Method and composition for generating basal forebrain cholinergic neurons (BFCNs).

Abstract

The present invention provides a method and composition for generating basal forebrain cholinergic neurons (BFCNs). [Solution] A highly reproducible protocol for efficiently obtaining BFCNs from pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs), provides a method and composition for generating BFCNs from stem cells, particularly BFCNs in which electrophysiological abnormalities associated with one or more mutations in PSEN2 have been repaired, as well as the use of such BFCNs in cell-based therapies for treating Alzheimer's disease. TIFF2026108846000021.tif41148

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 62 / 511,271 filed on 25 May 2017, U.S. Provisional Patent Application No. 62 / 571,741 filed on 12 October 2017, U.S. Provisional Patent Application No. 62 / 574,639 filed on 19 October 2017, and U.S. Provisional Patent Application No. 62 / 586,571 filed on 15 November 2017, the entire contents of each of these applications being incorporated herein by reference as a whole.

[0002] Government support description This invention was made with government support in part under grants R21AG042965, U01AG046170, U01AG046170, NS049442, and U01AG046170 from the National Institutes of Health (NIH). The U.S. Government has certain rights in this invention.

[0003] Incorporation of sequence lists The data in the attached sequence listing are incorporated herein by reference. The attached sequence listing text file, named NYSC1390_4WO_Sequence_Listing.txt, was created on May 24, 2018, and is 53kb in size. The file can be accessed using Microsoft Word on a computer using the Windows OS.

[0004] Field of Invention The present invention relates, as a whole, to pharmaceuticals, more specifically, to methods and compositions for generating basal forebrain cholinergic neurons (BFCNs), in particular BFCNs in which electrophysiological abnormalities associated with one or more mutations in the presenilin 2 gene (PSEN2) have been repaired, from stem cells, and to the field of use of such BFCNs in cell-based therapies for the treatment of Alzheimer's disease. [Background technology]

[0005] Alzheimer's disease (AD) is a progressive disease that causes senile dementia. Broadly speaking, the disease is classified into two categories: late-onset, which occurs in old age (above 65 years), and early-onset, which occurs well before old age, i.e., between 35 and 60 years of age. In both types of disease, the pathology is the same, but the abnormalities tend to be more severe and widespread when they begin at a younger age. The disease is characterized by at least two types of lesions in the brain: senile plaques and neurofibrillary tangles. Senile plaques are disordered areas of neural networks up to 150 μm in size with central extracellular amyloid deposits visible in microscopic analysis of sections of brain tissue. Neurofibrillary tangles are intracellular deposits of microtubules and associated tau protein, consisting of two pairs of filaments that are intertwined with each other.

[0006] The main component of plaques is a peptide called Aβ, or β-amyloid peptide. Aβ peptide is an internal fragment of 39-43 amino acids from a precursor protein called amyloid precursor protein (APP). Several mutations in the APP protein correlate with the presence of Alzheimer's disease. Such mutations are thought to cause Alzheimer's disease by increasing or altering the processing of APP to Aβ (particularly the processing of APP to an increased amount of the long form of Aβ (i.e., Aβ1-42 and Aβ1-43)). Mutations in other genes, such as the presenilin genes PSEN1 and PSEN2, are thought to indirectly affect the processing of APP, resulting in an increased amount of the long form of Aβ. These findings suggest that Aβ (especially its long form) is a contributing factor to Alzheimer's disease.

[0007] In the United States, 5 million people are currently affected by Alzheimer's disease, and according to Alzheimer's disease organizations, this number will increase to 16,000,000 by the 2050s. Unfortunately, the inventors have only direct evidence for a hereditary cause, which accounts for 3–5% of these patients. This percentage includes autosomal dominant early-onset familial Alzheimer's disease (EOFAD) variants caused by hereditary fully penetrant autosomal dominant mutations in APP, or PSEN1, PSEN2, which constitute the gamma-secretase apparatus

[87] , in which alterations in their function increase the production of Aβ42 oligomers and / or the deposition of amyloid plaques.

[0008] After decades of studies of mouse models of AD that do not fully replicate the pathophysiology of the disease in the human brain [5, 57, 58], a new complementary concept for in vitro AD modeling has emerged in the breakthrough by

[81] , which will enable adult human tissue reprogramming into iPSCs using predetermined factors, followed by in vitro differentiation into specific brain cell types.

[0009] BFCNs are one of the most vulnerable neuronal populations, and their deterioration partly explains cognitive decline in AD patients. Apart from evidence of BFCN failure and atrophy, other studies have shown that human embryonic stem cell-derived BFCNs transplanted into AD mouse models may be associated with improved behavioral learning in transplanted mice

[94] . These findings highlight the potential of iPSC and ESC-derived BFCNs not only as early clinical indicators but also as a potential strategy for subtype-specific cell-based therapies for AD

[39] . To advance this cell-based therapeutic strategy, sophisticated differentiation protocols for generating human ESC and / or iPSC-derived BFCNs are urgently needed. [Overview of the Initiative]

[0010] This invention provides a highly reproducible protocol for efficiently obtaining BFCNs from pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs).

[0011] Accordingly, in one embodiment, the present invention provides a method for generating BFCNs. The method comprises culturing PSCs in a basal medium containing an inhibitor of transforming growth factor beta (TGF-β) signaling and an activator of sonic hedgehog (Shh) signaling to induce neuroectodermal differentiation. In some embodiments, the basal medium is a modified mTeSR1 composition lacking pluripotency-supporting factors, including basic fibroblast growth factor (bFGF), TGF-β, lithium chloride (Li-Cl), GABA, and pipecolic acid. In some embodiments, the culture is carried out in the presence of a dual SMAD inhibitor such as SB431542 and LDN193189, along with a smoothed agonist (SAG) and one or more agonists of smoothed proteins such as purmorphamine. After culturing for about 9, 10, 11, or 12 days, CD271+ cells are selected and generate neurocytes (NEBs) in a basal medium for nerve cells such as Brainphys®. The basal medium for nerve cells is optionally supplemented with one or more of the following: B27 supplement, rho-related protein kinase (ROCK) inhibitor, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF). After culturing CD271+ cells for approximately 7, 8, 9, or 10 days, the formed NEBs are harvested, dissociated, seeded as monolayer cultures, and further cultured in basal medium for nerve cells (optionally supplemented with B27 supplement, NGF, and BDNF). To validate differentiation into BFCNs, the cultured cells are analyzed for positive expression of Tuj1, MAP2, BF1, Nkx2.1, and p75.

[0012] In another embodiment, the method utilizes iPSCs that can be processed using a gene editing system to repair one or more mutations, such as a mutation in presenilin 1 (PSEN1) or presenilin 2 (PSEN2). In one embodiment, the mutation is PSEN2 N141I Therefore, its repair restores neuronal excitability in the BFCN.

[0013] Accordingly, in another embodiment, the present invention provides a method for treating a disease or disorder in a subject. The method comprises administering BFCN produced using the culture method described herein to the subject. In certain embodiments, the disease or disorder is an amyloidogenic disorder such as systemic amyloidosis, Alzheimer's disease, adult-onset diabetes mellitus, Parkinson's disease, Huntington's disease, frontotemporal dementia, and prion-associated spongiform encephalopathy. In embodiments, a mutation that impairs neuronal excitability (e.g., PSEN2) is used. N141I A BFCN having the ) can be obtained from a subject, and it may be used to generate iPSCs, which can then be processed with a gene editing system to repair the mutation. The gene-edited iPSCs can then be cultured as described herein to generate BFCNs with restored neuronal excitability, which can then be administered to a subject to treat the disease or disorder. In certain embodiments, the disease or disorder is Alzheimer's disease (AD).

[0014] In a related embodiment, the present invention provides a method for restoring neuronal excitability in BFCNs in a subject. The method includes a) isolating BFCNs from a subject, wherein the BFCNs have a mutation in PSEN2 that causes impaired neuronal excitability of the BFCNs; b) generating iPSCs using the BFCNs from (a); c) repairing the PSEN2 mutations in the iPSCs; d) culturing the iPSCs from (c) using the differentiation protocol described herein to generate BFCNs with the mutations repaired; and e) administering the iPSCs from (d) to a subject, thereby restoring neuronal excitability in the BFCNs in the subject.

[0015] A method is also provided for identifying compounds for the treatment or prevention of diseases or disorders associated with reduced neuronal excitability in BFCNs. The method comprises: a) contacting BFCNs or neurocytes (NEBs) produced using the differentiation protocol described herein with a candidate compound, wherein the BFCNs contain a mutation in PSEN2 that causes impaired neuronal excitability in the BFCNs; and b) detecting the neuronal excitability of the BFCNs after contact with the candidate compound. An increase in neuronal excitability of the BFCNs after contact with the candidate compound identifies the compound as potentially having the ability to restore neuronal excitability in the BFCNs.

[0016] The present invention further provides BFCNs generated using the differentiation protocols described herein. The BFCNs may include PSEN2 gene-edited repair and detectable markers recombinantly introduced into the BFCN genome.

[0017] The present invention also provides a kit for generating BFCNs. The kit comprises a medium having a TGF-β signaling inhibitor and a Shh signaling activator. In embodiments, the medium is a modified mTeSR1 composition lacking pluripotency-supporting factors, including bFGF, TGF-β, lithium chloride (Li-Cl), GABA, and pipecolic acid. In embodiments, the medium comprises a dual SMAD inhibitor, such as SB431542 and LDN193189, along with one or more agonists of smoothed proteins, such as a smoothed agonist (SAG) and purmorphamine. The kit may also optionally include a basal medium for nerve cells, such as Brainphys®, with the addition of one or more of the B27 supplement, a ROCK inhibitor, NGF, and BDNF. In embodiments, the kit comprises reagents for detecting CD271+ cells, as well as cells positively expressing Tuj1, MAP2, BF1, Nkx2.1, and p75. [Invention 1001] A method for generating basal forebrain cholinergic neurons (BFCNs), A step of culturing pluripotent stem cells (PSCs) in a basal medium containing an inhibitor of transforming growth factor beta (TGF-β) signaling and an activator of sonic hedgehog (Shh) signaling to induce neuroectodermal differentiation, wherein the basal medium lacks basic fibroblast growth factor (bFGF), TGF-β, lithium chloride (Li-Cl), GABA, and pipecolic acid. A method that includes, thereby generating a BFCN. [Invention 1002] The method of the present invention 1001, wherein the inhibitor is a SMAD inhibitor. [Invention 1003] The method of the present invention 1002, wherein the inhibitor is SB431542, LDN193189, or a combination thereof. [Invention 1004] The method of the present invention 1001, wherein the activating factor is an agonist of smoothed protein. [Invention 1005] The method of the present invention 1004, wherein the activating factor is a smoothed agonist (SAG), purmorphamine, or a combination thereof. [Invention 1006] The method of the present invention 1001, wherein the culture is carried out for approximately 4 to 9 days. [Invention 1007] The method of the present invention 1002, wherein the inhibitor is present in the culture medium from approximately day 2 to day 8. [Invention 1008] The method of the present invention 1001, further comprising the step of selecting CD271+ cells. [Invention 1009] The method of the present invention 1008, wherein the selection step is performed after approximately 9 to 12 days of culture. [Invention 1010] The method of the present invention 1009, further comprising the step of culturing the CD271+ cells in a basal medium for nerve cells to generate neurocytes (NEBs). [Invention 1011] The method of the present invention 1010, wherein the CD271+ cells are cultured for approximately 7 days to generate NEB. [Invention 1012] a) A step of recovering the NEB; and b) Dissociating the cells of the NEB and reseeding the dissociated cells as a monolayer. The method of the present invention 1011, further comprising: [Invention 1013] The method of the present invention 1012, wherein the reseeded cells are cultured for a further duration in a medium containing growth factors to maintain the survival of the cells, and the cells express Tuj1, MAP2, BF1, Nkx2.1, and p75. [Invention 1014] The method of the present invention 1001, further comprising the step of culturing the cells in mTeSR1 basal medium until confluent before contacting the cells with the activator or inhibitor. [Invention 1015] The method of the present invention 1001, wherein the PSC is a human cell. [Invention 1016] The method of the present invention 1001, wherein the PSC is an induced pluripotent stem cell (iPSC). [Invention 1017] The method of the present invention 1016, wherein the iPSC is derived from a subject diagnosed with or at risk of having Alzheimer's disease (AD). [Invention 1018] The method of the present invention 1017, wherein the PSC is derived from a BFCN having a mutation in presenilin 2 (PSEN2). [Invention 1019] The aforementioned mutation is PSEN2 N141I The method of the present invention 1018. [Invention 1020] The method of the present invention 1018, wherein the mutation is repaired after the generation of the iPSC. [Invention 1021] The method of the present invention 1021, wherein the mutation is repaired using a gene editing system selected from the group consisting of the CRISPR / Cas system, the Cre / Lox system, the TALEN system, and homologous recombination. [Invention 1022] The method of the present invention 1001, wherein the cultured cells exhibit uniform expression of Nkx2.1 by the 8th day of culture. [Invention 1023] The method of the present invention 1001, wherein the cultured cells exhibit recordable action potentials by the 8th day of culture. [Invention 1024] The method of the present invention 1012, wherein the cultured cells exhibit mature action potentials. [Invention 1025] The method of the present invention 1001, wherein the cultured cells exhibit mature action potentials by the 38th day of culture. [Invention 1026] The method of the present invention 1020, wherein the cultured cells show normalization of the Aβ42 / 40 ratio compared to a control. [Invention 1027] The method of the present invention 1020, wherein the cultured cells show a reduction in electrophysiological abnormalities compared to a control. [Invention 1028] The method of the present invention 1027, wherein the reduction of the electrophysiological abnormalities includes, compared to a control, a recovery of the maximum number and spike height in response to the depolarizing current. [Invention 1029] A method for treating a disease or disorder in a subject, comprising administering a BFCN produced using the method of the present invention 1001 to the subject, wherein the disease or disorder is treated. [Invention 1030] The method of the present invention 1029, wherein the disease or disorder is an amyloid-forming disease. [Invention 1031] The method of the present invention 1029, wherein the disease or disorder is associated with a decrease in neuronal excitability in the BFCN of the subject. [Invention 1032] The method of the present invention 1030, wherein the disease or disorder is selected from the group consisting of systemic amyloidosis, Alzheimer's disease, adult-onset diabetes mellitus, Parkinson's disease, Huntington's disease, frontotemporal dementia, and prion-associated transmissible spongiform encephalopathy. [Invention 1033] The method of the present invention 1029, wherein the BFCN contains a genome in which the PSEN2 mutation has been repaired. [Invention 1034] The aforementioned PSEN2 mutation is PSEN2 N141I The method of the present invention 1033. [Invention 1035] A method for restoring the excitability of basal forebrain cholinergic neurons (BFCNs) in a subject, a) A step of isolating BFCN from the subject, wherein the BFCN has a mutation in presenilin 2 (PSEN2) that causes impaired neuronal excitability of the BFCN; b) A step of generating induced pluripotent stem cells (iPSCs) using the BFCN described in (a); c) A step of repairing the mutation in PSEN2 in the iPSC; d) A step of culturing the iPSCs of (c) using the method of the present invention 1001 to produce BFCNs in which the mutations have been repaired; and e) A step of administering the iPSCs described in (d) to the subject, wherein the excitability of the BFCN neurons in the subject is restored. Methods that include... [Invention 1036] The aforementioned mutation is PSEN2 N141I The method of the present invention 1035. [Invention 1037] The method of the present invention 1035, wherein the mutation is repaired using a gene editing system selected from the group consisting of the CRISPR / Cas system, the Cre / Lox system, the TALEN system, and homologous recombination. [Invention 1038] The method of the present invention 1035, wherein the subject has or is at risk of having Alzheimer's disease. [Invention 1039] A method for identifying compounds for the treatment or prevention of diseases or disorders associated with decreased neuronal excitability in cholinergic neurons (BFCNs) in the basal forebrain, a) A step of contacting a BFCN or neurocyte (NEB) produced by the method of the present invention 1001 with a candidate compound, wherein the BFCN contains a mutation in presenilin 2 (PSEN2) that causes impaired neuronal excitability of the BFCN; and b) A step of detecting the neuronal excitability of the BFCN after contact with the candidate compound. Methods that include... [Invention 1040] The aforementioned mutation is PSEN2 N141I The method of the present invention 1039. [Invention 1041] The method of the present invention 1039, wherein the disease or disorder is Alzheimer's disease. [Invention 1042] The method of the present invention 1039 is a high-throughput method. [Invention 1043] Forebral basal cholinergic neurons (BFCNs) generated using the method of the present invention 1001. [Invention 1044] BFCN of the present invention 1043, wherein the genome has recombinantly introduced markers. [Invention 1045] A kit for generating basal forebrain cholinergic neurons (BFCNs), comprising a medium containing an inhibitor of transforming growth factor beta (TGF-β) signaling and an activator of sonic hedgehog (Shh) signaling, wherein the basal medium lacks basic fibroblast growth factor (bFGF), TGF-β, lithium chloride (Li-Cl), GABA, and pipecolic acid; the inhibitors include SB431542 and LDN193189; and the activators include a smoothed agonist (SAG) and purmorphamine. [Invention 1046] A kit according to the present invention 1045, further comprising reagents for generating induced pluripotent stem cells (iPSCs). [Invention 1047] A kit according to the present invention 1045, further comprising a gene editing reagent. [Invention 1048] A kit according to the present invention 1045, further comprising reagents for detecting gene mutations. [Invention 1049] The aforementioned mutation is PSEN2 N141I The kit of the present invention 1048. [Brief explanation of the drawing]

[0018] [Figure 1]Figures 1A-1E: Schematic overview of the basic cholinergic differentiation protocol. (A) Cells were plated and brought to 100% confluence (day 0) before initiation of dual SMAD inhibition and subsequent introduction of ventralization factors (day 2). The monolayer was dissociated on day 10 to isolate p75+ cells, which were maintained as NEBs until day 19. The culture was then dissociated again to form a monolayer (see Methods for more details). (B) The left panel shows sustained EGFP expression in NKx2.1-EGFP hESCs, induced by Nkx2.1 during SHH+purumorphamine or SAG+purumorphamine treatment and maintained on day 14 (after removal on day 8 of treatment). The right panel shows the relative gene expression of Nkx2.1, Lhx8, and BF1 to GAPDH as measured by qPCR in the presence of the indicated ventralization factor or in unpatterned (UNP)NKx2.1-EGFP cell lines at day 12. n=3, technical triplicate. (C) Confocal microscopy images of immunostaining for nestin, Sox2, and DRAQ5 in f control and control lines at day 11 showing a typical neuronal rosette (left panel); or confocal microscopy images of Tuj1, Nkx2.1, and DRAQ5 (right panel). Representative images from three independent experiments. (D) Fluorescence microscopy images of immunostained NEB frozen sections or NEB dissociated into monolayers using the BFCN markers Nkx2.1 / Tuj1 / p75 / BF1 / MAP2 / ChAT. (E) Immunostaining of NEB dissociated into a monolayer on day 50 using MAP2, ChAT, and Hoechst. Fluorescence microscopy images showing the effect of NGF addition compared to SAG + purmorphamine treatment alone. Images are representative of at least three independent experiments.

[0019] [Figure 2]Figures 2A-2F: Basic cholinergic markers in PSEN2N141I neural progenitor cells. (A) Table showing cell lines used. Four iPS cells reprogrammed from fibroblasts; two controls (949 and 050643, labeled f control and f control, respectively) that did not have the PSEN2N141I mutation or the ε4 allele; and two AD patients (948 and 950, labeled AD1 and AD2, respectively) that had the mutation and the ε4 allele. Three of the four iPS cells were family-related (f control, AD1, and AD2). (B) Representative Sanger sequencing chromatogram showing exon 5 fragments of PSEN2. Arrows mark the site of the missense point mutation Chr1:227,073,304 A>T. (C) Immunocytochemistry and RT-PCR for markers of early neurons and basal forebrain. n=3, three independent experiments in technical triplicates. (D) Multiple change of RTPCR for TUJ1 and BF1. n=3, 3 independent experiments using technical triplicates. (E) Representative histogram of P75 staining. n>6. (F) ELISA quantification of Aβ40 and Aβ42. n=3, 3 independent experiments using technical triplicates. ***, p<0.001, *, p<0.05.

[0020] [Figure 3] Figures 3A-3B: Neuronal cell markers and basic cholinergic markers obtained by immunocytochemistry. (A) Immunostaining for TrkA in DIV21. (B) Immunostaining for ChAT and vAChT, as well as Tuj1 and MAP2, at different magnifications in DIV65. Images are representative of at least three independent experiments.

[0021] [Figure 4]Figures 4A-4D: CRISPR / Cas9-mediated modification of the PSEN2N141IiPS strain. (A) Schematic diagram showing the guide RNA used in CRISPR / Cas9 targeting and the donor ssODN used to introduce the wild-type genotype. Sequence identifiers from top to bottom: SEQ ID NO: 26-31. (B) The two left panels show GFP-positive HEK293T cells exhibiting a Cas9 system with guide RNA expression, where NT indicates untransfected cells; the two right panels show samples of GFP-positive iPSCs after lipofection with the pCas9-gN141I-GFP vector. (C) Sanger sequencing obtained from iPSC strains showing modification of the N141I mutation. (D) Aβ42 / 40 ratio (DIV34) detected by ELISA in the 72-hour culture supernatant from mutant, control, or Cispr-Cas9-modified BFCN. n=4, four independent experiments using technical triplicates. *, p<0.05; **, p<0.01, Student's t-test.

[0022] [Figure 5] Figures 5A-5B: BFCNs with various PSEN mutations are not consistently more susceptible to Aβ42 oligomer toxicity. (A) Sample images of BFCNs from the indicated genotypes, treated with propidium iodide to visualize cell death in response to 72 hours of exposure to Aβ42 oligomer (5 μM). (B) Recorded LDH release (%) from culture medium collected after 72 hours of exposure. n=3, 3 independent experiments in technical triplicates. Detected by two-way ANOVA Bonferroni after hoc assay. *, p<0.05; **, p<0.01.

[0023] [Figure 6]Figures 6A-6F: NLRP2 inflammasome mRNA levels are overexpressed in some PSEN2N141I cells, but this is not due to mutation. (A) RT-PCR expression of NLRP2, (B) NLRP3, and (C) ASB9 in cholinergic neural progenitor cells. (D) Western blot showing NLRP2, PSEN2, and β-actin. RT-PCR expression of NLRP2 in neural progenitor cells (E) and BFCN (F). n=3, three independent experiments using technical triplicates for all panels. ***, p<0.001.

