Alzheimer's disease model with human cell/tissue physiological characteristics using human cells.

1 is a conceptual diagram illustrating a method for producing a gene expression cassette and brain organoids of the present invention.
2 is a graph showing the results of confirming the levels of Aβ 1-40 and 1-42 in CmCPP control, CAPPP and CACPP brain organoids.
3 is a view showing the Western blot results confirming the phosphorylation of tau in CmCPP control, CAPPP and CACPP brain organoids.
4 and 5 are photographs showing the results of immunochemical analysis of CmCPP control, CAPPP and CACPP brain organoids and 5xFAD model.
Figure 6 is to investigate the usefulness of the FAD brain organoids (COs) model as a means for drug development, CACPP brain organoids (CO) BACE1 inhibitor IV (BSI), aβ-secretase inhibitor or compound E (CE), a It is a conceptual diagram showing the process of treatment with γ-secretase inhibitor.
7 is a diagram showing changes in the levels of Aβ 1-40 and 1-42 in CACPP brain organoids (CO) treated with BSI or CE.
8 is a diagram showing the change in p-tau expression in CACPP brain organoids (CO) treated with BSI or CE.
9 is a view showing the results of ELISA analysis showing that the levels of Aβ 1-40 and 1-42 significantly increased in CACPP neuronal cells compared to the CmCPP control group.
FIG. 10 is a view showing Western blot results showing that the level of p-tau in CACPP neuronal cells is significantly changed compared to CmCPP control.
11 is a view showing the results showing the change in PHF-1 expression in CACPP neurons compared to the CmCPP control.
12 is a conceptual diagram illustrating an experimental procedure for confirming the effect of BSI or CE on a neuron model.
13 is a diagram showing the results of changing the levels of Aβ 1-40 and 1-42 by treatment with BSI or CE in CACPP neurons.
14 and 15 are diagrams showing the results of changing the level of p-tau by treatment with BSI or CE in CACPP neurons.

Hereinafter, one or more specific examples will be described in more detail through examples. However, these examples are for illustrative purposes of one or more embodiments, and the scope of the present invention is not limited to these examples.

Materials and Methods

Human embryonic stem cell culture

Human embryonic stem cell lines (hESC, SNUES31) were provided by the 'Institute of Reproductive Medicine and Population', a medical research center at Seoul National University Hospital in Korea. hESCs were cultured in mitomycin-C-treated mouse embryonic fibroblasts at 10 μg/mL, hESC medium (20% knockout serum replacement, 1% minimum essential media-nonessential amino acids (MEM-NEAA) (Invitrogen), 1% Glutamax (Invitrogen), 20 ng/mL basic fibroblast growth factor (bFGF), 7 μL/L β-mercaptoethanol in DMEM/F-12). For feeder-free hESC culture, hESCs were isolated from feeder cells using 1 mg/mL dispase® (Invitrogen), and cultured in Essential-8 medium (Invitrogen) on Geltrex (Invitrogen)-coated culture plate. became hESCs were subcultured into small clusters using 0.5 mM EDTA solution every 4 days.

Preparation of FAD hESC cell lines

Full-length human amyloid precursor protein (APP) containing K670M/N671L (Swedish), I716V (Florida) and V717I (London) mutations (APPSweFlLon) and presenilin containing M146L and L286V mutations (PSEN1M146L/L286V) The construct encoding -1 (PSEN1) was obtained from brain tissue of an 8-month-old 5xFAD mouse (The Jackson Laboratory).

First, RNA was extracted using the RNeasy Plus RNA extraction kit (Qiagen). Reverse transcription was performed using the iScript™ cDNA synthesis kit (Bio-Rad). APPSweFlLon and PSEN1M146L/L286V cDNAs were PCR-amplified with restriction enzymes using KOD-PlusNeo (Toyobo). The primers used for replication were as follows:

APP-BamH I-F, 5'-GAATGGATCCATGCTGCCCGGTTTGGCACTG-3' and

APP-Mlu I-R, 5'-GAATACGCGTCTAGTTCTGCATCTGCTCAAA-3';

PSEN1-BamH I-F, GAATGGATCCATGACAGAGTTACCTGCACCG-3' and

PSEN1-Mlu I-R, GAATACGCGTCTAGATATAAAATTGATGGAA-3'.

