Patent Publication Number: US-2021180029-A1

Title: Novel human induced pluripotent stem cell lines for modeling alzheimer&#39;s disease and usage thereof

Description:
CROSS REFERENCE TO RELATED APPLICATIONS 
     This application is a National Phase application under 35 U.S.C. § 371 of International Application No. PCT/CN2017/109547, filed Nov. 6, 2017, the disclosures of which are herein incorporated by reference in their entirety. 
    
    
     SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE 
     The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: FA00005US-sequence_listing.txt, date recorded: May 4, 2020, size: 32.5 KB). 
     FIELD 
     The present disclosure relates to biomedical field, particularly to creation of physiologically relevant cellular models for Alzheimer&#39;s disease (AD). Specifically, the present invention relates to a method for preparing a cellular model of AD by genetically modifying human induced pluripotent stem cell (hiPSC). 
     BACKGROUND 
     Alzheimer&#39;s disease (AD) is a chronic neurodegenerative disease and the most common cause of dementia. The genetic heritability of Alzheimer&#39;s disease, based on reviews of twin and family studies, ranges from 49% to 79% (ref 1-2). Around 0.1% of the cases are familial forms of autosomal (not sex-linked) dominant inheritance, which have an onset before age 65 (ref 3). This form of the disease is known as early onset familial Alzheimer&#39;s disease. Most of autosomal dominant familial AD can be attributed to mutations in one of three genes: those encoding amyloid precursor protein (APP) and presenilins 1 and 2 (ref 4). Most mutations in the APP and presenilin genes increase the production of a small protein called A342, which is the main component of senile plaques (ref 5). 
     Most cases of Alzheimer&#39;s disease do not exhibit autosomal-dominant inheritance and are termed sporadic AD, in which environmental and genetic differences may act as risk factors. The best known genetic risk factor is the inheritance of the ε4 allele of the apolipoprotein E (APOE) (ref 6-7). Between 40 and 80% of people with AD possess at least one APOEε4 allele (ref 7). The APOEε4 allele increases the risk of the disease by three times in heterozygotes and by 15 times in homozygotes (ref 3). 
     Mutations in the TREM2 gene have been associated with a 3 to 5 folds higher risk of developing Alzheimer&#39;s disease (ref 8-9). A suggested mechanism of action is that when TREM2 is mutated, white blood cells in the brain are no longer able to control the amount of beta amyloid present. 
     AD is characterized by two distinct features: one is presence of extracellular plaques containing amyloid β-protein (Aβ), and the other is intracellular neurofibrillary tangles (NFTs). Although the true pathology of AD is still not clear, accumulated knowledge from decades of research has provided strong evidence for the amyloid cascade hypothesis. According to this hypothesis, the key event leading to AD appears to be the formation of a specific peptide during the process of amyloid precursor protein (APP). This peptide, known as amyloid beta peptide 42 (Aß-42), is a sticky peptide that can easily aggregate to form Aß 42  oligomers and subsequently amyloid plaques, the first feature of AD. Presence of Aß 42  aggregates and amyloid plaques can trigger inflammation in neurons and increase expression of calcium-activated kinases that, in turn, induce excessive phosphorylation of tau protein, a protein that stabilizes the cytoskeletal microtubules in neurons. Hyperphosphorylation of tau protein leads to formation of neurofibrillary tangles in neurons, the second feature of AD, and subsequently causes neuron death (ref 10-14). 
     Three enzymes, α-secretase, β-secretase, and γ-secretase, are involved in cleavage of APP. In normal process, APP is first cleaved by either α-secretase or β-secretase, whereas γ-secretase will further process the cleaved products into a mixture of peptides with different lengths. If APP is cleaved by β-secretase, the products will be further cleaved by γ-secretase to produce either a 40 amino acid amyloid peptide (Aβ40) which is soluble or a 42 amino acid peptide (Aβ 42 ) which clumps together to form insoluble aggregates and thus amyloid plaques. Strong evidence for amyloid cascade hypothesis comes from studies of familial AD (FAD), which show that all familial AD patients have mutation in either APP or Presenilin (PS) gene. The mutations in APP gene result in abnormal APP protein that is preferentially cleaved by beta-secretase to produce more Abeta peptides; whereas mutations in PS genes leads to preferentially production of Abeta42 peptide, the constituent of amyloid plaques. Another important observation is that beta-secretase expression and the enzyme activity is significantly elevated in AD patients, particularly in sporadic AD (SAD) patients that represent greater than 90% of AD population. The beta-secretase protein and activity levels are increased in brain regions affected by amyloid deposition and remain increased despite significant neuronal and synaptic loss in AD (ref 15-17). 
     Beta-secretase 1 (BACE1), also known as beta-site amyloid precursor protein cleaving enzyme 1, beta-site APP cleaving enzyme 1, membrane-associated aspartic protease 2, memapsin-2, aspartyl protease 2, and ASP2, is an aspartic-acid protease important in the formation of myelin sheaths in peripheral nerve cells (ref 18). In humans it is encoded by the BACE1 gene. Extracellular cleavage of APP by BACE1 creates a soluble extracellular fragment and a cell membrane-bound fragment referred to as C99. Cleavage of C99 within its transmembrane domain by γ-secretase releases the intracellular domain of APP and produces amyloid-β. Since gamma-secretase cleaves APP closer to the cell membrane than BACE1 does, it removes a fragment of the amyloid-β peptide. Initial cleavage of APP by α-secretase rather than BACE1 prevents eventual generation of amyloid-β. 
     Unlike APP and the Presenilin (PS) proteins, no known mutations in the gene encoding BACE1 cause early-onset, familial Alzheimer&#39;s disease, which is a rare form of the disorder. However, levels of this enzyme have been shown to be elevated in the far more common late-onset sporadic Alzheimer&#39;s. The physiological purpose of BACE&#39;s cleavage of APP and other transmembrane proteins is unknown. BACE2 is a close homolog of BACE1 with no reported APP cleavage in vivo. 
     Since production of Abeta-42 peptides play a major role in formation of amyloid plaques, all drug development effort in the past two decades or so have been focused on reduction of Abeta-42 peptides, either through inhibition of beta-secretase activity or by inhibition of Abeta-42 aggregation (ref 19-26). However, one of the major problems for AD drug development is lack of suitable AD models. The most known AD model so far is the 5×FAD transgenic mouse that is created by inserting mutant APP and PS1 genes into mouse genome to display AD phenotype in aged mice (ref 27). Although widely used, the 5×FAD mouse model has two major drawbacks regarding its utility in drug development. First, the human central nervous system is very different from that of mouse and thus drug candidates tested on the 5×FAD model usually have poor predictability for their effect on human. Second, it takes six to eight months or longer for 5×FAD mouse to develop phenotype so this model is obviously not suitable for early screening of AD drug candidates. 
     Pluripotent stem cells hold great promise in preparing cellular model for they can propagate indefinitely, as well as give rise to every other cell type in the body, such as neurons, heart, pancreatic, and liver cells. Human induced pluripotent stem cell (hiPSC), is a type of pluripotent stem cell that can be generated directly from adult cells. The iPSC technology was pioneered by Shinya Yamanaka&#39;s lab in Kyoto, Japan, who showed in 2006 that the introduction of four specific genes encoding transcription factors could convert adult cells into pluripotent stem cells. This breakthrough technology is called somatic cell reprogramming technology that enables researchers to convert terminally differentiated cells such as skin fibroblasts back to embryonic stem cell stage (ref 28-29). 
     hiPSC can be obtained from patients with all kinds of diseases and be propagated indefinitely and therefore provide unlimited cell source. More importantly, those patient-derived hiPSC can be re-differentiated into disease-relevant primary cells and display disease phenotype at cellular level (ref 30-36). This constitutes one common strategy for creation of hiPSC-based cellular disease model. The strategy is to generate hiPSC from patients who are clearly diagnosed with a disease and then differentiate the patient-derived hiPSC into targeted cell type to display the disease phenotype in vitro. 
     Besides the patient-derived hiPSC, researchers have been experimenting another strategy to establish cellular disease models. They generate hiPSC from non-diseased person and then introduce disease-causing genes into hiPSC by genome editing technology. 
     In order to make a suitable cellular model of AD from hiPSC, it has to meet several criteria: it has to be physiologically equivalent to human functional neurons; it has to display AD phenotype in vitro in a relatively short period of time; and it has to be feasible to make large quantities of AD-relevant human neurons consistently and reproducibly. Despite decades of research, there was no real success to make such a cellular model. 
     SUMMARY 
     The present invention satisfies at least some of the aforementioned needs by providing a method of preparing a cellular model of Alzheimer&#39;s disease (AD), comprising genetically modifying the hiPSC to produce higher levels of beta-secretase and beta-42 peptide compared to those isogenic hiPSC from which the cellular model is derived. 
     For the present disclosure, the inventors introduced AD-related genes into non-diseased hiPSC resulting in isogenic lines, i.e. the diseased cell line and the non-diseased cell line have the same genetic background, and the diseased cell line manifests AD phenotype at cellular level in vitro in a relatively short period of time because of the stacking effect of AD-related genes introduced by genome editing technology. 
     In one aspect, the present invention provides a method of generating a cellular model of Alzheimer&#39;s disease (AD), comprising integrating AD-related gene to hiPSC to induce increased beta-secretase level and/or Abeta-42 peptides. In one embodiment, the AD-related gene is constitutively overexpressed in hiPSC. In one embodiment, the AD-related gene is mutant APP or PS gene attributing to the onset of AD, especially PS1dE9 gene. In one embodiment, the AD-related gene is BACE1 gene. In one embodiment, the AD-related gene is selected from the group consisting of mutant APP attributing to the onset of AD, PS1dE9 gene and BACE1 gene. 
     In certain embodiments, the AD-related gene is integrated into hiPSC by a site-specific manner. Preferably, the AD-related gene is integrated into hiPSC at AAVS1 site. 
     In one aspect, the present invention provides the cellular model of Alzheimer&#39;s disease (AD) generated by above methods. 
     In one aspect, the present invention encompasses a method of modifying hiPSC, comprising introducing an AD-related gene into hiPSC to make it constitutively overexpressed. In one embodiment, the AD-related gene is introduced to the hiPSC by an expression vector comprising a nucleotide acid sequence encoding an AD-related protein and a reporter, wherein the nucleotide acid sequence operably linked with a promoter for driving high level of gene expression in a mammalian expression vector. In one embodiment, the promoter is PGK-1 promoter or CAG promoter. In one embodiment, a drug selection gene controlled by a promoter for driving high level of gene expression in a mammalian expression vector is further introduced into the hiPSC in the same expression vector comprising the nucleotide acid sequence encoding an AD-related protein and a reporter or in a separate expression vector. In one embodiment, the promoter is PGK-1 promoter or CAG promoter. In one embodiment, the expression vector further comprises a nucleotide acid sequence for site specific integration, preferably, a nucleotide acid sequence homologous to human AAVS1 site. In one aspect, the present invention provides an expression vector comprising a nucleotide acid sequence encoding an AD-related protein and a reporter, wherein the nucleotide acid sequence operably linked with a promoter for driving high level of gene expression in a mammalian expression vector. In one embodiment, the promoter is PGK-1 promoter or CAG promoter. In one embodiment, the vector further comprises a nucleotide acid sequence encoding a drug selection gene controlled by a promoter for driving high level of gene expression in a mammalian expression vector. In one embodiment, the promoter is PGK-1 promoter or CAG promoter. In one embodiment, the vector further comprises a nucleotide acid sequence for site specific integration, preferably, a nucleotide acid sequence homologous to human AAVS1 site. In one embodiment, the drug selection gene is antibiotic resistance gene, preferably, puromycine, neomycine, kanamycine, or geneticine resistance gene. In one embodiment, the reporter gene is a gene encoding green fluorescent protein or red fluorescent protein. In one embodiment, all the elements in the vector is in an order beneficial to expression of the AD-related gene. Preferably, all the elements in the vector is in a cis order. 
     In an aspect, the present invention provides a genetic construct, comprising a nucleic acid sequence coding for: a first promoter; a drug selection gene controlled by the first promoter; a second promoter; an AD-related gene linked with a reporter gene controlled by the second promoter; and sequence homologous to human AAVS1 site, wherein all said elements are in a cis order. In one embodiment, the first promoter is human PGK-1 or CAG promoter; the drug selection gene is antibiotic resistance gene; the second promoter is human PGK-1 or CAG promoter; the AD-related gene is BACE1 and the reporter gene is a gene encoding green fluorescent protein or red fluorescent protein, preferably, a gene encoding green fluorescent protein. In one embodiment, the first promoter is human PGK-1 or CAG promoter; the drug selection gene is antibiotic resistance gene; the second promoter is human PGK-1 or CAG promoter; the AD-related gene is PS1dE9 and the reporter gene is GFP. In one embodiment, the antibiotic resistance gene is puromycine, neomycine, kanamycine, or geneticine resistance gene. In one aspect, the present invention provides a modified hiPSC line transformed by any one of the expression vector as stated above, or any one of the genetic construct as stated above. In one embodiment, the hiPSC line is used for generating cellular AD model. 
     In one aspect, the present invention provides a genetically modified hiPSC for use as a cellular model of AD, which is integrated BACE1 gene at the human AAVS1 site and constitutively overexpresses the integrated BACE1 gene. In one embodiment, the modified hiPSC displays increased beta-secretase level and/or Abeta-42 peptides compared to an isogenic hiPSC without the integration of the BACE1 gene. In one embodiment, the modified hiPSC is integrated PS1dE9 gene at the human AAVS1 site and constitutively overexpresses the integrated PS1dE9 gene. 
     In one aspect, the present invention encompasses the use of the genetically modified hiPSC for high throughput screening of a drug for AD treatment. 
     In one aspect, the present invention provides a high throughput method for screening a therapeutic agent for treatment of AD, comprising 
     i) Preparing a cellular model of Alzheimer&#39;s disease (AD) from a hiPSC by introducing the expression vector as above stated or the genetic construct as above stated to the hiPSC,
 
