Disease models and uses thereof

Abstract

The invention provides a disease model and application thereof. Particularly, the invention provides an engineered human cell with a mutated APP gene, a cell model derived from the engineered human cell and application of the engineered human cell.

Description

Background Art

[0001] Alzheimer's disease (AD) is the leading cause of dementia and is rapidly becoming one of the most expensive, lethal, and burdensome diseases worldwide. AD is now biologically defined by the presence of senile plaques and neurofibrillary tangles, progressive neuronal death, and neuroinflammation. Disease-modifying therapies that can prevent or slow the rate of disease progression are urgently needed, but unfortunately, no such therapies are currently available, and many phase 3 clinical trials have failed to demonstrate benefit of investigational drugs. Potential reasons include 1) the translational gap between animal species involved in the process from initial discoveries in rodent models to human studies, and 2) suboptimal patient stratification, meaning that heterogeneous patient populations are grouped based solely on disease severity due to the lack of phenotypic and genetic markers.

[0002] Many factors may have contributed to this, but one of the major obstacles is the lack of disease models that adequately capture the pathogenesis of the disease. Animal models play an important role in identifying key disease-related mechanisms and have been widely used in the development and evaluation of new treatments. Many of the treatments currently in clinical trials have their origins in studies that were initially conducted in rodents. However, translational issues are increasingly evident due to potential inconsistencies between preclinical drug studies and human clinical trials. Significant species differences between humans and rodents, such as differences in the number of tau protein isoforms between species, prevent animal models from accurately reproducing the pathological features of human AD.

[0003] On the other hand, in the currently available human cell-derived models, although ectopic expression or overexpression of APP protein has been adopted, APP overexpression interferes with axonal transport due to interaction with kinesin. In addition, in these APP transgenic models, not only full-length APP, but also other APP fragments (such as sAPP, CTF-β, CTF-α and AICD) are overproduced, which may affect normal physiological functions. The use of artificial promoters usually results in transgenic expression in cells different from those expressing endogenous APP, and artificial promoters can compete with endogenous promoters for common transcription factors. Sometimes, transgenes are inserted into the locus of host cells in a multi-copy manner, which may destroy the function of endogenous genes. Therefore, these models can hardly reveal the AD pathology of human patients.

[0004] Therefore, there is an urgent need to develop tools and models that can more accurately reproduce or simulate the pathogenesis of AD in humans. Summary of the invention

[0005] The inventors of the present application used gene editing technology to introduce familial mutations (Swedish, Beyreuther / Iberian and / or Arctic) into the APP gene of human embryonic stem cells (hESC), and then differentiated them into brain organoids to establish a fully humanized AD model. Although brain organoids only contain certain types of cells in the human brain (for example, brain organoids do not contain blood vessels or microglia, which are generally considered to be important for neuronal function), surprisingly, the cell model of the present application successfully reproduced a variety of AD-related phenotypes, including β-amyloid accumulation, tau protein hyperphosphorylation and aggregation, glial cell proliferation, chronic neuroinflammation, synaptic loss and neuronal death. In addition, the inventors introduced the APOE3Christchurch (APOE3ch) mutation into the AD model of the present application and found that APOE3ch APP NL-G-F Organoids (with Swedish double mutations K670N and M671L (ie NL), Beyreuther / Iberian mutations I716F (ie F) and Arctic mutations E693G (ie G)) resist a variety of AD-related pathologies, even in the presence of high Aβ load. The mechanism of this resistance was studied, and it was found that APOE3ch enhanced the cellular phagocytosis and lysosomal degradation of neurotoxic substances such as tau aggregates and apoptotic cells by reducing lysosomal cholesterol load. In short, the cell model of the present application provides a new platform for understanding AD and drug development. In addition, protective APOE3ch mutations have been identified as promising new therapeutic targets for the treatment of AD.

[0006] In diseases characterized by toxic protein aggregation, such as AD, the presence of true interstitial cavities is important for pathological modeling. Previous three-dimensional (3D) tissue engineering methods have embedded the desired neural progenitor cells or cell types in a matrix or scaffold. Although these methods can mimic AD phenotypes, they cannot reproduce the spontaneous pathology caused by endogenous cell characteristics, but require overexpression of AD genes. Some other studies use iPSCs derived from familial AD patients to generate brain organoids. These patients usually carry only one AD mutation or risk gene and require a relatively long time to form a pathological phenotype in vivo. Currently, iPSC-derived brain organoids can only be maintained in vitro for a relatively short time. AD-related phenotypes are limited to toxic soluble Aβ, pathological aggregation of Aβ, and increased phosphorylated Tau protein in patient iPSC-derived brain organoids. Pathological aggregation of Tau protein has not been reported in AD brain organoid models. Moreover, pathological phenotypes that only appear in the middle and late stages of AD, such as abnormal gliosis and neuronal cell death, were not observed in these reports, which limits the understanding of the mechanism of AD.