Next, edit the pLV-mcherry, pSico-PGK-Puro and pCDH-EF1α-MCS-IRES-RFP (System Bioscience) plasmids to create the lentiviral dual promoter plasmid vectors CAG-MCS-PGK-Puro and CAG-MCS-PGK-mCherry was produced. pLV-mCherry was provided by Pantelis Tsoulfas (Addgene plasmid # 36084; http://n2t.net/addgene:36084 ; RRID:Addgene_36084) and pSico-PGK puro was provided by Tyler Jacks (Addgene plasmid # 11586; http://n2t) .net/addgene:11586 ; RRID:Addgene_11586). And, APP ^SweFlLon , PSEN1 ^M146L/L286V , APP ^SweFlLon -IRES-PSEN1 ^M146L/L286V was inserted, which was prepared by introducing the amplified genes of APP ^SweFlLon or PSEN1 ^M146L/L286V , or the mCherry gene into MCS, and four constructs of CAG-APP ^SweFlLon -IRES-PSEN1 ^M146L/L286V -PGK-Puro (CAPPP), CAG-APP ^SweFlLon -PGKPuro (CAPP), CAG-PSEN1 ^M146L/L286V -PGK-mCherry (CPPmC) and CAG-mCherry-PGK -Puro (CmCPP) was obtained. All newly constructed vectors were confirmed by sequence analysis (Enzynomics, Seoul, Korea). For CAPPP, CAPP, CPPmC or CmCPP lentivirus construction, each plasmid is a viral packaging plasmid [VSV-G expression envelope plasmid and a plasmid containing gag, pol and rev genes, which are prepared by Dr. HEK293T (ATCC) cells cultured in 100 mm tissue culture plates were transfected using X-tremeGene HP DNA transfection reagent (Roche) together with Yibing Qyang (provided by Yale Cardiovascular Research Center, Yale School of Medicine, CT, USA)]. was introduced, and incubated at 37° C., 5% CO ₂ . The culture medium was changed 24 hours after transduction, and 10 mL of the virus-containing culture medium was collected every day for 3 days. Each 30mL of the collected culture solution was concentrated to 200μL by ultracentrifugation (Hitachi) at 55,200g and 4℃ for 2 hours. Next, by changing the culture medium of hESCs cultured without a feeder in a 12-well cell culture plate (corning) to an Essential-8 culture medium containing 20 μL of concentrated virus and 2 μg/mL polybrene (hexadimethrine bromide, sigma-aldrich). Transduction of CmCPP, CPPmC or CAPPP genes via viral infection was induced. The culture medium was changed every 3 days. hESCs introduced with the CAPPP gene were selected after treatment with 5 μg/ml puromycin (Invitrogen). This cell line was designated as the CAPPP hESC line. hESCs introduced with CmCPP and CPPmC viruses were separated into single cells, and then 500 cells were inoculated into a 6-well plate STO-plated well (corning). After 7 days of culture, single cell-derived mCherry-expressing colonies were manually isolated, transferred to a Matrigel-coated 12-well tissue culture plate, and cultured in a feeder-free state. These cell lines were named CmCPP and CPPmC hESC lines, respectively. After the cells were stabilized, CPPmC hESCs were cultured for 24 hours by replacing the culture medium with Essential-8 medium containing 20 µL of concentrated CAPP virus and 2 µg/mL polybrene, thereby induced CAPP gene introduction through virus infection. Thereafter, CPPmC hESCs into which the CAPP gene was introduced were selected using 5 μg/ml puromycin. The hESC cell line double-infected with CPPmC and CAPP lentivirus was named CACPP hESC line. Each hESC was imaged using a fluorescence microscope (DM IL LED Fluo, Leica).

Brain organoid culture conditions

Cerebral organoids (COs) were prepared from hESCs using modified protocol 7.

-mercaptoethanol in 1:1 mixture of DMEM/F-12 and neurobasal medium]과 함께 옮겨주었다. 그 이후 배양액은 7일에 한 번씩 바꿔주었다. Briefly, hESCs cultured without a feeder were digested into single cells by incubation in 0.5 mM EDTA for 4 min, and incubated in Accutase® for 4 min. And the cells were 96-well ultra low-attachment U- at a density of 9x10 ⁴ cells/cm ² using low bFGF hESC culture medium containing 4 ng/ml bFGF and 50 μM Y27632, a Rho-associated protein kinase inhibitor (Tocris). Cultured on a bottom plate (Corning). The corresponding day was designated as day 0, and on day 2, the culture medium was changed to a hESC culture medium containing 5 ng/ml bFGF and 50 μM Y27632. On day 4, it was changed to hESC culture without bFGF and Y27632. On day 6, each embryoid body (EBs) was treated with 500 μl neural induction medium [1% N2 supplement (Invitrogen), 1% Glutamax (Invitrogen), MEM-NEAA, 1 μg/ml Heparin (Sigma-) aldrich) in DMEM/F-12] and transferred to a 24-well ultra low-attachment plate. On day 8, 500 μl of neural induction medium was added. On day 10, neuroepithelial tissues were transferred to 20 μl matrigel (BD Bioscience) droplets on a 3 mm grooved Parafilm, and left at 37° C. for 1 hour. The matrigel droplet contains 5ml brain organoid (CO) differentiation culture medium without vitamin A [0.5% N2 supplement, B27 supplement minus vitamin A (Invitrogen), 2.5 μg/mL human insulin (Roche), 1% Glutamax, 0.5% MEMNEAA, 3.5 μl/l β-mercaptoethanol in 1:1 mixture of DMEM/F-12 and neurobasal medium (Invitrogen)] was transferred to a 60 mm tissue culture dish (corning). The culture medium was changed every day, and on day 14, matrigel droplets containing brain organoids) were placed in a 125 ml bioreactor with a brain organoid differentiation culture medium [0.5% N2 supplement, B27 supplement (Invitrogen), 2.5 μg/mL human insulin 1% , Glutamax, 0.5% MEM-NEAA, 3.5 μl/l