ii) Culturing the candidate compounds with the cellular model for two days to two weeks,
 
iii) Measuring beta-secretase level, Abeta-42 concentration, and Abeta-42/Abeta-40 ratio before and after adding the candidate compounds; and
 
iv) Reduction of one or more measurements selected from beta-secretase level, Abeta-42 concentration and Abeta-42/Abeta-40 ratio indicates the candidate compound is a potential therapeutic agent for treatment of AD.
 
     In one embodiment, the hiPSCs come from a human donor and are converted to hiPSC by a conventional reprogramming method in vitro. In one embodiment, the method is for screening of early AD drug. 
     In one aspect, the present invention provides a drug screening process for screening beta-secretase or Abeta-42 inhibitor, comprising 
     i) Modifying hiPSC line by constitutively over-expressing BACE1 gene or PS1dE9 gene,
 
ii) Re-differentiating hiPSC line into functional neurons,
 
iii) Culturing the functional neurons in presence of candidate drug compounds, and
 
iv) Measuring beta-secretase level and/or Abeta-42 concentration and selecting compounds that can reduce beta-secretase level and/or Abeta-42 concentration.
 
     In an embodiment, the method comprises culturing the functional neurons in presence of candidate drug compounds for two days to two weeks. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and other aspects and advantages of embodiments of the present disclosure will become apparent and more readily appreciated from the following descriptions made with reference the accompanying drawings, in which: 
         FIGS. 1A-1F  Example for generation and characterization of the parental hiPSC lines (UCIS3007): Urine cells from a healthy non-diseased donor are isolated and converted into hiPSC cell by the standard reprogramming method using 4 transcription factors (Oct4, Sox2, Klf4, and cMyc).  FIG. 1A . The process of reprogramming of urine cells, showing morphological changes and alkanin phosphotase (AP) staining;  FIG. 1B . Immunostaining of biomarkers for pluripotent stem cells (Nanog, Tra-1-60, Tra-1-81, SSEA3, and SSEA4);  FIG. 1C-1E . Additional pluripotency test (transgene silencing, expression of endogenous pluripotency genes, FACS of surface markers, promoter demythelation, karyotyping, embryoid body formation and expression of germ layer markers); and  FIG. 1F . Teratoma formation, showing three germ layers. 
         FIG. 2  Neural stem cells and neuronal cells (NSC, mixed neuron, dopaminergic neuron and motor neuron) derived from the parental hiPSC lines. 
         FIGS. 3A-3C  Map of donor vector used for generating hiPSC lines with ectopic expression of AD-related gene at AAVS1 site. The nucleic acid sequence involved in targeted integration comprises a homologous sequence to human AAVS1 site (HA-L and HA-R), a human PGK-1 promoter, a drug selection marker gene, a CAG promoter, an AD-related gene and a reporter gene, all in a cis fashion. Different AD-related genes can be easily replaced by restriction enzyme digestion using Xho-I and BglII.  FIG. 3A . The backbone vector CIB-PCBEB;  FIG. 3B . Donor vector for targeted integration of BACE1 gene; and  FIG. 3C . Donor vector for targeted integration of PS1dE9 gene. 
         FIGS. 4A-4C . Targeted integration of BACE1 gene into AAVS1 site. AAVS1 site is located in the intron region of Exon 1 and Exon 2 of PPP1R12C locus. F1, F2, F3, R1, R2, and R3 are primers used for verification of insertion by junction PCR.  FIG. 4A . The targeted integration strategy;  FIG. 4B . Screening of single cell clones by junction PCR (upper panel: 5′end junction PCR using F1+R1 primers. The clone 21 is iPSN0041-21 with insert and the clone 24 is a negative clone without insert; lower panel: 3′ end junction PCR);  FIG. 4C . Sequences of 5′end and 3′end of the insert in iPSN0041-21. 
         FIGS. 5A-5C . Targeted integration of PS1dE9 gene into AAVS1 site. AAVS1 site is located in the intron region of Exon 1 and Exon 2 of PPP1R12C locus. F1, F2, F3, R1, R2, and R3 are primers used for verification of insertion by junction PCR.  FIG. 5A . The targeted integration strategy;  FIG. 5B . Screening of single cell clones by junction PCR (upper panel: 5′end junction PCR using F1+R1 primers. 74 is the selected clone with insert for further characterization and 67 is a negative clone without insert; lower panel: 3′ end junction PCR);  FIG. 5C . Sequences of 5′end and 3′end of the insert in clone 74;  FIG. 5D . Over-expression of PS1dE9 gene in iPSC (UCIS3007-74). 
         FIG. 6 . Manufacturing process for making large quantities of neuronal cells from hiPSC, showing that having neural stem cells (NSCs) as an intermediate stage is a critical step for shortening the assay process and reducing variation. Neural stem cells are immunostaining with NSC-specific biomarkers sox-1 (red, staining nuclei) and nestin (green, staining cytoplasm). Functional neurons are immunostaining with neuron-specific biomarker Tujl (red) and nuclear staining dye Dapi (blue). 
         FIGS. 7A-7B . Over-expression of BACE1 gene in iPSN0041-21 in comparison with iPSN0041, showing that iPSN0041-21 has significantly higher expression of BACE1 gene at RNA and protein levels compared to those of the parental line iPSN0041.  FIG. 7A . mRNA expression in hiPSC and in neurons derived from hiPSC between iPSN0041-21 and iPSN0041;  FIG. 7B . BACE1 protein expression in neural stem cells and neurons derived from the hiPSC line. 
         FIGS. 8A-8B . Over-expression of Abeta-42 peptides in iPSN0041-21 in comparison with iPSN0041.  FIG. 8A . Abeta-42 concentration in hiPSC-derived neurons, showing that neurons derived from iPSN0041-21 have higher Abeta-42 concentration than that from iPSN0041;  FIG. 8B . Time course study of Abeta-42 expression between iPSN0041-21 and iPSN0041, showing the different expression patterns. 
         FIGS. 9A-9B . Example of drug screening using iPSN0041-21, showing that two small molecule compounds not being reported before, B3 and B16, reduce production of both Abeta-40 and Abeta-42 peptides in hiPSC-derived neurons, but not affect the Abeta-42/40 ratio. A known beta-secretase inhibitor (10 uM) is used as the positive control. Data are from three biological repeats.  FIG. 9A . Reduction of B3 and B16 on Abeta-40 production;  FIG. 9B . Reduction of B3 and B16 on Abeta-42 production; and  FIG. 9C . Abeta-42 vs. Abeta-40 ratio. 
     
    
    