[0024] [Figure 7] Figures 7A-7B: Electrophysiological and morphological characteristics of BFCNs. (A) Sodium and potassium compound currents in the top row, generated by the potential protocol shown in the bottom row. Recording of currents generated by potential steps down to -20mV, shown in red. The inset shows the current for the first 25ms generated by the potential steps down to -20mV (scale bar, 200pA, 5ms). (B) Differential interference contrast image of the patch BFCN recorded in (A). 94 neurons (22 wild-type controls, 21 familial controls, 18 AD1, 28 AD2, and 5 iAD1 controls). Scale bar is 30μm.

[0025] [Figure 8]Figures 8A-8C: Electrophysiological abnormalities in BFCNs derived from AD strains. (A) Colocalization of biocitin-labeled neurons with cholinergic markers ChAT and VAChT. Arrows indicate the location of the recorded neuronal cell body, and the scale bar is 50 μm. (B) Representative firing patterns of BFCNs produced by negative and positive rectangular current injection for 1 second. A total of 94 individual neurons were electrophysiologically studied: 22 wild-type control neurons, 21 familial control neurons, 18 AD1 neurons, 28 AD2 neurons, and 5 iAD1 (CRISPR-modified) neurons. The experiments on the 94 neurons took several days to several weeks. Each experimental day included representatives of each genotype, along with at least three samples from each genotype studied on that day. (C) Summary data for the maximum number of action potentials a neuron can sustain (left) and the height of a single action potential at the baseline current (right) across all conditions. Individual data points are shown as circles, and group means are shown as bars. **, p<0.01, Tukey HSD test.

[0026] [Figure 9] Figures 9A-9D: Intrinsic electrophysiological properties of BFCNs. Summary of data for all recorded BFCNs from five groups. 94 neurons (22 wild-type controls, 21 familial controls, 18 AD1, 28 AD2, and 5 iAD1 controls). Histograms show individual values ​​(circles) and group means (bars) for each neuron for membrane resistance (A), capacitance (B), resting potential (C), and baseline current (D). Statistical significance was tested using ANOVA and Tukey's post-hoc comparisons.

[0027] [Figure 10] Figures 10A-10B: Quality control of iPSC strains. (A) Immunofluorescence shows the expression of pluripotency markers SSEA4, Nanog, and Tra160 in the 7889(S)B iPSC strain. (B) Three germ layers (endoderm, mesoderm, and ectoderm) derived from a teratoma produced by the 7889(S)B iPSC strain.

[0028] [Figure 11] Figures 11A-11B: Amyloid-beta levels in mature BFCN. (A) Levels of Aβ40 in BFCN (DIV34). *, P<0.01 compared to other strains in the study, according to one-way ANOVA Bonferroni post-hoc test. (B) Levels of Aβ42 in BFCN (DIV34). n=3, 3 independent experiments with technical triplicates. *, P<0.01 based on Student's t-test. [Modes for carrying out the invention]

[0029] Detailed description of the invention This invention is based on the discovery of a robust, rapid, and reproducible differentiation protocol for generating BFCNs from PSCs using a chemically defined culture medium.

[0030] The following is a detailed description of the invention provided to assist those skilled in the art in carrying out the invention. Those skilled in the art may modify and vary the embodiments described herein without departing from the spirit or scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the invention pertains. Technical terms used in the description of the invention herein are used solely to describe specific embodiments and are not intended to limit the invention. All publications, patent applications, patents, drawings and other references mentioned herein are expressly incorporated by reference in their entirety.

[0031] Any methods and substances similar or equivalent to those described herein may also be used in the practice or testing of the present invention, but preferred methods and substances are described herein. All publications mentioned herein are incorporated herein by reference in connection with the citation of such publications to disclose and describe methods and / or substances.

[0032] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art to the extent of the present invention. The following references, incorporated herein by reference as the entire disclosure, provide those skilled in the art with many general definitions (unless otherwise defined herein) of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (Part 2, 1994); The Cambridge Dictionary of Science and Technology (edited by Walker, 1988); The Glossary of Genetics, Part 5, edited by R. Rieger et al., Springer Verlag (1991); and Hale & Marham, the Harper Collins Dictionary of Biology (1991). Generally, the methods of molecular biological methods described herein or specifically described herein are general methods used in the art. Such standard techniques can be found in reference manuals such as Sambrook et al. (2000, Molecular Cloning—A Laboratory Manual, Part 3, Cold Spring Harbor Laboratories) and Ausubel et al. (1994, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0033] The technical terms used in this description are solely for the purpose of illustrating specific embodiments and are not intended to be limitations of the invention. Where a wide range of values ​​is provided, unless the context explicitly indicates otherwise, it is understood that the values ​​between the upper and lower limits of that range, up to one-tenth of a unit of the lower limit (for example, in the case of a group containing many carbon atoms, where the number of each carbon atom within the range is provided), and any other formulas or intermediate values ​​within the range of that formula are included in the invention. These narrower upper and lower limits are independently included in the narrower ranges also included in the invention and may be subject to any specifically excluded limits within a given range. Where the range of a formula includes one or both of the limits, the range excluding both of these included limits is also included in the invention.

[0034] The following terms are used to describe the present invention. If a term is not specifically defined herein, it shall be given the meaning recognized in the art by those skilled in the art in the context of its use in describing the present invention.

[0035] In this specification and the attached claims, the articles “a” and “an” refer to one or more (i.e., at least one) grammatical objects of the articles, unless the context otherwise clearly indicates. For illustrative purposes, “component” means one or more constituent elements.

[0036] The phrase "and / or" as used in the specification and claims should be understood to mean "either or both" of the linked components, i.e., components that exist together in some cases and separately in other cases. Multiple components listed using "and / or," i.e., "one or more" of the linked components, should be interpreted in the same manner. Other components, other than those specifically identified by the "and / or" clause, may exist, whether or not they are related to these specifically identified components. Thus, as a non-restrictive example, when used in conjunction with an unrestrictive phrase such as "including," a reference to "A and / or B" may, in one embodiment, refer to A only (including components other than B); in another embodiment, refer to B only (including components other than A); and in yet another embodiment, refer to both A and B (including other components).

[0037] As used in the specification and claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” should be interpreted as inclusive, that is, including at least one of a number of components or a list of components, but more than one, and in some cases even more unrelated items. Only terms that are explicitly indicated in the opposite way, such as “one of” or “exactly one of” or, when used in the claims, “consisting of,” refer to including exactly one of a number of components or a list of components. In general, as used herein, the term “or” should be interpreted as indicating exclusive substitutes (i.e., “one or the other, but not both”) when preceded by exclusive terms such as “either,” “one of,” “one of” or “exactly one of.”

[0038] In the description and claims, the phrase “at least one” in a list of one or more components should be understood to mean at least one component selected from any one or more components in the list of components, not necessarily including at least one of each and all components specifically listed in the list of components, but not excluding any combination of components in the list of components. This definition also makes it possible, in some cases, for components other than those specifically listed in the list of components to which the phrase “at least one” refers to to exist, whether related to or unrelated to these specifically identified components. Therefore, as a non-limiting example, “at least one of A and B” (or equivalently, “at least one of A or B” or equivalently, “at least one of A and / or B”) may mean, in one embodiment, at least one comprising A and B absent (and possibly comprising components other than B), which may optionally include one or more; in another embodiment, at least one comprising B and A absent (and possibly comprising components other than A); and in yet another embodiment, at least one comprising A and B (and possibly comprising other components), which may optionally include one or more A.

[0039] In any particular method described herein that includes multiple steps or operations, it should be understood that the order of the steps or operations of the method is not necessarily limited to the order in which the steps or operations of the method are listed, unless the context otherwise indicates.

[0040] In this specification, the term "PSEN2 gene" refers to the gene encoding the PSEN2 polypeptide. The PSEN2 gene is represented by the NCBI reference sequence NC_000001.11 (SEQ ID NO: 1) and known orthologs. In this specification, the term "PSEN2 polypeptide" refers to the polypeptide represented by the NCBI reference sequence NP_000438.2 (SEQ ID NO: 2) and known orthologs.

[0041] The term “amyloidogenic disorders” encompasses any disease associated with (or caused by) the formation or deposition of insoluble amyloid fibrils. Typical amyloidogenic disorders include, but are not limited to, systemic amyloidosis, Alzheimer's disease, adult-onset diabetes mellitus, Parkinson's disease, Huntington's disease, frontotemporal dementia, and prion-associated transmissible spongiform encephalopathy (Kuru and Creutzfeldt-Jakob disease in humans, and scrapie and BSE in sheep and cattle, respectively). Various amyloidogenic disorders are defined or characterized by the nature of the polypeptide components of the deposited fibrils. For example, in subjects or patients with Alzheimer's disease, β-amyloid protein (e.g., wild-type, variant, or cleaved β-amyloid protein) is the polypeptide component characterized by the amyloid deposition. Therefore, Alzheimer's disease is an example of a “disease characterized by Aβ deposition” or “disease associated with Aβ deposition” in the brain of a subject or patient. The terms “β-amyloid protein,” “β-amyloid peptide,” “β-amyloid,” “Aβ,” and “Aβ peptide” are used interchangeably herein.

[0042] Throughout this specification, the terms “patient” or “subject” are used to describe animals (preferably humans or domesticated animals) to which treatment (including prophylactic treatment) using the compositions of this disclosure is provided. For the treatment of a condition or disease specific to a particular animal (e.g., a human patient), the term patient refers to these particular animals, including domesticated animals (e.g., dogs or cats) or livestock (e.g., horses, cattle, sheep, etc.). In general, in this disclosure, the term patient refers to a human patient unless otherwise specified or inferred from the context of the use of the term.

[0043] The terms “β-amyloid protein,” “β-amyloid peptide,” “β-amyloid,” “Aβ,” and “Aβ peptide” are used interchangeably herein. Aβ peptides (e.g., Aβ39, Aβ40, Aβ41, Aβ42, and Aβ43) are approximately 4 kDa internal fragments of APP consisting of 39 to 43 amino acids. For example, Aβ40 consists of residues 672 to 711 of APP, and Aβ42 consists of residues 672 to 713 of APP. Aβ peptides include peptides resulting from the cleavage of APP by secretase and synthetic peptides having the same or essentially the same sequence as the cleavage product. Aβ peptides can originate from various sources, e.g., tissues, cell lines, or bodily fluids (e.g., serum or cerebrospinal fluid). For example, Aβ is derived from APP, as described, for example, by Walsh et al. (2002), Nature, 416, pp. 535-539. 717VAβ preparations can be derived from APP-expressing cells such as Chinese hamster ovary (CHO) cells that have been stably transfected with F. Aβ preparations can be obtained from tissue samples using methods already described (see, e.g., Johnson-Wood et al., (1997), Proc. Natl. Acad. Sci., USA 94: p. 1550). Alternatively, Aβ peptides can be synthesized using methods well known in the art. See, e.g., Fields et al., Synthetic Peptides: A User's Guide, Grant, WH Freeman & Co., New York, NY, 1992, p. 77. Thus, peptides can be synthesized, for example, using side-chain protected amino acids in Applied Biosystem's peptide synthesizer model 430A or 431, using automated Merrifield techniques for solid-phase synthesis with α-amino groups protected by either t-Boc or F-moc chemistry. Longer peptide antigens can be synthesized using well known recombinant DNA techniques. For example, polynucleotides encoding peptides or fusion peptides can be synthesized or molecularly cloned and inserted into appropriate expression vectors for gene transfer and heterologous expression by suitable host cells. Aβ peptides also refer to the associated Aβ sequences resulting from mutations in the Aβ region of a normal gene.

[0044] As used herein, the term “substantially pure” refers to a population of cells in which at least 95% of the cells have the enumerated phenotypes. In all embodiments referring to a “substantially pure” cell population, alternative embodiments in which the cell population has a lower or higher level of purity are also considered. For example, in some embodiments, instead of a given cell population that is “substantially pure,” the cell population may have at least 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the cells, or 100% of the cells, having the enumerated phenotypes.

[0045] The terms “co-administration,” “co-administered,” and “co-administering” or “combination therapy” refer to both simultaneous administration (administering two or more drugs at the same time); and administration at different times (administering one or more drugs at different times than when additional drugs are administered), as long as the drugs are present to some extent (preferably in effective amounts) at the area to be treated simultaneously.

[0046] The term "therapeutically effective dose" refers to the amount necessary to achieve a therapeutic effect. A therapeutic effect can range from prevention, symptom improvement, or treatment of symptoms to the termination or cure of a disease, such as the treatment of Alzheimer's disease or related conditions.

[0047] As used herein, the term “administration” refers to a means of bringing a composition into a subject in a manner that the composition is present within the subject’s body. Such administration may be by any route, including but not limited to subcutaneous, intradermal, intravenous, intraarterial, intraperitoneal, and intramuscular.

[0048] The term “effective” is used to describe the amount of compound, composition, or component that produces the intended result when used in the context of its intended use. The term “effective” encompasses all other effective amounts or effective concentration periods described or used otherwise in this application.

[0049] As used herein, the term “contains” is intended to mean that compositions and methods include the listed components but do not exclude other components. “Essentially consisting of” means, when used to define compositions and methods, to exclude any other components that are essentially important to the combination. Thus, compositions essentially consisting of the components defined herein do not exclude trace contaminants from isolation and purification methods, as well as pharmaceutically acceptable carriers (e.g., phosphate-buffered saline, preservatives, etc.). “Consists of” means to exclude other components and, other than trace elements, the steps of a substantial method for administering the compositions of the present invention. Embodiments defined by each of these transitional clauses are within the scope of the present invention.

[0050] The ranges provided herein are understood to omit all values ​​within that range. For example, the range 1–50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, and 50.

[0051] As used herein, “kit” is understood to contain at least the non-standard experimental reagents of the present invention, and one or more non-standard experimental reagents for use in the method of the present invention.

[0052] In this specification, the term “obtain” is understood to mean to manufacture, purchase, or otherwise acquire.

[0053] As used herein, the terms "treated," "treating," or "treatment" include the attenuation or alleviation of at least one symptom associated with or caused by a treated condition, disorder, or disease. A treated subject may exhibit partial or total alleviation of symptoms (e.g., Alzheimer's disease or related conditions), or the symptoms may remain static after treatment according to the present invention. The term "treatment" is intended to encompass prevention, therapy, and cure.

[0054] As used herein, the term "control" refers to a sample or standard used for comparison with an experimental sample. In some embodiments, the control is a sample obtained from a healthy patient. In other embodiments, the control is a historical control or a standard reference value or range of values (e.g., a sample, subject, or group of samples or subjects that has already been tested).

[0055] Method BFCNs are thought to be one of the first cell types affected in all forms of AD, and their dysfunction clinically correlates with impairments in short-term memory formation and recall. As detailed in the examples of the present disclosure, the inventors present an optimized in vitro protocol for generating human BFCNs from iPSCs using cell lines derived from carriers of PSEN2 mutations and controls. PSEN2 N141I mutant cell lines showed an increase in Aβ42 / 40 in BFCNs derived from iPSCs. PSEN2 N141I The maximum number of action potentials generated by neurons derived from the PSEN2 cell line in response to rectangular depolarizing current injection was lower. The magnitude of the first action potential in basal current injection was also significantly reduced in PSEN2 N141I BFCNs. CRISPR / Cas9 correction of the PSEN2 point mutation abolished the electrophysiological abnormalities and restored both the maximum number of action potentials and the action potential height to levels recorded in controls. The increased Aβ42 / 40 was also PSEN2 N141IThe mutations were normalized after CRISPR / Cas-mediated correction. The genome editing datasets described herein demonstrate strong consistency in mutation-associated changes in the Aβ42 / 40 ratio, while also showing PSEN2 mutation-associated changes in electrophysiology.

[0056] Accordingly, in one embodiment, the present invention provides a method for generating BFCN. The method may first include preparing PSC colonies. The PSCs are seeded (plated) at a low density and grown in adherent culture for about 1-2 days. "Low density" refers to about 8,000-11,000 cells / cm². 2 This means that the cells are preferably about 9,500 to 10,500 cells / cm². 2 More preferably, about 10,000 cells / cm² 2 They are sown. After about 1 to 2 days (or longer, i.e., 3, 4, 5, 6, 7, 8, 9, 10 or longer), the PSCs form colonies, preferably with a diameter of about 75 μm to about 300 μm, more preferably with a diameter of about 100 μm to about 250 μm.

[0057] The term "PSC" has its common meaning in the art, namely, a self-replicating cell that has the ability to develop into endodermal cells, ectoderm cells, and mesodermal cells. Preferably, the PSC is hPSC. PSCs include ESCs and iPSCs, preferably hESCs and hiPSCs. PSCs can be seeded on a surface containing a substrate (e.g., a gel or basement membrane substrate). Preferred substrates are protein mixtures secreted by Angel's Breath-Home-Swan (EHS) mouse sarcoma cells, marketed under trademark names including MATRIGEL®, CULTREX®, and GELTREX®. Other suitable substances include, but are not limited to, collagen, fibronectin, gelatin, laminin, polylysine, vitronectin, and combinations thereof.

[0058] In some embodiments, a culture medium suitable for use in maintaining pluripotent stem cells is used. In some embodiments, such a medium is mTeSR1 medium from Stem Cell Technologies. However, those skilled in the art will recognize that there are several other types of culture media that are equivalent to mTeSR medium in terms of suitability for use in maintaining pluripotent stem cells, and any of them may be used. Typically, such media contain one or more pluripotency factors to promote the maintenance of cells in a pluripotent state. The composition of mTeSR1 medium is known in the art and is described, for example, in Ludwig et al., 2006 (Nat Methods, August 2006; 3(8): pp. 637-46; "Feeder-Independent Culture of Human Embryonic Stem Cells"), the contents of which are incorporated herein by reference.

[0059] The pluripotent stem cells used in the method of the present invention may be any suitable type of pluripotent stem cell. If iPSCs are used, such cells may be "reprogrammed" from a non-pluripotent state to a pluripotent state using any suitable means known in the art, including but not limited to modified RNA-based methods and Sendai virus-based methods. Furthermore, such cells may be reprogrammed to a pluripotent state using any suitable cocktail of reprogramming factors known in the art.

[0060] In one embodiment, after preparing PSCs and growing them to confluence in, for example, mTeSR1 medium, the method comprises culturing the PSCs in a basal medium containing an inhibitor of transforming growth factor beta (TGF-β) signaling and an activator of sonic hedgehog (Shh) signaling to induce neuroectodermal differentiation. The basal medium used is a modified mTeSR1 medium, which is a variant of mTeSR1 medium that does not contain lithium chloride, GABA, pipecolic acid, bFGF, or TGFβ1 (sometimes referred to herein as “mTeSR1 custom” medium). The inhibitor of TGFβ signaling includes, for example, one or more of SB431542, GW788388, LDN193189, LY2109761, LY2157299, and LY364947. Activators of Shh signaling include smoothed agonists such as SAG (3-chloro-N-[(1r,4r)-4-(methylamino)cyclohexyl]-N-[3-(pyridine-4-yl)benzyl]benzo[b]thiophene-2-carboxamide) and purmorphamine.

[0061] After culturing for approximately 6, 7, 8, 9, 10, 11, or 12 days (or longer, i.e., 15, 16, 17, 18, 19, 20, 25, 30, or longer), CD271+ cells are selected and cultured in a basal medium for nerve cells (e.g., Brainphys®) to generate neuroid bodies (NEBs). The basal medium for nerve cells may optionally be supplemented with one or more of the following: B27 supplement, rho-related protein kinase (ROCK) inhibitors, nerve growth factor (NGF), and brain-derived neurotrophic factor (BDNF). ROCK inhibitors include, for example, GSK269962, GSK429286, H-1152, HA-1077, RKI-1447, thiazovibin, Y-27632, or their derivatives.

[0062] To select CD271+ cells, overconfluent cells are detached from the culture surface, purified by FACS, and replated. This process allows for the formation of cell aggregates or spheres, also referred to herein as NEBs. For the purposes of this invention, the terms “NEB,” “aggregate,” and “sphere” are used interchangeably and refer to multicellular three-dimensional structures of at least about 100 cells, although this is not essential.

[0063] Delamination can be performed mechanically or chemically using a cell scraper or other suitable tool. Chemical delamination can be achieved using proteolytic enzymes, such as collagenase, trypsin, trypsin-like proteolytic enzymes, recombinant enzymes such as those sold under the trademark TRYPLE®, naturally occurring enzymes such as those sold under the trademark ACCUTASE®, and combinations thereof. Chemical delamination can also be performed using chelating agents such as EDTA or compounds such as urea. Mechanical or delamination offers the advantage of minimal cell death but produces aggregates of variable size, thus requiring the selection of appropriate spheres through a manual selection process. Good spheres are defined as spherical, golden / brown in color with a darker center, and with a diameter of approximately 300 μm to 800 μm. Delaminating cells using chemical methods such as enzymatic cleavage primarily produces spheres suitable for further culture. Therefore, manual selection of spheres is not required, and the delamination process can be adapted to automation and used in high-throughput research. However, enzymatic cleavage increases cell death and produces fewer spheres.

[0064] Selected CD271+ cells are cultured for approximately 5, 6, 7, 8, 9, or 10 days (or longer, i.e., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 days, or longer), after which the formed NEB is harvested, dissociated, plated as a monolayer culture, and further cultured in a basal medium for nerve cells such as Brainphys® (optionally supplemented with B27 supplement, NGF, and BDNF). The surface on which the cells are plated and cultured may contain extracellular matrix proteins (e.g., collagen, fibronectin, laminin) and / or positively charged polyamino acids (e.g., polyarginine, polylysine, polyornithine). Preferably, the surface contains laminin and / or polyornithine.

[0065] To confirm differentiation into BFCN, cultured cells are analyzed for positive expression of Tuj1, MAP2, BF1, Nkx2.1, and p75.