-mercaptoethanol in 1:1 mixture of DMEM/F-12 and neurobasal medium]. After that, the culture medium was changed once every 7 days.

nerve cell differentiation

receptor inhibitor 및 2 μM XAV939, a tankyrase inhibitor가 첨가된 신경세포 배양액으로 교체하였고 매 7일마다 배양액을 교체하였다. day19에 배양액은 신경세포 성숙 배양액 (neuronal maturation medium) (neurobasal medium supplemented with 1% N2 supplement, 2% B27 supplement, 1% Glutamax and 25 ng/ml BDNF) 으로 바꿔주었고, 이후 매일 바꿔주었다. day25에, 세포들은 Accutase®로 단일세포 분리 후, 5 x 10⁴ cells/cm² 밀도로 재접종하였다. 약물 처리의 경우, 1 μM β-secretase inhibitor IV (BSI) 또는 10 nM compound E (CE)를 함유하는 신경세포 성숙 배양액을 이용하였다 (도 12). To induce neuronal differentiation, feeder-free hESC cell lines were separated into single cells by incubation with Accutase® for 5-8 minutes, and 10 μM Y27632, a Rhoassociated protein kinase inhibitor was added at a density of 5 x 10 ⁴ cells/cm ² Inoculated on Matrigel-coated 24-well plates containing mTeSR ^TM 1. This time was designated as day-4. The next day, the culture medium was changed to mTeSR ^TM 1 without Y27632, and the culture medium was changed daily for additional 2 days. In order to induce neuronal differentiation, the mTeSR ^TM 1 culture medium is a neuronal cell induction medium (1:1 mixture of DMEM/F12 medium and neurobasal medium supplemented with 1% N2 supplement, 2% B27 supplement, 10 μg/ml human Insulin, 1% Glutamax, 1% NEAA and 3.5 μl/l β-mercaptoethanol) were changed (day 0) and the culture medium was changed every day for 10 days. Cells differentiated on day 11 were single-celled with Accutase®, and then inoculated again on Matrigel-coated 24-well plates at a density of 1.5x10 ⁵ cells/cm ² . On day 13, the culture medium was 100 nM LDN193189, selective BMP type I receptor inhibitor, 10 μM SB431542, a selective TGF-

The receptor inhibitor and 2 μM XAV939, a tankyrase inhibitor were added to the neuron culture medium, and the culture medium was replaced every 7 days. On day 19, the culture medium was changed to a neuronal maturation medium (neurobasal medium supplemented with 1% N2 supplement, 2% B27 supplement, 1% Glutamax and 25 ng/ml BDNF), and thereafter changed daily. On day 25, cells were re-inoculated at a density of 5 x 10 ⁴ cells/cm ² after single cell isolation with Accutase®. For drug treatment, a neuronal maturation culture medium containing 1 μM β-secretase inhibitor IV (BSI) or 10 nM compound E (CE) was used ( FIG. 12 ).

Immunocytochemistry

Cell clusters on Matrigel-coated coverslip were fixed with 3.2% paraformaldehyde phosphate buffer solution (PBS) (Wako) at 4°C for 2 hours. Then, cells were permeabilized with PBST (0.1% [vol/vol] Triton X-100 in PBS) and blocked with 10% normal goat serum (Vector). Cells were diluted with the primary antibody in PBST containing 2% normal goat serum and left overnight at 4°C. The primary antibodies used were:

Oct4 (mouse, Santa Cruz sc- 365509, 1:100),

nanog (rabbit, Cell Signaling 3580,1:100),

SSEA4 (mouse, Santa Cruz sc-21704,1:100),

Tra-1-60 (mouse, Santa Cruz sc-21705,1:100),

PHF-1 (mouse, Biolegend 836101, 1:500)

After washing 3 times with PBST, the cells were left with secondary antibody formation at room temperature for 3 hours. After washing three times with PBST, a coverslip was attached to a slide glass using Vectashield mounting medium (Vector Laboratories) and imaged using a confocal microscope (Leica TCS SP5 II, Leica).

Histology and immunofluorescence

Brain organoids were fixed with a 3.2% paraformaldehyde phosphate buffer solution at 4° C. for 2 hours, and sequentially immersed in 15% and 30% sucrose solutions until the brain organoids subsided. Brain organoid samples were placed in OCT compound (Leica) and cryosectioned (cryosectioned) to a thickness of 20 μm using a Leica CM1850 cryostat (Leica). For immunochemical staining, tissue sections were immersed in PBST (0.1% [vol/vol] Triton X-100 in PBS) containing 10% normal goat serum at room temperature for 1 hour. Tissue sections were also incubated overnight at 4°C with primary antibody in PBST containing 2% normal goat serum. The primary antibodies used were:

PHF-1 (mouse, Biolegend 836101, 1:500),

TUJ1 (rabbit, Cell Signaling 5666, 1:100),

SOX2 (rabbit, Cell Signaling 3579, 1:100),

TUJ1 (mouse, R&D systems MAB1195, 1:200),

TBR2 (mouse, R&D systems MAB6166, 1:100),

DCX (rabbit, Cell Sgnaling 4604, 1:100),

PAX6 (rabbit, Biolegend 901301, 1:200),

MAP2 (rabbit, Cell Signaling 4542, 1:100),

MAP2 (mouse, abcam ab11267, 1:300) and

N-cadherin (rabbit, Santa Cruz sc-7939, 1:100)

After washing 3 times with PBST, the cells were incubated with the secondary antibody for 3 hours at room temperature. As a secondary antibody, Donkey Alexa Fluor 488 and 647 conjugates (Life technologies, 1:500) were used. After washing 3 times with PBST, coverslips were attached to slides using Vectashield mounting medium (Vector Laboratories) and imaged using a confocal microscope (Leica TCS SP5 II, Leica).

Western blot analysis

Each brain organoid or nerve cell was subjected to sonication (Vibra-Cell) with a lysis buffer (iNtRON biotechnology) on ice. Cell lysates were separated by 10-12% sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride transfer membranes (Millipore). The blot was washed with TBST (10 mM Tris-HCl [pH 7.6], 150 mM NaCl, 0.1% Tween-20), blocked with 5% skim milk (Millipore) after 1 hour, and left overnight at 4° C. together with the primary antibody. . The primary antibodies used were:

anti-phospho-tau (mouse, Biolegend, 836101, 1:1000),

anti-Tau (mouse, Cell Signaling 4019, 1:1000),

anti-amyloid precursor protein (APP) (moouse Cell Signaling 2450, 1:1000),

anti-presenilin (PSEN)1 (rabbit, Cell Signaling 5643, 1:1000) and

anti-β-actin (mouse, Santa Cruz sc-47778, 1:1000).

The primary antibody was goat anti-rabbit (Santa Cruz sc-2004, 1:10000) or goat anti-mouse (Santa Cruz sc-2005, 1:10,000) conjugated to horseradish peroxidase (HRP). It was detected with IgG. The bands were visualized with an enhanced chemiluminescence solution (Thermo Scientific), and images were acquired using an ImageQuant LAS 4000 Mini (GE Healthcare).

ELISA analysis

Aβ 1-40 and Aβ 1-42 levels were measured with a commercially available human amyloid-β ELISA Kit (Immuno-Biological laboratories Inc.). Each brain organoid (COs) was homogenized by ultrasonication with a lysis buffer on ice, and the supernatant was collected after centrifugation. ELISA was performed according to the manufacturer's instructions. Aβ 1-40 and Aβ 1-42 ELISA signals were quantified using an ELISA plate reader (BioTek, Winooski, VT, USA).

statistical analysis

All results were expressed as mean ± standard error of mean (S.E.M). Significance was analyzed using Student's t-test. Values of p<0.05 were considered statistically significant.

Experiment result

The pathological phenotypic expression of Aβ and tau phosphorylation was confirmed in FAD brain organoids. Aβ 1-40 and 1-42 levels were measured and compared using 70-day-old controls and FAD brain organoids. As confirmed in FIG. 2 , the levels of Aβ 1-40 and 1-42 were significantly increased in CAPPP and CACPP brain organoids compared to CmCPP control brain organoids. In addition, a significant increase in tau phosphorylation (phosphorylated at Ser396, Ser404) was confirmed in CAPPP and CACPP brain organoids through Western blot analysis (FIG. 3). These results were also confirmed by immunochemical analysis ( FIGS. 4 and 5 ), and Aβ aggregation was confirmed in CACPP and CAPPP brain organoids. These brain organoids showed extensive PHF-1 expression.

In order to evaluate the usefulness of the FAD brain organoid model as a means for drug development, as shown in FIG. 6, CACPP brain organoids were treated with BACE1 inhibitor IV (BSI), aβ-secretase inhibitor or compound E (CE), a γ-secretase inhibitor treated with BSI or CE treatment significantly lowered the levels of Aβ 1-40 and 1-42 in CACPP brain organoids ( FIG. 7 ). Western blot analysis showed that p-tau expression of CACPP brain organoids was also decreased by BSI or CE (FIG. 8). These results suggest that the well-known theoretical results (tau phosphorylation is induced by excessive expression of Aβ by FAD mutation) are reproduced in the FAD brain organoid model of the present invention, and at the same time, the model will be utilized as a useful means for drug development research. imply that it can

In addition, if the FAD brain organoid model can be generated from FAD hPSCs, we hypothesized that FAD neurons can also be produced from FAD hPSCs, and that FAD neurons can also be utilized as a two-dimensional FAD model. CmCPP control and CACPP FAD hPSCs were differentiated into neurons, and Aβ and tau phosphorylation in FAD neurons, ie, pathological phenotyping of AD, was performed. ELISA analysis showed that the levels of Aβ 1-40 and 1-42 were significantly increased in CACPP neurons compared to the CmCPP control ( FIG. 9 ). And as a result of Western blot analysis, it was confirmed that the expression level of p-tau was significantly increased in CACPP neurons ( FIG. 10 ). Extensive expression of PHF-1 in CACPP neurons was also confirmed by immunostaining ( FIG. 11 ).