[0007] The inventors introduced Swedish, Beyreuther / Iberian and / or Arctic mutations into the APP gene of host hESCs by knock-in technology to help establish an AD brain organoid model. The model successfully reproduced several AD-related phenotypes, including accumulation of β-amyloid protein, hyperphosphorylation and aggregation of Tau protein, glial cell proliferation, chronic neuroinflammation, synaptic loss and neuronal death, providing a promising new tool for the study of AD disease mechanisms and high-throughput drug screening.

[0008] Among people with familial AD Colombia mutation, a patient carrying Christchurch (APOE3ch) mutation was accidentally found to be able to resist the onset of AD caused by AD Colombia mutation. In order to study the potential protective effect of APOE3ch, the present invention introduces this mutation into APP NL-G-F hESCs and differentiated them into brain organoids. Discovery of APOE3chAPP NL-G-F Brain organoids resist multiple AD-related pathologies, including attenuated Tau aggregation and neuronal cell death. Mechanistically, APOE3ch can alleviate endolysosomal dysfunction and then enhance cellular phagocytosis and degradation of neurotoxic substances such as Tau aggregates and apoptotic cell debris, which may partially explain its disease-modifying effects.

[0009] APOE is well known for its role in regulating cellular cholesterol metabolism. It is reported that APOE3ch significantly reduces cholesterol levels in neuronal cells. Cholesterol accumulation can cause damage to endolysosomal function in neuronal cells, especially in lysosomal storage disease C1 Niemann-Pick disease (NPC1), in which mutations in cholesterol transporters cause cholesterol to accumulate in lysosomes. In addition, the use of cyclodextrin can reduce intracellular cholesterol, thereby partially restoring the acidity of lysosomes. Therefore, the inventors also identified reducing intracellular cholesterol as a potential therapeutic strategy for treating AD.

[0010] On the one hand, the present application provides engineered human cells, wherein the APP gene is mutated to encode a mutant APP protein, which comprises amino acid substitutions at the following residues compared to the wild-type human APP protein shown in SEQ ID NO:1: K670, M671, I716 and / or E693.

[0011] In some embodiments, the mutant APP protein comprises amino acid substitutions at the following residues: K670, M671, I716, and E693.

[0012] In some embodiments, the mutant APP protein further comprises one or more additional mutations that can affect the amount of Aβ produced, the amount of Aβ fragments produced, and / or the ratio of Aβ42 / Aβ40 produced.

[0013] In some embodiments, the mutant APP protein further comprises one or more additional or alternative mutations selected from the group consisting of: I716V and V717I.

[0014] In some embodiments, the engineered human-derived cell is derived from a subject that has had, is currently suffering from, or is at risk of developing a disease or condition.

[0015] In some embodiments, the engineered human cells are human stem cells.

[0016] In some embodiments, the engineered human cells include pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells and / or neuronal stem cells. In some embodiments, the embryonic stem cells are from established cell lines. In some embodiments, the embryonic stem cells are from human embryonic stem cells (hESC) system H1. In some embodiments, the embryonic stem cells are not obtained by the process of destroying human embryos.

[0017] In some embodiments, the mutant APP protein comprises a Swedish double mutation. In some embodiments, the Swedish double mutation comprises a K670N substitution and a M671L substitution.

[0018] In some embodiments, the mutant APP protein comprises a Beyreuther / Iberian mutation. In some embodiments, the Beyreuther / Iberian mutation comprises an I716F substitution.

[0019] In some embodiments, the substitution at position I716 is I716V or I716F.

[0020] In some embodiments, the mutant APP comprises an Arctic mutation. In some embodiments, the Arctic mutation comprises an E693G substitution.

[0021] In some embodiments, the engineered human cells are homozygous for one or more mutations at the following residues of the APP protein: K670, M671, I716, and / or E693.

[0022] In some embodiments, the expression level of CTF-α derived from the engineered human cells is not significantly different compared to the expression level of CTF-α in the corresponding wild-type human cells.

[0023] In some embodiments, the expression level of CTF-β derived from the engineered human cell is increased compared to the expression level of CTF-β in a corresponding wild-type human cell.

[0024] In some embodiments, the expression level of Aβ42 derived from the engineered human cells is increased compared to the expression level of Aβ42 in corresponding wild-type human cells.

[0025] In some embodiments, the ratio of Aβ42 / Aβ40 derived from the engineered human cells is increased compared to the ratio of Aβ42 / Aβ40 of a corresponding wild-type human cell.

[0026] In some embodiments of the engineered human-derived cells, hyperphosphorylation of Tau is detectable.

[0027] In some embodiments of the engineered human cells, the hyperphosphorylation of Tau in the engineered human cells comprises increased phosphorylation of Ser396, Thr181, Thr205 and / or Ser202 in Tau compared to the hyperphosphorylation of Tau in the corresponding wild-type human cells.