[0066] Many embodiments of the present invention include, for example, certain factors used (or excluded) in the compositions and methods described herein, such as culture medium supplements. These factors include, but are not limited to, bFGF, GABA, pipecolic acid, lithium chloride, TGF-β, NGF, and BDNF. Each of these factors, including their full names where acronyms or other abbreviations are used, is well known in the art. Furthermore, all of these factors are publicly available from multiple sources, including commercial sources. Typical amounts / concentrations for the use of each of these factors in the methods and compositions of the present invention are provided in the Examples section of this patent disclosure. For all embodiments in which a specific amount is mentioned, an "approximate" specified amount is also intended. Furthermore, those skilled in the art will recognize that in some situations further deviations of a specific amount may be used, and the amount to use can be determined by performing routine testing, optimization, dose-response studies, etc., to suggest or increase a predetermined amount, as long as the amount used still achieves a predetermined effect, e.g., a predetermined differentiation effect. For example, in some embodiments, a certain amount of a particular agent may be reduced to 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of a given amount. Similarly, in some embodiments, a certain amount of a particular agent may be increased by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, or 500% of a given amount. Likewise, where a particular factor is mentioned, those skilled in the art will recognize that analogues, variants, or derivatives of the factor may also be used, as long as they have the same general function / activity as the particular factor.

[0067] As discussed herein, the inventors discovered that mutations in PSEN2 in the BFCN result in impaired neuronal excitability. As discussed in the examples herein, the inventors discovered that PSEN2 mutations restore neuronal excitability. N141I Through mutation repair, we observed significant differences in BFCN excitability related to mutations that can be reversed by editing. Therefore, the method of the present invention may utilize iPSCs that can be processed using a gene editing system to repair one or more mutations, such as mutations in presenilin 1 (PSEN1) or presenilin 2 (PSEN2). In one embodiment, the mutation is PSEN2 N141I Therefore, this repair restores neuronal excitability in the BFCN.

[0068] As used herein, the terms “gene editing” or “genome editing” refer to a type of genetic manipulation in which DNA is inserted, replaced, or removed from target DNA (e.g., the genome of a cell) using one or more nucleases and / or nickases. Nucleases produce specific double-strand breaks (DSBs) at desired locations in the genome and utilize the cell’s endogenous mechanisms for repairing introduced breaks by homologous recombination repair (HDR) (e.g., homologous recombination) or non-homologous end joining (NHEJ). Nickases produce specific single-strand breaks at desired locations in the genome. In one non-limiting example, two nickases may be used to produce two single-strand breaks on opposite strands of target DNA, thereby producing blunt or sticky ends. Any suitable nuclease (including, but not limited to, CRISPR-related protein (Cas) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, other endo- or exo-nucleases, their variants, their fragments, and combinations thereof) can be introduced into cells to induce genome editing of a target DNA sequence. In certain embodiments, nuclease-mediated genome editing of a target DNA sequence (e.g., a safe harbor gene) by homologous recombination repair (HDR) (e.g., homologous recombination) is used to generate genetically modified human neural stem cells according to the methods described herein.

[0069] The term "DNA nuclease" refers to an enzyme capable of cleaving phosphodiester bonds between nucleotide subunits of DNA, and may be an endonuclease or an exonuclease. According to the present invention, the DNA nuclease may be an engineered (e.g., programmable or targetable) DNA nuclease that can be used to induce genome editing of a target DNA sequence, such as a safe harbor gene. Any suitable DNA nuclease may be used, including but not limited to CRISPR-related protein (Cas) nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, other endo- or exonucleases, their variants, their fragments, and combinations thereof.

[0070] In various embodiments of the present invention, gene editing systems utilize DNA nucleases to edit genes and repair mutations. In specific embodiments, mutations are repaired using one or more of the following gene editing systems: CRISPR / Cas system, Cre / Lox system, TALEN system, and homologous recombination.

[0071] The differentiation protocol of the present invention may be used to treat a disease or disorder in a subject. The method includes administering BFCN produced using the culture method described herein to the subject. In various embodiments, the disease or disorder is an amyloidogenic disorder such as systemic amyloidosis, Alzheimer's disease, adult-onset diabetes mellitus, Parkinson's disease, Huntington's disease, frontotemporal dementia, and prion-associated spongiform encephalopathy. In embodiments, a mutation that impairs neuronal excitability (e.g., PSEN2) is used. N141I BFCNs having ) can be obtained from a subject, which can be used to generate iPSCs, which can then be processed using a gene editing system to modify the mutations. Next, the gene-edited iPSCs are cultured as described herein to produce BFCNs with restored neuronal excitability, which are administered to a subject to treat a disease or disorder.

[0072] In the related method, the neuronal excitability of BFCN may be restored in the subject. This method includes a) isolating BFCN from a subject, wherein the BFCN has a gene mutation that causes impaired neuronal excitability of BFCN; b) generating iPSCs using the BFCN from (a); c) repairing the mutation in the iPSCs; d) culturing the iPSCs from (c) using the differentiation protocol described herein to generate BFCN with the mutation repaired; and e) administering the iPSCs from (d) to a subject, thereby restoring the neuronal excitability of BFCN in the subject. In embodiments, the mutation is a mutation in PSEN1 or PSEN2, for example, PSEN2 N141I That is the case.

[0073] The present invention also includes BFCNs produced using the differentiation protocols described herein. The BFCNs may include PSEN2 gene-edited repair and a detectable marker recombinantly introduced into the BFCN genome. In some embodiments, the BFCNs differentiate from PSCs, in which embodiments the PSCs may be iPSCs. The iPSCs may originate from somatic cells of the subject. In one embodiment, the subject has an amyloid-forming disease or disorder.

[0074] iPSC technology, in addition to its potential for autologous cell transplantation, is emerging as a tool for developing novel drugs and gaining insights into disease etiology. Han, SSW et al., Neuron, 70: pp. 626-644 (2011). The methods and cells of the present invention may be aimed at developing high-throughput in vitro screening for compounds that promote the restoration of neuronal excitability. To that end, the present disclosure provides a method for identifying compounds that may be used for the treatment or prevention of diseases or disorders associated with reduced neuronal excitability in BFCNs. The method comprises a) contacting a BFCN or neurocyte (NEB) produced using the differentiation protocol described herein with a candidate compound, wherein the BFCN contains a mutation in PSEN2 that causes impaired neuronal excitability of the BFCN; and b) detecting the neuronal excitability of the BFCN after contact with the candidate compound. Beneficial effects on neuronal excitability are evidence of partial or complete recovery of neuronal excitability, and therefore, such effects serve as an indicator for candidate therapeutic agents for treating diseases or disorders associated with reduced neuronal excitability in BFCNs (e.g., amyloidogenic diseases or disorders). Preferably, the method is carried out in a high-throughput format.

[0075] The present invention also provides a model system for diseases or disorders associated with reduced neuronal excitability in BFCNs, such as neurological disorders, preferably amyloidogenic disorders or disorders. In one embodiment, the model system comprises BFCNs differentiated from iPSCs derived from a subject having a disease or disorder associated with reduced neuronal excitability in BFCNs, such as amyloidogenic disorders or disorders. The model system may further comprise a non-human mammal to which myelin-producing cells have been transplanted. In one embodiment, the non-human mammal is a mouse or a rat. Diseases or disorders can be studied using the model system provided by the present invention, which includes understanding the underlying mechanisms and identifying therapeutic targets.

[0076] In some embodiments, the present invention provides tissue culture media, tissue culture media supplements, and various kits useful for carrying out the various methods described herein.

[0077] In one embodiment, the present invention provides a kit for generating BFCNs by the differentiation protocol of the present invention. The kit comprises a medium having a TGF-β signaling inhibitor and a Shh signaling activator. In an embodiment, the medium is a modified mTeSR1 composition lacking pluripotency-supporting factors, comprising bFGF, TGF-β, lithium chloride (Li-Cl), GABA, and pipecolic acid. In an embodiment, the medium comprises a dual SMAD inhibitor, such as SB431542 and LDN193189, along with one or more agonists of smoothed proteins, such as a smoothed agonist (SAG) and purmorphamine.

[0078] The kit may also include additional neuronal cell basal media, such as Brainphys® (which may optionally be supplemented with one or more of the following: B27 supplement, ROCK inhibitor, NGF, and BDNF).

[0079] The kit may also include reagents for the detection and isolation of CD271+ cells by FACS, as well as for the detection of Tuj1, MAP2, BF1, Nkx2.1, and p75 expression.

[0080] The kit may include, depending on the circumstances, instructions for use, one or more containers, one or more antibodies, or any combination thereof. Typically, a label is attached to the kit, which may include any written or recorded information, which may be in an electronic or computer-readable form (e.g., disk, optical disc, memory chip, or tape) that provides instructions for using the kit contents or other information. [Examples]

[0081] The following embodiments are provided to further illustrate the advantages and characteristics of the present invention, but are not intended to limit the scope of the invention. While these embodiments are typical of what may be used, other methods, methodologies, or techniques known to those skilled in the art may be used instead.

[0082] Example I Alzheimer's disease originating from iPSCs (presumably referring to a specific type of microorganism) is related to PSEN2. N141I Molecular and physiological phenotypes associated with CRISPR / Cas9-modifiable mutations in mutations Basal forebrain cholinergic neurons (BFCNs) are thought to be one of the first cell types affected in all forms of AD, and their dysfunction clinically correlates with impairments in short-term memory formation and retrieval. We present an optimized in vitro protocol for generating human BFCNs from iPSCs using cell lines derived from carriers and controls of the presenilin 2 (PSEN2) mutation. As expected, PSEN2 N141I Cell lines containing the mutation showed increased Aβ42 / 40 levels in BFCN derived from iPSCs. PSEN2 N141I The maximum number of spikes generated by neurons derived from the strain in response to the injection of a rectangular depolarizing current was lower. The height of the first action potential at the time of injection of the baseline current was also lower in PSEN2. N141I A significant decrease was observed in BFCN. CRISPR / Cas9 correction of the PSEN2 point mutation eliminated the electrophysiological abnormality and restored both the maximum number of spikes and spike height to levels recorded in the control. In addition, increased Aβ42 / 40 was corrected in PSEN2 N141I The mutation was normalized after CRISPR / Cas-mediated correction. Genome editing data demonstrates strong consistency in mutation-related changes in the Aβ42 / 40 ratio, while also showing PSEN2 mutation-related changes in electrophysiology.

[0083] The “amyloid hypothesis” is one of the most supported explanations for the pathogenesis of Alzheimer’s disease (AD). Recent examples of clinicopathological and / or clinical radiological degradation have led to consideration of alternative models to explain why neuropathological AD is not always associated with dementia

[24] and why about one-third of patients with clinical AD have negative amyloid brain scans

[40] . It has been proposed that clinical AD may be caused by one of several “feedforward” scenarios linked to amyloidosis, tauopathy, neuroinflammation, and neurodegeneration

[22] . Mutations in the gene encoding presenilin 2 (PSEN2) are associated with autosomal dominant early-onset familial Alzheimer’s disease (EOFAD). The combination of the gene locus on human chromosome 1q31–42 linked to EOFAD was identified in 1995 in the Volga German blood relationship. N141I This led to the identification of a point mutation

[43] . This mutation causes an increase in the Aβ42-43 / 40 ratio, thereby promoting the assembly of Aβ oligomers and fibrils

[83] .

[0084] When considering the progression of AD, human basal forebrain cholinergic neurons (BFCNs) are one of the first cell types whose dysfunction underlies the early loss of short-term memory recall in all forms of AD. The "cholinergic hypothesis of AD" was summarized in the mid-1970s [6, 20, 61], and Meynert's discovery of reduced acetylcholine release from basal ganglia neurons established the presence of presynaptic cholinergic abnormalities in the basal forebrain of AD patients [1, 71]. Based on these findings, acetylcholinesterase inhibitors were developed and remain the most widely used symptomatic treatment for AD [21, 28, 33, 82]. Ultimately, biochemical and volumetric studies of post-autopsy brains at different stages of the disease have identified several other areas of the brain that were similarly affected early in the course of AD

[63] . These results have traditionally focused on the hippocampus and cortex, but more recently, attention has begun to shift back to the basal forebrain and to add other areas such as the striatum [27, 62]. Recent analyses suggest that cholinergic basal forebrain volume measurements may be a better predictor of the transition from MCI to AD than conventional standard hippocampal volume

[10] .

[0085] The inventors of the present invention have identified PSEN1 A246E and PSEN1 M146L We have previously reported the generation of neurons derived from iPSCs from conserved fibroblasts of a mutated subject

[77] . In characterizing the gene expression characteristics of these iPSC-derived neurons, we observed an unexpected association between increased expression of the inflammasome gene NLRP2 in undifferentiated PSEN1 mutant iPSCs and their differentiated offspring

[77] . Accordingly, we investigated NLRP2 expression in our PSEN2 mutant strains and used CRISPR / Cas9

[15] to examine whether inflammasome activation is strongly linked to pathogenic mutations in PSEN2. We did not find any changes in NLRP2 expression in our genetically modified PSEN2 strains, but we observed a significant difference in BFCN excitability that was mutation-related and reversible by editing.

[0086] material and method Creation and maintenance of iPSC strains iPSC strains 7889(s)B, 050643(control), 948(AD1), 949(f control), and 950(AD2) were obtained via the NYSCF storage facility in accordance with the guidelines in

[60] . The induction and characterization of the Nkx2.1-GFP ESC strains have been previously published

[30] . Expansion and maintenance of ES and iPS cell lines were performed in serum-free mTeSR1® medium (Stem Cell Technologies). Cells were detached using StemPro® Accutase (ThermoFisher), and 10 μM ROCK inhibitor (Y27632, Stemgent) was added to the medium during cell passage.

[0087] For all studies in this document, cell lines underwent at least three independent differentiations from the iPSC stage to the mature neuronal stage. Data were typically compared across identical neurons (or, in some cases, neural progenitor cells) from these independently derived genotypes. If the independently derived genotypes yielded equivalent results across identical cells, they were considered certified representatives of those genotypes and therefore transmitted for genotype-specific experiments.

[0088] Aβ42 oligomer preparation Aβ42 oligomers were prepared as previously reported [23, 78]. Briefly, the inventors dissolved 1 mg of Aβ42 (American Peptide Company) in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) (Sigma). This preparation was divided into equal parts and dried using a SpeedVac® centrifuge. The pellet was then resuspended in DMSO and sonicated in a water bath for 10 minutes to obtain a 5 mM solution. From this, aliquots were stored at -20°C and used within two weeks by diluting with 100 μl of PBS and allowing to stand at 4°C for 12 hours to promote oligomer formation. This final solution was diluted 1:16 in study cell medium and cells were exposed to 5 μM Aβ42 oligomers. Control wells were diluted with 1:16 PBS. Cells were exposed to the oligomer or PBS for 3 days without changing the medium.

[0089] Cell death assay Cells were assayed in a 96-well plate format. Oligomer or vehicle solution was added to the culture medium and incubated for 3 days. The medium was then collected and assayed using a lactate dehydrogenase toxicity assay (Thermo Fisher Scientific). 50 μl of medium and an equal volume of reaction buffer were incubated for 30 minutes. A further set of wells per experiment was incubated with 2% Triton® X-100 for 5 minutes to lyse all cells, and the medium from these wells was also collected and incubated as described. After incubation, the signal and background absorbances were recorded at 490 nm and 680 nm, respectively. The background was subtracted from the signal value, and the value was adjusted to match the total LDH concentration determined by the wells treated with Triton X-100. Propidium iodide (Thermo Fisher Scientific) was added to the cell medium to a final concentration of 1 μM and incubated for 5 minutes. The cells were then washed twice with medium and imaged. Images were acquired using the CELIGO® image hemocytometer and its accompanying software (Nexcelom Bioscience). Each biological variable was evaluated in technical triplicates within its designated "experiment," and each designated "experiment" was performed in at least three complete "start-to-end" iterations.

[0090] Differentiation of basal forebrain cholinergic neurons from iPS and ES cells Human ES or iPSCs were chemically dissociated using Accutase® (Sigma-Aldrich), and then 4-8 × 10⁶ cells per well in a 6-well plate or Petri dish were processed onto a Cul-trex® (Trevigen) coated plate. 5Cells were plated as single cells at a specific cell density, with the number of cells equalized. The cells were initially maintained in mTeSR1® medium (Stem Cell Technologies) until they reached full confluence. On day 0 of differentiation, the medium was replaced with a custom mTeSR1® medium (Stem Cell Technologies) lacking pluripotency-promoting factors, namely bFGF, TGF-β, Li-Cl, GABA, and pipecolic acid. Addition of dual SMAD inhibitors (10 μM SB431542 and 250 nM LDN193189, Selleckchem) on day 0 directed the cells toward neuroectoderm identification. On day 2 of differentiation, the medium was replaced with custom mTeSR1 containing dual SMAD inhibitors and two ventralizing factors: 500 nM SAG® and 2 μM purmorphamine (Stemgent®). When gradually switching the culture medium to Brainphys® medium (Stemcell Technologies) supplemented with B27 (Life Technologies), cells were given this medium every two days until day 9 [3]. Neural progenitor cells were harvested on day 11 using Accutase, p75+ (CD271)NPC was purified by FACS, and plated at a cell density of 80,000 cells per well in Brainphys®+B27 supplemented with 10 μM ROCK inhibitor (Y27632, Stemgent), nerve growth factor, NGF (Alamone Labs, 50 ng / mL), and brain-derived neurotrophic factor, BDNF (R&D, 50 ng / mL) in non-adherent 96-well V-bottom plates. Cells were aggregated to form neuroid bodies (NEBs) and added every other day until day 19. On day 19, NEB was dissociated using Accutase (Sigma-Aldrich), and the monolayer culture was plated onto plates coated with branched polyethinylimine (0.1%, Sigma-Aldrich) and laminin (10 mg / mL, Life Technology) in Brainphys® medium + B27 supplemented with BDNF and NGF. The medium was changed every two days until analysis.Alternatively, 3D NEBs were manually dissected into 3-4 smaller pieces for further growth or cryopreservation. The initial version of the protocol used Neurobasal® as the basal medium instead of Brainphys®.

[0091] Isolation and sequencing of genomic DNA Following the manufacturer's instructions, genomic DNA was isolated from PSEN2 mutant iPSC strains or control iPSC strains using the High Pure® PCR template preparation kit (Roche). Prior to amplification, the genomic samples were treated with RNAse (QIAGEN). The following primers were used: forward: TIFF2026108846000001.tif4128, reverse: Using TIFF2026108846000002.tif4128, PSEN2 N141I A fragment derived from PSEN2 exon 5 containing the mutation was amplified, yielding a 173 bp fragment regardless of genotype. For the detection of ApoE allele variants, primers were used prior to sequencing: forward: TIFF2026108846000003.tif5133, reverse: A 244 bp fragment was amplified using TIFF2026108846000004.tif4128. Both PCRs were performed using the following settings: 10 minutes at 94°C; 40 cycles (30 seconds at 94°C, 20 seconds at 62°C, 10 seconds at 72°C); 7 minutes at 72°C. The PCR products were run on a 2% agarose gel to check the size of the amplified fragment. After amplification, the samples were washed using EXOSAP-it (Trademark) (Thermo Fisher Scientific), and then the following primers were applied: PSEN2 primers from

[53] (forward: TIFF2026108846000005.tif4128, reverse: Primer for ApoE from TIFF2026108846000006.tif5128);

[36] (forward: TIFF2026108846000007.tif4128, reverse: The sequence was determined using TIFF2026108846000008.tif5128.

[0092] Genetic modification using CRISPR / Cas9 iAD1 control stock and iAD2 control stock are PSEN2 WT / WT PSEN2 N141I / WT CRISPR / Cas9-mediated modification of heterozygous point mutations was generated from 948(AD1) and 950(AD2)iPSC strains. The g1N141I single guide RNA (sgRNA) was cloned into the pSpCas9(BB)-2A-GFP(PX458) vector, thereby producing a pSpCas9-g1N141I-GFP vector to direct gene editing to the sequence in exon 5 of PSEN2 where the Volga mutation is located. Single-stranded oligonucleotides (ssODNs) are efficient templates for CRISPR / Cas9 modification [13, 66]. ssODN#A-N141I (sequence details below) was used as the donor sequence for gene modification. The inventors designed an asymmetric ssODN sequence having a 91 bp long homology arm and a 36 bp short homology arm because asymmetric ssODNs showed higher efficiency in homology-directed repair using CRISPR / Cas9

[68] .

[0093] [Table 1] Underlined = ssODN bases that correct point mutations

[0094] AD1 and AD2 iPSC lines plated to 50-70% confluence using the Amaxa Human Stem Cell Nucleofector® Kit (Lonza VPH-5002) were transduced with donor sequences and the pSpCas9-g1N141I-GFP vector, and replated for recovery. GFP+ cells were sorted using a BD FACSAria IIu Cell Sorter®, seeded at a rate of 30-50 cells per well in a 96-well format, and single colonies were detected and picked. Positive colonies were expanded, qDNA was extracted, and good HDR was analyzed using a specially designed TaqMan® genotyping assay with a probe specific to a SNP located at Chr1:227,073,304 A>T (dbSNP ID:rs63750215). The selected clones were analyzed by Sanger sequencing to confirm the correction at position Chr1:227,073,304, and any potential inserts or deletions in the surrounding region were discarded.

[0095] Fluorescent cell sorting (FACS) Neural progenitor cells on day 12 of differentiation were dissociated using Accutase (Sigma-Aldrich) at 37°C for 5 minutes and inactivated in neuronal basal medium. The cells were spun at 1000 rpm for 4 minutes, and the pellet was resuspended in FACS buffer (DPBS, 0.5% BSA fraction V solution, 100 U / mL penicillin-streptomycin, 0.5% EDTA, and 20 mM glucose) containing PE mouse anti-human CD271 antibody (clones C40-1457, BD), also known as p75 or NGFR, in a 1:100 ratio, and incubated at room temperature (RT) in the dark for 20 minutes. After incubation, the cells were washed with FACS buffer, and the pellet was resuspended in 2 mL of FACS buffer containing 10 μM ROCK inhibitor (Y27632, Stemgent). p75-positive cells were purified using BD FACSAria IIu Cell Sorter (trademark), and the data were analyzed using FlowJo (trademark) software.

[0096] Real-time quantitative polymerase chain reaction (RT-qPCR) Human iPSCs derived from PSEN2 mutant patients or control patients were grown in monolayers and directly lysed in cell culture wells containing RLT buffer. Total RNA purification was performed using the RNeasy® Microkit (Qiagen) according to the manufacturer's instructions. cDNA was synthesized using SuperScript® III reverse transcriptase (RT) (Invitrogen, Carlsbad, CA). Semi-quantitative real-time PCR was performed using the StepOnePlus® Real-Time PCR System (Applied Biosystems, Foster City, CA) with the primers listed in Table 2 below. The inventors standardized the expression levels for GAPDH. The PCR cycle parameters were 40 cycles of 2 minutes at 50°C, 10 minutes at 95°C, followed by 15 seconds at 95°C, and 1 minute at 60°C. Each biological variable was evaluated in technical triplicates in each designated “experiment,” and each designated “experiment” was performed in at least three complete “start-to-end” replicates. Expression levels were standardized relative to the control strain, and the results were expressed as AVG±SEM.