And the effect of BSI or CE on the neurons was confirmed (FIG. 12). In an experiment to confirm the usefulness of the FAD neuron model as a means for drug screening, the levels of Aβ 1-40 and 1-42 were significantly inhibited by treatment with BSI or CE in CACPP neurons ( FIG. 13 ). . Western blot and immunostaining also significantly reduced p-tau levels by BSI or CE ( FIGS. 14 and 15 ).

In conclusion, the human-derived two-dimensional neuronal three-dimensional brain organoid system of the present invention can successfully reproduce the pathological characteristics of amyloid β and tau, and serves as a platform for drug screening and pathological mechanism study of Alzheimer's disease (AD). can be used In addition, the model of the present invention can provide a novel human disease model platform for the study of molecular mechanisms of Alzheimer's disease (AD). The present invention, which reproduces the Alzheimer's (AD) pathological phenotype in two-dimensional and three-dimensional models, may contribute to the development of precise human models of other diseases.

So far, the present invention has been looked at with respect to preferred embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the presented embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within the scope equivalent thereto should be construed as being included in the present invention.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> EXPRESSION CASSETTE AND VECTOR WITH GENES RELATED ALZHEIMER'S DISEASE AND TRANSGENIC HUMAN PLURIPOTENT STEM CELL LINE MADE FROM IT AND ALZHEIMER'S CELL DISEAE ORGANOID AND 00ER46 MODEL D19IS CELLLINE <NEURON MODEL 160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 2088 <212> DNA <213> Artificial Sequence <220> <223> APP mutant sequence <400> 1 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccccatccaga t gtaatgct ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aaga gtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag ccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttcca gaaagccaaa 960 gagaggcttg aggccaagca ccgagagaga atgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaa gaa agccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaag tgaatctgga tgcagaattc 1800 cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcat agcgacagtg 1920 gtcatcatca ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc agaactag 2088 <210> 2 <211> 2088 <212> DNA <213> Artificial Sequence <220> <223> APP normal sequence <400> 2 atgctgcccg gt ttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct a caacagcag ccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttcca gaaagccaaa 960 gagaggcttg aggccaagca ccgagagaga atgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccga g gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaag tgaatctgga tgcagaattc 1800 cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcat agcgacagtg 1920 gtcatcatca ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc agaactag 2088 <210> 3 < 211> 1403 <212> DNA <213> Artificial sequence <220> <223> PSEN1 mutant sequence <400> 3 atgacagagt tacctgcacc gttgtcctac ttccagaatg cacagatgtc tgaggacaac 60 cacctgagca atactgtacg tagccagaat gacaatagag aacggcagga gcacaacgac 120 agacggagcc ttggccaccc tgagccatta tctaatggac gaccccaggg taactcccgg 180 caggtggtgg agcaagatga ggaagaagat gaggagctga cattgaaata tggcgccaag 240 catgtgatct tgctctttgt ccctgtgact ctctgcatgg tggtggtcgt ggctaccatt 300 aagtcagtca gcttttatac ccggaaggat gggcagctaa tctatacccc attcacagaactg cactcaattgaga 360 gataccatt c tgaatgctgc catcatgatc 420 agtgtcattg ttgtcatgac tatcctcctg gtggttctgt ataaatacag gtgctataag 480 gtcatccatg cctggcttat tatatcatct ctattgttgc tgttcttttt ttcattcatt 540 tacttggggg aagtgtttaa aacctataac gttgctgtgg actacattac tgttgcactc 600 ctgatctgga attttggtgt ggtgggaatg atttccattc actggaaagg tccacttcga 660 ctccagcagg catatctcat tatgattagt gccctcatgg ccctggtgtt tatcaagtac 720 ctccctgaat ggactgcgtg gctcatcttg gctgtgattt cagtatatga tttagtggct 780 gttttgtgtc cgaaaggtcc