[0028] In some embodiments of the engineered human cell, the cell is a knock-in cell or is derived from a knock-in cell, and wherein at least a portion of the endogenous APP gene is replaced by a heterologous nucleic acid sequence encoding at least a portion of the mutant APP protein.

[0029] In some embodiments, the at least a portion of the endogenous APP gene comprises at least a portion of exon 16 and / or at least a portion of exon 17 of the endogenous APP gene.

[0030] In some embodiments, the heterologous nucleic acid sequence encodes or comprises a mutated exon 16 and / or a mutated exon 17.

[0031] In some embodiments of the engineered human cells, the mutant APP protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-10.

[0032] In some embodiments of the engineered human-derived cells, the APOE gene is mutated to encode a mutant APOE3 protein, and the residue R136 in the mutant APOE3 protein is substituted compared to the wild-type human APOE3 protein shown in SEQ ID NO: 11. In some embodiments, the residue R136 in the mutant APOE3 protein is substituted by S.

[0033] In some embodiments of the engineered human cells, the mutant APOE3 protein comprises the amino acid sequence shown in SEQ ID NO:12.

[0034] In some embodiments, the engineered human cells are homozygous for the mutant APOE gene.

[0035] In some embodiments, the engineered human-derived cells further comprise mutations in one or more additional genes selected from the group consisting of: Tau, APOE2, APOE4, and BDNF.

[0036] In another aspect, the present application provides a cell model of dementia-related neurological diseases, comprising an in vitro culture of a plurality of cells derived from the engineered human-derived cells of the present application.

[0037] In some embodiments of the cell model, the dementia-related neurological disease is Alzheimer's disease.

[0038] In some embodiments of the cell model, the plurality of cells are cultured in two dimensions.

[0039] In some embodiments of the cell model, the plurality of cells are cultured in three dimensions.

[0040] In some embodiments, the cell model has one or more of the following features: 1) is a fully humanized cell model; 2) exhibits accumulation of β-amyloid protein; 3) exhibits hyperphosphorylation of Tau protein; 4) exhibits aggregation of Tau protein; 5) exhibits glial cell proliferation; 6) exhibits chronic neuroinflammation; 7) exhibits synaptic loss and neuronal death; 8) exhibits mitochondrial dysfunction and oxidative damage; 9) exhibits autophagy defects; 10) exhibits neurotransmitter imbalance; and / or 11) exhibits glucose metabolism dysfunction.

[0041] In some embodiments, the cell model produces amyloid plaques and Tau tangles.

[0042] In some embodiments of the cell model, the cells are cultured in a 3D Matrigel matrix.

[0043] In some embodiments, the cell model is a brain organoid. In some embodiments, the cell model is a forebrain organoid. In some embodiments, the cell model includes astrocytes, neurons, neural progenitor cells and / or oligodendrocytes. For example, in some cases, the cell model includes astrocytes and neurons.

[0044] In another aspect, the present application provides a method for producing engineered human cells. The method comprises introducing a mutation into an endogenous APP gene of a human cell to produce a mutant APP gene, wherein the mutant APP gene encodes a mutant APP protein, and the mutant APP protein comprises amino acid substitutions at the following residues compared to the wild-type human APP protein shown in SEQ ID NO: 1: K670, M671, I716 and / or E693.

[0045] In some embodiments of the method, the mutant APP protein comprises amino acid substitutions at the following residues: K670, M671, I716, and E693.

[0046] In some embodiments of the method, introducing a mutation into the APP gene comprises knocking a heterologous nucleic acid sequence into an endogenous APP locus, wherein the knock-in replaces at least a portion of the endogenous APP gene with a heterologous nucleic acid sequence encoding at least a portion of the mutant APP protein.

[0047] In some embodiments of the method, the knock-in comprises contacting the genome of the human cell with: 1) a CRISPR-associated (Cas) protein; and 2) one or more ribonucleic acid (RNA) sequences comprising: i) a sequence complementary to a target portion of the endogenous APP gene; and ii) a binding site for the Cas protein in the presence of a donor nucleic acid molecule comprising the heterologous nucleic acid sequence.

[0048] In some embodiments of the method, the knock-in further comprises maintaining the cell under conditions wherein the one or more RNA sequences hybridize to the target portion of the endogenous APP gene and the Cas protein cleaves the endogenous APP gene nucleic acid sequence upon hybridization of the one or more RNA sequences.

[0049] In some embodiments of the method, the Cas protein is Cas9.

[0050] In some embodiments of the method, the binding site of the Cas protein comprises a tracrRNA sequence.

[0051] In some embodiments of the method, the Cas protein is introduced into the cell in the form of a protein, a messenger RNA (mRNA) encoding the Cas protein, or a DNA encoding the Cas protein.

[0052] In some embodiments of the method, the one or more RNA sequences are introduced into the cell in the form of one or more RNA molecules or one or more DNA molecules encoding the RNA sequences.