[0097] [Table 2]

[0098] Aβ assay Cells were conditioned for 3 days after 8 days of dual SMAD inhibition, and Aβ secretion by neural progenitor cells was measured in vitro. Aβ levels were quantified using the Human / Rat β-Amyloid 40 ELISA Kit and the β-Amyloid 42 ELISA Kit High Sensitivity (Wako). Each biological variable was evaluated using technical triplicates in each designated "experiment," and each designated "experiment" was performed in at least three complete "start-to-end" replicates.

[0099] Immunostaining / ICC Cells were fixed directly into the wells of 12, 48, or 96-well plates with 4% PFA for 20 minutes and washed three times with 1× DPBS (ThermoFisher). For staining, cells were incubated at room temperature for 2 hours in blocking solution (1× DPBS containing 0.1% Triton® X-100 plus 5% donkey serum). The corresponding primary antibody was diluted to an appropriate concentration in the blocking solution and incubated overnight at 4°C. The primary antibodies used are shown in the table below. Cells were washed three times with DPBST (1× DPBS + 0.1% Triton® X-100), and an appropriate secondary antibody was added in the blocking solution at room temperature for 1 hour. Next, cells were washed three times with DPBST, and for nuclear counterstaining, they were incubated at room temperature for 10 minutes with DRAQ5 or Hoescht 33,342 (1 μg / mL, diluted in 1× DPBS). Cells were visualized using an inverted fluorescence microscope (Olympus® IX71 microscope) or a confocal microscope (Zeiss® LSM5 Pascal microscope) at magnifications of 10×, 20×, or 63×.

[0100] Western blot Human iPSCs derived from PSEN2 mutant patients or control patients were grown in monolayers and directly lysed in cell culture using RIPA buffer (Thermo Scientific) containing protease and phosphatase inhibitors. Protein concentrations were measured using the BCA protein assay kit (Thermo Scientific). After protein identification, 20 μg of cell lysate was separated by SDS-PAGE electrophoresis on 4-12% Bis-Tris gel (Bolt® protein gel) and transferred to a nitrocellulose membrane by electrophoretic blotting. The membrane was blocked for 1 hour at room temperature with agitation using blocking buffer 1X TBST (Tris-buffered saline + 0.1% Tween) and 5% non-fat dried milk, and washed three times with TBST. After washing, the membrane was incubated overnight at 4°C with primary antibodies against NLRP2 (1:1000), PSEN2 (1:200), or 3-actin (1:1000) with agitation. After washing, the membranes were incubated with a suitable secondary antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. Finally, the protein bands were visualized using a chemiluminescent reagent according to the manufacturer's instructions. 3-actin was used as a control.

[0101] Electrophysiology Whole-cell patch-clamp recordings were obtained from single neurons at 38–55 days of differentiation. Cells were seeded at low density on plastic coverslips placed in a perfusion-based recording chamber. Neurons were recorded using Hamamatsu Orca®® 2The signal was localized using differential interference contrast optics under an Olympus BX61WI microscope equipped with a CCD camera. Recording was performed at room temperature using a MultiClamp® 700B amplifier (Molecular Devices, Sunnyvale, CA, USA). The signal was sampled at 10 kHz and filtered at 6 kHz using a Digidata® 1440A, similar to a digital converter (Molecular Devices). Amplifier control and data acquisition were performed using pClamp® 10.0 software (Molecular Devices).

[0102] During recording, neurons were perfused with oxidized Brainphys® medium (StemCell Technologies Inc.). A medium resistance recording pipette (4–6 MΩ) was titrated to pH 7.1 and filled with an intracellular solution consisting of 130 K-gluconate, 10 KCl, 2 Mg-ATP, 0.2 Na-GTP, 0.6 CaCl2, 2 MgCl2, 0.6 EGTA, and 5 HEPES at a molar osmotic concentration of 310 mOsm (mM). In some experiments, the intracellular solution also contained 4 mg / mL biocitin (Sigma-Aldrich) for individual neurons after hoc identification, which was visualized using streptavidin-conjugated Alexa 488 (Life Sciences) as described elsewhere

[42] . After the initial interruption, access resistance (Rs) was consistently monitored, and the record was discarded if Rs exceeded 20 MΩ or changed by more than 30%. The potential protocol for characterizing compound Na+ and K+ currents was as follows: Cells were maintained at a potential of -80mV and then subjected to 500ms steps of increasing the potential by 10mV at a frequency of 0.1Hz from -100mV to 30mV. After transitioning to a current-fixed mode, the resting membrane potential was recorded, and the cells were hyperpolarized by injecting negative DC current down to -70mV to validate the consistency of the excitability measurements. Action potentials were induced using 1-second rectangular current steps of 1pA from -10pA to 40pA.

[0103] Electrophysiological records were analyzed using ClampFit® software (Molecular Devices, Sunnyvale, CA, USA), and the statistical significance of the results was measured using an ANOVA test with Tukey post-hoc comparisons. Salts and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA).

[0104] statistical analysis qPCR gene expression experiments and Aβ42 / 40 ELISA were analyzed for statistical significance using Student's t-test. LDH release assays were analyzed using a two-way ANOVA Bonferroni post-hoc test. ANOVA tests with Tukey post-hoc comparisons were used for the analysis of electrophysiological results. The experiments required the detection of each of the 94 neurons recorded for electrophysiological analysis, which took several days to several weeks. On each experimental day, a representative of each genotype was included, along with at least three samples from each genotype studied on that day. * p<0.05; ** p<0.01; *** p<0.001.

[0105] result Optimization of BFCN differentiation protocols The BFCN differentiation scheme is shown in Figure 1A. iPSCs derived from control subjects or AD patients were plated without feeder and allowed to reach 100% confluence before differentiation using mTeSR1 basal medium. On day 0, both branchings of TGF-β signaling were inhibited (dual SMAD inhibition) to induce neuroectoderm fate

[12] . Differentiation (days 2-10) was performed using a modified mTeSR1 composition lacking pluripotency-supporting factors (bFGF, TGF-β, Li-Cl, GABA, and pipecolic acid). Ventralization for globus pallidus primordium (MGE) induction was required to identify these cells as forebrain basal cholinergic neurons [19, 85, 91]. The cells themselves were treated with 500 nM sonic hedgehog (Shh) analog (SAG) and 2 μM purmorphamine on days 2-8. SAG is less expensive than recombinant Shh and has several advantages over Shh itself in terms of neuronal viability [7, 35], ChAT + As demonstrated during the differentiation of motor neurons and glutamatergic interneurons, it is a suitable alternative for activating Shh signaling

[91] . The inventors used an Nkx2.1-GFP embryonic stem cell (ESC) reporter strain as a tool to modulate the combination, dosage, and timing of ventralization factors to be more beneficial for identifying BFCNs derived from induced Nkx2.1 forebrain basal precursors.

[0106] However, given Nkx2.1's ability to generate multiple neuronal subtypes, including TH+ and GABA+ hypothalamic neurons, from intermediate neural progenitor cells, we analyzed the expression of the downstream cholinergic specificity factor Lhx8 relative to the expression of the GABAergic interneuron-specific transcription factor Lhx6

[26] under different specificity conditions (Figure 1b). These data are consistent with data supporting the existence of a synergistic effect of SAG and pulmorphamine on Nkx2.1 induction, although the effect is less significant than that of Shh+ pulmorphamine

[50] (Figure 1B). GFP levels derived from Nkx2.1 were maintained beyond day 14 and even after discontinuation of SAG+ pulmorphamine on day 8 (Figure 1B). We observed higher Lhx8 levels when treated with SAG+ pulmorphamine than with SAG alone, and even higher than with Shh+ pulmorphamine (Figure 1B). Interestingly, intermediate Nkx2.1 levels induced by SAG + purmorphamine correlated with higher gene expression induction of Lhx8 and BF1 (Figure 1B). Our choice to initiate SHH pathway-induced ventralization on day 2 was based on reports indicating other MGE-derived populations generated by earlier (e.g., hypothalamic neurons) or later (e.g., GABAergic interneurons) ventralization in association with dual smad inhibition protocols.

[0107] Following the pattern formation phase, the inventors gradually switched from custom mTESR1® medium to Brainphys® medium containing a B27 supplement to support neuronal survival and growth [3]. On day 11, the inventors observed neuronal rosettes positive for nestin and Sox2 markers (Figure 1C); and also observed Tuj1+ neurites as early as day 11 (Figure 1C). To obtain a higher purity cholinergic population, the inventors developed a P75+ FACS strategy to isolate cholinergic neuronal-specific offspring, due to the fact that BFCNs are the only CNS neuronal cell type that expresses robust levels of P75 under non-pathogenic conditions in the adult brain. Support for this strategy includes a previously published protocol using FACS to isolate high-expression P75+ cells from embryonic mouse septa

[65] . This population correlated with the highest expression of cholinergic-related markers.

[0108] On days 11 and 12, the inventors isolated cells using chemical dissociation (Accutase), purified p75+ (CD271) neural progenitor cells from days 11-12, and generated 3D neuroid bodies (NEBs) by spinning down the neural progenitor cells in a V-bottom 96-well plate. On day 19, the NEBs were dissociated and replated as a monolayer on a plate coated with branched polyethyleneamine (Aldrich catalog number 408727) and laminin. The monolayer culture was maintained by adding growth factors BDNF, NGF, and DAPT until day 26 (when the culture no longer contained the added DAPT). Immunostaining of frozen sections of both NEB structures and fixed monolayers, obtained from chemical dissociation of NEB from several control iPSC and H9 hESC strains, showed expression of BFCN lineage markers Tuj1, MAP2, BF1, Nkx2.1, and p75 at the final stage of the differentiation protocol (Figure 1D). Addition of NGF to neuronal cell cultures showed favorable effects on maturation, neurite outgrowth, and the presence of ChAT (Figure 1E).

[0109] PSEN2 N141IiPSC strain creation and QC PSEN2 N141I Mutant iPSCs and control cells were obtained from fresh skin biopsies. Established fibroblast cells were identified as having the presenilin 2-Volga familial AD mutation (PSEN2). N141I The cells were grown from skin punches donated from the families of two carriers and one unaffected member of the ) family. In addition, the inventors included non-familial-related controls. Fibroblasts were reprogrammed using a modified RNA method to introduce Yamanaka factors (Oct4, KLF4, SOX2, and c-Myc), and the resulting iPSC lines were subjected to several quality control processes to confirm robust cell regeneration and pluripotency, including alkaline phosphatase (AP) enzyme activity, gene expression analysis, immunostaining for pluripotency markers, and karyotype analysis for detection of chromosomal abnormalities, according to an automated iPSC reprogramming and QC method developed by

[60] . The genotypes, sexes, and ages of the subjects included in the study are shown in Figure 2a. Also, two types of PSEN2 N141I The iPSC strains were heterozygous (ε3 / ε4) for APOE ε4, while the control iPSC strain was homozygous ε3 / ε3. The results of iPSC strain characterization, pluripotency marker expression, and quality control are shown in Figure 10. Briefly, all selected iPSC clones exhibited pluripotency through embryoid body formation and differentiation into three germ layers (Figure 10A), which is incorporated herein by reference. Finally, the strains were fingerprinted (Cell Line genetics) to ensure they matched the parent fibroblast cell lines (data not shown). All parent fibroblast cell lines and iPSC strains were subjected to Sanger sequencing to determine the PSEN2 and APOE genotypes. PSEN2 N141IA 173 bp fragment from exon 5 of PSEN2, surrounding the region where the point mutation is located (Chr1:227,073,304 A>T), was amplified by PCR and sequenced using primers published in

[53] ; similarly, a 244 bp fragment from the APOE locus containing two SNPs determining the three allele variants was amplified by PCR from genomic DNA, subsequently sequenced, and distinguished between the ε2 / ε3 / ε4 variants using primers from

[36] . PSEN2 N141I Chromatograms of samples showing the presence of point mutations are shown in Figure 2B, and all genotypes are outlined in Figure 2A.

[0110] PSEN2 N141I Characterization of neural progenitor cells PSEN2 in the early stages of cholinergic neuronal differentiation N141I To study the effects of mutations, the inventors analyzed neural progenitor cells (NPCs) obtained in DIV11-16 according to the BFCN differentiation protocol. Analysis of this intermediate, immature population allows the inventors to detect potential early changes in BFCN formation that would not be detected in fully differentiated cholinergic neurons if this analysis were not performed. Such abnormalities may potentially play a role in mature neurons and contribute to the pathophysiology of AD. The inventors used gene expression and immunofluorescence to analyze PSEN2 N141I The expression of early neuronal markers in mutant NPCs and control NPCs was analyzed. The inventors found that the RNA expression of Tuj1 (βIII-tubulin), a common neuronal marker, was lower in mutant NPCs at day 11 of differentiation. However, at approximately days 16-21, the inventors did not detect any differences quantifiable by immunocytochemistry (Figures 2C and D). Furthermore, immunostaining of NPC monolayer cultures at day 11 for typical NPC markers: Sox2 and Pax6 showed a decrease in Pax6 levels, as predicted with Nkx2.1 introduction (not shown). The inventors also found that at day 11, PSEN2... N141IEquivalent expression of Sox2 and nestin was observed in the culture (Figure 2C, upper panel). On day 21, mutant NPCs expressed equivalent levels of Nkx2.1 (MGE marker), but qPCR showed reduced levels of BF1 (forebrain marker); however, immunohistochemistry at this differentiation stage appeared unaffected by BF1 protein expression (Figure 2C, lower panel, and D). The inventors compared the percentage of positive cells (%) or the fluorescence median peak value of PSEN2 in DIV11-12. N141I No difference was observed in the surface expression of NGFR(p75 / CD271) in cells (Figure 2E).

[0111] As previously published by [59, 73], variant PSEN2 N141I Expression of leads to an increase in the Aβ42 / 40 ratio in the brains of transgenic mice; in addition, this enhanced Aβ42 production is associated with mutant PSEN2 N141I Neuronal cell lines when protein overexpression is induced

[83] and PSEN2 N141I This was observed in iPSCs derived from mutant patients

[93] . Consistently, the inventors have identified PSEN2 of DIV11. N141I In the culture supernatant of neural progenitor cells, a twofold increase in the Aβ42 / 40 ratio, a 50% increase in the amount of secreted Aβ40, and a 2.5-fold increase in the types of Aβ42 were observed. *** (p<0.001) (Figure 2e). When different neuronal differentiation methods were applied to FAD1 / PS2 iPSC lines derived from Coriel's Reservoir fibroblasts, the secretion levels of Aβ40 and 42 observed in our study, as well as the levels seen in

[93] , were very similar in both absolute numbers and multiples.

[0112] PSEN2 N141I Characterization of mature BFCNs derived from iPSC strains and controls. PSEN2 for BFCN differentiation, gene expression, function, and communication N141ITo determine the effects of mutations, the inventors characterized cells at a later time point for appropriate expression markers; the inventors' goal was to identify PSEN2 N141I The objective was to investigate whether the iPSCs were able to complete the BFCN maturation process, and if so, whether any abnormalities along later stages of BFCN differentiation would explain the pathophysiology of EOFAD (Figure 3). In addition to p75, which preferentially binds to proNGF, we also investigated PSEN2 N141I We analyzed the expression of TrkA, a primary mature NGF receptor, expressed in BFCN and the control (Figure 3a). This is PSEN2 N141I This suggests that BFCNs, as expected, receive and benefit from NGF's survival-promoting and differentiation signals, further supporting their unique identity. The inventors of PSEN2 N141I Equivalent expression of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (vAChT), further cholinergic neuron-specific markers, was observed in BFCN and controls (Figure 3b). Other common neuronal markers, such as Tuj1 and the maturation marker microtubule-associated protein 2 (MAP2), showed clear differences by immunofluorescence (Figure 3b).

[0113] PSEN2 mediated by CRISPR / Cas9 N141I Effects on mutation correction and Aβ42 / 40 ratio As previously observed in PSEN1 mutants

[77] , molecular changes in APP processing and cleavage, as well as / or impaired NLRP2 inflammasome activation, are observed in PSEN2 N141I To determine whether mutations alone are involved, the inventors used CRISPR / Cas9 technology to modify the PSEN2 locus in their iPSC strains. The inventors then modified the PSEN2 locus in two PSEN2 mutant iPSC strains (AD1, AD2). N141IThis was done by correcting point mutations. For this purpose, an online tool (tools.genome-engineering.org) was used to modify PSEN2 N141I A specific guide RNA (g1N141I) was designed to induce Cas9 in the PSEN2 exon 5 region surrounding the mutation (Chr1:227,073,304 A>T 23bp upstream). g1N141I was cloned into the pSpCas9(BB)-2A-GFP(PX458) vector. pSpCas9-g1N141I-GFP was transduced into HEK293T, and GFP expression was evaluated by fluorescence (Figure 4a).

[0114] Since asymmetric donor sequences in which the shorter arm is positioned closer to the PAM side have shown superior efficiency in homologous recombination repair using the CRISPR / Cas9 system

[13] , we designed an asymmetric ssODN HDR (homologous recombination repair) template, ssODN#A-N141I, having a 91 bp longer homology arm and a 36 bp shorter homology arm, to correct mutations. Next, we treated iPSC strains to transduce pSpCas9-g1N141I-GFP and ssODN#A-N141I using Amaxa® nucleofection (Figure 4a). 48 hours after nucleofection, the cells were dissociated and GFP was transduced. + The population was purified by FACS and replated at low density in a feeder-free manner to isolate a single gene-modified clone (Figure 4b). Subsequently, the clone was amplified, and gDNA was extracted after expansion. Screening of positive clones demonstrating successful HDR was performed by qPCR using a specially designed TaqMan® genotyping assay with a probe specific to the SNP located at Chr1:227,073,304 A>T (dbSNP ID: rs63750215). By this method, the inventors identified homozygous PSEN2 derived from the original iPSC strain. N141I heterozygous PSEN2 N141I , and PSEN2 WTDistinguishing between single clones is possible, and pre-selected clones are subjected to Sanger sequencing to confirm the location of Chr1:227,073,304, and to detect possible insertions, deletions, or mismatches introduced by CRISPR / Cas9 modifications in the surrounding region, demonstrating the success of HDR (Figure 4c).

[0115] The successfully modified clones were expanded and subjected to the BFCN differentiation protocol in parallel with the other four strains used in the study. The inventors collected culture medium from BFCN(DIV34) and retested for amyloid-beta production. Supporting the inventors' previous findings in NPCs of DIV11-12 (Figure 2f), the inventors observed that mature BFCNs also exhibited a significant increase in the Aβ42 / 40 ratio (Figure 4d) and overall Aβ production (Figure 11). Importantly, these results also showed normalization of the Aβ42 / 40 ratio to control levels in the modified strains (iAD1 control and iAD2 control are modified clones of AD1 and AD2, respectively) (Figure 4d). These results also indicated abnormal APP processing and PSEN2 N141I This supported previous findings that the mutations linked presenilin to the presence of a γ-secretase catalytic site

[90] .

[0116] PSEN2 derived from iPSC N141I Evaluation of the sensitivity of nerve cells to Aβ42 oligomer toxicity Previous reports have shown that iPSC strains with FAD mutations may exhibit enhanced sensitivity to harmful irritants such as high concentrations of Aβ42 oligomers [2]. Therefore, we have identified PSEN2 N141IWe tested whether our BFCN derived from the mutant exhibited enhanced toxicity to Aβ42 oligomers in culture medium (Figure 5). We assessed neurotoxicity by measuring the percentage of lactate dehydrogenase (LDH) released by dead cells (this provides an indirect measure of toxicity). Using this method by two-way ANOVA, we detected a significant effect on toxicity induced by 5 μM Aβ42 oligomers added to culture medium after 72 hours of exposure. *** (p<0.01). Post-hoc Bonferroni analysis revealed a significant difference between the AD2 strain and its modified isogenic control (iAD2 control). However, this clearly enhanced susceptibility to Aβ42 oligomer toxicity was not observed in the AD1 strain and its corresponding control. These results suggest that the difference in susceptibility to Aβ42 cannot be attributed solely to the mutant PSEN2 genotype, and that further genetic factors between AD1 and AD2 subjects may influence susceptibility to this stress, further supporting the importance of multiple isogenic models.

[0117] PSEN2 derived from iPSC N141I Evaluation of NLRP2 mRNA in nerve cells The inventors have previously reported that NLRP2 mRNA was elevated in PSEN1 mutant iPSCs and NPCs, similar to the case of PSEN1 mutant cortical neurons (unpublished findings)

[77] . Therefore, the inventors have reported that PSEN2 N141IWe wanted to analyze the state of inflammasome components in relation to mutations. When we assayed NLRP2 mRNA levels in NPCs using qPCR in DIV12, we observed a more than 100-fold increase in AD1 and AD2 strains compared to the control strain (Figure 6a). This correlated with a significant increase in NLRP2 protein observed by SDS-PAGE in whole cell lysates from PSEN2 mutants on day 11 (Figure 6d). However, remarkably, we did not detect a band for NLRP2 by immunoblotting in AD2 strain lysates. Furthermore, we were unable to demonstrate several other transcriptional phenomena already observed in PSEN1 mutant iPS neural progenitor cells, such as an increase in ASB9, which encodes an E3 ligase that induces mitochondrial creatinine kinase degradation. Instead, we observed a tendency for levels to decrease by 20-30% in PSEN2 mutant carriers.