acttcgtatg ctggttgaaa cagctcagga gagaaatgaa 840 acgctttttc cagctgtcat ttactcctca acaatggtgt ggttggtgaa tatggcagaa 900 ggagacccgg aagctcaaag gagagtatcc aaaaattcca agtataatgc agaaagcaca 960 gaaagggagt cacaagacac tgttgcagag aatgatgatg gcgggttcag tgaggaatgg 1020 gaagcccaga gggacagtca tctagggcct catcgctcta cacctgagtc acgagctgct 1080 gtccaggaac tttccagcag tatcctcgct ggtgaagacc cagaggaaag gggagtaaaa 1140 cttggattgg gagatttcat tttctacagt gttctggttg gtaaagcctc agcaacagcc 1200 agtggagact ggaacacaac catagcctgt ttcgtagcca tattaattgg ttt gtgcctt 1260 acattattac tccttgccat tttcaagaaa gcattgccag ctcttccaat ctccatcacc 1320 tttgggcttg ttttctactt tgccacagat tatctttttat 213 normal Sequence EN 213 Sequence tatctttttat cta <ggaccaatta <3 210 DNA Sequence 4400 g400211 <3 <3 aatttttat artificial atgacagagt tacctgcacc gttgtcctac ttccagaatg cacagatgtc tgaggacaac 60 cacctgagca atactgtacg tagccagaat gacaatagag aacggcagga gcacaacgac 120 agacggagcc ttggccaccc tgagccatta tctaatggac gaccccaggg taactcccgg 180 caggtggtgg agcaagatga ggaagaagat gaggagctga cattgaaata tggcgccaag 240 catgtgatca tgctctttgt ccctgtgact ctctgcatgg tggtggtcgt ggctaccatt 300 aagtcagtca gcttttatac ccggaaggat gggcagctaa tctatacccc attcacagaa 360 gataccgaga ctgtgggcca gagagccctg cactcaattc tgaatgctgc catcatgatc 420 agtgtcattg ttgtcatgac tatcctcctg gtggttctgt ataaatacag gtgctataag 480 gtcatccatg cctggcttat tatatcatct ctattgttgc tgttcttttt ttcattcatt 540 tacacttgtgtgggg aagacttgttgtgt aacctg tgt ggtgggaatg atttccattc actggaaagg tccacttcga 660 ctccagcagg catatctcat tatgattagt gccctcatgg ccctggtgtt tatcaagtac 720 ctccctgaat ggactgcgtg gctcatcttg gctgtgattt cagtatatga tttagtggct 780 gttttgtgtc cgaaaggtcc acttcgtatg ctggttgaaa cagctcagga gagaaatgaa 840 acgctttttc cagctctcat ttactcctca acaatggtgt ggttggtgaa tatggcagaa 900 ggagacccgg aagctcaaag gagagtatcc aaaaattcca agtataatgc agaaagcaca 960 gaaagggagt cacaagacac tgttgcagag aatgatgatg gcgggttcag tgaggaatgg 1020 gaagcccaga gggacagtca tctagggcct catcgctcta cacctgagtc acgagctgct 1080 gtccaggaac tttccagcag tatcctcgct ggtgaagacc cagaggaaag gggagtaaaa 1140 cttggattgg gagatttcat tttctacagt gttctggttg gtaaagcctc agcaacagcc 1200 agtggagact ggaacacaac catagcctgt ttcgtagcca tattaattgg tttgtgcctt 1260 acattattac tccttgccat tttcaagaaa gcattgccag ctcttccaat ctccatcacc 1320 tttgggcttg ttttctactt tgccacagat tatcttgtac agccttttat ggaccaatta 1380 gcattccatc aattttatat cta 1403 <210> 5 <211 > 7602 <212> DNA <213> Artificial Sequence <220> <223> exp ression cassette <400> 5 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc 60 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 120 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360 tagtcatcgc tattaccatg gtcgaggtga gccccacgtt ctgcttcact ctccccatct 420 cccccccctc cccaccccca attttgtatt tatttatttt ttaattattt tgtgcagcga 480 tgggggcggg gggggggggc gcgcgccagg cggggcgggg cggggcgagg ggcggggcgg 540 ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt 600 tatggcgagg cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt 660 cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg 720 gctctgactg accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg 780 ctgtaattag cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct 840 tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg 900 tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg 960 gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg 1020 tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt 1080 gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc 1140 tccccgagtt gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc 1200 ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc 1260 gcctcgggcc ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg 1320 tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg 1380 acttcctttg tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc 1440 tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct 1500 tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg 1560 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg 1620 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg 1680 caac gtgctg gttattgtgc tgtctcatca ttttggcaaa gaattccggt gccaccatgc 1740 tgcccggttt ggcactgctc ctgctggccg cctggacggc tcgggcgctg gaggtaccca 1800 ctgatggtaa tgctggcctg ctggctgaac cccagattgc catgttctgt ggcagactga 1860 acatgcacat gaatgtccag aatgggaagt gggattcaga tccatcaggg accaaaacct 1920 gcattgatac caaggaaggc atcctgcagt attgccaaga agtctaccct gaactgcaga 1980 tcaccaatgt ggtagaagcc aaccaaccag tgaccatcca gaactggtgc aagcggggcc 2040 gcaagcagtg caagacccat ccccactttg tgattcccta ccgctgctta gttggtgagt 2100 ttgtaagtga tgcccttctc gttcctgaca agtgcaaatt cttacaccag gagaggatgg 2160 atgtttgcga aactcatctt cactggcaca ccgtcgccaa agagacatgc agtgagaaga 2220 gtaccaactt gcatgactac ggcatgttgc tgccctgcgg aattgacaag ttccgagggg 2280 tagagtttgt gtgttgccca ctggctgaag aaagtgacaa tgtggattct gctgatgcgg 2340 aggaggatga ctcggatgtc tggtggggcg gagcagacac agactatgca gatgggagtg 2400 aagacaaagt agtagaagta gcagaggagg aagaagtggc tgaggtggaa gaagaagaag 2460 ccgatgatga cgaggacgat gaggatggtg atgaggtaga ggaagaggct gaggaaccct 2520 acgaagaagc cacagagaga accaccagca ttgccaccac caccaccacc accacagagt 2580 ctgtggaaga ggtggttcga gttcctacaa cagcagccag tacccctgat gccgttgaca 2640 agtatctcga gacacctggg gatgagaatg aacatgccca tttccagaaa gccaaagaga 2700 ggcttgaggc caagcaccga gagagaatgt cccaggtcat gagagaatgg gaagaggcag 2760 aacgtcaagc aaagaacttg cctaaagctg ataagaaggc agttatccag catttccagg 2820 agaaagtgga atctttggaa caggaagcag ccaacgagag acagcagctg gtggagacac 2880 acatggccag agtggaagcc atgctcaatg accgccgccg cctggccctg gagaactaca 2940 tcaccgctct gcaggctgtt cctcctcggc ctcgtcacgt gttcaatatg ctaaagaagt 3000 atgtccgcgc agaacagaag gacagacagc acaccctaaa gcatttcgag catgtgcgca 3060 tggtggatcc caagaaagcc gctcagatcc ggtcccaggt tatgacacac ctccgtgtga 3120 tttatgagcg catgaatcag tctctctccc tgctctacaa cgtgcctgca gtggccgagg 3180 agattcagga tgaagttgat gagctgcttc agaaagagca aaactattca gatgacgtct 3240 tggccaacat gattagtgaa ccaaggatca gttacggaaa cgatgctctc atgccatctt 3300 tgaccgaaac gaaaaccacc gtggagctcc ttcccgtgaa tggagagttc agcctggacg 3360 atctccagcc gtggc attct tttggggctg actctgtgcc agccaacaca gaaaacgaag 3420 ttgagcctgt tgatgcccgc cctgctgccg accgaggact gaccactcga ccaggttctg 3480 ggttgacaaa tatcaagacg gaggagatct ctgaagtgaa tctggatgca gaattccgac 3540 atgactcagg atatgaagtt catcatcaaa aattggtgtt ctttgcagaa gatgtgggtt 3600 caaacaaagg tgcaatcatt ggactcatgg tgggcggtgt tgtcatagcg acagtggtca 3660 tcatcacctt ggtgatgctg aagaagaaac agtacacatc cattcatcat ggtgtggtgg 3720 aggttgacgc cgctgtcacc ccagaggagc gccacctgtc caagatgcag cagaacggct 3780 acgaaaatcc aacctacaag ttctttgagc agatgcagaa ctagacgcgt cccctctccc 3840 tccccccccc ctaacgttac tggccgaagc cgcttggaat aaggccggtg tgcgtttgtc 3900 tatatgtgat tttccaccat attgccgtct tttggcaatg tgagggcccg gaaacctggc 3960 cctgtcttct tgacgagcat tcctaggggt ctttcccctc tcgccaaagg aatgcaaggt 4020 ctgttgaatg tcgtgaagga agcagttcct ctggaagctt cttgaagaca aacaacgtct 4080 gtagcgaccc tttgcaggca gcggaacccc ccacctggcg acaggtgcct ctgcggccaa 4140 aagccacgtg tataagatac acctgcaaag gcggcacaac cccagtgcca cgttgtgagt 4200 tggatagttg tggaaagagt caaatggctc tcctcaagcg tattcaacaa ggggctgaag 4260 gatgcccaga aggtacccca ttgtatggga tctgatctgg ggcctcggtg cacatgcttt 4320 acatgtgttt agtcgaggtt aaaaaaacgt ctaggccccc cgaaccacgg ggacgtggtt 4380 ttcctttgaa aaacacgatg ataatctaga atgacagagt tacctgcacc gttgtcctac 4440 ttccagaatg cacagatgtc tgaggacaac cacctgagca atactgtacg tagccagaat 4500 gacaatagag aacggcagga gcacaacgac agacggagcc ttggccaccc tgagccatta 4560 tctaatggac gaccccaggg taactcccgg caggtggtgg agcaagatga ggaagaagat 4620 gaggagctga cattgaaata tggcgccaag catgtgatct tgctctttgt ccctgtgact 4680 ctctgcatgg tggtggtcgt ggctaccatt aagtcagtca gcttttatac ccggaaggat 4740 gggcagctaa tctatacccc attcacagaa gataccgaga ctgtgggcca gagagccctg 4800 cactcaattc tgaatgctgc catcatgatc agtgtcattg ttgtcatgac tatcctcctg 4860 gtggttctgt ataaatacag gtgctataag gtcatccatg cctggcttat tatatcatct 4920 ctattgttgc tgttcttttt ttcattcatt tacttggggg aagtgtttaa aacctataac 4980 gttgctgtgg actacattac