[0053] In some embodiments of the method, the one or more ribonucleic acid (RNA) sequences comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 13-16.

[0054] In some embodiments of the method, the at least a portion of the endogenous APP gene comprises at least a portion of exon 16 and / or at least a portion of exon 17 of the endogenous APP gene.

[0055] In some embodiments of the method, the heterologous nucleic acid sequence comprises a mutated exon 16 and / or a mutated exon 17 of the human APP gene.

[0056] In some embodiments of the method, the mutation in the mutated exon 16 and / or the mutated exon 17 results in one or more amino acid substitutions in the polypeptide encoded thereby, and wherein the one or more amino acid substitutions include amino acid substitutions at the following residues: K670, M671, I716 and / or E693.

[0057] In some embodiments of the methods, the one or more amino acid substitutions comprise substitutions K670N, M671L, I716F, and / or E693G.

[0058] In some embodiments of the method, the mutant APP protein further comprises one or more additional mutations capable of affecting the amount of Aβ produced, the amount of Aβ fragments produced, and / or the ratio of Aβ42 / Aβ40 produced.

[0059] In some embodiments of the method, the mutant APP protein further comprises one or more additional or alternative mutations selected from the group consisting of: I716V and V717I. For example, in some cases, the mutant APP protein may comprise the following amino acid substitutions: K670N, M671L, I716F, E693G, and V717I. In some cases, the mutant APP protein may comprise the following amino acid substitutions: K670N, M671L, I716V, E693G, and V717I. In some cases, the mutant APP protein may comprise the following amino acid substitutions: K670N, M671L, I716V, E693G, and V717I. In some cases, the mutant APP protein may comprise the following amino acid substitutions: K670N, M671L, I716V, and E693G.

[0060] In some embodiments of the methods, the human-derived cell is derived from a subject who has had, is currently suffering from, or is at risk of developing a disease or disorder.

[0061] In some embodiments of the method, the human-derived cell is a human stem cell.

[0062] In some embodiments of the method, the human-derived cells are pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells and / or neuronal stem cells.

[0063] In some embodiments of the methods, the embryonic stem cells are from an established cell line.

[0064] In some embodiments of the method, the embryonic stem cells are from the human embryonic stem cell (hESC) line H1.

[0065] In some embodiments of the methods, the embryonic stem cells are not obtained by a process that destroys a human embryo.

[0066] In some embodiments of the method, the mutant APP protein comprises a Swedish double mutation.

[0067] In some embodiments of the method, the Swedish double mutation comprises a K670N substitution and a M671L substitution.

[0068] In some embodiments of the methods, the mutant APP protein comprises a Beyreuther / Iberian mutation. In some embodiments of the methods, the Beyreuther / Iberian mutation comprises an I716F substitution.

[0069] In some embodiments of the method, the mutant APP comprises an Arctic mutation. In some embodiments of the method, the Arctic mutation comprises an E693G substitution.

[0070] In some embodiments of the method, the engineered human-derived cell is homozygous for one or more mutations at the following residues of the APP protein: K670, M671, I716, and / or E693.

[0071] In some embodiments of the method, the mutant APP protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-10.

[0072] In some embodiments, the method further comprises introducing a mutation into the APOE gene to produce a mutant APOE3 protein, wherein residue R136 in the mutant APOE3 protein is substituted compared to the wild-type human APOE3 protein shown in SEQ ID NO:11.

[0073] In some embodiments of the method, the residue R136 in the mutant APOE3 protein is substituted with S.

[0074] In some embodiments of the method, the mutant APOE3 protein comprises the amino acid sequence shown in SEQ ID NO:12.

[0075] In some embodiments of the method, the engineered human cell is homozygous for the mutant APOE gene. In some embodiments of the method, the engineered human cell further comprises a mutation in one or more additional genes selected from the group consisting of Tau, APOE2, APOE4, and BDNF.

[0076] In another aspect, the present application provides engineered human cells produced by the method of the present application.

[0077] In another aspect, the present application provides a method for producing a cell model, the method comprising contacting the engineered human-derived cell of the present application with a differentiation medium to obtain the cell model.

[0078] In some embodiments of the method, the cell model is a model of a dementia-related neurological disease.

[0079] In some embodiments of the methods, the dementia-related neurological disease is Alzheimer's disease.

[0080] In some embodiments of the method, the engineered human-derived cells are cultured in a two-dimensional in vitro culture. In some embodiments of the method, the engineered human-derived cells are cultured in a three-dimensional in vitro culture.

[0081] In some embodiments of the method, the engineered human-derived cells are cultured in a gel, in a bioreactor, under ultra-low adhesion conditions, or on a microchip.

[0082] In some embodiments of the method, the engineered human cells are cultured in a matrix. In some embodiments of the method, the matrix is ​​an extracellular matrix and / or wherein the matrix comprises one or more of natural molecules, synthetic polymers, biological-synthetic hybrids, metals, ceramics, bioactive glass and / or carbon nanotubes.