[0118] PSEN2 derived from iPSC N141I Evaluation of BFCN excitability The inventors of this invention used the BFCN differentiation protocol to introduce PSEN2 from day 35 of differentiation. N141IElectrophysiologically active cholinergic neurons were successfully generated in dishes derived from two mutant AD patients, a wild-type control, and a familial control. Initially, BFCNs grown in neuronal basal medium did not yield mature action potential waveforms at this stage, but switching to Brainphys® medium significantly improved the electrophysiological properties of the cultured neurons [3]. These findings are consistent with electrophysiological characterization of neurons generated from other iPSCs used to compare both media [3]. The benefits of the Brainphys® medium protocol were replicated in two further cell lines (including an H9 embryonic stem cell line) with comparable ChAT and VAChT endpoint expression and electrophysiological responses. To investigate the electrophysiological properties of BFCNs, the inventors recorded a total of 94 neurons (22 wild-type controls, 21 familial controls, 18 AD1, 28 AD2, and 5 iAD1 controls) using the whole-cell patch-clamp method. In all experimental groups, the recorded neurons exhibited the ability to generate voltage-activated currents, or action potentials, via sodium and potassium ion channels, and showed classical neuronal morphology (Figure 7). In a subset of the experiments, the recorded neurons were labeled with biocytin via a patch pipette, enabling post-hoc cell identification and ICH characterization. We found that all biotin-labeled cells were also immunopositive for ChAT and VAChT (n=12, Figure 8a).

[0119] No significant differences were observed between groups regarding neuronal membrane resistance and membrane capacity, and membrane resting potential and minimum current required the generation of a single action potential (Figure 9). However, significant differences in BFCN excitability were observed, which were mutation-related and reversible by editing.

[0120] The maximum number of action potentials that neurons from AD1 and AD2 strains could generate in response to rectangular depolarizing current injection was lower (compared to wild-type and familial controls) (ANOVA test with Tukey's post-hoc comparison, Figure 8b, c). The height of the first action potential in basal current injection was also significantly reduced in AD1 and AD2 BFCNs (Figure 1c). Importantly, CRISPR / Cas9 modification of the PSEN2 point mutation in AD1 mutant iPSC strains eliminated the observed electrophysiological abnormalities, thereby restoring both the maximum number of action potentials and action potential height to levels recorded in wild-type and familial controls (ANOVA test with Tukey's post-hoc comparison, Figure 8).

[0121] Consideration In the United States, 5 million people are currently affected by Alzheimer's disease, and according to Alzheimer's disease organizations, this number will increase to 16,000,000 by the 2050s. Unfortunately, the inventors have only direct evidence for a genetic cause, which accounts for 3–5% of these patients. This percentage includes EOFAD variants caused by genetically fully penetrating autosomal dominant mutations in the amyloid protein precursor (APP), or PSEN1, PSEN2, which constitute the γ-secretase apparatus

[87] , and alterations in their function increase the production of Aβ42 oligomers and / or the deposition of amyloid plaques.

[0122] After decades of research on mouse models of AD that do not fully replicate the pathophysiology of the disease in the human brain [5, 57, 58], a new complementary concept for in vitro AD modeling has emerged following a breakthrough by

[81] that enables the reprogramming of adult human tissue into iPSCs using predetermined factors, followed by their subsequent differentiation into specific brain cell types in vitro.

[0123] BFCNs are one of the most vulnerable neuronal populations, and their deterioration partly explains cognitive decline in AD patients. Apart from evidence of BFCN failure and atrophy, other studies have shown that human embryonic stem cell-derived BFCNs transplanted into AD mouse models may be associated with improved behavioral learning in transplanted mice

[94] . These findings highlight the validity of iPSC and ESC-derived BFCNs not only as early clinical indicators but also as a potential strategy for subtype-specific cell-based therapies of AD

[39] . To advance this cell-based therapeutic strategy, sophisticated differentiation protocols for generating human ESC and / or iPSC-derived BFCNs are urgently needed.

[0124] Our initial goal was to develop an improved protocol for generating BFCNs and intermediate neural progenitor cells (NPCs), and subsequently, a control and PSEN2 N141I These methods were used when differentiating cell lines derived from both individuals with the mutation. iPSCs were developed using fibroblasts isolated from three sisters (two with the PSEN2 mutation and cognitive impairment, and the third wild-type for the mutation)

[60] . To investigate the accuracy of the association between various phenotypes and pathogenic mutations, the inventors used a fluorescent cell sorter (FACS) to extract intermediate CD271 + (p75) Starting by optimizing a published BFCN protocol [4, 17, 46, 50, 89] including the purification of a forebrain progenitor cell population, we generated 3D ventralized neurocytes (vNEBs), which were subsequently dissociated to observe monolayer neuronal populations.

[0125] After inducing BFCN differentiation in these cell lines, the inventors (1) performed Tuj1 in vitro + / BF1 + / ChAT +We analyzed (1) the ability to generate nerve cells; (2) the expression of target genes / proteins related to nerve cell differentiation or inflammation; (3) the production of soluble and oligomeric Aβ40 and 42; (4) electrophysiological (ePhys) properties; and (5) the selective vulnerability of BFCNs to one or more innate or microenvironmental factors within or adjacent to these cells.

[0126] Several studies in AD mouse models have revealed electrophysiological abnormalities associated with the later stages of AD pathology. Synaptic function in the hippocampus was reduced in the APP23 mouse model

[70] . Similarly, cholinergic neurons derived from the prefrontal cortex of TgCRND8 mice were unable to maintain cholinergic excitation compared to control mice

[64] . In this specification, we describe the in vitro PSEN2 N141I We report abnormalities in the electrophysiological properties of BFCNs derived from iPSCs. Notably, correction of this point mutation restored neuronal excitability to the level of neurons derived from control iPSCs.

[0127] The inventors have optimized an in vitro BFCN differentiation protocol from human iPSCs, which focuses on generating a homogeneous population of electrophysiologically active ChAT+ / VAChT+ neurons in a reproducible and rapid manner. The innovations introduced into the protocol conferred homogeneous expression of Nkx2.1, a transcription marker for the MGE subregion, as early as day 8 and very strongly up to day 11, compared to day 20 as suggested in previously published protocols

[38] , without forcing overexpression of cholinergic fate-related factors under predetermined serum-free culture conditions. The inventors were able to record mature action potentials in neurons at day 38 of culture, accompanied by co-expression of cholinergic-specific markers, which is an earlier time point compared to other existing protocols using ES or iPSCs [4, 17, 46, 50, 89]. Therefore, the inventors' protocol has applicability to high-throughput drug screening in homogeneous cholinergic cultures. In addition, the 3D structure of the NEB itself, if it remains in its undissociated organoid form, allows for mechanistic analysis in a more physiological setting.

[0128] This optimized protocol is for PSEN2 N141I After application to mutant iPSC strains, the inventors found an increase in the Aβ42 / 40 ratio in the culture supernatant. The inventors did not observe any abnormalities that would provide any evidence of abnormalities in the neuronal differentiation process or the expression of BFCN markers. Interestingly, the inventors found that PSEN2 N141I A decrease in BDNF gene expression was observed in NPCs, which is related to homozygous and heterozygous APPs. swe / PSEN1 M146VBDNF alterations are observed in mice, similar to the results described in the report

[18] . Both mutants are also carriers of one APOE ε4 allele. The presence of this allele variant, the most common and well-characterized risk factor polymorphism for LOAD

[16] , can modulate age onset and phenotypic severity

[49] . Therefore, these iPSC strains combining both the EOBAD PSEN2 Volga mutation (or modification by CRISPR / Cas9) and the APOE E4 allele constitute a very useful tool for studying the pathophysiology of early-onset AD in vitro, especially when astrocytes derived from apoE-secreting iPSCs are also present.

[0129] Searching for adjacent mechanisms or phenomena that may cause or result from increased β-amyloid production, researchers found hyperinflammatory and electrophysiological abnormalities associated with AD mutations. The concept of these abnormalities independent of β-amyloid deposition and their demonstration of correcting EOFAD mutations using CRISPR / Cas9 technology sparks discussion about the need for combination AD treatments to overcome not only β-amyloid plaques (Gandy et al., in press) but also parallel inflammatory processes or excitotoxic / neuronal firing abnormalities.

[0130] NLRPs are components of the inflammasome that induce the secretion of the mature pro-inflammatory cytokine IL-13 in response to pathogens and toxic stimuli [11, 41]. In association with Alzheimer's disease and other neurological disorders such as Parkinson's disease [14, 32], NLRP2 appears to be unregulated in astrocytes [45, 51] and NLRP3 in microglia

[34] ; in addition, NLRP2 / 3 are altered in the pathology of AD: obesity and type 2 diabetes comorbidity. The inventors have developed PSEN1 A246E and PSEN1 M146LWe have previously reported an unpredictable association between increased expression of the inflammasome gene NLRP2 in neurons derived from iPSCs originating from conserved fibroblasts of a mutated subject

[77] . This association reminded us of the association between mutations in presenilin 1, nicatrin, APH-1, and PEN-2 and the inflammatory skin disease acne (AI), which led us to wonder whether mutations in certain γ-secretase components may be associated not only with amyloidogenesis but also with pro-inflammatory mechanisms.

[0131] PSEN2 N141I Despite our finding that mutant cells had higher NLRP2 levels compared to controls, gene modification did not significantly reduce NLRP2 levels, so we could not conclude that this upregulation was caused by familial PSEN2 mutations. Our results suggest that inflammasome dysregulation can occur in the brains of EOFAD patients, but that there may be factors other than any effect of PSEN on inflammasome biology that are reversed in the reprogrammed PSEN2 mutant cell lines that cause this phenomenon. Several possible explanations for this PSEN2-independent NLRP2 upregulation include the effect of the apoE4 allele, which is present in both PSEN2 subjects (but not in controls), or epigenetic effects in fibroblasts collected from EOFAD subjects that are maintained through the reprogramming process.

[0132] Electrophysiological abnormalities in neurons are associated with mutations in PSEN1 and PSEN2. Some of these abnormalities are caused by changes in the function of voltage-gated K+ channels, which potentially occur through cleavage of channel components mediated by the PS / γ-secretase apparatus [44, 72]. Presenilin mutations also disrupt calcium signaling by increasing the levels of calcium stored in the endoplasmic reticulum (and thus increasing stimulus-induced release into the cytoplasm), rather than by changing calcium influx. One mechanism underlying calcium regulation abnormalities in neurons has been described in cortical neurons from PSEN1M146V mice, which is mediated by inositol trisphosphate (IP3)

[79] ; more directly, it is the formation of a dual-functional protein-ion channel by unprocessed PSEN1 and PSEN2 themselves, which regulates calcium efflux from the endoplasmic reticulum [29, 55, 80, 84]. Considering the important role of presenilins in potassium and calcium influx and neuronal excitability, mutations in PSEN1 and PSEN2 can lead to reduced neuronal excitability and neuronal toxicity. Mice with mutant APP showed abnormal action potentials associated with a decrease in sodium current without a change in potassium current only after a substantial amyloid burden [9]. There is evidence that APP overexpression causes hyperexcitability in mouse cortical neurons [75, 86, 92].

[0133] Mucke and Selkoe

[52] revealed the toxic effects of Aβ that cause synaptic and network dysfunction. In fibroblasts and neuronal cell lines, deposition of mitochondrial Ca 2+ was increased when mutant PS1 was expressed

[31] . The neuronal firing pattern in mouse hippocampal neurons was changed by exposure to Aβ [67, 69]. Aβ exposure was also associated with changes in K + channel conductance in pyramidal neurons

[54] . PSEN1 mutations lead to mitochondrial Ca 2+It has been observed that this is associated with channel changes, which in turn lead to a reduction in spike activity in Purkinje cells in the absence of amyloid plaque deposition

[74] . Aβ42, through further modulation of voltage-gated ion channels, Ca 2+ It can exacerbate abnormalities in homeostasis [8, 25, 76, 88].

[0134] Except for mouse data and immortalized neuronal cell lines, electrophysiological abnormalities were observed in iPSC-derived neurons upon exposure to Aβ: hiPSC-derived cortical pyramidal neurons and GABAergic interneurons exhibited abnormal action potentials upon exposure to Aβ

[56] , and PS1 A426E Neurons differentiated from hiPSCs with the mutation also exhibited abnormal firing patterns

[47] . However, there is no published data to characterize the electrophysiological properties of BFCNs derived from PSEN2 mutant iPSCs.

[0135] The differences in high or low excitability effects and firing frequencies vary with gene mutations and are highly dependent on neuronal subtypes [37, 48]. All of these phenomena may be involved in the progressive neurodegeneration present in the pathogenesis of AD, and we specifically describe phenomena that may explain the neuronal abnormalities associated with the early stages of the human pathogenesis of EOFAD. In this specification, we describe PSEN2 N141I We report abnormalities in the electrophysiological properties of BFCNs derived from iPSCs specifically associated with familial mutations. Interestingly, while some previous studies attributed this dysfunction in neuronal activity to plaque accumulation in the brains of AD mice, we found, consistent with other reports

[18] , that action potential induction was substantially reduced simply in the presence of excess Aβ42 oligomers isolated in the culture medium, in the absence of amyloid plaques. Correction of this point mutation restored the firing pattern to that of neurons derived from wild-type iPSCs.

[0136] Modulators of potassium channels in neurons have proven effective in improving memory in the AD mouse model

[44] . Ca 2+ Regulation of Ca channels and excitotoxicity could open up a new wave of AD drugs. Understanding the mechanism by which PSEN2 mutations affect electrophysiological activity in different subsets of neuronal populations, and elucidating the link between PSEN2, other gene regulatory factors, and inflammation, could lead to new drugs that not only treat other symptoms, but also reduce vulnerability to ROS mediated by Ca 2+ when administered at an early stage, and halt neuronal loss and disease progression.

[0137] It is clear that mutant presenilins alter neuronal excitability even before the formation of Aβ plaques [18, 74]. One plausible hypothesis is that APP and presenilins may exert an effect on regulating neuronal excitability through mechanisms that are not yet understood at present, separate from their roles in Aβ biosynthesis. The accumulation of Aβ may act synergistically with altered electrophysiological mechanisms in the pathway leading to AD. Further studies focusing on elucidating the possible roles of PSEN and / or Aβ in physiological or pathological phenomena, using abundant data supporting neuronal excitotoxicity as a key mechanism associated with AD, are desired.

[0138] Conclusion The inventors optimized an in vitro protocol to generate human BFCNs from iPSCs derived from presenilin 2 (PSEN2) carriers and controls. As expected, PSEN2 N141I was associated with an increase in Aβ42 / 40 in BFCNs derived from iPSCs, which was restored by gene editing mediated by CRISPR / Cas9. Unexpectedly, BFCNs or cortical neurons derived from iPSCs from PSEN2 N141I carriers showed a reduction in basal excitability, quantified by a reduction in both action potential frequency and action potential amplitude. Also, this electrophysiological phenotype was PSEN2N141I The mutation disappeared after CRISPR / Cas9 correction. Gene editing data demonstrates strong consistency in mutation-associated changes characterizing all expected findings and all cell-derived genotypes.

[0139] Abbreviation AD: Alzheimer's disease; ApoE: Apolipoprotein E; APP: Amyloid protein precursor; AVG: Average; Aβ: Amyloid beta; BDNF: Brain-derived neurotrophic factor; BF1: Brain Factor 1; BFCN: Forebrain basal cholinergic neurons; CHAT: Acetylcholinetransferase; DAPT:(N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester); DIV: Number of days in vitro; DNA: Deoxyribonucleic acid; DPBS: Dulbecco's phosphate-buffered saline; DPBST: Dulbecco's phosphate-buffered saline + 0.1% Triton X-100; EGTA: Ethylene-bis(oxyethylenenitrilo)tetraacetic acid; EOFAD: Early-onset familial Alzheimer's disease; ESC: Embryonic stem cells; FACS: Fluorescent Cell Sorting; GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; GFP: Green fluorescent protein; HDR: Homologous Recombination Repair; HFIP: 1,1,1,3,3,3-Hexafluoro-2-propanol; HRP: Horseradish peroxidase; IPSC: induced pluripotent stem cells; LDH: Lactate dehydrogenase; MAP2: Microtubule-associated protein 2; MGE: globus pallidus primordia; NEB: Neurocyte (neuroid body); NGF: Nerve Growth Factor; NLRP2: NLR familial pyrine domain 2; NPC: Neural progenitor cell; PFA: Paraformaldehyde; PSEN: Presenilin; RNA: ribonucleic acid; Rock:Rho-related, coiled-coil-containing protein kinase; RT: Reverse transcriptase; RT-qPCR: Real-time quantitative polymerase chain reaction; SAG: Smooth and Agonist; SDS-PAGE: Sodium dodecyl sulfate polyacrylamide gel electrophoresis; SEM: Standard error of the mean; sgRNA: Single guide RNA; Shh: Sonic Hedgehog; SNP: Single nucleotide polymorphism; ssODN: Single-stranded oligonucleotide; TBST: Tris-buffered saline + 0.1% Tween; VACht: vesicular acetylcholine transporter; WT: wild type

[0140] References (each incorporated herein by reference) TIFF2026108846000011.tif238160TIFF2026108846000012.tif245157TIFF20261088460 00013.tif237158TIFF2026108846000014.tif238158TIFF2026108846000015.tif245159 TIFF2026108846000016.tif238158TIFF2026108846000017.tif245159TIFF20261088460 00018.tif245158TIFF2026108846000019.tif245159TIFF2026108846000020.tif187158

[0141] Although the present invention is described with reference to the examples herein, it is understood that modifications and variations are included within the spirit and scope of the invention. Accordingly, the present invention is limited only by the following claims:

[0142] Sequence information SEQUENCE LISTING <110> NEW YORK STEM CELL FOUNDATION, INC. ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI <120> METHOD AND COMPOSITION FOR GENERATING BASAL FOREBRAIN CHOLINERGIC NEURONS (BFCNS) <150> US 62 / 511,271 <151> 2017-05-25 <150> US 62 / 571,741 <151> 2017-10-12 <150> US 62 / 574,639 <151> 2017-10-19 <150> US 62 / 586,571 <151> 2017-11-15 <160> 31 <170> PatentIn version 3.5 <210> 1 <211> 33258 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> chromosome 1, GRCh38.p12 Primary Assembly <400> 1 ggggcctggg ccggcgccgg gtccggccgg gcgctcagcc agctgcgtaa actccgctgg agcgcggcgg cagagcaggt gagcggggcgg tgccgggggg tgcccaggcc agggccctgt 120 cgcctgcggc gctgagggcc cggggtgggg ctgcgccctg agggccctgc cctgccctcc 180 gcacgcctct ggccacggtc ccttccccgg ctgtgggtct gcggcccctg cgtgcgcagc 240 gctcctggcc tctgcggcca gcgcgggggc ggagagaga gagtgcccgg caggcggcgg 300 ctgggccggc ccggaactgg gtcgtggaag gatcgcgggg agcggccctc aggccttcgg 360 cctcactgcg tccccacttc cctgcgcccg cctgccgccg agccccggct gggggtgggc 420 gcggcgcgag cggttaaagg gccggtgcat ttaaaggagc ggtgcacgtg 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agctgcgtcc tcacgacgcc cacgttgagc cttgggttcc agggcagaga ctggagtgag 1380 ggcttgggg catgttgctt tgaagtggga tggatgtatc aggtttttgg ggaaaactct 1440 gtaccctttg gtgttgaagt gcccatgtgc caagtcttga gtccagcatg ttcacatgtg 1500 gggagtgagt ggcttgttcc tgtctatttg aaagagcagc aaggaggagg aggagcaagg 1560 gctaggggct gctgctgggg tgcctggagc tgtggtgcat aatgtcacac ctgtctcccc 1620 tccgtagctg ctcaccgtcc ccccaagggg ggtttgcctc ttgcctactt tggccttct 1680 ctgttatcga tgttaataat gacatatatc tcgcttatga gttggtcata ataaaaagct 1740 atcttgtaca gaatattaga atttaagatc ttaagaattt caatgacact gaaatagttt 1800 attattacct ttttacagaa gaggaaacaa gttcagggag ttaagcagct agtccagtta 1860 tgtggcttca gtgctttaag caccaggatt tgaacatagc tggctacact gtctttatct 1920 cttgagtttt tgcgcaggag gttcctgtat tcaactccta cccgtgtctc tccactactg 1980 ctgggaaagt tttgtggagt ccccatgagc aacttcctga caaacaaaca aaattttttt 2040 aaagaaacca aagcagtgtg tgtaggtcac atgcagtgtg tctaatgaaa acatctctgg 2100 cgggttttca gctgttgctt tgactttcgg acactgttta gttggggact gataagacag 2160 caaatatttc tgcaagtatt cccacctgtt ctattcccag ctgccacagc tgcggaaagg cgggggtgag gctgagaggc cccgagaga acattttcca ctgggctcca atcctggaga 2280 tgggatgacc atcatgttaa tgtctggaga aaagaatgat ttcaggctgg gtgctgtggc tcatgtctgt aatcccagca ctttggggagg ccgaggtggg tggatcacct gaggtcgga 2400. gtttgagacc agcctgacca acatggaga accccatctt tactaaaat acaaaattag ccggggcgtgg tggcacatgc ctgtaatccc agctactcag gaggctgagg caggagaatc gcttgaaccc aggaggcgga ggttgcagtg agccgagatc gggccatggc actccagcct 2580 gggcaacaag agcaaaactc catctcaaaa aaaaaaaaat ggttcacat cagtcctcag gaagatcag atgtcagtga gggagatcat ttcttgagag cctcttcact gagtgggaga atgggctgct tgttcatctt tgtgaaaatt ctagaacggg aagaacaatt caaagggtgt ccaccattct gctgtacctt aaccagaaac ttactggact ctttttaaaa taaagtaat tcatgtttat tctagaaaat taggaaaaa aaattttttt tgagatggag tttcactctt gttgcccagg ctggagtgcg atggtatgat ctcagctaac tgcaacctct gcttgccggg 2940 ttctagcgac tctcctgcct cagcctcctg ggtagctggg attacaggtg cctgccacca ctcccagcta atttttgtat ttttattaga ggcggggttt caccatgttg gccaggctgg 3060. 3120. tctcaaactc ctgacctcag gtgatccgcc aaccttggcc tcccaaaatg ctggtattac aagcataagc cattgcgctg ggctgagata accactctta acatgcattt ccttccacac tgttcacata tgcatattta tctattaaaa caatagaggg aggaccgt aggtgaagtt tgtgtatcct gttttttctg ttatgccttc agaatttcc ttttattgag tactcatgga 3300 aaagcagatt tgatggctgt gtagagcatt tgaattattt atcctaccag tcccacagca ggacactttc ccaccctccc ttttgcccca ggacagcagt gccctctgtc ctccccatgc 3420. ccatatgtgg gcactcccca ccatggagcc aaacctacct gggcaagtag cagagggaga 3480 gcagagtgag ccctgggggc aggagaga cttgagagtt ttgaggtgac agatgagctg 3540 gtgagtgagt gattagggag catttcttga cacatacctg cccgtggtga aggcatgtgt cttgtgagtg tgctcccaga aagcctgtgt agtgtgtggt gggcctgcct gtgtgaccaa 3660 accctggcca ctgggtacgt gaccctcaca agtgctgact gggctgagaa gagctccttg 3720 atgggcagtt tggagacttg agttgtaact gtggcttttg gccatgggac attaactgat 3780 tacttttgcc cctctaggct tcagttgtcc ttaattataa tacagggagc tgactaggtg 3840 gcgttgatgg cctttacggt ccttgccagc tctgacattg tcctatggat atgtcctttc 3900 atttgataat atgtttacgt ggccatagtg cctggggctg ggccgggaat ggaaacttga 3960 tctctggggc ctggcctttg aagccagttc atgtgtctgg tggttcagca gatccgtaac 4020 tttccaagag gcacatccat aggctaccgt gtcctttctc actgtgtccc tcctccattt 4080 catcttcttt ataactacga cttattgaac atctactgtg tgctggacac tttacaggtt 4140 atctctaggt tttacgataa tcttgcaagg tatgcctgtt ctgcttttta cagcagagga 4200 aatgagctgt gtcagattag actgtctgag gcctcttggc cagggagtat gtggttcaaa 4260 tcacataggc aggcgatctg aaccctgtca gtctccaaag cctctgcttt tgaccgctga 4320 cttgctgctg cttgtttaaa aataaatgtg tttctggagc ctactccaga ggggcgtgct aggggctccc tctcccactt ccccacaaac cacccttttc cctggctgct tcaggaaatg agagaactct gcctgggccc caggcacttc tgagtgggac agggctgtta gaggtagtc 4500 tagagcctgg cccaaaattc aggaggcccc atcagagggc ccctggggcc tgtggtccgg 4560 gagggtggta gggcagtacc tcacttccct ttgagactca ggccccagct ctggcttagg 4620 ccagggagaa ccatccccaa gtggtatgtg ttactatatg agctgagatg gatggtcagc tggaccaaat acatagtcgg gtacccaggg ccagggggag gaaggtgagc aggaagctg tgggcaattg tctgggtatc acctgacctt agcaaactct tccttgtttt aagcgaggac 4800. gtgggacttc tcagacgtca ggagagtgat gtgagggagc tgtgtgacca tagaaagtga cgtgttaaaa accagcgctg ccctctttga aagccaggga gcatcattca tttagcctgc tgagaagag aaaccaagtg tccgggattc agacctctct gcggccccaa gtgttcgtgg tagtgcagt gactcccaac ctgcttttga accctctttt tccattagga ttttctccgt ggaggcagat ttccatggga gtttgctgtg gcattttgaa atctgtttct tacctagttc 5100 cattggcctt aaatgttaag gccaaagcct ttacatttct ctgtaatgaa aagaaggtcg 5160 aggaaattgg gtcattgggt ttccataatg attgcaggaa ctgctgacac aagcacggct 5220 ggggagattc tctaggtcag actcccttgg tttggctaat tcagcagttt gatcccattc 5280 agctgattaa tgggaatgtg cagtggcttc tttggatgtt tgattttgca tcctaatcca 5340 aagcagctat cagcctcagc acttccttgt tggaaggctt tccagaacgt agtctatgtt 5400 ggacacttcc ttctgcctct ctgcattttc ctgccacttc tctagagaat ggggtgcagg 5460 gggtgggaga cggggaaagc tggtcgctga gtggctgatg ggacttgaca tcacccagcc 5520 ccacccccac ctgcccgtga gtcagcctcc ggggagagtt catcgcgtca ccggcactct 5580 aatgtggaca gacacctagc agtgttgttt atctgcacac gtttgggtgg tgatttttcc 5640 ctccaaggat ttcagagcac cagcaggctt cagagcagac ttaggtggct tgcaaagcag 5700 gccctcagga attcagaggg tagcagaagt ccatcccaga tgctctgttt tccttcagga 5760 gctaggtaaa tcagaggggc tgagggacaa atgaaaaaag ttacagcctt tgagtcccat 5820 ctgctcctcc tggccaatga gaggggatct gggaggggca gatgtagagg aaaatctgtc 5880 5940 atacagtctg tgtttattat ggcgacttag agaaatgcag aaaaatatca agaaataaa 6000 aaccactctt ggttctacca tgcaaagata atcatcttta atgttttgta atatttccga 6060 tcttttatat acatacattt tataaggaca ttcagatgat caggttcgta aagtttatg 6120 ttcggttaaa tttaacagcg tgtcattgtt caggttatta aatgtttgaa ataagatttt 6180 tggtggtcct gtcacagtct ccatgaagta gcatttcagg atcgaaaggt atgctgtgtt 6240 taaagtgttg attcttactc ctttcagtta aggccagtgc agtttgtcca ggtagtgact 6300 gagacccagt ttttccacac tctcctccgc agtgggcatt gttttgggcc tttttcagcc 6360 caagagctct cttctcccca tgccgctctg ctggtctgag atttttccac tcctctcct 6420 ccctagttgc tctctgacca gactctaggt attcaggaga aagtgttcat tgtctcactc 6480 tctcatgtgg caatcaagta gtgccaagca gtgagagggt gaaggtgggt gggtgaggga 6540 cactcacctt gctgagaaag ggccccagcc tgttcgggtg attataaagc agagacagtg 6600 ccaggaaaag tctgacactg gctgagaatc acccggggac caaccatccc gaatgcggat 6660 ccctgacact gggtgaggat ggagcttgga gatctgcatt gttaataagc agcctagcag 6720 agtggtgaag agtccagaca cactacctag gtccaagggt aaccttgagc taattacttt 6780 ttgagcctct gtttcctcat cagtaccatg gggaagaata gtagcacctt gctccaggat 6840 gttagtgcc ggctaagggc tcagcaggtg ctggtccatc tccaccagcc cccagtggcc 6900 tgggccacct ttgagaaaca gtgatcctaa gggattcagc atttcctaag ttggtgcctc 6960 ccacctgtca cccccacccc accaggctag gagggttgtg attagagggt gcccttgctg 7020 tgacagctga gactagctct tccctgatta ttccttaatg acagctctct ccttccctgc 7080 tttcttgaag tcttggtcct cgttgttgtg ggcacagctt caggggaggc cttggaggaa 7140 ttttgaaag tggaatgagg gaagcagcct gctcaaggga acacttgttt tctggtgagg 7200 aggccgcatg tatgaatgac gtttgtgggt tagaaagcat gttttgtagt ttttccttgt ttcttcctga agacatgtca ggtcttgatg agaccgggcc tgggcacagg gcaggcagtc agcgagtgtg gatgatgacg acagtggtca ccaggtcact gtctagacca ggtcactgtc tagcgcagtg tcacatggaa agggtatggt cctttaaccc taccctcccc agcacaacta tcacagatgt caggacct ctgctcacag aactgctttc cagggattgt cttttttttc tttttctttt tctttctttt ttttttgaga cagagtctca ctctgtcgcc caggctgag 7560 tgcagtggtg cgatctcagc tcactgcagc ctccgcctcc tgggttcaag tgattatctt 7620 gctacagcct tctgagtagc tgggattaca ggtgcctgcc accatgtcca gctaattttt 7680 gtatttttat tagagacggg gtttccccat gttggctagg ctggtcttga actcctgacc 7740 tcaggtgatc tgcccacctc agacaggcat gagcaccgca cccagcccca gggagcgtct tattagtggt tggcaactga atggagacgt gggaattgta aggaactgat tctacttgat cctgggtccc ctgcttctcc atcttcaccc acccatcagc tccctttctc ctttaaacag gcacctttgc tctctgctta tccatttttg ttgtgcattg ctatttggga gcctaagaaa 7980 cacaacatcc tctgaatgct ccagctgttg tgggtctgaa gggtgagcct gccctctgtc 8040 attggaggct gcagcctgtg gctttttagg tacagggact cccagaactg ctcctccagt 8100 catagcagag ataaatcaca ggagcttaag aggcatggga agaacagagg gaggagatcg 8160 tagcttccct gttcattcac acccaaaaca aaactgtcat actagaaaag gaggtattaa 8220 aagagccacc tgtacagcct cgtatctcat ccagcacact gctgcagatg gaatattatg 8280 atttagcttg agaaaatgca gcaactcttt gttgtggtgc ccctctttga gtaagagtga 8340 attccccatt gccagagtgg atagtgaggg aaaccctggg tccaggcagg agtctgttta 8400 ggatttatct agtgaggctg agccagagga ggaccttaca gttttttctc ttcaatttct 8460 tttatttatt tatttatttt tgtagagatg gggttttgcc atgttaccca ggctggtctt 8520 gaactcctgg gctcaagcga tctgtctgcc tcagcctccc aaactgttgg gattacaggc 8580 gtgagccgag ccaccatacc cggcccttct cctgcatttc cacctgataa tttctctcat 8640 ttccatagat gatgaagga ctaaagccaa gaactttcca aggtcctgca gctctttggg ggatgtgaag ctgtgctcta tttgtatgga ttttgctggt tcccagaact tccctgtggc 8760. cctggggcct agtctgaggg tactctgagt gaagagggag gaggcccac acctcttctg caaaggctgc ttttgtaaag ttcacttcag ttcacatctt cctcctggtc agaaagcttc gggggctctc ctctgctgca ttagctctt actcctccat caggcacca actcctccct ggcatggccc atcctaccag gtccccacac ttgagccaca tccaattgct cgatattatc aggataggtt atgttatgtt cccaactcat atgtttactt aagtggttac ctctttccag 9060 aatgagcccc ctcctccaaa ctctgcctgg tgaatattc ctaacctttg cagcttcaca tccctcttac ttcttgtgac ctgaggcatc tactcctgac aactgataga ctgtgtcccc tcctgtcggg tgcattgtcc ttgtcactac cctcctggct tttagctggc tttgcttccc 9240 gctgttgtta ctcctgtact tgtctcatct atcctaaaca gaaggtgctg caggctgggg agtttgttca tgttgaaatc cctgtgatgg aggtgagcag aggcagtctc tgcctgtgcc tcttatttgg ggatgaagtt aaagtccctg taggaataat ccaggccata gccggggttg 9420 ctgtcttcag aaagaagggc agccacaggt cttgttaagg ggattgaaat tggctgactt 9480 ggtggaagga acctgcctgc tttgtttaaa aaccacatat agctgagtgt agtggttcac 9540 actctgtaat accagtgctt tgggaggctg aggcaggagg atcacttgag gccaggagtt 9600 tgagactagc ttgggcaaca acgtgagacc ctcatttcta caaaatattt taaaaattag 9660 cctagtatgg tggcgtgcat ctgcagccct agctactgag gaggctgaag tgggagaatt 9720 gcttgaaccc aggagttcaa ggctgcagtg agctatgatt gcaccactgt actccaacgt 9780 aagtgaccag tgagaccctg tctctagaaa taaaaataaa aaaaatcaca tatattgtgg 9840 ggtgacttac ttggagacga actttcagca gagcgcacac ctgctatccc tgcccagggt 9900 gtgaagctca gccctgaggg tctctggaca gcgatcactc agcctctgga cagcgatcac 9960 tcagcctctg gacagacagc gatcactcag cctctggaca gcgatcactc agcctctgga 10020 cagcgatcac tcagcctctg gacagcgata actcagcctc tgtccccgtc tgagatgttg 10080 gcagggactg tcagatttgc caggcattgt ttgaagttct tcccagccca gaaacctgca 10140 tgtgtagatt ttggtacact gggtccccca cttggtacta ctgtgtgaaa ccccacttgg 10200 cactgtttta gggggcaggc ttccctcctg tccccttggc cttggccttc ccctgggtcc 10260 cgccctcagt ggcacttccc cacctcacac gtctgctctc atggcttagg tctccacttc 10320 taacctcagg agacctggtc ctcagacacc tcccagacag cttccccatt ttatcccata 10380 gacactcaaa gggtgaaatt catggtcttt cccgagactc tcttctccgg tcttccctgt 10440 cttagtcccc acctggctgc cattctagac tgtgttttct ctcctgcgtc aggtcctgcc 10500 cttgacctct tgaccccttc tggactctgc cctcacttgc atccatcctg ctgctgtcct 10560 cagctctcct cacctgccac agttgtctct gggggtcact ttccctctct ggtcagtggc 10620 cagactgact tttataaacc tggttcagat cttgtctgtc aagattgtcc tcagggtgat 10680 gtgtgtctcc ttaacatggt gcctgagacc ctgatcatct ccactgcccg ccccacagtg 10740 tgaggccctc actggaacat tgtgccttct gcccttccct cctcctggga aaaccagtct 10800 10860. ggctcttctc tcgtgatctc tgagatttgg ttccactttt gtgcttctgc acctaccatc aaacacccgg attgtatcat ttgtcacatt agatgatttt tgttttgttt tagcaagg gtgtttctta ctcatctttt tatccccaga gcccagcatg atctttggtg cataatagat gcaccacaga tgtttgctga ttgaatgaat gagcacactg acagtttgga gctgccctga ctttcgtggc tatgcgtttt gccccctggg atgtgagtca 11100 cctcaggcca gccccaggca aggccgctgc tgcctccatg gtaactctca aggcctcttg ttttatggca gtcgtttgat tgacaggcat ctcttggaag cttttggggc aggacttgtg tccaagtctc caggtcgcct ccagccaccc cctgagtcct ccactgcctt tgtctcacag gaaagtgga caaggtcctt gtgctccttt ttccaggtgc ttccagaggc agggctatgc tcacattcat ggcctctgac agcgaggag aagtgtgtga tgagcggacg tccctaatgt cggctgagag ccccacgccg cgctcctgcc aggagggcag gcagggccca gaggatggag 11460 agaacactgc ccagtgggta ggtcccacca gcagctgggg gccttcaaac aggtccctgc 11520 ggctactgta ccttacagat gaaaaccaga cattcattcc ctgatgcggg agggagaagg 11580 gaagtaatga tgaggattgg ccgaaaaggt gggtggctgg ccatgatgga ccttccatct 11640 gcagggtttc ataggactgc gcattcacag ccagagatgg acttggcagt gggctgaagg 11700 acgctgtcca ctctgccacc ttgggtttac ctctctcatg caggtcactg tttccactgt 11760 aataggagag tttgtttgga tgcctgggtg ctaggacagg taacacagaa gcttaggatg 11820 gtagcagggg aagcattttt tggcagatgg ccagacatgg taagtgtgag aggagtctgc 11880 ctgatacacg attgactttt gagctgggga tatttgggct tcactgtgat cattcagccc 11940 ccaggggagg agattgtaac gttagaaaga gtaggatatc gttgggagag ccacttagtt 12000 gtgtcctttc tctcccgatc agggcagaac atctgaattt gcctgaaccc tgttctctgt 12060 tttgcccatt atagaattaa aaaatgtctc tgtgtggact gttttcttgc agccagtctt 12120 aatcctgctt gctgaaattt gagctcactt ctccatgttc tccttgagaa cggaaccatc 12180 gtccctaagc cctgagtgaa atcacaccag cttaaggcca ctgctctgcc actcctcagc 12240 cttttcttgt ttgttatctc cgggaagttt tgtacacttt ggttgtttca gtttctgttc 12300 atgagtagtc ttctttcttg gctgaacgtc tagattggga ctctctctgc agagaaccgg 12360 tactgaagca actgtcattt tcagtttttg tttcatttgg ctttttcttt agctgttcac 12420 ctcattagca aggcagccca tgaccttgac ttgccacagt tccaaaacac aaattcttac 12480 agatcggttt gtgctagtgt ctggcaggtg tcctgccctc cctcgttacc tcctcatttg 12540 tgcctgccca ccttcccaga gcctgcgtct tctcagatgc ttaacacctg tttagcctct 12600 ctagttcaga gctacaaatt tacatgcttg attctgtggg gcagaaagtt caaagtaatt 12660 tcttcctctg caaattccca gtatcttagt cacacgcaaa gagagtgtcc ctgtgcactg 12720 actcctctag ctagtgattt gtcagccaaa aatgtttatt tatctcctgg cctgtttcct 12780 cccatatcag tatggccaca tgaacagaat tgagtgacct cctgagtccc tgtattagga 12840 12900 12900 gcctgcagac catagcaac ttccttcctt catttatggt gctcatcca gctccaaatc 12960 ttctctactt tgtcctcaca aactttcat atgccctagt agctcataga ctgctcctta 13020 tatctggaaa gcacattca aacttcat ttctgttcc aaaaatccgt gcattacatg 13080 gataggctgc cgtgggggac attctgcggc cctcacgatg tggttccca cagagaagcc 13140 aggagaacga ggaggacggt gaggaggacc ctgaccgcta tgtctgt ggggttcccg 13200 ggcggccgcc aggctggag gagagctga ccccaata cggagcgaag cacgtgatca 13260 tgctgttgt gcctgtcact ctgtgcatga tcgtgttgt agccaccatc aagtctgtgc 13320 gcttctacac agagagaat ggacagctgt gagttggggg gctgggggga gcagggttgggg 13380 gtgaggggctg agttgccagg gggtgggggg cgcagcagcc tgtgttggtc actgtacctg 13440 cagctccaca ccagcagcgg taagagcag ggatgaagaa ccgcccaggt tcatgcctg 13500 gctcactgcc tcctggattg tgacctactt gggcatgctt tcatccc tatgcctcag 13560 cttccttgtt cgtataatgg gttgataacg cagttactgg gagaattaag tgagttaata 13620 tgagtgaagg gcttagaaga gtgtctactg cacgtgagtg ctcaggcaag ctggatcctg 13680 ctgcagaaag caagctcttg atcctgggca tggctgtgcc actgatccct gtgtgactgc 13740 aaacaaatca cttcctctct gagtctctgc ttccctgaat gtgaaacaag gtggttggac 13800 cagatatttc tcagctcact tccagccttg tgaggaagac ttataaagcc tttcgtttat 13860 tttagtaaaa tacatgcaga ggcagcagcg tagaaaaatg agaagcttcc tccacttctt 13920 ccccctcccc tttctgtggt cctcactgct aagcaccttc tgtaaacttt tttttttttt 13980 tttaaagtta gggatttttg tttcatttcg tgtgtgttgg ttttttttgt tgttgttgtt 14040 tcttttaaag aaaggaataa ggccaggtgt ggtgtctcat gcctgtaatc ccagcacttt 14100 gggagactga ggtgagagga ttatttgagc ccaggagttt gagaccagcc tgggaaatgt 14160 ggcgagaccc tgtctgtaca aaaaatgcaa aaattagcca ggtgtggtgg tacatgcctg 14220 tagtctcagc tacttgggag actgaggtgg aagaacacct gagcccagaa gtcgaggctg 14280 cagtgagcca tgattgcgcc actgcactgc agcctcagca acagagtgag accctgtctc 14340 aaaatttttt taaaaaatta aaaaagaagt agagtcccat cctcagaaag cttatagtgt 14400 gtgggggatt cagcgcagaa caggtgaaag catggagaga atgcagccag cggtttgttt 14460 gcagcagtcc aggctgggaa gagtgaggtt tgagtgaatt gcttcctgtg tctgcttcct 14520 gagcttatga gctgcaagga cagcagttgc ttcagcggat gggggtcggg tagtagcagg 14580 tggaggagtg ctgggctggg tggagctggt ggagaggtgt gggtgggtgg gggaatgaga 14640 actggatggg tgagagaagt gcctagggag cctttaatcc ctgtgggggt ggggaaagca 14700 gcagggaggt catctagccc tcgtcctcac tgctgcactg ggcccagttg gcaggctgag 14760 agccacaggt ctgtggtcag ggtgccagga aatgagctgg aggacaggaa ctgctcatgg 14820 ggatggtgcc cgcactccat cagggcagca tgtgggcagc atgggcatcc caggcacctc 14880 ccctagcagg tccagaatca ctcaaggtgg ggagcctcga ggagcagtca gggccgggag 14940 catcagccct ttgccttctc cctcagcatc tacacgccat tcactgagga cacaccctcg 15000 gtgggccagc gcctcctcaa ctccgtgctg aacaccctca tcatgatcag cgtcatcgtg 15060 gttatgacca tcttcttggt ggtgctctac aagtaccgct gctacaaggt gaggccctgg 15120 ccctgccctc cagccacgct tctctccgtc tgccccacac catggcggca gggcccgtga 15180 aacagccgcc tttagaaaaa cacaaattag aggaaaatag acccagattt tttgtactcc 15240 tccccacccc atcctgtctc ccaccgtgga tgacctaata ctgttgtctt ttatttttat 15300 ttattttctt tttcttgaaa catggtctca ctccattgcc caggctggag tgcagtggtg 15360 cgatcatgac tcactgcagc ctcaacctcc tgggctcaag ttctcccacc cagcccctca 15420 agtagctagg actacaggtt tgcaccacca tacctggcta attaaaaaat ttttttttgt 15480 gcaggctaga tctcacagtg ttgcccaggc tggtctcaaa ctcctggact caagtgatct 15540 cccaccttgg cctcccaaag ttctgggatt acatgtgtga gccattgcat ccagcctgtt 15600 gtcttttaaa tttacacatt atcccacttg agttcctcat tgcagtgttc caagcatcat 15660 ttctcatatt tcaaagttaa ttttgttttg cttctctttc tgaagttcta ttttaggctc 15720 ccctcacccc gatacttccc ctgaagattt atttttagtt ttccttttcc ttttcgggca 15780 aggatgtgca gaggccatgc tgaggtcttg cagccctggg agactttttgg gttgtagctg 15840 cctatagctg ccgagtagcc ccagggagta gtggaagggc agatcccatc tggccagaat 15900 catgggcact gcctgtcccc aaagatgcca tagctttta gacagcggct tcaggcttt 15960 ctcccaggta aggggttgaa cccctaacga tggaaaggaa attaagctgg gcattaccta 16020 ttttaaaact gttacacac aggtgcctca cagcattttt tgttcaggcc gctgccatcc 16080 atggagcagg tagatagaag tgcagagtgc ccaggctaga gggatgggac agggacagtg 16140 cagggaggga gctgagcccc cttccagcgg gggcagcaga ggggaaagcc atgggagggg 16200 ctgcaggatg tgtcctgagc tgaagcttat caacaagtaa tgagtaccag ctgggcattg 16260 tggtgcacgc ctgtggtccc aactacttgg gagactgagg caggaggatc gcctgacccc 16320 aggagttcaa gtctagcctg ggcaatgtaa gaccctgtct ctaaaaaaat aataataaaa 16380 tagtaacaa ttacctgtgt aactgtgacg aggcagggtt tgaacattgc cgctgggagg 16440 ttggcagatg gtgggaagca gggtggaggg ctgctggttt ggagcagagg atacagattg 16500 catggggtca agctagaaat tgcgtggcag atgtgaagag ctggccccac tgcgggcagt 16560 aggtgtctgg tggccagtcc cagaggctgt gaagaggggc tcagccatct gtctagtagg 16620 gcttccttgg aggttccacg atacaggcag atggtggtgg cccgggcagc caggtggtgg 16680 ctgggatgaa ggggttggc aggtcccaga ggcagcccct tccccttttg gctgtgtgtg 16740 cagcagggcc gtggaggctg cttttagtcc aggtagacca gggccacgct gaggtcccag 16800 tgggctgagc tggtgactga tgagttggtc ctcaggggtg aggctggtgg gaagtgatgt 16860 cactgtcccg ccgatggcca gctaagggac tgggttagga tcagccccct cttgtccttc 16920 actctcccat ccttggccag gagaagagga acaggtcttt ctgaggacct gcttgtagac 16980 ctttgggtag gaggggactt cccaggttct ctgttgaggc cactctatct aaaatagcac 17040 cccagtgagt ctcctatcac tgtatcctaa cattattttc tccatggccc tcatcattac 17100 ctgctgatat actgtatgtt tgtctatatg tcatctaaca cccctcacac tggaacacaa 17160 tgcccgtggg cagagacttt gctagccttg gttccagagc ctagaacagt gcctggcaag 17220 taggagacac ccagcattac ctttctaagt gaaccagtag agatgggggg agaccgcaag 17280 gctatgccgg cagacctgag ggagtcctgt ctgcatgcgc tgcaggatga cctgagggga 17340 actccttgga cttctgtgcc ctctttatct gtaaggtggc cacctgatcc cttccagcgt 17400 aggcatgaag tagcctaatg aagagcattc aggcttgggt atcagtctca ggatcctggg 17460 ggccttagaa tttgtggcgc ttggggacac cttgtgatcg tgcaatttct gttgtctagt 17520 tcatccatgg ctggttgatc atgtcttcac tgatgctgct gttcctcttc acctatatct 17580 accttgggta agtgacagat aagcagcagg gtccctggga gcccctctcc atgtggcaca 17640 agtggacatg ggcatgagga cctgggcggg gaaagatgac catcgagctc cagtcttccc 17700 cagtgccagc cgttttggga acccaggcct ccgtcgccct ctctcatggc cttgacacag 17760 gggagtggaa gtggggctgc atggtggacc acatgtttct gtctcgttcc tgatttaaaa 17820 tgaacccttc atggagaagg ctctctgtga accccagggg gatagaaacc ccccaaaatt 17880 tacattctga tttttaggct aggcctgggt actttctggt ttgtgggaaa aattatctgt 17940 tctatcgccc cttgatttgg gatatcagcc tgacccaggg gcccaaagag actgggagga 18000 caagagaaaa cactttccca aggacctttc catgtgcaca gggtcttcca ggtcatgccc 18060 atgcacattt ctgtgatctg ttccaagcat ccccaccttg ttttagaaaa tgctgcaaat 18120 ggtaaattgt aaggacagtg aaggtcgggg aaggaaatgt tagtaaagag ggccaggttt 18180 ggactgaatg gtggtaaact gctaggctgt aatgcctcca ctgagtccca gtcacaggct 18240 ccaccttggt cctgcaggga agtgctcaag acctacaatg tggccatgga ctaccccacc 18300 ctcttgctga ctgtctggaa cttcggggca gtgggcatgg tgtgcatcca ctggaagggc 18360 cctctggtgc tgcagcaggc ctacctcatc atgatcagtg cgctcatggc cctagtgttc 18420 atcaagtacc tcccagagtg gtccgcgtgg gtcatcctgg gcgccatctc tgtgtatggt 18480 aggtgggcag caaggctggt gggggcagtg ggggcgatgt ccagggccaa atcgtcccca 18540 gtgctgcaca aggagggcag gtgctgaagg gcttgcatcc ctttctgcag aggcctgggt 18600 gggatccctc ctgagagagt cgcctttgta aaacagaggg gggtccacta tttctggaac 18660 actcctggtg gtctagataa aacgcagtag tcactgagct cctcatttac tttttttttt 18720 tttgagatgg agtcttgctc tgtcgcccag gctggagtgt agtggcgcca tcttggctga 18780 ctgcaacctc cgcctcccgg gttcaagtga ttctcctgcc tcagcctcct gagtagatag 18840 gattataggc atgtgccacc acgctgggct aatttttgta tttttagtag agatggggtt 18900 tcaccatgtt ggccaggctg atctcgaact cctgaccttg tgatcggccc gcctcagcct 18960 cccaaagtac tgggattaca ggcatgagcc actacaccca gcctcatttt ccattattac 19020 tgctatgctg attgagcaag tgcactgtta agcactggac acgctgtaag tgatttgttc 19080 atcaagacag tcctttgggt accatgcata tacataaccc caaatgttag ctgctatttg atattagcat gattatcatt gccagtattg ttacttccat tttaaggtta aagaattgga ggctcagaga agtggactc cccagcctgg ccaccgcgtc tcgggtgcac agctcctcca 19260 tgcttgcagt tgcctgcgag gccctactct ggctcacacc agggcctgct ctaagttgtg 19320 actggagaat gagaatttgg gatgccagcc cagaggcaag gcatgctctg agagctccac ccggggctcc tgtgctacag ggcaggctct tcttcagggg gctgcccggg gatagtttga 19440. caaggatgtc tctgtcttcc tagatctcgt