     DETAILED DESCRIPTION 
     All technical and scientific terms used herein have the same meaning as is commonly understood by one of ordinary skill in the art unless defined otherwise. For example, for terms in the biological field provided herein, practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 nd  ed., Cold Spring Harbor Press, Plainsview, N.Y. (1989); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 47), John Wiley &amp; Sons, New York (1999). Those terms should not be construed to have a scope less than understood by a person of ordinary skill in the art. 
     The term “comprising” is used in the present description and claims, it does not exclude other elements or steps. Where an indefinite or definite article is used when referring to a singular noun e.g. “a” or “an”, “the”, this includes a plural of that noun unless something else is specifically stated. 
     “AD-related gene” indicates any gene associated with the onset of Alzheimer&#39;s disease (AD). Although the cause of Alzheimer&#39;s disease is poorly understood, about 70% of the risk is believed to be genetic with many genes usually involved, for example, mutant genes encoding amyloid precursor protein (APP) and presenilins (PS) 1 and 2. The mutations in APP gene result in abnormal APP protein that is preferentially cleaved by beta-secretase to produce more Abeta peptides; whereas mutations in PS genes leads to preferentially production of Abeta42 peptide (Abeta-42 peptide), the constituent of amyloid plaques. Although there is no mutation in the gene encoding beta-secretase was found in either FAD or SAD patients, it is clear that elevated BACE1 expression results in elevated expression of Abeta peptides and thus increases the level of Abeta-42 peptides in AD patients. These genes, together with other genes found or will be found closely related to the onset of AD, are collectively denoted as AD-related gene in the present invention. 
     Mutant amyloid precursor protein (APP) or presenilins (PS) 1 and 2 gene herein indicates APP or PS1 or PS2 gene with mutations attributing to the onset of AD. The mutations of these genes may be naturally occurring in the AD patients or genetically created in vitro. When the term “BACE1 gene” is stated in the present invention, it encompassed not only the naturally occurring gene, but also its functional variants. The functional variants may be slightly different from the naturally occurring gene in amino acid sequence, for example, having at least 80%, preferably at least 90%, more preferably at least 95%, 96%, 97%, 98%, 99% homology with the amino acid sequence of the naturally occurring protein, but nevertheless, they still have the same function as the natural protein. 
     “Constitutive expression” means a gene that is transcribed continually compared to a facultative gene which is only transcribed as needed. “Overexpress” means an excessively high level of gene expression which produces a pronounced gene-related phenotype. As far as the present invention, the AD-related gene integrated into hiPSC is constitutively overexpressed in the hiPSC and leads to the appearance of phenotype of AD in the process of amyloid precursor protein. 
     “Phenotype of AD” in the present invention is related to the phenotype in the process of amyloid precursor protein induced by the integration of AD-related gene and constitutive overexpression of the integrated AD-related gene, including but not limited to increased beta-secretase level, amyloid beta peptide, Abeta-42 concentration and/or Abeta-42/Abeta-40. 
     In the present invention, the AD-related gene is integrated into hiPSC for constitutively overexpressing the AD-related gene to generate AD phenotype. The integration is carried out preferably by site-specific manner, most preferably, by using a sequence homologous to safe harbor site, i.e., human AAVS1 site. Being “homologous” is required to achieve homologous recombination so that the foreign gene can be integrated into the target site. A skilled person in the art, based on common knowledge, knows how to design a sequence homologous to the sequence at the target integration site to achieve the purpose of homologous recombination at the human AAVS1 site. Therefore, for such sequence, the embodiments encompassed by the present invention include but not limit to the exemplified. 
     Vectors 
     The cellular AD model of the present invention can be produced using genetic recombinant methods to introduce AD-related gene to the hiPSC derived from a non-diseased person. For recombinant production of the cellular model of AD, nucleic acids encoding the AD-related proteins, for example, mutant APP or PS protein, or BACE1 enzyme, is isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. DNA encoding said proteins may be readily isolated and sequenced using conventional procedures in the art. 
     Many vectors are available for further arrangement for the purpose of the present invention. The components in the vectors generally include, but are not limited to, one or more of the following: an origin of replication, one or more marker genes, one or more report genes, an enhancer element, a promoter, and a transcription termination sequence. 
     Origin of Replication 
     Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in the selected host cell. 
     Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2p plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter). 
     Selection Gene Component 
     Expression and cloning vectors may contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, uromycine, kanamycine, and geneticine (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli. 
     One example of a selection scheme utilizes a drug to arrest growth of a host cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection use the drugs neomycin, uromycine, kanamycine, and geneticine and hygromycin. 
     Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up antibody encoding nucleic acid, such as DHFR, glutamine synthetase (GS), thymidine kinase, metallothionein-1 and -11, preferably primate metallothionein genes, adenosine 20 deaminase, ornithine decarboxylase, etc. 
     For example, cells transformed with the DHFR gene are identified by culturing the transformants in a culture medium containing methotrexate (Mtx), a competitive antagonist of DHFR. Under these conditions, the DHFR gene is amplified along with any other co-transformed nucleic acid. 
     Alternatively, cells transformed with the GS gene are identified by culturing the transformants in a culture medium containing L-methionine sulfoximine (Msx), an inhibitor of GS. Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acid. The GS selection/amplification system may be used in combination with the DHFR selection/amplification system described above. 
     Report Gene Component 
     A reporter gene (often simply reporter) is a gene that researchers attach to a regulatory sequence of another gene of interest in organisms. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and measured, or because they are selectable markers. Reporter genes are often used as an indication of whether a certain gene has been taken up by or expressed in the cell or organism population. 
     To introduce a reporter gene into an organism, scientists place the reporter gene and the gene of interest in the same DNA construct to be inserted into the cell or organism. Commonly used reporter genes that induce visually identifiable characteristics usually involve fluorescent and luminescent proteins. Examples include the gene that encodes jellyfish green fluorescent protein (GFP), which causes cells that express it to glow green under blue light, and the red fluorescent protein from the gene dsRed. 
     Promoter Component 
     Expression and cloning vectors generally contain a promoter that is recognized by the host cell and is operably linked to nucleic acid encoding the interested protein. 
     Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately to bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3′ end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the polyA tail to the 3′ end of the coding sequence. All of these sequences are suitably inserted into eukaryotic expression vectors. 
     The AD disease-causing protein transcription from vectors in hiPSC can be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40 (SV40), or from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, from heat-shock promoters, provided such promoters are compatible with the host cell systems. 
     The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Pat. No. 4,419,446. A modification of this system is described in U.S. Pat. No. 4,601,978. See also Reyes et al., Nature 297:598-601 (1982) on expression of human p-interferon cDNA in mouse cells under the control of a thymidine kinase 5 promoter from herpes simplex virus. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter. 
     The CAG promoter is a strong synthetic promoter frequently used to drive high levels of gene expression in mammalian expression vectors (ref 37-38). It was constructed from the following sequences: 
     (C) the cytomegalovirus (CMV) early enhancer element,
 
(A) the promoter, the first exon and the first intron of chicken beta-actin gene,
 
(G) the splice acceptor of the rabbit beta-globin gene
 
     The resulting synthetic element was used in the pCAGGS expression vector. 
     Although the whole construct is commonly referred to as the “CAG promoter”, it is not a promoter in a strict sense, as it includes a part of the transcribed sequence and an intron) and enhancer elements. In addition to the CMV immediate early enhancer, the intron of the chicken beta actin gene contains an enhancer element, which is highly conserved among vertebrates. The 3′ part of the promoter has high GC content and is thus refractory to PCR amplification. 
     The PGK-1 gene encodes the housekeeping enzyme, 3-phosphoglycerate kinase, and is ubiquitously expressed. This gene resides on the X chromosome in mammals and is always expressed except where it is silenced along with most other genes on the inactive X chromosome of female somatic cells or male germ cells. The PGK-1 promoter is in a region rich in nucleotides G and C. This promoter can efficiently drive high levels of expression of reporter genes such as  E. coli  lacZ and neo. The 120 bp upstream of the transcription start site functions as a core promoter. Upstream of this is a 320 bp region which enhances transcription from the core promoter in an orientation and position independent fashion. This 320 bp region does not enhance transcription from the core promoter of the SV40 early region. Nuclear proteins bind to this 320 bp fragment although the restricted regions to which binding can be demonstrated with gel mobility shift assays suggests that the activity of the enhancer may be mediated by factors which bind at multiple sites each with low affinity (ref 39). 
     Enhancer Element Component 
     Transcription of a DNA encoding the interested protein by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one will use an enhancer from a eukaryotic cell virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elements for activation of eukaryotic promoters. The enhancer may be spliced into the vector at a position 5′ or 3′ to the antibody-encoding sequence, but is preferably located at a site 5′ from the promoter. 
     Transcription Termination Component 
     Expression vectors used in eukaryotic host cells, for example human cells, will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5′ and, occasionally 3′, untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the interested protein. 
     In one embodiment, the vector of the present invention is designed for targeted integration of genes associated with Alzheimer&#39;s disease (AD)-related genes into human pluripotent stem cell (hiPSC) with high efficiency. Therefore, the vector is also arranged to contain a sequence for site-specific integration. 
     The insertion of genes at known locations by enzymes with target recognition capacity is a kind of technology targeting genome edition, which enables researchers to delete, insert, or modify any genes or DNA segments at genome level with high efficiency and precision (ref 40-42). Such technology also includes recently wide-used CRISPRcas9 gene editing technique disclosed in for example, U.S. Pat. Nos. 8,697,359, 8,771,945, 8,795,965. In certain embodiments of the present invention, the AD-related genes are integrated specifically to the safe harbor site, i.e., AAVS1 site, by CRISPRcas9 gene editing technique. 
     AAVS1 site is a natural AAV integration site on human chromosome 19. This region (AAVS1) has characteristics that make it an ideal target for transgenesis. 
     In one specific embodiment of the present invention, the vector comprises a drug selection marker gene controlled by a first promoter, an AD-related gene linked with a reporter gene controlled by a second promoter, and sequences homologous to those at human AAVS1 site. 
     In one specific embodiment of the present invention, the donor vector backbone contains a left arm for targeted integration (HA-L), PGK-1 promoter, a puromycine gene, a CAG promoter, a GFP reporter gene and a right arm for targeted integration (HA-RL) in a cis arrangement ( FIG. 4A ). 
     As used herein, the left arm for targeted integration (HA-L) comprises the sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO. 1) 
               
               
                 TGCTTTCTCTGACCAGCATTCTCTCCCCTGGGCCTGTGCCGCTTTCTGTC 
               
               
                   
               
               
                 TGCAGCTTGTGGCCTGGGTCACCTCTACGGCTGGCCCAGATCCTTCCCTG 
               
               
                   
               
               
                 CCGCCTCCTTCAGGTTCCGTCTTCCTCCACTCCCTCTTCCCCTTGCTCTC 
               
               
                   
               
               
                 TGCTGTGTTGCTGCCCAAGGATGCTCTTTCCGGAGCACTTCCTTCTCGGC 
               
               
                   
               
               
                 GCTGCACCACGTGATGTCCTCTGAGCGGATCCTCCCCGTGTCTGGGTCCT 
               
               
                   
               
               
                 CTCCGGGCATCTCTCCTCCCTCACCCAACCCCATGCCGTCTTCACTCGCT 
               
               
                   
               
               
                 GGGTTCCCTTTTCCTTCTCCTTCTGGGGCCTGTGCCATCTCTCGTTTCTT 
               
               
                   
               
               
                 AGGATGGCCTTCTCCGACGGATGTCTCCCTTGCGTCCCGCCTCCCCTTCT 
               
               
                   
               
               
                 TGTAGGCCTGCATCATCACCGTTTTTCTGGACAACCCCAAAGTACCCCGT 
               
               
                   
               
               
                 CTCCCTGGCTTTAGCCACCTCTCCATCCTCTTGCTTTCTTTGCCTGGACA 
               
               
                   
               
               
                 CCCCGTTCTCCTGTGGATTCGGGTCACCTCTCACTCCTTTCATTTGGGCA 
               
               
                   
               
               
                 GCTCCCCTACCCCCCTTACCTCTCTAGTCTGTGCTAGCTCTTCCAGCCCC 
               
               
                   
               
               
                 CTGTCATGGCATCTTCCAGGGGTCCGAGAGCTCAGCTAGTCTTCTTCCTC 
               
               
                   
               
               
                 CAACCCGGGCCCCTATGTCCACTTCAGGACAGCATGTTTGCTGCCTCCAG 
               
               
                   
               
               
                 GGATCCTGTGTCCCCGAGCTGGGACCACCTTATATTCCCAGGGCCGGTTA 
               
               
                   
               
               
                 ATGTGGCTCTGGTTCTGGGTACTTTTATCTGTCCCCTCCACCCCACAGTG 
               
               
                   
               
               
                 GGGC. 
               
            
           
         
       
     
     The PGK-puromycin cassette comprises the sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO. 2) 
               
               
                 ATAACTTCGTATAATGTATGCTATACGAAGTTATTACCGGGTAGGGGAGG 
               
               
                   
               
               
                 CGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGC 
               
               
                   
               
               
                 ACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCCC 
               
               
                   
               
               
                 CGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTT 
               
               
                   
               
               
                 CTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCG 
               
               
                   
               
               
                 TGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGAT 
               
               
                   
               
               
                 GGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCC 
               
               
                   
               
               
                 AATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGT 
               
               
                   
               
               
                 GGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCG 
               
               
                   
               
               
                 AAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTG 
               
               
                   
               
               
                 CCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGGAATTCATGACC 
               
               
                   
               
               
                 GAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCCGGGC 
               
               
                   
               
               
                 CGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACA 
               
               
                   
               
               
                 CCGTCGACCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTC 
               
               
                   
               
               
                 TTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGA 
               
               
                   
               
               
                 CGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGG 
               
               
                   
               
               
                 CGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGG 
               
               
                   
               
               
                 CTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAA 
               
               
                   
               
               
                 GGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGG 
               
               
                   
               
               
                 GCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAG 
               
               
                   
               
               
                 CGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCC 
               
               
                   
               
               
                 CTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCG 
               
               
                   
               
               
                 AAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGATAGAAC 
               
               
                   
               
               
                 TTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAA 
               
               
                   
               
               
                 TTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCA 
               
               
                   
               
               
                 AACTCATCAATGTATCTTATCATGTCTGATAACTTCGTATAATGTATGCT 
               
               
                   
               
               
                 ATACGAAGTTAT. 
               