[0083] In some embodiments of the methods, the cell model is a brain organoid. In some embodiments of the methods, the cell model is a forebrain organoid.

[0084] In another aspect, the present application provides a cell model produced by the method of the present application.

[0085] In another aspect, the present application provides a composition comprising the engineered human cells or cell models of the present application.

[0086] In another aspect, the present application provides a kit for screening substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of dementia-related neurological diseases, wherein the kit comprises the engineered human cells or cell models of the present application.

[0087] In some embodiments of the kit, the dementia-related neurological disease is Alzheimer's disease.

[0088] In some embodiments of the kits, the substances, devices, and / or compositions comprise molecules, membrane-bound vesicles, and / or cells.

[0089] In some embodiments, the kit further comprises one or more additional components selected from the group consisting of assay buffers, controls, substrates, standards, detection materials, experimental supplies, devices, machines, cells, organs, tissues, and user manuals or instructions.

[0090] On the other hand, the present application provides a method for screening substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of dementia-related neurological diseases. The method comprises: exposing the engineered human cells or cell models of the present application to candidate substances, devices and / or compositions; evaluating the engineered human cells or cell models for one or more characteristics of the dementia-related neurological diseases in the presence of the candidate substances, devices and / or compositions; and selecting candidate substances, devices and / or compositions that can prevent, inhibit and / or alleviate one or more of the characteristics of the dementia-related neurological diseases.

[0091] On the other hand, the present application provides a method for identifying potential substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of dementia-related neurological diseases, the method comprising: (i) contacting the engineered human cells or cell models of the present application with the candidate substances, devices and / or compositions to be tested; and (ii) evaluating the activity of the candidate substances, devices and / or compositions on one or more characteristics of the dementia-related neurological diseases.

[0092] On the other hand, the present application provides a method for designing a substance, device and / or composition useful in the treatment, prevention and / or prognosis of a dementia-related neurological disease. The method comprises the following steps: (i) exposing the engineered human cell or the cell model to a candidate substance, device and / or composition; (ii) evaluating the engineered human cell or the cell model for one or more characteristics of the dementia-related neurological disease; (iii) selecting a candidate substance, device and / or composition that can prevent, inhibit and / or alleviate one or more of the characteristics of the dementia-related neurological disease; and (iv) modifying the structure and / or composition of the candidate substance, device and / or composition of step (iii) to obtain a modified substance, device and / or composition having improved activity in the treatment, prevention and / or prognosis of the dementia-related neurological disease.

[0093] In some embodiments of the methods, the dementia-related neurological disease is Alzheimer's disease.

[0094] In some embodiments of the method, the features of the dementia-related neurological disease include: accumulation of β-amyloid protein; hyperphosphorylation of Tau protein; aggregation of Tau protein; glial cell proliferation; chronic neuroinflammation; synaptic loss; neuronal death; the appearance of amyloid plaques; the appearance of Tau tangles; increased expression level of CTF-β; increased expression level of Aβ42; increased ratio of Aβ42 / Aβ40; mitochondrial dysfunction and oxidative damage; autophagy defect; neurotransmitter imbalance; and / or glucose metabolism dysfunction.

[0095] In some embodiments of the method, the hyperphosphorylation of the Tau protein comprises increased phosphorylation of Ser396, Thr181, Thr205 and / or Ser202 in Tau compared to the hyperphosphorylation of the Tau protein in a corresponding wild-type human cell.

[0096] In another aspect, the present application provides use of the engineered human-derived cells or cell models of the present application as models of dementia-related neurological diseases.

[0097] In another aspect, the present application provides use of the engineered human cells or cell models of the present application in preparing a dementia-related neurological disease model.

[0098] In some embodiments of the use, the dementia-related neurological disease is Alzheimer's disease.

[0099] On the other hand, the present application provides a method for identifying potential biological targets and / or biomarkers of substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of dementia-related neurological diseases. The method comprises the following steps: (i) performing quantitative proteomic comparative analysis, lipidomic comparative analysis and / or genomic comparative analysis on the engineered human cells or cell models of the present application and control human cells or control cell models; (ii) identifying genes, proteins and / or lipids with altered sequences, quantities, expression levels, modifications and / or activities; (iii) wherein the genes, proteins and / or lipids identified in step (ii) are potential biological targets and / or biomarkers of the substances, devices and / or compositions for the treatment, prevention and / or prognosis of the dementia-related neurological diseases.

[0100] In some embodiments of the methods, the dementia-related neurological disease is Alzheimer's disease.

[0101] On the other hand, the present application provides a method for screening biological targets and / or biomarkers useful in diagnosing and / or monitoring dementia-related neurological diseases, the method comprising determining the presence and / or level of a substance in a sample obtained from the engineered human cells or cell model of the present application before and after detecting characteristics of the dementia-related neurological disease, and identifying a substance that exhibits a change in presence and / or level before and after the detection.