ggctgtgctg tgtcccaaag ggcctctgag 19500 aatgctggta gaaactgccc aggagagaaa tgagcccata ttccctgccc tgatatactc atgtgagtga gccccccgtg cctctgcctg actcggggtc agcaggcagc ctgtgggggg 19620. acaggggcct gcttcctggc cgtggctttc agagttgact gggcgatccc aggagggtct 19680 ccactttcag aagccaggga gggcagtatc ttgttattac acagtaagaa gcttagaaag ttaggacagg aagcaggcat ctgctgggat gtgctgcagt ccctgacttc atcccgtcca 19800 tcctccagcg gcatgctgcg gtgcaggttg cattcctgtg atcccgcagc cacccctcag 19860 ctctccaggc tcttgagaag ggactttgga gagggattct tcagggcagg gggtcgggga 19920 gcaaggagct tctgggcttc cttgacagca gcgtggctga ttggcattaa tcctaactga 19980 agggaaggca cacgggatgg cccctggcct cggggtcaat gtgtagagat ttggacttac 20040 acatgcagtc aacaaaggca catcaagtcc ccattttgtg acaggcactg tgctaggcat 20100 tgggggaccc agcaggaaag aagaccacag ggtcccaggc ctcatggagc tcacggccct 20160 gtgattgtga tgccctcggt ctgttgatgg cggggcttaa atagcctgaa tttctggagc 20220 tctggcgtct gcaaggtggc ctgggaaaga gtttatggaa cagctacaga gttctaggta 20280 ccttcatgca gttgaggatt cgagcccgta gaggagaatc gcctgcagcg tggccccacg 20340 ggaaagcaca ttccaggcgc attccgagga tgagcggaga ccatgtatgg aaaggtagtg 20400 ccaggactgt catgagtgtc ccagggctcg ggggattcac ccgtgaactg tgaggtcttg 20460 gctctgatag acctggttct tatgctttag gaggggagac aaacagtaac agaatagaca aatgcaagag agagtgactc tggacccctc ccacaacggc ctcctaacaa tggagcatga gcagatacct gcaggatgga gggtcctgtg caggctttct gggacgcaga ctggccacct 20640 cccccaggcc ctgcaggcag ccctgttag cccgcctga gatgtgaacc ttttctcctc ccccagctgc catggtgtgg acggttggca tggcgaagct ggacccctcc tctcagggtg 20760 ccctccagct cccctacgac ccggagatgg gtgagtatct tggggagcta acagcctctc 20820 atcactgggg ggcagctccc tacctgcacc cagctctgct cggcctggct tccctgagag 20880 gcatgagttc aggaggggca gaggaagg tccgttgaaa accagccgga cacatgcggc ttgaagattc agcaagtgtt ggaccctcgg tcctctgcca gcctctgttg catcgttctg ctggggcgtgg gtgggtggag tggggggagc cctggtgtca ggtgctggtg ctcaggggga 21060 ccccttcttg gagctttgtt ccctggtaac actctgacca gctgttgttt ctctctcttg ttgtcccctc ctcacggtga tgacggacat cttctcttcc tggacaccca gaagaact 21180 cctatgacag ttttggggag ccttcatacc ccgaagtctt tgagcctccc ttgactggct 21240 acccaggga ggagctgggag gaaggagg aaagtaaggt gcccatgttc acaccggcctg 21300 cttcagccta cggcgggagc ggagacagag ggtggaggct ccctgcagcc tgggtggagg 21360 agggcatgag gggaggggcc ccttttccca tcagaggcat ctctgtgaaa gtagaagaatg 21420 cctgcagcgc tggggtcttc tcagcaggcc ccatgtagtt gtccggcatg tattgagtat 21480 gggccacgtg cccgtgctgt gctgggtgag gcccagccct ggtgggaccc acaggctaag 21540 gagacacggg cagtaatcac atagactgag aagccaagga ctatgaaggg ggccatgggg 21600 ttggggaggg gcggcaggag agcatgccac ggggcttctt gacctggttg gcaggggtga 21660 gagaagtca gctgaggaag taactgctga gctgagctct gaaggttgag tcacagcagt 21720 cacataggagagaggcaca gggtggggagag catttcctga cagacagact cagagaatcag 21780 aggaagccgg ggcgggatgc aggagcaga agtgtgggag agccttgcaa acaggcctgg 21840 agacatgcga agataggagt tcatcctggc gtcagtacac ggtgcctgcc taacacccaa 21900 tgccagccca ctgctgcgtg ccaggcagca ccctggagca gggagatgct gcactgtcgt 21960 aacagcccct gccttgagag gtgccttacg ggagcagcct ggtgacagtg gcttggcata 22020 caggactcca gtgacacggg aggggcaagc tagggaaaga tcactctgcg gtgggtctgg 22080 aaggaggagc aggtgcgcac cctccaggca ggcttggggg aggtatttat tccaaggcca 22140 actggtgtgc tgcagaccag gagttagcac agatcccacg gggcccgcag gactggcctc 22200 cctccagaca ccagccacaa gctctagagg gtctagatgc cacttgtgct tctgaccggc 22260 tgcaaattta gggctcccat gaccccctta ggttcaataa cttgctagaa tgactcacag 22320 aactcaggaa agcactacac ttaaaattgc agtttgtttt ttgttgtcgt tttgttttgg 22380 agacagggtc tcgctctgtt gcccaggctg gagtgcagta gcacgattgt ggctcactgc 22440 aaccttgact tcctgggctc aagtgatcct cccaccttgg cctcctgagt agctgggatt 22500 acaggcacgt gctaccacac ctggctcatt tatatttta gtagagacaa ggttttgact 22560 tgttgccgg gctgtttcg aactcctggg ctcaagtgat ccacctgcct tggccttcca 22620 aagtactggg attataggtc tgagccacag cacccggcca aaattagtttt tattataga 22680 gatgcaactc aggaccagcc aaatgaagg acagtgaga agtaatgctg atggatcaca 22740 cctggtgggg gaggaggac agctggggcc aggaggcagg gggacacctg caggggctgga 22800 agggcagggg aggtgggcct ccatggtttg tgtttattgc ataccattt ttattgtcta 22860 cagtgagcaa agttatccta windowsgtg tcagggacca ttgcactaa gaaaacaac 22920 gagagcattt tggaagctct aatttcctga tcagtaatgg gtagactaat tcccagttat 22980 atttacctgt tgtaggtga aaggttctc agaggaccctc tgtcttggtg ttatatgggc 23040 ttttgaatgt actgaaatta aattccctaa aaatctgtga ttcagacttc atactaaatt 23100 gtacagcagt gcccagccca aggccttgca tttctttg ttgttttctt tactctctaa 23160 gtgcccaaca ctggttttac ctgagtttca gaacgcccg cttttctg cccaggttgt 23220 aagtcaccca gtccacaggt gtcccctgct ttcccactgg ccactgattt ggggaggcag 23280 ctgtccatgt ccccagtcca catcttagct tctagaggcc aggtggggtg ggctgggctg 23340 ggcaagagca gctgggcctt ctgggccaga gtttctcttc tttttccatt ctgtgcacgc 23400 ctcttcagta cgggttactg tctctcctca cacagggggc gtgaagcttg gcctcgggga 23460 cttcatcttc tacagtgtgc tggtgggcaa ggcggctgcc acgggcagcg gggactggaa 23520 taccacgctg gcctgcttcg tggccatcct cattgtgagt ggctggggat gcgtccagct 23580 gcctcgtggt gggggccccc agggtcctca ttgtggtggg ggcaggtctc aggatcccta 23640 gggatttttc atttcttctc ttccctctga gggacaagag cagggagcgg ggctggaagg 23700 gtcagcttga gaccaaggct cacaggaggt gtgctcgccc ctaggtgggc tccagcctgt 23760 ggaggacagt gcaggggagg gtgaggagtg taccggcccc agcgtggctg agcacacagc 23820 ctccaggccg aggacccagc tgacagcttt gcgcagtgat gataccctcg aggtggttgt 23880 23940. gatgacatca gatttgcaga aaagaaaatt gcttaagggc cttgcccatg ggcgcaaagc tagtgaggac catgttttcc ccctcctcca tgccattggg acaccacagg gtctgaatct ggggcactag gggtggcccc gttactgtga accacagcag tgaatgtgg aggccctgta gtcagttaac gtgaccagat acacataatg gggagacgtc ctgccgtgac ttcatctcag agatttcgct gtcacgttag aggagga gcgtctgagc cgtgcgcttg gcatctgccc 24180 cttagtgaaa accctgggca tggcatgatt aaggttgatg ctccagtgtc cagaaggttt tctttttgcc cacaagtata tcagggatgg gatggtggac ccaggctcct ccaccaccag actgccttac ctgagccctg ctggccccaa agatatagaa ggcaccctgg ttccctgtgc 24360 tcacctggac cactgcctgc atcagctggg tcaggggagg atgggcagcc cccacacctg 24420 cttcccaggg gcaggttgcc tggcggctct gattcccttg gtgccagctg ctgagaacct 24480 24540. tactgccatt tcagttgagc ccacctagct ctcatataaa tacatgttcc ctgagggcat cttaccatcc catgtgacca ctccagccag acaggggagg cagcacggcc tcggggcaca gcactgctcc aggagtcagg aggcctgcct tctggttcac tcactaacag gtgaggtgat 24660 ctaatggggg tgagaacttc tgcccttaac acctcaagag ctgttgcagg accagggaag 24720 ataatggggt gtctagcgcc gttatccgac tggtcctcga acaagctcct gtgcccaggg 24780 actagaccat gactcacagc tcctgtccac accagggatc accacgctca ccctcccctc 24840 catgtcctgc agggcttgtg tctgaccctc ctgctgcttg ctgtgttcaa gaaggcgctg 24900 cccgccctcc ccatctccat cacgttcggg ctcatctttt acttctccac ggacaacctg 24960 gtgcggccgt tcatggacac cctggcctcc catcagctct acatctgagg gacatggtgt 25020 gccacaggct gcaagctgca gggaattttc attggatgca gttgtatagt tttacactct 25080 agtgccatat atttttaaga cttttctttc cttaaaaaat aaagtacgtg tttacttggt 25140 gaggaggagg cagaaccagc tctttggtgc cagctgtttc atcaccagac tttggctccc 25200 gctttgggga gcgcctcgct tcacggacag gaagcacagc aggtttatcc agatgaactg 25260 agaaggtcag attagggcgg ggagaagagc atccggcatg agggctgaga tgcgcaaaga 25320 gtgtgctcgg gagtggcccc tggcacctgg gtgctctggc tggagaggaa aagccagttc 25380 cctacgagga gtgttcccaa tgctttgtcc atgatgtcct tgttatttta ttgcctttag 25440 aaactgagtc ctgttcttgt tacggcagtc acactgctgg gaagtggctt aatagtaata 25500 tcaataaata gatgagtcct gttagaatct tggagtttgg tccgttgtaa atgttgaccc 25560 ctctccctgc atcttgggca cccctgggat aacttgtgct gtgagcccag gatggaggca 25620 gtttgccctg tttgaaggaa cttttaatga tctcgcctct ctgcacacat ttctttaact 25680 agaaagtttc ctaagcaaag gagttaggag agcagggtgg cctgacatct gccagccctg 25740 agctgtaagg ctgtggatgc tgagcaggtc cctggactca gttgtgcacg gtggcacaga 25800 cactgccagg tggttgccaa aacatccagt ggttccttca gcaagtgttc accctctgca 25860 gaagcctgtg agggcctgag ctcagaaacc actctccttt ccttctctgg ctttggccct 25920 gggcactgtg gtgggagagt ggacagtttg gctttgcctt ctctgtacat caatcatggg 25980 26040. ttgcaaagag aatctcagaa gtgcctcttc ctgagcacag tggctcacac ctgtaatccc aatacttcgg gaggtcgagt cggaaggatc acttgagccc aggagtttga gaccagccgg ggcaacatag tgagactttg tacaaaaaaa aatttaaaaa ttagccaagc atggtggcat gcatctgtag tctcagctac tctggaggct gaggtgggag gatcacttaa gcccaggagg ctgaggctgc aatgagccga gatcaagcag gtgttaggta tatcagacag ctgagaagac gcaagtgtgc cctggggttc aaactggtac ccctgtctcc ctgttccagg grandfathercatga gtgccgggac aatgcatctt tattatgaga ggaatgaga ttgtgtatct tgacatttga caggagcttg ctttccccca ggctgtttga ggaagggcag aggaaaatgt ggtgccctaa gaggaga cagaggaggc cgaacactgg cgggtggaat cccactgatt agtagtgcag gtcagagacc tgggatgggg ggcattgccg tcatggaagc cacagcgggg agcgggtaaa 26580. gcagacaggg atggtccctg atggtgacaa ctcgcaagag gttaagggga aagaaaaact 26640 gaaaagctta ttcaatttgg caattatggc agtgtttatc ttcagaagag cagttttagg 26700 gtggggtttc caaagatggg attggacata tattttgaat cattaagctt gaggtctttc 26760 aaaggcctgg ccaagggttg ctgggtggag accacattca gagtaaagg cagaaattgg 26820 gggcccttaa gtagacagcg agggagaag aaatgaaggg gcctggtgat ggttagggtg 26880 aagtgttaag actgagaaaa caaggacatg tgagaagacg agggaagagc attggagaga 26940 acaaagacac tggaggagat gctacttgga ggtccccaga gagcaggag acaaatgaac 27000 ccagaacaca aatggcaaag aagaaaaatg agagaatttg taaaagacag cattcgaaca 27060 tgccgaacaa gagcagggta ctggtgttca aacacctgta tctcccccgt gtaacccgtc 27120 aactaatatc tttccatatt tgctccagat ttgtctttag aaataaaacc cacgttctga 27180 agtcctgttt gtatgtggcc ccagtcctgt tgcctccgcc tcctgtcctg aagtcgattt 27240 ctgcccttct catctatggt tagtttgtt ttgtatgttg gcatgttttc ttaactttac 27300 agaaatggta tcatactgta catatttgat aattttttaa aatattgcat tctggaggca 27360 tgtataaatg tagctccagt tcatttattt tatttatttt ttgagatgga gttttgctct 27420 tgtcacccag gctagagtgc aatggcgtga tgttggctca ctgcaacctc tgcctcctgg 27480 gttcaagcaa ttctcctgtc tcaatttcct gagtagctgg gattacagtt gcccgccacc 27540 atgcctggct aattttgtat tttagtagag acggggtttc accacgttag ccaggctggt 27600 ctcaaactcc tgactgcagg tgatccacgc accttggcct ccaaaagtgc tgggattaca 27660 ggcgtgagcc accgtgccca gcccagttat tttaactatt gtatagtgtt ccattgtatg 27720 agttctactg tttatatgct attgatcgac ctgtaggggt tttgcagtgt ttctgtatta 27780 cagctgtgct gcagtgagca tcccatcaca ttgtgtggat ttgaggaagt attggaattc 27840 ccccaattga ctggacattc ccaattaccc tccaagtatg tgtctgttta tccttccatc 27900 cgcaatctga gagttcccca actctataat acttggtgtc atcagacttt tcatcttgtc 27960 tgattggatg ggtgtcattt cctttaggtt ttataattat cttttcatat gtgtattggc 28020 tgtacaaggt tccttctctg ttcattatta ttaattttt tagacagagt ctcgcgctgt 28080 cgcccaggct ggagtgcagc agcgtgatct tggctcactg caagctccgc ctcccgggtt 28140 catgccattc tcctgcctca gcctcctgag tagctgggat tacaggtgcc tgccatcacg 28200 cccggctagt ttttttgtat tttgagtaga gatggggttt caccgtgtta gccaggaggg 28260 tctcgatctc ctgacctcgt gatccacccg cctcggcctc ccaaagtgct gggattacag 28320 gtgtgagtca ctgcgcccag cccaagttttc cttctctgtt acttgttcat atcctctgcc 28380 catttttcac ttggatttt tgtcttacgg atattaagc ctcttaaaat atatattctg 28440 gagagatgct aatctttgat taattatatg cattgcaaat gtctggtaca ttgtggcttg 28500 cctctcttcc ctgcctttag gagtgttttg ctggacccaa gtaattttta aatgttaatg 28560 ttattaaatc tatcagtttt ttgcttgtat ggcttatgcc attgaatctt gttttaagag 28620 atccttccct accctcaagg ttttctaaat ttttattttc ataataagat ttttagttca 28680 tctgaaatgt atttttatga ttgtatttag tagggaccta atttgtttt tctttgtaac 28740 caggtgtccc agcactgttt actgaacagt ctctcctttc tcgctggtct gtagaactct 28800 cctgacatat accaagtttc cataagtggg tggatgggtt cctgagctct ctactgttaa 28860 tagaacttgc tctctcgcag gccaatgcct caccaggtga ttgaagcaga gaaacttagg 28920 tggtgaaagg agaagatggg gcctgtcctg agagtttctg ttcctgagat gctagaggca 28980 gagggtatgt aaatctgaag ttacactgga tctcctaaaa cagtataaag ctacagaagt 29040 ataatagtgt ggaatggtgg tgggagtcag taagggttag gtcactgcag tggtttaaac 29100 aagatgggct agaatccttt cacaggcaca ggcagcttgg agagggtgca atagtgcatg 29160 gtatcagggg tcagatgcct ctttttcctt tgagatcagt aagtggcttt cacctcatga 29220 cctaggctgg ctgctgtgtg ctagccgtca agtcacactc catccagcat gaaaggaggt 29280 tagaaaaggg tgcatttcct cttcttaaa acatgtctca aagttgcaca cagcacttttt 29340 gcctatattc aattggccat tagtcccacg gccatacctg tctgagactg agagactggg 29400 aaatgtcttt atttcaagtg gccatatatc cacctaaa agataaggga tacgtggtta 29460 tggcgtgtct tttggtttac caatgcagat aatgaagtta ccaaaacaat gagaaaatgg 29520 ggtcgtgagg gatcatgtga atcacaagct gatgtcttca aagacggtgg aaatgggccc 29580 cgggaggcag cagatgacag cagtggggat taaggtagac ctccatcctg gggttaaaat 29640 gaggggaagg tgatggagct ggaccagcag tcagaatggt cagtggttag gagaccctct 29700 gccccccacc gctgccacca ttggctctct agaatgcc tgcgagtggc ttagagtgac 29760 caaggatgag gtgcagatcc atgtgcaccc ccctgccccc tctgtggaca attttcatgc 29820 ctgacagcac agtctatgtg gattgcaagc cgatgaaact atgcaaagta gaagcatgcc 29880 tgcagtttgt gattcggtga tgtgttttat gcttatgtga gtcgaatggg gcggcagggt 29940 cctgtggtca cccgctgaga aggaagggtc ctgtaaccac tgcctttctt tcagctactt 30000 gagaaggtg ttgtgaggga ccgtggattt tgggacagct ttgaatggtg gtagggagga 30060 agggtccggt ctgagtgaat ggccagaaag ctgtgggggaa gcttttagga cattggccaa 30120 gagctccctg aaggcagcca gggagatact tgtcagtaca tgtgactaat ggccaactga atataagcag aagtgctgtg ttgctgtgtg caacactgga caccttagga aggacctcga gacagtggtt gtggactctg tagagagtaa cagtgacagt agcaaaccct tacccagtgc caaccttgtg ctaggctcgc actaaatgag tttaccttca attctcgtaa caataggagg 30420. tctaatttcc attttataga tgaggaaact aaggcacaga gatcactgac ttgcccaaaa tcaagcaggg agtagttagt fatherccc acggtatgtg gtttgtagaa 30540. tagtgctct tgactagcag aaataggtcc tccctgcagt gtgtaattga taacaagcat gggctgccat cttcctgtcg aggccactca aaacacccaa caggctacgc acggtggctc acacctgtaa tcccagcact gtgggaggcc gaggtggggcg ggtcacctga ggtcaggagt 30660 30720. tcgaccag cctggccgac atggtgaaac tccgtctcta ctaacagtac aaaaattagc tgggcgtggt ggcgggcacc tgtaatccca gctactcagg aggctgagac agaagaatca cttgaaccag ggaggcagag gttgcagtga gacaagatca cgccattgca ctccagcctg 30840 tgtgacaaaa gcgaaactgt ctcaaaaaaa aaaaaaaaaa aagtatgatt ttataatccc 30900 agcaccttgg gaggctgagt cgtgagaatc acttgagccc aggagtttaa gaccaatcta 30960 ggcaacatgg caagacccca tctctgccaa aaataaaaaa tagtctaatt ttagctattc 31020 atgtgtgtgt gaagtggtgt ctcttcgtgg ctttgatctg catttcccta atgctgacta 31080 atgacgttgg gcacctgttc atgtgcttac tggtcagata tctttctttt gttacatttt 31140 attaagtttt aaaatttaaa gtcaaagatt tccctatgag aatgactttt aaaatgacca 31200 aaaaggggaa gataacatta attcttgaag agaaggcctc tgagaaaaat acagttgtag 31260 caagctgcta ctttgcaaat gacccatgca ttttaatttt cccctaagga aggccaagga 31320 agagtcttat cacctcaggg caggagatgt agggacttgg gtcatttaat aagagtggta 31380 ggtttgaaaa ctcaaaccca gaagactcct tagagtttct cccaggaggt agggaagggg 31440 ccgcatccat ggagagagga ggatgtgact tagagcagtg gtccccaatc tttagggacc 31500 agggactggt gtcatggtag acagtttttc cacagatagg ggttgggggg atgatttgga 31560 gctgaaactg ctccacctca ggtcatcagg cattagattc tcatgtggag tgtgccactt 31620 agatccttgg cgtgcacagt tcacaatggg gttcgaggtc ctatgagaat ccgatgccac 31680 tgatttgaca ggaggcggag ctcaggtggt aatgctcatc tccaccgctt accacctgct 31740 gtgcagcctg gttcctaata ggctatagac tggtactggt ccatggcctg ggggttgggg 31800 acccctgatt tagaggaagt aagggcatgg cttaccgtgg gccctggggt gttctgggaa 31860 tggggaggat ggagagaaga gaggaggtag ggaagacctc cccttgctcc ccatttggga 31920 tttggggaga aagtcaggtc tcaggctcaa cagtacctga tcctgtacca tcttccaaag 31980 ggaagtcagt ggggttggaa ggtaggcagg ggttatcttc tctgagccac ggcacaagac 32040 agaagtttcc caccattcct gagggggcag gtggtaggtc cccaagcaga gagccagcag 32100 tccctctctg aggcctgcaa tggaatgggg tggggtgtcc actgagccaa gggtctgtca 32160 gtgagagctg gggaggctgggctggcttgc aagcacctgt tataaccaaa ccaggaaatc 32220 aggttccgag tcttgccagc aagggcctac agctgccagc agagatggac agccaggaga 32280 ccccaattgg ccacccagag ccaccctcct ctgcctaccc caccctccag tactccagag 32340 cctactcgga ggggaacaga aacctgagag gctgaacaca cacacatgga gaacaaacg 32400 tagtaaaata tttggggaat caggaaat tatttgtact attcctgcaa cctttctata 32460 ggcttgaaat tatcaaaata aatttttaaa aattgtaata acattctcat actaaaacac 32520 tgagtttttt tctttcattt tttgattttt tctttttgac tccagcatga cttactctaa 32580 caatgggtgg tctcgatttt gaaatacttt cttctccaag cctttcatga caccctgtct 32640 ctgttggttc tgaaaatgtt ggattttgtc tcagcccttg cttctggaaa cagccaaggt 32700 tagaaaacc ccccatgctt tgtgttctag cagacagctt cctgcaaaga gccatcttcc 32760 cagagcactt aggcctctta gatgtctccc ttgtttaatt atgacaagag cacacacaca 32820 gaccctccaa attcccattc ttagtcttct aaatgattag ctgagctgct tttccccact 32880 gattaatcgg aataaaatgc tcattaacca aacttccctc ctttccccag gtccctaaac 32940 tttcctgagt cggcagacat cccctctgga gaagaggttg gccccagagt cgaacatcct 33000 ctgatctacc tgatcctgct gcccttccat tccacttccc cacatctgtt ctttctggtc 33060 gtgtttactc ccctattaaa aaaacaaaac cagaaaacgt gtttgcctag atcttgagac 33120 tctggaagat cttaacagtc agaggttccc cctatttgca atgatctcct ttcctgcccc 33180 ttcctatcct tgcaataatc cttttgaata aagtctctcc ttactaaatc cagttcctaa 33240 aaattaattt ttttagag 33258 <210> 2 <211> 448 <212> PRT <213> Homo sapiens <220> <221> misc_feature <223> Presenilin-2 isoform 1 <400> 2 Met Leu Thr Phe Met Ala Ser Asp Ser Glu Glu Glu Val Cys Asp Glu 1 5 10 15 Arg Thr Ser Leu Met Ser Ala Glu Ser Pro Thr Pro Arg Ser Cys Gln 20 25 30 Glu Gly Arg Gln Gly Pro Glu Asp Gly Glu Asn Thr Ala Gln Trp Arg 35 40 45 Ser Gln Glu Asn Glu Glu Asp Gly Glu Glu Asp Pro Asp Arg Tyr Val 50 55 60 Cys Ser Gly Val Pro Gly Arg Pro Pro Gly Leu Glu Glu Glu Leu Thr 65 70 75 80 Leu Lys Tyr Gly Ala Lys His Val Ile Met Leu Phe Val Pro Val Thr 85 90 95 Leu Cys Met Ile Val Val Val Ala Thr Ile Lys Ser Val Arg Phe Tyr 100 105 110 Thr Glu Lys Asn Gly Gln Leu Ile Tyr Thr Pro Phe Thr Glu Asp Thr 115 120 125 Pro Ser Val Gly Gln Arg Leu Leu Asn Ser Val Leu Asn Thr Leu Ile 130 135 140 Met Ile Ser Val Ile Val Val Met Thr Ile Phe Leu Val Val Leu Tyr 145 150 155 160 Lys Tyr Arg Cys Tyr Lys Phe Ile His Gly Trp Leu Ile Met Ser Ser 165 170 175 Leu Met Leu Leu Phe Leu Phe Thr Tyr Ile Tyr Leu Gly Glu Val Leu 180 185 190 Lys Thr Tyr Asn Val Ala Met Asp Tyr Pro Thr Leu Leu Leu Thr Val 195 200 205 Trp Asn Phe Gly Ala Val Gly Met Val Cys Ile His Trp Lys Gly Pro 210 215 220 Leu Val Leu Gln Gln Ala Tyr Leu Ile Met Ile Ser Ala Leu Met Ala 225 230 235 240 Leu Val Phe Ile Lys Tyr Leu Pro Glu Trp Ser Ala Trp Val Ile Leu 245 250 255 Gly Ala Ile Ser Val Tyr Asp Leu Val Ala Val Leu Cys Pro Lys Gly 260 265 270 Pro Leu Arg Met Leu Val Glu Thr Ala Gln Glu Arg Asn Glu Pro Ile 275 280 285 Phe Pro Ala Leu Ile Tyr Ser Ser Ala Met Val Trp Thr Val Gly Met 290 295 300 Ala Lys Leu Asp Pro Ser Ser Gln Gly Ala Leu Gln Leu Pro Tyr Asp 305 310 315 320 Pro Glu Met Glu Glu Asp Ser Tyr Asp Ser Phe Gly Glu Pro Ser Tyr 325 330 335 Pro Glu Val Phe Glu Pro Pro Leu Thr Gly Tyr Pro Gly Glu Glu Leu 340 345 350 Glu Glu Glu Glu Glu Arg Gly Val Lys Leu Gly Leu Gly Asp Phe Ile 355 360 365 Phe Tyr Ser Val Leu Val Gly Lys Ala Ala Ala Thr Gly Ser Gly Asp 370 375 380 Trp Asn Thr Thr Leu Ala Cys Phe Val Ala Ile Leu Ile Gly Leu Cys 385 390 395 400 Leu Thr Leu Leu Leu Leu Path Val Phe Lys Lys Path Leu Pro Path Leu 405 410 415 Pro Ile Ser Ile Thr Phe Gly Leu Ile Phe Tyr Phe Ser Thr Asp Asn 420 425 430 Leu Val Arg Pro Phe Met Asp Thr Leu Ala Ser His Gln Leu Tyr Ile 435 440 445 <210> 3 <211> 19 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Forward 5'-3' Primer <400> 3 catcagccct ttgccttct 19 <210> 4 <211> 20 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Reverse 3'-5' First <400> 4 ctcaccttgt 20 <210> 5 <211> 26 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Forward 5'-3' First <400> 5 acagaattcg ccccggcctg gtacac <210> 6 <211> 25 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Reverse 3'-5' First <400> 6 tagcttggc acggctgtcc aagga <210> 7 <211> 20 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Forward 5'-3' First <400> 7 tcagcatcta cacgccattc <210> 8 <211> 20 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Reverse 3'-5' Primer <400> 8 agcaccacca agaagatggt 20 <210> 9 <211> 26 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Forwad 5'-3' Primer <400> 9 attcgccccg gcctggtaca ctgcca 26 <210> 10 <211> 27 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Reverse 3'-5' Primer <400> 10 ctgtccaagg agctgcaggc ggcgcag 27 <210> 11 <211> 25 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> g1N141I guide RNA F <400> 11 caccgcatca tgatcagcgt catcg 25 <210> 12 <211> 25 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> g1N141I guide RNA R <400> 12 aaaccgatga cgctgatcat gatgc 25 <210> 13 <211> 100 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Donor ssODN#A N141I <400> 13 gagagaagcg tggctggagg gcagggccag ggcctcacct tgtagcagcg gtacttgtag 60 agcaccacca agaagatggt cagggtgttc agcacggagt 100 <210> 14 <211> 21 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Forward 5' Primer <400> 14 taacggcggc agacaaaaag a 21 <210> 15 <211> 22 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Reverse 5'-3' Primer <400> 15 gaagtattgc ttcagttggc ct 22 <210> 16 <211> 21 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Forward 5' Primer <400> 16 agaagaacgg caagtacgag a 21 <210> 17 <211> 21 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Reverse 5'-3' Primer <400> 17 tgttgaggga cagattgtgg c 21 <210> 18 <211> 21 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Forward 5' Primer <400> 18 taacggcggc agacaaaaag a 21 <210> 19 <211> 22 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Reverse 5'-3' Primer <400> 19 gaagtattgc ttcagttggc ct 22 <210> 20 <400> 20 000 <210> 21 <400> 21 000 <210> 22 <211> 20 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Forward 5' Primer <400> 22 acgaatctcc gaccaccact 20 <210> 23 <211> 20 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Reverse 5'-3' Primer <400> 23 ccatggccac aacaactgac 20 <210> 24 <211> 22 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Forward 5' Primer <400> 24 gaagtgtccc aggacatgat aa 22 <210> 25 <211> 22 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Reverse 5'-3' Primer <400> 25 ctcttgagta gctgggattg ag 22 <210> 26 <211> 46 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Forward 5'-3' Primer <400> 26 ctccgtgctg atcaccctca tcatgatcag cgtcatcggt tatgac 46 <210> 27 <211> 48 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Reverse 3'-5' Primer <400> 27 gaggcacgac tagtgggagt agtactagtc gcagtagcac caatactg 48 <210> 28 <211> 20 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Forward 5'-3' Primer <400> 28 catcatgatc agcgtcatcg 20 <210> 29 <211> 25 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> g1N2411 guide RNA F <400> 29 caccgcatca tgatcagcgt catcg 25 <210> 30 <211> 25 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> g1N2411 guide RNA R <400> 30 aaaccgatga cgctgatcat gatgc 25 <210> 31 <211> 118 <212> DNA <213> Homo sapiens <220> <221> misc_feature <223> Donor ssODN#A N141I <400> 31 gagagaagcg tggctggagg gcagggccag ggcctcacct tgtagcagcg gtacttgtag 60 agcaccacca agaagatggt cataaccacg atgacgctga tcatgatgag ggtgttca 118