            
           
         
       
     
     The CAG promoter comprises the sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO. 3) 
               
               
                 ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTT 
               
               
                   
               
               
                 CATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCC 
               
               
                   
               
               
                 GCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGT 
               
               
                   
               
               
                 ATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTG 
               
               
                   
               
               
                 GAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT 
               
               
                   
               
               
                 GCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC 
               
               
                   
               
               
                 ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATC 
               
               
                   
               
               
                 TACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTG 
               
               
                   
               
               
                 CTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTAT 
               
               
                   
               
               
                 TTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGC 
               
               
                   
               
               
                 GCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGG 
               
               
                   
               
               
                 AGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTT 
               
               
                   
               
               
                 TATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGC 
               
               
                   
               
               
                 GGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCC 
               
               
                   
               
               
                 GCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGT 
               
               
                   
               
               
                 GAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTT 
               
               
                   
               
               
                 AATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTC 
               
               
                   
               
               
                 CGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTG 
               
               
                   
               
               
                 TGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTG 
               
               
                   
               
               
                 AGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAG 
               
               
                   
               
               
                 GGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGG 
               
               
                   
               
               
                 AACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGT 
               
               
                   
               
               
                 GGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTG 
               
               
                   
               
               
                 CTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCG 
               
               
                   
               
               
                 GGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGG 
               
               
                   
               
               
                 GGCGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCC 
               
               
                   
               
               
                 CCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGTCGCAGCCATTGCCTT 
               
               
                   
               
               
                 TTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTG 
               
               
                   
               
               
                 TGTGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCG 
               
               
                   
               
               
                 GGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTT 
               
               
                   
               
               
                 CGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTG 
               
               
                   
               
               
                 TCCGCGGGGGGGCGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCG 
               
               
                   
               
               
                 GCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCAT 
               
               
                   
               
               
                 GCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCT 
               
               
                   
               
               
                 GTCTCATCATTTTGGCAAAGAATTCCTCGACCTCGAG. 
               
            
           
         
       
     
     The GFP reporter gene comprises the sequence: 
     
       
         
           
               
            
               
                 (SEQ ID NO. 4) 
               
               
                 AGATCTGGCAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGT 
               
               
                   
               
               
                 GGAGGAGAATCCCGGCCCTAGGTTCGAAATGGTGAGCAAGGGCGAGGAGC 
               
               
                   
               
               
                 TGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAAC 
               
               
                   
               
               
                 GGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGG 
               
               
                   
               
               
                 CAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCT 
               
               
                   
               
               
                 GGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGC 
               
               
                   
               
               
                 TACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGA 
               
               
                   
               
               
                 AGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACA 
               
               
                   
               
               
                 AGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATC 
               
               
                   
               
               
                 GAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAA 
               
               
                   
               
               
                 GCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAAGC 
               
               
                   
               
               
                 AGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGAC 
               
               
                   
               
               
                 GGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGA 
               
               
                   
               
               
                 CGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCC 
               
               
                   
               
               
                 TGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTC 
               
               
                   
               
               
                 GTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTGA. 
               
            
           
         
       
     
     The right arm for targeted integration (HA-RL) comprises the sequence (SEQ ID NO.5): 
     
       
         
           
               
            
               
                 TACTAGGGACAGGATTGGTGACAGAAAAGCCCCATCCTTAGGCCTCCTCC 
               
               
                   
               
               
                 TTCCTAGTCTCCTGATATTGGGTCTAACCCCCACCTCCTGTTAGGCAGAT 
               
               
                   
               
               
                 TCCTTATCTGGTGACACACCCCCATTTCCTGGAGCCATCTCTCTCCTTGC 
               
               
                   
               
               
                 CAGAACCTCTAAGGTTTGCTTACGATGGAGCCAGAGAGGATCCTGGGAGG 
               
               
                   
               
               
                 GAGAGCTTGGCAGGGGGTGGGAGGGAAGGGGGGGATGCGTGACCTGCCCG 
               
               
                   
               
               
                 GTTCTCAGTGGCCACCCTGCGCTACCCTCTCCCAGAACCTGAGCTGCTCT 
               
               
                   
               
               
                 GACGCGGCTGTCTGGTGCGTTTCACTGATCCTGGTGCTGCAGCTTCCTTA 
               
               
                   
               
               
                 CACTTCCCAAGAGGAGAAGCAGTTTGGAAAAACAAAATCAGAATAAGTTG 
               
               
                   
               
               
                 GTCCTGAGTTCTAACTTTGGCTCTTCACCTTTCTAGTCCCCAATTTATAT 
               
               
                   
               
               
                 TGTTCCTCCGTGCGTCAGTTTTACCTGTGAGATAAGGCCAGTAGCCAGCC 
               
               
                   
               
               
                 CCGTCCTGGCAGGGCTGTGGTGAGGAGGGGGGTGTCCGTGTGGAAAACTC 
               
               
                   
               
               
                 CCTTTGTGAGAATGGTGCGTCCTAGGTGTTCACCAGGTCGTGGCCGCCTC 
               
               
                   
               
               
                 TACTCCCTTTCTCTTTCTCCATCCTTCTTTCCTTAAAGAGTCCCCAGTGC 
               
               
                   
               
               
                 TATCTGGGACATATTCCTCCGCCCAGAGCAGGGTCCCGCTTCCCTAAGGC 
               
               
                   
               
               
                 CCTGCTCTGGGCTTCTGGGTTTGAGTCCTTGGCAAGCCCAGGAGAGGCGC 
               
               
                   
               
               
                 TCAGGCTTCCCTGTCCCCCTTCCTCGTCCACCATCTCATGCCCCTGGCTC 
               
               
                   
               
               
                 TCCTGCCCCTTCCCTACAGGGGTTCCTGGCTCTGCTCT. 
               
            
           
         
       
     
     In another specific embodiment, the donor vector comprises a BACE1 gene that is inserted into the vector backbone at the BglII and XhoI site ( FIG. 5B ); cDNA sequence for BACE1 gene is disclosed in Genbank under locus NM_138973.3 ( Homo sapiens  beta-secretase 1, transcript variant d, mRNA). As used herein, “BACE1” denotes a transmembrane protease encoded by BACE1 gene. BACE1 catalyzes the first step in the formation of amyloid beta peptide from amyloid precursor protein. Amyloid beta peptides are the main constituent of amyloid beta plaques, which accumulate in the brains of human Alzheimer&#39;s disease patients. 
     The DNA sequence of BACE1 gene in the donor vector is described as: 
     
       
         
           
               
            
               
                 (SEQ ID NO. 6) 
               
               
                 ATGGCCCAAGCCCTGCCCTGGCTCCTGCTGTGGATGGGCGCGGGAGTGCT 
               
               
                   
               
               
                 GCCTGCCCACGGCACCCAGCACGGCATCCGGCTGCCCCTGCGCAGCGGCC 
               
               
                   
               
               
                 TGGGGGGCGCCCCCCTGGGGCTGCGGCTGCCCCGGGAGACCGACGAAGAG 
               
               
                   
               
               
                 CCCGAGGAGCCCGGCCGGAGGGGCAGCTTTGTGGAGATGGTGGACAACCT 
               
               
                   
               
               
                 GAGGGGCAAGTCGGGGCAGGGCTACTACGTGGAGATGACCGTGGGCAGCC 
               
               
                   
               
               
                 CCCCGCAGACGCTCAACATCCTGGTGGATACAGGCAGCAGTAACTTTGCA 
               
               
                   
               
               
                 GTGGGTGCTGCCCCCCACCCCTTCCTGCATCGCTACTACCAGAGGCAGCT 
               
               
                   
               
               
                 GTCCAGCACATACCGGGACCTCCGGAAGGGTGTGTATGTGCCCTACACCC 
               
               
                   
               
               
                 AGGGCAAGTGGGAAGGGGAGCTGGGCACCGACCTGCTTTGTGGTGCTGGC 
               
               
                   
               
               
                 TTCCCCCTCAACCAGTCTGAAGTGCTGGCCTCTGTCGGAGGGAGCATGAT 
               
               
                   
               
               
                 CATTGGAGGTATCGACCACTCGCTGTACACAGGCAGTCTCTGGTATACAC 
               
               
                   
               
               
                 CCATCCGGCGGGAGTGGTATTATGAGGTGATCATTGTGCGGGTGGAGATC 
               
               
                   
               
               
                 AATGGACAGGATCTGAAAATGGACTGCAAGGAGTACAACTATGACAAGAG 
               
               
                   
               
               
                 CATTGTGGACAGTGGCACCACCAACCTTCGTTTGCCCAAGAAAGTGTTTG 
               
               
                   
               
               
                 AAGCTGCAGTCAAATCCATCAAGGCAGCCTCCTCCACGGAGAAGTTCCCT 
               
               
                   
               
               
                 GATGGTTTCTGGCTAGGAGAGCAGCTGGTGTGCTGGCAAGCAGGCACCAC 
               
               
                   
               
               
                 CCCTTGGAACATTTTCCCAGTCATCTCACTCTACCTAATGGGTGAGGTTA 
               
               
                   
               
               
                 CCAACCAGTCCTTCCGCATCACCATCCTTCCGCAGCAATACCTGCGGCCA 
               
               
                   
               
               
                 GTGGAAGATGTGGCCACGTCCCAAGACGACTGTTACAAGTTTGCCATCTC 
               
               
                   
               
               
                 ACAGTCATCCACGGGCACTGTTATGGGAGCTGTTATCATGGAGGGCTTCT 
               
               
                   
               
               
                 ACGTTGTCTTTGATCGGGCCCGAAAACGAATTGGCTTTGCTGTCAGCGCT 
               
               
                   
               
               
                 TGCCATGTGCACGATGAGTTCAGGACGGCAGCGGTGGAAGGCCCTTTTGT 
               
               
                   
               
               
                 CACCTTGGACATGGAAGACTGTGGCTACAACATTCCACAGACAGATGAGT 
               
               
                   
               
               
                 CAACCCTCATGACCATAGCCTATGTCATGGCTGCCATCTGCGCCCTCTTC 
               
               
                   
               
               
                 ATGCTGCCACTCTGCCTCATGGTGTGTCAGTGGCGCTGCCTCCGCTGCCT 
               
               
                   
               
               
                 GCGCCAGCAGCATGATGACTTTGCTGATGACATCTCCCTGCTGA. 
               
            
           
         
       
     
     And the protein sequence (SEQ ID NO.7) of BACE1 is described as: 
     
       
         
           
               
            
               
                 MAQALPWLLLWMGAGVLPAHGTQHGIRLPLRSGLGGAPLGLRLPRETDEE 
               
               
                   
               
               
                 PEEPGRRGSFVEMVDNLRGKSGQGYYVEMTVGSPPQTLNILVDTGSSNFA 
               
               
                   
               
               
                 VGAAPHPFLHRYYQRQLSSTYRDLRKGVYVPYTQGKWEGELGTDLLCGAG 
               
               
                   
               
               
                 FPLNQSEVLASVGGSMIIGGIDHSLYTGSLWYTPIRREWYYEVIIVRVEI 
               
               
                   
               
               
                 NGQDLKMDCKEYNYDKSIVDSGTTNLRLPKKVFEAAVKSIKAASSTEKFP 
               
               
                   
               
               
                 DGFWLGEQLVCWQAGTTPWNIFPVISLYLMGEVTNQSFRITILPQQYLRP 
               
               
                   
               
               
                 VEDVATSQDDCYKFAISQSSTGTVMGAVIMEGFYVVFDRARKRIGFAVSA 
               
               
                   
               
               
                 CHVHDEFRTAVEGPFVTLDMEDCGYNIPQTDESTLMTIAYVMAAICALFM 
               
               
                   
               
               
                 LPLCLMVCQWRCLRCLRQQHDDFADDISLLK. 
               