[0102] In some embodiments of the methods, the dementia-related neurological disease is Alzheimer's disease.

[0103] In some embodiments of the method, the features of the dementia-related neurological disease include: accumulation of β-amyloid protein; hyperphosphorylation of Tau protein; aggregation of Tau protein; glial cell proliferation; chronic neuroinflammation; synaptic loss; neuronal death; the appearance of amyloid plaques; the appearance of Tau tangles; increased expression level of CTF-β; increased expression level of Aβ42; increased ratio of Aβ42 / Aβ40; mitochondrial dysfunction and oxidative damage; autophagy defect; neurotransmitter imbalance; and / or glucose metabolism dysfunction.

[0104] On the other hand, the present application provides the use of the engineered human cells or cell models of the present application in the preparation of a system for screening for substances, devices, compositions, biological targets and / or biomarkers useful in the treatment, diagnosis, prevention, monitoring and / or prognosis of dementia-related neurological diseases. In some embodiments of the use, the dementia-related neurological disease is Alzheimer's disease.

[0105] On the other hand, the present application provides an engineered human cell or cell model of the present application for screening for substances, devices, compositions, biological targets and / or biomarkers useful in the treatment, diagnosis, prevention, monitoring and / or prognosis of dementia-related neurological diseases. In some embodiments, the dementia-related neurological disease is Alzheimer's disease.

[0106] According to the following detailed description, other aspects and advantages of the present application will become apparent to those skilled in the art, in which only exemplary embodiments of the present application are shown and described. As will be appreciated, the present application can have other embodiments and different embodiments, and its several details can be modified in various obvious aspects, all of which do not deviate from the present application. Therefore, the drawings and description should be considered as exemplary in nature, rather than restrictive.

[0107] Incorporation by Reference

[0108] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0109] The novel features of the present invention are specifically set forth in the appended claims. The features and advantages of the present invention may be better understood by reference to the following detailed description, which sets forth exemplary embodiments employing the principles of the present invention, and the accompanying drawings (also referred to herein as "figures"), in which:

[0110] Figures 1A to 1G Shows the APP NL-G-F Accumulation of extracellular amyloid-β deposits is increased in knock-in brain organoids.

[0111] FIG. 2A to FIG. 2J Shows the APP NL-G-F Tau pathology in brain organoids.

[0112] FIG. 3A to FIG. 3E Shows the APP NL-G-F Enhanced gliosis and neuroinflammation in brain organoids.

[0113] 4A to 4H Shows the APP NL-G-F Increased synaptic loss, apoptosis, and necrosis in brain organoids.

[0114] FIG. 5A to FIG. 5H APOE3ch showed a reduction in APP NL-G-F Multiple AD-associated phenotypes in brain organoids.

[0115] 6A to 6H Shows APOE3ch activation APP NL-G-F Endolysosomal function in brain organoids.

[0116] FIG. 7A to FIG. 7D Shows the use of APP NL-G-F Effects of TrkB antibodies on brain organoids.

[0117] Figure 8 The knock-in targeting strategy is shown.

[0118] Fig. 9 Differentially expressed proteins in AD brain organoids at 3 months are shown. Red (right), proteins increased in the AD group; blue (left), proteins decreased in the AD group. -log10(p.adj)>0.5, fold change>1.

[0119] Fig.10 GSEA analysis of biological processes enriched using the KEGG database is shown. NES>0 represents enriched pathways in the AD group; NES<0 represents enriched pathways in the control group.

[0120] Fig.11 GSEA analysis of biological processes enriched using GO database is shown. NES>0 represents enriched pathways in the AD group; NES<0 represents enriched pathways in the control group. DETAILED DESCRIPTION

[0121] Although various embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that these embodiments are provided by way of example only. Without departing from the present invention, those skilled in the art may conceive of many variations, changes and replacements. It should be understood that various alternatives to the embodiments of the present invention described herein may be employed.

[0122] As used herein, the term "CRISPR" generally refers to clustered regularly spaced short palindromic repeats. The difference between CRISPR loci and other SSRs is generally the structure of the repeats, which are called short regularly spaced repeats (SRSR). Typically, repeats are short sequence structures that occur in regularly spaced clusters of unique intervening sequences with substantially constant length. Repeats are highly conserved between strains, but the number of spaced repeats and spacer sequences is generally different between strains.

[0123] As used herein, the terms "sgRNA," "guide RNA," "single guide RNA," and "synthetic guide RNA" are used interchangeably and generally refer to a polynucleotide sequence comprising a guide sequence. The guide sequence is approximately 20 bp and is located within the guide RNA at a specified target site.