tgttgcactc ctgatctgga attttggtgt ggtgggaatg 5040 atttccattc actggaaagg tccact tcga ctccagcagg catatctcat tatgattagt 5100 gccctcatgg ccctggtgtt tatcaagtac ctccctgaat ggactgcgtg gctcatcttg 5160 gctgtgattt cagtatatga tttagtggct gttttgtgtc cgaaaggtcc acttcgtatg 5220 ctggttgaaa cagctcagga gagaaatgaa acgctttttc cagctgtcat ttactcctca 5280 acaatggtgt ggttggtgaa tatggcagaa ggagacccgg aagctcaaag gagagtatcc 5340 aaaaattcca agtataatgc agaaagcaca gaaagggagt cacaagacac tgttgcagag 5400 aatgatgatg gcgggttcag tgaggaatgg gaagcccaga gggacagtca tctagggcct 5460 catcgctcta cacctgagtc acgagctgct gtccaggaac tttccagcag tatcctcgct 5520 ggtgaagacc cagaggaaag gggagtaaaa cttggattgg gagatttcat tttctacagt 5580 gttctggttg gtaaagcctc agcaacagcc agtggagact ggaacacaac catagcctgt 5640 ttcgtagcca tattaattgg tttgtgcctt acattattac tccttgccat tttcaagaaa 5700 gcattgccag ctcttccaat ctccatcacc tttgggcttg ttttctactt tgccacagat 5760 tatcttgtac agccttttat ggaccaatta gcattccatc aattttatat ctagatcacc 5820 gtcctcgaga atctagagga tcgtacaagt aaagcggccg cgtcgagggg gttggggttg 5880 cgccttttcc aaggcagccc tgggtttgcg c agggacgcg gctgctctgg gcgtggttcc 5940 gggaaacgca gcggcgccga ccctgggtct cgcacattct tcacgtccgt tcgcagcgtc 6000 acccggatct tcgccgctac ccttgtgggc cccccggcga cgcttcctgc tccgccccta 6060 agtcgggaag gttccttgcg gttcgcggcg tgccggacgt gacaaacgga agccgcacgt 6120 ctcactagta ccctcgcaga cggacagcgc cagggagcaa tggcagcgcg ccgaccgcga 6180 tgggctgtgg ccaatagcgg ctgctcagca gggcgcgccg agagcagcgg ccgggaaggg 6240 gcggtgcggg aggcggggtg tggggcggta gtgtgggccc tgttcctgcc cgcgcggtgt 6300 tccgcattct gcaagcctcc ggagcgcacg tcggcagtcg gctccctcgt tgaccgaatc 6360 accgacctct ctccccaggg ggatccaccg gagcttacca tgaccgagta caagcccacg 6420 gtgcgcctcg ccacccgcga cgacgtcccc agggccgtac gcaccctcgc cgccgcgttc 6480 gccgactacc ccgccacgcg ccacaccgtc gacccggacc gccacatcga gcgggtcacc 6540 gagctgcaag aactcttcct cacgcgcgtc gggctcgaca tcggcaaggt gtgggtcgcg 6600 gacgacggcg ccgcggtggc ggtctggacc acgccggaga gcgtcgaagc gggggcggtg 6660 ttcgccgaga tcggcccgcg catggccgag ttgagcggtt cccggctggc cgcgcagcaa 6720 cagatggaag gcctcctggc gccgcaccgg cccaaggagc ccgcgtggtt cctggccacc 6780 gtcggcgtct cgcccgacca ccagggcaag ggtctgggca gcgccgtcgt gctccccgga 6840 gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg agacctccgc gccccgcaac 6900 ctccccttct acgagcggct cggcttcacc gtcaccgccg acgtcgagtg cccgaaggac 6960 cgcgcg acct ggtgcatgac ccgcaagccc ggtgcctgag cggccgcgtc gacaatcaac 7020 ctctggatta caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta 7080 cgctatgtgg atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt 7140 tcattttctc ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg 7200 ttgtcaggca acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg 7260 gcattgccac cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca 7320 cggcggaact catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca 7380 ctgacaattc cgtggtgttg tcggggaagc tgacgtcctt tccatggctg ctcgcctgtg 7440 ttgccacctg gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag 7500 cggaccttcc ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc 7560gccctcagac gagtcggatc tccctttggg ccgcctcccc gc 7602

Claims (10)

a) APP mutant gene of SEQ ID NO: 1,
b) PSEN1 mutant gene of SEQ ID NO: 2 and
An Alzheimer's disease-related gene expression cassette comprising a CAG promoter.
delete delete delete A recombinant expression vector comprising the expression cassette of claim 1 .
The recombinant expression vector of claim 5, wherein the recombinant expression vector has the sequence of SEQ ID NO: 5.
A cell line transformed using the recombinant expression vector of claim 5 .
According to claim 7, wherein the cell line is human adult stem cells (human adult stem cells, hASC), human embryonic stem cells (human embryonic stem cells, hESCs) and human induced pluripotent stem cells (human induced pluripotent stem cell, hiPSC) of any cell line.
A nerve cell derived from the cell line of claim 7.
An organoid derived from the cell line of claim 7.

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