            
           
         
       
     
     In another specific embodiment, the donor vector comprises PS1dE9 gene that is inserted into the vector backbone at the BglII and XhoI site ( FIG. 4B ). PS1dE9 is a mutant of PS1 gene with Exon 9 of PS1 gene being deleted. As used herein, the term “PS1” denotes a protein encoded by the presenilin 1 gene. cDNA sequence for PS1 gene is disclosed in Genbank under locus NM_000021.3 ( Homo sapiens , presenilin 1, transcript variant 1, mRNA). Presenilin 1 is one of the four core proteins in presenilin complex, which mediate the regulated proteolytic events of several proteins in the cell, including gamma secretase. The term “PS1dE9” denotes a mutant gene of PS1, which causes abnormal Gamma-secretase cleavage that is favorable for production of Abeta-42 peptides and thus results in early onset of Alzheimer&#39;s disease. 
     The DNA sequence of PS1dE9 in the donor vector is described below (highlighted is the deleted sequence): 
     
       
         
           
               
            
               
                 (SEQ ID NO. 8) 
               
               
                 ATGACAGAGTTACCTGCACCGTTGTCCTACTTCCAGAATGCACAGATGTC 
               
               
                   
               
               
                 TGAGGACAACCACCTGAGCAATACTGTACGTAGCCAGAATGACAATAGAG 
               
               
                   
               
               
                 AACGGCAGGAGCACAACGACAGACGGAGCCTTGGCCACCCTGAGCCATTA 
               
               
                   
               
               
                 TCTAATGGACGACCCCAGGGTAACTCCCGGCAGGTGGTGGAGCAAGATGA 
               
               
                   
               
               
                 GGAAGAAGATGAGGAGCTGACATTGAAATATGGCGCCAAGCATGTGATCA 
               
               
                   
               
               
                 TGCTCTTTGTCCCTGTGACTCTCTGCATGGTGGTGGTCGTGGCTACCATT 
               
               
                   
               
               
                 AAGTCAGTCAGCTTTTATACCCGGAAGGATGGGCAGCTAATCTATACCCC 
               
               
                   
               
               
                 ATTCACAGAAGATACCGAGACTGTGGGCCAGAGAGCCCTGCACTCAATTC 
               
               
                   
               
               
                 TGAATGCTGCCATCATGATCAGTGTCATTGTTGTCATGACTATCCTCCTG 
               
               
                   
               
               
                 GTGGTTCTGTATAAATACAGGTGCTATAAGGTCATCCATGCCTGGCTTAT 
               
               
                   
               
               
                 TATATCATCTCTATTGTTGCTGTTCTTTTTTTCATTCATTTACTTGGGGG 
               
               
                   
               
               
                 AAGTGTTTAAAACCTATAACGTTGCTGTGGACTACATTACTGTTGCACTC 
               
               
                   
               
               
                 CTGATCTGGAATTTTGGTGTGGTGGGAATGATTTCCATTCACTGGAAAGG 
               
               
                   
               
               
                 TCCACTTCGACTCCAGCAGGCATATCTCATTATGATTAGTGCCCTCATGG 
               
               
                   
               
               
                 CCCTGGTGTTTATCAAGTACCTCCCTGAATGGACTGCGTGGCTCATCTTG 
               
               
                   
               
               
                 GCTGTGATTTCAGTATATGATTTAGTGGCTGTTTTGTGTCCGAAAGGTCC 
               
               
                   
               
               
                 ACTTCGTATGCTGGTTGAAACAGCTCAGGAGAGAAATGAAACGCTTTTTC 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
               
                 CAGAAAGCACAGAAAGGGAGTCACAAGACACTGTTGCAGAGAATGATGAT 
               
               
                   
               
               
                 GGCGGGTTCAGTGAGGAATGGGAAGCCCAGAGGGACAGTCATCTAGGGCC 
               
               
                   
               
               
                 TCATCGCTCTACACCTGAGTCACGAGCTGCTGTCCAGGAACTTTCCAGCA 
               
               
                   
               
               
                 GTATCCTCGCTGGTGAAGACCCAGAGGAAAGGGGAGTAAAACTTGGATTG 
               
               
                   
               
               
                 GGAGATTTCATTTTCTACAGTGTTCTGGTTGGTAAAGCCTCAGCAACAGC 
               
               
                   
               
               
                 CAGTGGAGACTGGAACACAACCATAGCCTGTTTCGTAGCCATATTAATTG 
               
               
                   
               
               
                 GTTTGTGCCTTACATTATTACTCCTTGCCATTTTCAAGAAAGCATTGCCA 
               
               
                   
               
               
                 GCTCTTCCAATCTCCATCACCTTTGGGCTTGTTTTCTACTTTGCCACAGA 
               
               
                   
               
               
                 TTATCTTGTACAGCCTTTTATGGACCAATTAGCATTCCATCAATTTTATA 
               
               
                   
               
               
                 TCTAG. 
               
            
           
         
       
     
     And the protein sequence of PS1dE9 is described below (highlighted is the miss-translated part due to deletion of Exon9): 
     
       
         
           
               
            
               
                 (SEQ ID NO. 9) 
               
               
                 MTELPAPLSYFQNAQMSEDNHLSNTVRSQNDNRERQEHNDRRSLGHPEPL 
               
               
                   
               
               
                 SNGRPQGNSRQVVEQDEEEDEELTLKYGAKHVIMLFVPVTLCMVVVVATI 
               
               
                   
               
               
                 KSVSFYTRKDGQLIYTPFTEDTETVGQRALHSILNAAIMISVIVVMTILL 
               
               
                   
               
               
                 VVLYKYRCYKVIHAWLIISSLLLLFFFSFIYLGEVFKTYNVAVDYITVAL 
               
               
                   
               
               
                 LIWNFGVVGMISIHWKGPLRLQQAYLIMISALMALVFIKYLPEWTAWLIL 
               
               
                   
               
               
                 
                   
                     
                     
                         
                         
                     
                   
                 
               
            
           
         
       
     
     In another embodiment of the present invention, the donor vector of the invention may comprise any functional variant of BACE1 and/or any variant of PS1 attributing to the onset of AD. The variants include, for instance, naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), alternative splicing forms, etc. Variants are preferably substantially homologous to sequences according to the present invention, i.e., exhibit a nucleotide sequence identity of typically at least about 80%, preferably at least about 90%, more preferably at least about 95%, 96%, 97%, 98%, 99% with sequences of the present invention. Variants of the genes of the present invention also include nucleic acid sequences, which hybridize to a sequence as defined above (or a complementary strand thereof) under stringent hybridization conditions. Typical stringent hybridization conditions include temperatures above 42° C. and salinity of equal or less than 200 mM. 
     hiPSC Lines 
     In one embodiment the present disclosure relates to creation of an hiPSC line that has an AD-related gene integrated at a safe harbor site (AAVS1 site) by the method of the present invention. The hiPSC line created by the present invention constitutively over-expresses the integrated gene and displays increased beta-secretase level and/or Abeta-42 peptides compared to an isogenic control hiPSC line without gene integration. 
     In one specific embodiment, an hiPSC line made by the method of the present invention has an exogenous nucleic acid sequence integrated at AAVS1 site. The nucleic acid sequence comprises a PGK-1 promoter, a puromycin gene, a CAG promoter, a BACE1 gene, and a GFP gene and sequences that link different segments. The inserted nucleic acid sequence is described below (the underlined art is BACE1 gene): 
     
       
         
           
               
            
               
                 (SEQ ID NO. 10) 
               
               
                 ATAACTTCGTATAATGTATGCTATACGAAGTTATTACCGGGTAGGGGAGG 
               
               
                   
               
               
                 CGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGC 
               
               
                   
               
               
                 ACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCCC 
               
               
                   
               
               
                 CGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTT 
               
               
                   
               
               
                 CTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCG 
               
               
                   
               
               
                 TGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGAT 
               
               
                   
               
               
                 GGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCC 
               
               
                   
               
               
                 AATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGT 
               
               
                   
               
               
                 GGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCG 
               
               
                   
               
               
                 AAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTG 
               
               
                   
               
               
                 CCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGGAATTCATGACC 
               
               
                   
               
               
                 GAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCCGGGC 
               
               
                   
               
               
                 CGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACA 
               
               
                   
               
               
                 CCGTCGACCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTC 
               
               
                   
               
               
                 TTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGA 
               
               
                   
               
               
                 CGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGG 
               
               
                   
               
               
                 CGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGG 
               
               
                   
               
               
                 CTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAA 
               
               
                   
               
               
                 GGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGG 
               
               
                   
               
               
                 GCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAG 
               
               
                   
               
               
                 CGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCC 
               
               
                   
               
               
                 CTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCG 
               
               
                   
               
               
                 AAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGATAGAAC 
               
               
                   
               
               
                 TTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAA 
               
               
                   
               
               
                 TTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCA 
               
               
                   
               
               
                 AACTCATCAATGTATCTTATCATGTCTGATAACTTCGTATAATGTATGCT 
               
               
                   
               
               
                 ATACGAAGTTATGCGGCCGCAATCGTCGACCTGCAGGCATGCAAGCTTAT 
               
               
                   
               
               
                 TGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCA 
               
               
                   
               
               
                 TAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGC 
               
               
                   
               
               
                 CTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTAT 
               
               
                   
               
               
                 GTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA 
               
               
                   
               
               
                 GTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC 
               
               
                   
               
               
                 CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCAT 
               
               
                   
               
               
                 TATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA 
               
               
                   
               
               
                 CGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCT 
               
               
                   
               
               
                 TCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTT 
               
               
                   
               
               
                 ATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGCGC 
               
               
                   
               
               
                 GCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAG 
               
               
                   
               
               
                 AGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTA 
               
               
                   
               
               
                 TGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGG 
               
               
                   
               
               
                 GCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGC 
               
               
                   
               
               
                 CTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGA 
               
               
                   
               
               
                 GCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAA 
               
               
                   
               
               
                 TGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCG 
               
               
                   
               
               
                 GGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTG 
               
               
                   
               
               
                 TGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAG 
               
               
                   
               
               
                 CGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGG 
               
               
                   
               
               
                 GAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAA 
               
               
                   
               
               
                 CAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGG 
               
               
                   
               
               
                 GCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCT 
               
               
                   
               
               
                 GAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGG 
               
               
                   
               
               
                 GCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGG 
               
               
                   
               
               
                 CGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCC 
               
               
                   
               
               
                 CGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGTCGCAGCCATTGCCTTTT 
               
               
                   
               
               
                 ATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTG 
               
               
                   
               
               
                 TGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGG 
               
               
                   
               
               
                 GCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCG 
               
               
                   
               
               
                 TGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTC 
               
               
                   
               
               
                 CGCGGGGGGGCGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGC 
               
               
                   
               
               
                 TTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGC 
               
               
                   
               
               
                 CTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGT 
               
               
                   
               
               
                 CTCATCATTTTGGCAAAGAATTCCTCGACCTCGAG ATGGCCCAAGCCCTG   
               
               
                   