Claims

1. A method for generating basal forebrain cholinergic neurons (BFCNs), A step of culturing pluripotent stem cells (PSCs) in a basal medium containing an inhibitor of transforming growth factor beta (TGF-β) signaling and an activator of sonic hedgehog (Shh) signaling to induce neuroectodermal differentiation, wherein the basal medium lacks basic fibroblast growth factor (bFGF), TGF-β, lithium chloride (Li-Cl), GABA, and pipecolic acid. A method that includes, thereby generating BFCN.

2. The method according to claim 1, wherein the inhibitor is a SMAD inhibitor.

3. The method according to claim 2, wherein the inhibitor is SB431542, LDN193189, or a combination thereof.

4. The method according to claim 1, wherein the activator is an agonist of smoothed protein.

5. The method according to claim 4, wherein the activating factor is a smoothed agonist (SAG), purmorphamine, or a combination thereof.

6. The method according to claim 1, wherein the culture is carried out for approximately 4 to 9 days.

7. The method according to claim 2, wherein the inhibitor is present in the culture medium from about day 2 to day 8.

8. The method according to claim 1, further comprising the step of selecting CD271+ cells.

9. The method according to claim 8, wherein the selection step is performed after approximately 9 to 12 days of culture.

10. The method according to claim 9, further comprising the step of culturing the CD271+ cells in a basal medium for nerve cells to generate a neurocyte (NEB).

11. The method according to claim 10, wherein the CD271+ cells are cultured for about 7 days to produce NEB.

12. a) A step of recovering the NEB; and b) A step of dissociating the cells of the NEB and reseeding the dissociated cells as a monolayer. The method according to claim 11, further comprising:

13. The method according to claim 12, wherein the reseeded cells are cultured for a further duration in a medium containing growth factors to maintain the survival of the cells, and the cells express Tuj1, MAP2, BF1, Nkx2.1, and p75.

14. The method according to claim 1, further comprising the step of culturing the cells in mTeSR1 basal medium until confluent before contacting the cells with the activator or inhibitor.

15. The method according to claim 1, wherein the PSC is a human cell.

16. The method according to claim 1, wherein the PSC is an induced pluripotent stem cell (iPSC).

17. The method according to claim 16, wherein the iPSC is derived from a subject diagnosed with or at risk of having Alzheimer's disease (AD).

18. The method according to claim 17, wherein the PSC is derived from a BFCN having a mutation in presenilin 2 (PSEN2).

19. The aforementioned mutation is PSEN2 Ｎ１４１Ｉ The method according to claim 18.

20. The method according to claim 18, wherein the mutation is repaired after the generation of the iPSC.

21. The method according to claim 21, wherein the mutation is repaired using a gene editing system selected from the group consisting of the CRISPR / Cas system, the Cre / Lox system, the TALEN system, and homologous recombination.

22. The method according to claim 1, wherein the cultured cells show uniform expression of Nkx2.1 by the 8th day of culture.

23. The method according to claim 1, wherein the cultured cells exhibit recordable action potentials by the 8th day of culture.

24. The method according to claim 12, wherein the cultured cells exhibit mature action potentials.

25. The method according to claim 1, wherein the cultured cells exhibit mature action potentials by the 38th day of culture.

26. The method according to claim 20, wherein the cultured cells show normalization of the Aβ42 / 40 ratio compared to a control.

27. The method according to claim 20, wherein the cultured cells show a reduction in electrophysiological abnormalities compared to a control.

28. The method according to claim 27, wherein the reduction of the electrophysiological abnormality includes, compared to a control, a recovery of the maximum number of spikes and spike height in response to the depolarizing current.

29. A method for treating a disease or disorder in a subject, comprising administering to the subject a BFCN produced using the method of claim 1, wherein the disease or disorder is treated.

30. The method according to claim 29, wherein the disease or disorder is an amyloid-forming disease.

31. The method according to claim 29, wherein the disease or disorder is associated with a decrease in neuronal excitability in the BFCN of the subject.

32. The method according to claim 30, wherein the disease or disorder is selected from the group consisting of systemic amyloidosis, Alzheimer's disease, adult-onset diabetes mellitus, Parkinson's disease, Huntington's disease, frontotemporal dementia, and prion-associated transmissible spongiform encephalopathy.

33. The method according to claim 29, wherein the BFCN comprises a genome in which the PSEN2 mutation has been repaired.

34. The aforementioned PSEN2 mutation is PSEN2 Ｎ１４１Ｉ The method according to claim 33.

35. A method for restoring the excitability of basal forebrain cholinergic neurons (BFCNs) in a subject, a) A step of isolating BFCN from the subject, wherein the BFCN has a mutation in presenilin 2 (PSEN2) that causes impaired neuronal excitability of the BFCN; b) A step of generating induced pluripotent stem cells (iPSCs) using the BFCN of (a); c) A step of repairing the mutation in PSEN2 in the iPSC; d) A step of culturing the iPSC of (c) using the method of claim 1 to produce a BFCN in which the mutation has been repaired; and e) A step of administering the iPSC of (d) to the subject, wherein the excitability of the BFCN neurons in the subject is restored. Methods that include...

36. The aforementioned mutation is PSEN2 Ｎ１４１Ｉ The method according to claim 35.

37. The method according to claim 35, wherein the mutation is repaired using a gene editing system selected from the group consisting of the CRISPR / Cas system, the Cre / Lox system, the TALEN system, and homologous recombination.

38. The method according to claim 35, wherein the subject has or is at risk of having Alzheimer's disease.

39. A method for identifying compounds for the treatment or prevention of diseases or disorders associated with decreased neuronal excitability in basal forebrain cholinergic neurons (BFCNs), a) A step of contacting a BFCN or neurocyte (NEB) produced by the method of claim 1 with a candidate compound, wherein the BFCN contains a mutation in presenilin 2 (PSEN2) that causes impaired neuronal excitability of the BFCN; and b) A step of detecting the neuronal excitability of the BFCN after contact with the candidate compound. Methods that include...

40. The aforementioned mutation is PSEN2 Ｎ１４１Ｉ The method according to claim 39.

41. The method according to claim 39, wherein the disease or disorder is Alzheimer's disease.

42. The method according to claim 39, which is a high-throughput method.

43. Forebral basal cholinergic neurons (BFCNs) generated using the method described in claim 1.

44. The BFCN according to claim 43, wherein the genome has a recombinantly introduced marker.

45. A kit for generating basal forebrain cholinergic neurons (BFCNs), comprising a medium containing an inhibitor of transforming growth factor beta (TGF-β) signaling and an activator of sonic hedgehog (Shh) signaling, wherein the basal medium lacks basic fibroblast growth factor (bFGF), TGF-β, lithium chloride (Li-Cl), GABA, and pipecolic acid; the inhibitors comprise SB431542 and LDN193189; and the activators comprise a smoothed agonist (SAG) and purmorphamine.

46. The kit according to claim 45, further comprising reagents for generating induced pluripotent stem cells (iPSCs).

47. The kit according to claim 45, further comprising a gene editing reagent.

48. The kit according to claim 45, further comprising a reagent for detecting gene mutations.

49. The aforementioned mutation is PSEN2 Ｎ１４１Ｉ The kit according to claim 48.

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