[0124] As used herein, the term "heterologous nucleic acid sequence" generally refers to a nucleic acid sequence that is derived from an exogenous source and / or exists in a non-endogenous form. For example, a heterologous nucleic acid sequence can be derived from an external subject, can be derived from an external species, can be artificially synthesized, can be located in an external locus, and / or can be substantially modified.

[0125] As used herein, the term "homologous recombination" generally refers to a type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical DNA molecules, called homologous sequences or homology arms.

[0126] As used herein, the term "endogenous APP gene" generally refers to an endogenous DNA fragment (such as an endogenous human DNA fragment) encoding the amyloid precursor protein or a fragment thereof.

[0127] As used herein, the term "CRISPR-associated protein 9" or "Cas9" protein generally refers to an RNA-guided DNA endonuclease associated with the CRISPR (clustered regularly interspaced short palindromic repeats) type II adaptive immune system found in certain bacteria, such as Streptococcus pyogenes and other bacteria. For example, the Cas9 protein may include not only the wild-type Cas9 found in Streptococcus pyogenes, but also various variants of the wild-type Cas9, such as those described in WO2013 / 176772A1. In some embodiments, the Cas9 protein may include Cas9 sequences from Streptococcus pyogenes, Neisseria meningitidis, Streptococcus thermophilus, and Treponema denticola, as described in Esvelt et al., Nature Methods, 10(11): 1116-1121, 2013.

[0128] As used herein, the term "Cas9 coding sequence" generally refers to a polynucleotide sequence that can be transcribed and / or translated to produce a Cas9 protein according to a functional genetic code in a host cell / host animal. The Cas9 coding sequence can be DNA (such as a plasmid) or RNA (such as mRNA).

[0129] As used herein, the term "Cas9 ribonucleoprotein" generally refers to a protein / RNA complex consisting of a Cas9 protein and an associated guide RNA.

[0130] As used herein, the term "CRISPR / Cas9 system" generally refers to a tool for site-specific genome targeting in an organism. For example, it can be a type II CRISPR / Cas system, which is a prokaryotic adaptive immune response system that uses non-coding RNA to guide Cas9 nuclease-induced site-specific DNA cutting. This DNA damage is repaired by the cell DNA repair mechanism, through the non-homologous end-joining DNA repair pathway (NHEJ) or homology-directed repair (HDR) pathway. The CRISPR / Cas9 system can be used to generate a simple RNA-programmable method to mediate genome editing in mammalian cells, and can be used to generate gene knockouts (by insertion / deletion) or knock-ins (by HDR).

[0131] As used herein, the term "knock-in" generally refers to a genetic engineering process involving a one-to-one replacement of DNA sequence information in a genetic locus or the insertion of sequence information not present in an endogenous locus. Knock-in can involve the insertion of a gene into a specific locus and can therefore be a "targeted" insertion.

[0132] As used herein, the term "vector" generally refers to a DNA molecule used as a vehicle for artificially carrying foreign genetic material into another cell or host where the DNA molecule can be replicated and / or expressed.

[0133] As used herein, the term "targeting vector" generally refers to a vector carrying a targeting sequence to be inserted or incorporated into a host genome and / or used to replace endogenous DNA segments.

[0134] As used herein, the term "embryonic stem cells" or "ES cells" generally refers to pluripotent stem cells derived from the inner cell mass (ICM) of a blastocyst (an early preimplantation mammalian embryo), which can be cultured in vitro for a period of time before being inserted / injected into a normal blastocyst cavity and induced to resume the normal embryonic development program to differentiate into various cell types of adult mammals, including germ cells.

[0135] As used herein, the term "homozygous" or "homozygous" is used with respect to a specific gene or DNA (e.g., a heterologous nucleic acid sequence that has been knocked in), and refers to a diploid cell or organism in which two homologous chromosomes have the same alleles or copies of the gene / DNA.

[0136] As used herein, the term "heterozygote" or "heterozygous" is used with respect to a specific gene or DNA (e.g., a heterologous nucleic acid sequence that has been knocked in), and refers to a diploid cell or organism in which two homologous chromosomes have different alleles / copies / variants of the gene or DNA.

[0137] As used herein, the term "mutated APP protein" or "mutant APP protein" generally refers to an amyloid precursor protein having an addition, deletion and / or substitution of at least one amino acid residue.

[0138] As used herein, the term "Swedish double mutation" or "Swedish mutation" generally refers to a double mutation in the APP gene originally discovered in a Swedish family, which is located before the amyloid β-peptide (Aβ) region of APP and increases the production and secretion of Aβ, which is described in Nat Genet. August 1992, 1(5):345-7. The Swedish double mutation is located in exon 16 of the human APP gene and is the only known mutation adjacent to the β-secretase site in APP. In some cases, the Swedish mutation results in the substitution of two amino acids, namely lysine (K) 670 and methionine (M) 671. Studies have shown that the Swedish double mutation increases total Aβ levels. Specifically, the production and secretion of Aβ40 and Aβ42 are increased, but the Aβ40 / Aβ42 ratio is generally unaffected. In some embodiments, the Swedish double mutation comprises amino acid substitutions K670N and M671L.