               
               
                 
                   CCCTGGCTCCTGCTGTGGATGGGCGCGGGAGTGCTGCCTGCCCACGGCAC 
                 
               
               
                   
               
               
                 
                   CCAGCACGGCATCCGGCTGCCCCTGCGCAGCGGCCTGGGGGGCGCCCCCC 
                 
               
               
                   
               
               
                 
                   TGGGGCTGCGGCTGCCCCGGGAGACCGACGAAGAGCCCGAGGAGCCCGGC 
                 
               
               
                   
               
               
                 
                   CGGAGGGGCAGCTTTGTGGAGATGGTGGACAACCTGAGGGGCAAGTCGGG 
                 
               
               
                   
               
               
                 
                   GCAGGGCTACTACGTGGAGATGACCGTGGGCAGCCCCCCGCAGACGCTCA 
                 
               
               
                   
               
               
                 
                   ACATCCTGGTGGATACAGGCAGCAGTAACTTTGCAGTGGGTGCTGCCCCC 
                 
               
               
                   
               
               
                 
                   CACCCCTTCCTGCATCGCTACTACCAGAGGCAGCTGTCCAGCACATACCG 
                 
               
               
                   
               
               
                 
                   GGACCTCCGGAAGGGTGTGTATGTGCCCTACACCCAGGGCAAGTGGGAAG 
                 
               
               
                   
               
               
                 
                   GGGAGCTGGGCACCGACCTGCTTTGTGGTGCTGGCTTCCCCCTCAACCAG 
                 
               
               
                   
               
               
                 
                   TCTGAAGTGCTGGCCTCTGTCGGAGGGAGCATGATCATTGGAGGTATCGA 
                 
               
               
                   
               
               
                 
                   CCACTCGCTGTACACAGGCAGTCTCTGGTATACACCCATCCGGCGGGAGT 
                 
               
               
                   
               
               
                 
                   GGTATTATGAGGTGATCATTGTGCGGGTGGAGATCAATGGACAGGATCTG 
                 
               
               
                   
               
               
                 
                   AAAATGGACTGCAAGGAGTACAACTATGACAAGAGCATTGTGGACAGTGG 
                 
               
               
                   
               
               
                 
                   CACCACCAACCTTCGTTTGCCCAAGAAAGTGTTTGAAGCTGCAGTCAAAT 
                 
               
               
                   
               
               
                 
                   CCATCAAGGCAGCCTCCTCCACGGAGAAGTTCCCTGATGGTTTCTGGCTA 
                 
               
               
                   
               
               
                 
                   GGAGAGCAGCTGGTGTGCTGGCAAGCAGGCACCACCCCTTGGAACATTTT 
                 
               
               
                   
               
               
                 
                   CCCAGTCATCTCACTCTACCTAATGGGTGAGGTTACCAACCAGTCCTTCC 
                 
               
               
                   
               
               
                 
                   GCATCACCATCCTTCCGCAGCAATACCTGCGGCCAGTGGAAGATGTGGCC 
                 
               
               
                   
               
               
                 
                   ACGTCCCAAGACGACTGTTACAAGTTTGCCATCTCACAGTCATCCACGGG 
                 
               
               
                   
               
               
                 
                   CACTGTTATGGGAGCTGTTATCATGGAGGGCTTCTACGTTGTCTTTGATC 
                 
               
               
                   
               
               
                 
                   GGGCCCGAAAACGAATTGGCTTTGCTGTCAGCGCTTGCCATGTGCACGAT 
                 
               
               
                   
               
               
                 
                   GAGTTCAGGACGGCAGCGGTGGAAGGCCCTTTTGTCACCTTGGACATGGA 
                 
               
               
                   
               
               
                 
                   AGACTGTGGCTACAACATTCCACAGACAGATGAGTCAACCCTCATGACCA 
                 
               
               
                   
               
               
                 
                   TAGCCTATGTCATGGCTGCCATCTGCGCCCTCTTCATGCTGCCACTCTGC 
                 
               
               
                   
               
               
                 
                   CTCATGGTGTGTCAGTGGCGCTGCCTCCGCTGCCTGCGCCAGCAGCATGA 
                 
               
               
                   
               
               
                   TGACTTTGCTGATGACATCTCCCTGCTGAAG GTCGACAGATCTGGCAGCG 
               
               
                   
               
               
                 GAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGACGTGGAGGAGAATCCC 
               
               
                   
               
               
                 GGCCCTAGGTTCGAAATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGT 
               
               
                   
               
               
                 GGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCA 
               
               
                   
               
               
                 GCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTG 
               
               
                   
               
               
                 AAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGT 
               
               
                   
               
               
                 GACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACA 
               
               
                   
               
               
                 TGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAG 
               
               
                   
               
               
                 GAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGA 
               
               
                   
               
               
                 GGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCA 
               
               
                   
               
               
                 TCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAAC 
               
               
                   
               
               
                 TACAACAGCCACAAGGTCTATATCACCGCCGACAAGCAGAAGAACGGCAT 
               
               
                   
               
               
                 CAAGGTGAACTTCAAGACCCGCCACAACATCGAGGACGGCAGCGTGCAGC 
               
               
                   
               
               
                 TCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTG 
               
               
                   
               
               
                 CTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCC 
               
               
                   
               
               
                 CAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCG 
               
               
                   
               
               
                 GGATCACTCTCGGCATGGACGAGCTGTACAAGTGAGAGCTCCTCGACTGT 
               
               
                   
               
               
                 GCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCT 
               
               
                   
               
               
                 TGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAA 
               
               
                   
               
               
                 ATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGT 
               
               
                   
               
               
                 GGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCGA 
               
               
                   
               
               
                 TCG. 
               
            
           
         
       
     
     Sequence at the 5′end junction between the insert and host genome is denoted as: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 11) 
               
               
                   
                 
                   ACCCCACAGTGGGGCAAGCTTGGATCCTC 
                 
               
               
                   
                 and its complementary sequence. 
               
            
           
         
       
     
     Sequence at the 3′end junction between the insert and host genome is denoted as: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 12) 
               
               
                   
                 
                   CGATCGGCGGCCGCTACTAGGGACAGGATT 
                 
               
               
                   
                 and its complementarysequence. 
               
            
           
         
       
     
     In one specific embodiment, an hiPSC line made by the method of the present invention has an exogenous nucleic acid sequence integrated at AAVS1 site. The nucleic acid sequence comprises a PGK-1 promoter, a puromycin gene, a CAG promoter, a mutant PS1 gene (PS1dE9), and a GFP gene and sequences that link different segments. The inserted nucleic acid sequence is described below (the underlined part is PS1dE9 gene): 
     
       
         
           
               
            
               
                 (SEQ ID NO. 13) 
               
               
                 ATAACTTCGTATAATGTATGCTATACGAAGTTATTACCGGGTAGGGGAGG 
               
               
                   
               
               
                 CGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGC 
               
               
                   
               
               
                 ACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCCC 
               
               
                   
               
               
                 CGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTT 
               
               
                   
               
               
                 CTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCG 
               
               
                   
               
               
                 TGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGAT 
               
               
                   
               
               
                 GGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCC 
               
               
                   
               
               
                 AATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGT 
               
               
                   
               
               
                 GGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCG 
               
               
                   
               
               
                 AAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTG 
               
               
                   
               
               
                 CCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGGAATTCATGACC 
               
               
                   
               
               
                 GAGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCCGGGC 
               
               
                   
               
               
                 CGTACGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACA 
               
               
                   
               
               
                 CCGTCGACCCGGACCGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTC 
               
               
                   
               
               
                 TTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGA 
               
               
                   
               
               
                 CGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGG 
               
               
                   
               
               
                 CGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGG 
               
               
                   
               
               
                 CTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAA 
               
               
                   
               
               
                 GGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGG 
               
               
                   
               
               
                 GCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAG 
               
               
                   
               
               
                 CGCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCC 
               
               
                   
               
               
                 CTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCG 
               
               
                   
               
               
                 AAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGCCTGATAGAAC 
               
               
                   
               
               
                 TTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAA 
               
               
                   
               
               
                 TTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCA 
               
               
                   
               
               
                 AACTCATCAATGTATCTTATCATGTCTGATAACTTCGTATAATGTATGCT 
               
               
                   
               
               
                 ATACGAAGTTATGCGGCCGCAATCGTCGACCTGCAGGCATGCAAGCTTAT 
               
               
                   
               
               
                 TGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCA 
               
               
                   
               
               
                 TAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGC 
               
               
                   
               
               
                 CTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTAT 
               
               
                   
               
               
                 GTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGA 
               
               
                   
               
               
                 GTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGC 
               
               
                   
               
               
                 CAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCAT 
               
               
                   
               
               
                 TATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTA 
               
               
                   
               
               
                 CGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCT 
               
               
                   
               
               
                 TCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTT 
               
               
                   
               
               
                 ATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGCGC 
               
               
                   
               
               
                 GCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAG 
               
               
                   
               
               
                 AGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTA 
               
               
                   
               
               
                 TGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGG 
               
               
                   
               
               
                 GCGGGAGTCGCTGCGCGCTGCCTTCGCCCCGTGCCCCGCTCCGCCGCCGC 
               
               
                   
               
               
                 CTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGA 
               
               
                   
               
               
                 GCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAA 
               
               
                   
               
               
                 TGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCG 
               
               
                   
               
               
                 GGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTG 
               
               
                   
               
               
                 TGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAG 
               
               
                   
               
               
                 CGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGG 
               
               
                   
               
               
                 GAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGCTGCGAGGGGAA 
               
               
                   
               
               
                 CAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGG 
               
               
                   
               
               
                 GCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGAGTTGCT 
               
               
                   
               
               
                 GAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGG 
               
               
                   
               
               
                 GCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGG 
               
               
                   
               
               
                 CGGGGCCGCCTCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCC 
               
               
                   
               
               
                 CGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGTCGCAGCCATTGCCTTTT 
               
               
                   
               
               
                 ATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTG 
               
               
                   
               
               
                 TGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGG 
               
               
                   
               
               
                 GCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAGGGCCTTCG 
               
               
                   
               
               
                 TGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTC 
               
               
                   
               
               
                 CGCGGGGGGGCGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGC 
               
               
                   
               
               
                 TTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGC 
               
               
                   
               
               
                 CTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGT 
               
               
                   
               
               
                 CTCATCATTTTGGCAAAGAATTCCTCGACCTCGAG ATGACAGAGTTACCT   
               
               
                   
               
               
                 
                   GCACCGTTGTCCTACTTCCAGAATGCACAGATGTCTGAGGACAACCACCT 
                 
               
               
                   
               
               
                 
                   GAGCAATACTGTACGTAGCCAGAATGACAATAGAGAACGGCAGGAGCACA 
                 
               
               
                   
               
               
                 
                   ACGACAGACGGAGCCTTGGCCACCCTGAGCCATTATCTAATGGACGACCC 
                 
               
               
                   
               
               
                 
                   CAGGGTAACTCCCGGCAGGTGGTGGAGCAAGATGAGGAAGAAGATGAGGA 
                 
               
               
                   
               
               
                 
                   GCTGACATTGAAATATGGCGCCAAGCATGTGATCATGCTCTTTGTCCCTG 
                 
               
               
                   
               
               
                 
                   TGACTCTCTGCATGGTGGTGGTCGTGGCTACCATTAAGTCAGTCAGCTTT 
                 
               
               
                   
               
               
                 
                   TATACCCGGAAGGATGGGCAGCTAATCTATACCCCATTCACAGAAGATAC 
                 
               
               
                   
               
               
                 
                   CGAGACTGTGGGCCAGAGAGCCCTGCACTCAATTCTGAATGCTGCCATCA 
                 
               
               
                   
               
               
                 
                   TGATCAGTGTCATTGTTGTCATGACTATCCTCCTGGTGGTTCTGTATAAA 
                 
               
               
                   
               
               
                 
                   TACAGGTGCTATAAGGTCATCCATGCCTGGCTTATTATATCATCTCTATT 
                 
               
               
                   
               
               
                 
                   GTTGCTGTTCTTTTTTTCATTCATTTACTTGGGGGAAGTGTTTAAAACCT 
                 
               
               
                   
               
               
                 
                   ATAACGTTGCTGTGGACTACATTACTGTTGCACTCCTGATCTGGAATTTT 
                 
               
               
                   
               
               
                 
                   GGTGTGGTGGGAATGATTTCCATTCACTGGAAAGGTCCACTTCGACTCCA 
                 
               
               
                   
               
               
                 
                   GCAGGCATATCTCATTATGATTAGTGCCCTCATGGCCCTGGTGTTTATCA 
                 
               
               
                   
               
               
                 
                   AGTACCTCCCTGAATGGACTGCGTGGCTCATCTTGGCTGTGATTTCAGTA 
                 
               
               
                   
               
               
                 
                   TATGATTTAGTGGCTGTTTTGTGTCCGAAAGGTCCACTTCGTATGCTGGT 
                 
               
               
                   
               
               
                 
                   TGAAACAGCTCAGGAGAGAAATGAAACGCTTTTTCCAGCTCTCATTTACT 
                 
               
               
                   
               
               
                 
                   CCTGCACAGAAAGGGAGTCACAAGACACTGTTGCAGAGAATGATGATGGC 
                 
               
               
                   
               
               
                 
                   GGGTTCAGTGAGGAATGGGAAGCCCAGAGGGACAGTCATCTAGGGCCTCA 
                 
               
               
                   
               
               
                 
                   TCGCTCTACACCTGAGTCACGAGCTGCTGTCCAGGAACTTTCCAGCAGTA 
                 
               
               
                   
               
               
                 
                   TCCTCGCTGGTGAAGACCCAGAGGAAAGGGGAGTAAAACTTGGATTGGGA 
                 
               
               
                   
               
               
                 
                   GATTTCATTTTCTACAGTGTTCTGGTTGGTAAAGCCTCAGCAACAGCCAG 
                 
               
               
                   
               
               
                 
                   TGGAGACTGGAACACAACCATAGCCTGTTTCGTAGCCATATTAATTGGTT 
                 
               
               
                   
               
               
                 
                   TGTGCCTTACATTATTACTCCTTGCCATTTTCAAGAAAGCATTGCCAGCT 
                 
               
               
                   
               
               
                 
                   CTTCCAATCTCCATCACCTTTGGGCTTGTTTTCTACTTTGCCACAGATTA 
                 
               
               
                   
               
               
                 
                   TCTTGTACAGCCTTTTATGGACCAATTAGCATTCCATCAATTTTATATCT 
                 
               
               
                   
               
               
                   AG AGATCTGGCAGCGGAGAGGGCAGAGGAAGTCTTCTAACATGCGGTGAC 
               
               
                   
               
               
                 GTGGAGGAGAATCCCGGCCCTAGGTTCGAAATGGTGAGCAAGGGCGAGGA 
               
               
                   
               
               
                 GCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAA 
               
               
                   
               
               
                 ACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC 
               
               
                   
               
               
                 GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCC 
               
               
                   
               
               
                 CTGGCCCACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCC 
               
               
                   
               
               
                 GCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCC 
               
               
                   
               
               
                 GAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTA 
               
               
                   
               
               
                 CAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCA 
               
               
                   
               
               
                 TCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCAC 
               
               
                   
               
               
                 AAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCGACAA 
               
               
                   
               
               
                 GCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGG 
               
               
                   
               
               
                 ACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGC 
               
               
                   
               
               
                 GACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGC 
               
               
                   
               
               
                 CCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGT 
               
               
                   
               
               
                 TCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTGA 
               
               
                   
               
               
                 GAGCTCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCT 
               
               
                   
               
               
                 CCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCC 
               
               
                   
               
               
                 TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT 
               
               
                   
               
               
                 TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACA 
               
               
                   
               
               
                 ATAGCAGGCATGCGATCG. 
               
            
           
         
       
     
     Sequence at the 5′end junction between the insert and host genome is denoted as: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 11) 
               
               
                   
                 
                   ACCCCACAGTGGGGCAAGCTTGGATCCTC 
                 
               
               
                   
                 and its complementary sequence 
               
            
           
         
       
     
     Sequence at the 3′end junction between the insert and host genome is denoted as: 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO. 12) 
               
               
                   
                 
                   CGATCGGCGGCCGCTACTAGGGACAGGATT 
                 
               
               
                   
                 and its complementary sequence. 
               
            
           
         
       
     
     In another embodiment, the inserted nucleic acid sequence may contain sequence that encodes any variant of BACE1 and/or any variant of PS1. The variants include, for instance, naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), alternative splicing forms, etc. Variants are preferably substantially homologous to sequences according to the present invention, i.e., exhibit a nucleotide sequence identity of typically at least about 80%, preferably at least about 90%, more preferably at least about 95% with sequences of the present invention. Variants of the genes of the present invention also include nucleic acid sequences, which hybridize to a sequence as defined above (or a complementary strand thereof) under stringent hybridization conditions. Typical stringent hybridization conditions include temperatures above 42° C. and salinity of equal or less than 200 mM. 
     Drug Screening Method 
     In one embodiment the present disclosure relates to a process of using hiPSC lines created by the method of the present invention to screen therapeutic agents for treatment of AD. The process may include multiple steps of re-differentiating hiPSC line into functional neurons, administrating drug compounds into the neuron culture media, culturing neurons in presence of drug compounds for a period of time, and measuring beta-secretase level, Abeta-40 concentration, Abeta-42 concentration, and Abeta-42/Abeta-40 ratio, etc. 
     The measurement of beta-secretase can be conducted by testing the expression of BACE1 gene at RNA and protein levels by conventional methods in the art. 
     In one specific embodiment, a hiPSC line that over-expresses BACE1 gene will be used to produce large quantities of functional neurons. Neuronal cells derived from the hiPSC line will be cultured in a 96-well plate. Compounds to be screened will be added to the neurons culture for 2 days to 2 weeks. The effect of a compound on reduction of beta-secretase and/or Abeta-42 peptides will be measured in parallel. 
     In another specific embodiment, an hiPSC line that over-expresses PS1dE9 gene will be used to produce large quantities of functional neurons. Neuronal cells derived from the hiPSC line will be cultured in a 96-well plate. Compounds to be screened will be added to the neurons culture for 2 days to 2 weeks. The effect of a compound on reduction of Abeta-42 peptides will be measured. 
     In another embodiment, a hiPSC line that over-expresses any variant of BACE1 gene and/or any variant of PS1 gene at AAVS1 site will be used to produce large quantities of functional neurons. The variants include, for instance, naturally-occurring variants due to allelic variations between individuals (e.g., polymorphisms), alternative splicing forms, etc. Variants are preferably substantially homologous to sequences according to the present invention, i.e., exhibit a nucleotide sequence identity of typically at least about 80%, preferably at least about 90%, more preferably at least about 95% with sequences of the present invention. Variants of the genes of the present invention also include nucleic acid sequences, which hybridize to a sequence as defined above (or a complementary strand thereof) under stringent hybridization conditions. Typical stringent hybridization conditions include temperatures above 42° C. and salinity of equal or less than 200 mM. 
     Reference will be made in detail to embodiments of the present disclosure. The embodiments described herein with reference to drawings are explanatory, illustrative, and used to generally understand the present disclosure. The embodiments shall not be construed to limit the present disclosure. The same or similar elements and the elements having same or similar functions are denoted by like reference numerals throughout the descriptions. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. 
     EXAMPLES 
     The examples are provided only with a purpose of illustration. 
     Example 1: Generation of Parental hiPSC Lines for Genome Editing 
     Two hiPSC lines, UCIS3007 and iPSN0041, were generated and used as parental hiPSC lines for making isogenic diseased hiPSC lines. UCIS3007 was derived from urine cells of a non-diseased female donor by a retrovirus vector-mediated reprogramming method that uses four transcription factors (Oct4, Sox1, Klf4, and cMyc). The donor urine epithelial cells were cultured with urine epithelial cell proliferation medium (CIB, cat #UC-0302). About 20,000 urine epithelial cells were inoculated into 12-well plates one day before the virus infection. The cells transfected with retroviral vectors were cultured with the hiPSC re-programming serum medium (CIB, Cat #. RE-0201). On day 6, the cells were plated onto a T25 flask containing feeder cells and treated with mitomycin C. About 10,000 cells were seeded in each T25 flask and cultured with reprogramming medium for about 20 days until hiPSC clones appears. Then, the clones were picked up onto Matrigel (Corning Cat #352477) coated plates for further purification and expansion. 
     iPSN0041 was derived from umbilical cord matrix cells of a non-diseased donor by a footprint-free method using episomal vector with six transcription factors. About one million umbilical cord matrix cells were electrotransfered with episome plasmids (3 μg pCEP4-EO2S-EN2K, 2.4 μg pCEP4-M2L, 3.2 μg pCEP4-EO2S-1) mixed with a nuclear transfer reagent (Lonza, Cat. No. VPI-1005) using Amaxa Nucleofector kit II. The transfected cells were immediately inoculated into two T25 flasks coated with Matrigel (Corning Cat #352477), cultured with hiPSC reprogramming medium (CIB, Cat. No. RE-0202). The medium was changed to mTeSR1 on the 15th day after transfection and cultured for additional 7 days until hiPSC colonies appear. Clones with typical hiPSC morphology were selected for further purification and expansion. 
     Characterization of hiPSC clones were followed the international standard (ref 43-44), including exogenous gene silencing and pluripotency marker expression detection, exogenous gene integration assays, promoter demethylation analysis, karyotyping test, differentiation potential test (embryoid body formation), teratoma test, and cell ID (STR) test (ref 43-44), etc. ( FIGS. 1A-1F ). In addition, the neural lineage differentiation potential for these two parental hiPSC lines was also validated before conducting genome editing work ( FIG. 2 ). 
     Example 2: Construction of Donor Vector and Targeting Vectors 
     hiPSC genetic engineering was conducted using the most commonly used genome editing technology, CRISPR/Cas9 system. For targeted integration of AD-related genes into a safe harbor site (AAVS1 site), a specific CRISPR sgRNA vector targeted the AAVS1 site was constructed. sgRNA design and construction followed the method described in literature (ref 29). The target site of the BpiI enzyme is located on the vector and the recognition site is at the cleavage moiety. Therefore, the target site spacer sequence can be cloned into the vector by one step digestion. The vector contains 5′GTGG3′ and 5′GTTT3′ cohesive ends after digestion. By synthesizing the cohesive end of the CACC+target spacer sequence and the cohesive terminal AAAC+reverse complement sequence, sgRNA becomes a double stranded oligo sequence with additional cohesive ends, which can be connected to the cas9 vector. CRISPR-cas9 plasmid is generally constructed by a two-step method. The first step is to treat the synthesis of the cleavage site of oligo sequence. The second step is to ligate the treated oligo sequence with the CRISPR-Cas9 vector treated by restriction enzyme digestion. 
     For donor construction, a backbone vector that comprises PGK-PURO-SV40PA, P2A-EGFP-BGHPA and CAG promoters, and a multiple cloning site (MSC) was generated first. To construct the final donor, five plasmids were generated: 1) T-HindIII-CAG-EcoRI; 2) T-SalI-P2A-EGFP-BGHPA-NotI; 3) T-HindIII-PGK-PURO-SV40PA-HindIII; 4) Synthetic PUC19-EcoRI-BACE1cds-SalI; and 5) PZD vector (Sigma-Aldrich). The first step is to digest plasmid 1, 2, 4, and 5, recover four fragments, HindIII-CAG-EcoRI-, -LALI-EGA-EGFP-BGHPA-NotI-, EcoRI-BACE1cds-SalI-, and HindIII-PZDonor-HindIII-, and then ligate all fragments together. After transformation into DH5α cell the positive clones were identified as the intermediate vector of PCBEB6. The final donor vector was generated by HindIII digestion and then ligation of PCBEB6 and T-HindIII-PGK-PURO-SV40PA-HindIII. This donor vector is named as CIB-PCBEB ( FIG. 3A ). All exogenous genes for over-expression at AAVS1 site was done by synthesizing the gene sequence with BglII and XhoI cleavage sites at both ends. The gene of interest is inserted into the vector by digestion of the vector with BglII and XhoI, and then ligation of the insert with the vector. The final donor plasmid was used to transform DH5α cell and then verified by sequencing. Donor constructs for over-expression of BACE1 gene and PS1dE9 gene were shown in  FIGS. 3B and 3C . 
     Example 3: Targeted Integration of AD-Related Genes at AAVS1 Site 
     For introducing AD-related genes into AAVS1 site, parental hiPSC was cultured with mTeSR1 (Stemcell Technologies, cat. no. 05850) on a six well plate coated with Matrigel (Corning, Cat. no. 354277) at 37° C., 5% C02. Cells were harvested at 80% confluence and were co-transfected with Cas9-sgRNA plasmid and donor plasmid by electroporelation method (Amaxa Nucleofector II, program A-024). The number of cells for each transfection reaction was 0.5 to 1×10 6 , and the amount of plasmids was: Cas9-sgRNA 2.5 μg and donor plasmid 4 μg. The transfected cells were immediately inoculated into a six-well plate coated with Matrigel, incubated with mTeSR1 and 10 uM Y-27632 (Sigma, cat. No. Y0503). After 48-hour incubation, 0.5 μg/ml of Puromycin (Xiya Reagent, cat. No. 1014553) was added to culture media for 24 hours for drug selection. Single cell clones were prepared by inoculating Puromycin-resistant cells onto Matrigel-coated T25 flasks at a density of 1000 cells/ml, incubated with mTeSR1 and 10 uM Y-27632. Single-cell clones were picked up 10 days later with assistant of microscopy and then transferred to 96-well plates coated with Matrigel. Each clone was divided into two aliquots and was cultured on two 96-well plates, one for propagation and one for identification. To verify gene integration, cells were lysed by DNA lysis kit (QuickExtract™ DNA Extraction Solution 1.0, Epicentre, Cat. #QE09050) and then presence of insert was identified by junction PCR ( FIG. 4B ). Positive clones identified by junction PCR were further verified by sequencing ( FIG. 4C ). In addition, selected clones were also subjected to pluripotency test and karyotyping to eliminate variation introduced by the single cell cloning process ( FIG. 4D ). Two hiPSC lines that constitutively overexpress BACE1 and PS1dE9 gene were created using a similar strategy ( FIG. 4A  and  FIG. 5A ). 
     Example 4: Large Scale Production of Neuronal Cells 
     As described above, being able to make large quantities of AD-relevant human neurons consistently and reproducibly is pivotal for building a physiological cellular platform for drug screening. In the current art, differentiation of hiPSC into functional neurons involves a lengthy process and is subjected to a series of changes of culture conditions. Therefore, directly seeding hiPSC in 96-well plate to make neurons will result in huge well-to-well variation in terms of number and quality of differentiated neurons. Such large variation is not suitable for drug screening. To solve this problem, a step-wise process has been developed to control variations. The first step is to produce high quality neural stem cells (NSCs) from hiPSC. This step shortens the whole process from hiPSC to mature neuron by 6-8 weeks. Second step is to differentiate NSC into neuronal progenitor cell (NPC), which further reduces the differentiation time for making mature neurons. For production of NSC stock, hiPSC were inoculated on a 12-well plate containing Neural Induction Medium (Stemcell Technologies, Cat. No. 05835) and 10 uM Y-27632 (Sigma, Cat. No. Y0503). After 5 days, the embryoid body ball formed was spread on a 12-well plate coated with Matrigel, incubated in a CO 2  incubator at 37° C., and changed daily with Neural Induction Medium. After 7 days, NSC-like rosette was picked and transferred into 24-well plates. The medium was replaced with NSC Proliferation Medium (CIB, Cat. No. NE-0603). After 7-8 days, NSC-like cells were picked up and transferred to 48-well plates. When NSC density reaches 90% confluence, transfer them into 6-well plates for further expansion and storage. For making functional neurons, about 8×10 4 /cm 2  NSC were transferred to a 6-well plate coated with Poly-L-Ornithine (Sigma, Cat. No. P4957) and Laminin (Sigma, Cat. No. 2020) and cultured in Neuron Induction Medium (Stemcell Technologies, Cat. No. 08500). After 7 days, change the culture medium into Neuron Maturation Medium (Stemcell Technologies, Cat. No. 08510) and continue culture for 2-3 weeks. Mature neurons were verified by immunostaining of neuron-specific biomarkers such as Tuj1 ( FIG. 6 ). 
     Example 5: Over-Expression of Beta Scretase in Modified hiPSC Line 
     As described above, two hiPSC lines have been made using the vectors created by the present invention. One hiPSC line (iPSN0041-21) contains a constitutively expressed BACE1 gene at AAVS1 site. For this line, expression of BACE1 gene at RNA and protein levels has been compared with those of its isogenic parental line (iPSN0041). For quantification of BACE1 mRNA expression, total RNA was extracted with TRIzol (Sigma, cat. No. T9424). qPCR was carried out using ABITM 7500 system and the fluorescent dye SYBR Premix EXTaqTMII (TaKaRa, cat. no. RR820A). The house keeping gene beta-actin was used as reference and each data point was average of three repeats. The result showed that BACE1 mRNA expression of iPSN0041-21 is much higher in iPSC and in iPSC-derived neurons compared to those of iPSN0041, indicating that the transgenic BACE1 gene is indeed constitutively over-expressed in iPSN0041-21 ( FIG. 7A ). Expression of BACE1 protein was carried out using an ELISA kit (Thermo Fisher, Cat. No. P0013). iPSN0041 and mature neurons were obtained using the method described above, the cells were lysed by protein lysis buffer (Biyuntian Biotechnology, Cat. No. P0013) and BACE1 protein concentration was measured using the ELISA kit according to manufacturer&#39;s instruction. Similar to the RNA result, BACE1 protein (Beta-secretase 1) level in iPSN0041-21 is significantly higher than that of the parental line iPSN0041 in hiPSC-derived NSC and neurons ( FIG. 7B ) 
     Example 6: Expression of Abeta-42 Peptides in Modified hiPSC Line 
     For Abeta-42 peptides detection, mature neurons were cultured in Neuron Maturation Medium (Stemcell Technologies, Cat. No. 08510) for up to 7 weeks. Supernatant was collected at 1 week intervals. Concentration of Abeta-42 peptides was measured using the Human/rat A42 ELISA kit (WAKO, Cat. No. 290-62601). For normalization, total protein from each collection was also measured (Thermo-Fisher, Pierce BCA Protein Assay Kit, Cat. No. 23225). As expected, a much higher expression of Abeta-42 peptides in iPSN0041-21 neuron culture was observed compared to those of iPSN0041 neurons ( FIG. 8A ). Further study over a six-week period of time showed that neurons derived from iPSN0041-21 have a different expression pattern compared to that of iPSN0041-derived neurons. Abeta-42 peptide level in iPSN0041-derived neurons (wild type) was very low at the first week of neuron culture and increased over time with a peak at the fifth week and decreased at the sixth week; whereas Abeta-42 expression in neurons derived from iPSN0041-21 was high at the first two weeks of neuron culture and decreased over time ( FIG. 8B ). Since neuronal cell culture in vitro for six weeks mimics the aging process of mature neurons, our observation suggested that for the wild type neurons, Abeta-42 level increased during the aging process of neurons, which is consistent with the disease mechanism for AD. The drop of Abeta-42 at sixth week might be due to neuron death in the culture. On the other hand, the high level expression of Abeta-42 in iPSN0041-21 neurons at early stage can be explained as over-expression of BACE1 transgene and the decrease in Abeta-42 expression after 3 weeks could be due to pre-mature neuron dysfunction or death as a result of too much beta-secretase. This observation suggested that elevated beta-secretase expression is harmful for neuron survival. 
     Example 7: Screening of Drug Compounds Using Modified hiPSC Lines 
     Large quantities of neuron progenitor cells (NPCs) have been produced from the modified hiPSC line IPSN0041-21. To conduct compound screening assay, NPCs were seeded in 96-well plates with a cell density at 50,000 cells/well. NPCs were cultured in Neuron Maturation Medium (Stemcell Technologies, Cat. No. 08510) for up to 4 weeks. Supernatant were collected at one-week intervals. Abeta-40 and Abeta-42 concentrations were measured at two, three, or four weeks using the Human/rat Aβ42 ELISA kit (WAKO, Cat. No. 290-62601). Compounds to be tested were added to neuron culture medium one week before measurement of Abeta-40 and Abeta-42 peptides. As shown in  FIGS. 9A-9C , effect of two novel chemical compounds (B3 and B16) on Abeta peptide production were tested. The result showed that B3 and B16 reduce production of both Abeta-40 and Abeta-42 peptides, but did not affect the Abeta-42 vs. Abeta-40 ratio. One interesting notion was that the positive control, a commonly used beta-secretase inhibitor (Sigma-Aldrich, cat #S4562), disproportionally reduced production of Abeta-40 peptides as indicated by a higher Abeta-42 vs. Abeta-40 ratio compared to those of B3 and B16 treatment. This observation suggested that elevated expression of Abeta peptides in IPSN0041-21 due to over-expression of BACE1 gene indeed increases assay sensitivity of Abeta-40 and Abeta-42 peptides measurement, which allows differentiation of effect of potential drug compounds on different classes of Abeta peptides. In particularly, the system enables screening of compounds that are more effective for reduction of Abeta-42 production. 
     CONCLUSION 
     The present invention described creation of physiologically relevant cellular models for AD by over-expression of AD-related genes, e.g. BACE1 and PS1 and variants thereof in hiPSC. Several unique donor vectors have been created for targeted integration of AD-related genes at a safe harbor site (AAVS1) in human genome with high efficiency. Targeted integration of AD-related genes at AAVS1 site provides a safe and controlled transgene expression that overcomes drawbacks of commonly used random integration method, such as unknown copy numbers and potential disruption to endogenous genes. hiPSC lines created by those donor vectors display high level of beta-seretase activity and/or high level of Abeta-42 peptides that is the major component for amyloid plaque formation. A novel cellular assay platform has been established using neuronal cells derived from hiPSC lines over-expressing AD-related genes. Preliminary screening of potential drug candidate compounds indicated that the cellular platform created by the present invention offers clear advantages for screening new beta-seretase inhibitors and/or Abeta-42 peptides inhibitors compared to current art in the field. 
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