[0139] As used herein, the term "Beyreuther / Iberian mutation" generally refers to a mutation in the APP gene (e.g., at residue I716) that affects APP protein cleavage by γ-secretase. Specifically, the Beyreuther / Iberian mutation is located in exon 17 of the human APP gene and can affect γ-secretase cleavage specificity and cause a significant increase in the Aβ42 / Aβ40 ratio. In some embodiments, the Beyreuther / Iberian mutation comprises the amino acid substitution I716F.

[0140] As used herein, the term "Arctic mutation" generally refers to a mutation in the APP gene (e.g., at residue E693) that results in an increased tendency and an accelerated rate of Aβ40 fibril formation. It is also referred to as "E22G" because it affects the 22nd amino acid of the Aβ peptide. The Arctic mutation is one of several pathogenic APP mutations found that confer resistance to enkephalinase-catalyzed proteolysis of Aβ40. The Arctic mutation is located in exon 17 of the human APP gene. In some embodiments, the Arctic mutation comprises the amino acid substitution E693G.

[0141] As used herein, the term "no significant difference" generally refers to the difference between two values ​​or two objects is not significant. For example, when comparing two values, less than about 10%, such as less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5.5%, less than about 5%, less than about 4.5%, less than about 4%, less than about 3.5%, less than about 3%, less than about 2.5%, less than about 2%, less than about 1.5%, less than about 1% or even less difference can be considered as no significant difference.

[0142] As used herein, the term "Aβ oligomer" generally refers to soluble amyloid β (Aβ) peptide aggregates, which generally form small clumps. The Aβ oligomer may be a dimer, trimer or other multimer of the Aβ peptide.

[0143] As used herein, the term "Aβ plaque" or "amyloid plaque" generally refers to fibrillar aggregates of Aβ peptides (e.g., Aβ42 and / or Aβ40) in which many copies of the Aβ peptide stick together to form fibrils or fibrous deposits (e.g., plaques).

[0144] As used herein, the term "massive accumulation of Aβ peptides" generally refers to the formation of Aβ oligomers or amyloid plaques, which can be detected using commonly used detection methods or tools such as specific Aβ antibody staining.

[0145] As used herein, the term "Aβ" or "amyloid-β" generally refers to amyloid-β peptides produced by regulated intramembrane proteolysis of amyloid precursor protein (APP). Continuous proteolytic cleavage events of β-secretase and γ-secretase produce Aβ peptides of different lengths, including Aβ40 and Aβ42. Aβ42 has two additional hydrophobic residues, which makes it easier to aggregate into soluble oligomers and insoluble deposits, which exceeds Aβ40 or a series of shorter peptides observed by cerebrospinal fluid (CSF) mass spectrometry in recent years. In dense neuritic plaques, there are a variety of Aβ aggregation forms, from dimers to β-folded sheet fibrils. Excessive Aβ can induce a variety of pathological processes. Aβ can impair neuronal and glial function, synaptic physiological function / process, neurotransmission and cognition. Evidence shows that transcellular diffusion and templated seeding and the resulting deposition of aggregated Aβ in extraneuronal amyloid plaques are pathological features of AD.

[0146] As used herein, the term "hyperphosphorylated" or "hyperphosphorylation" refers to a state of abnormal phosphorylation at one or more additional sites. For example, it was found that phosphorylation of tau protein negatively regulates its activity in promoting microtubule assembly, and abnormally hyperphosphorylated tau is considered to be the main component of PHF in AD. Normal brain contains 2-3 moles of phosphate per mole of tau. Studies on human brain biopsy tissues have shown that several serine and threonine residues of tau are usually phosphorylated at low substoichiometric levels. The phosphorylation level of tau isolated from dissected AD brains is as high as 3 to 4 times that of tau in normal human brains. Tau phosphorylation at different sites has different effects on its biological functions and its pathogenic effects. Studies on the binding between hyperphosphorylated tau and normal tau have shown that Ser199 / Ser202 / Thr205, Thr212, Thr231 / Ser235, Ser262 / Ser356, and Ser422 are key phosphorylation sites that convert tau into inhibitory molecules that sequester normal microtubule-associated proteins from microtubules.

[0147] As used herein, the term "neuronal loss" generally refers to a decrease in the amount or function of neuronal cells in an organism. Neuronal loss can be manifested as the death of neuronal cells.

[0148] As used herein, the term "Tau protein" generally refers to microtubule-associated tau protein (MAPT) that stabilizes microtubules. Tau protein is abundant in neurons of the central nervous system, less common elsewhere, but also expressed at very low levels in CNS astrocytes and oligodendrocytes. Tau protein can control microtubule stability in two ways: subtype and phosphorylation. For example, the NCBI accession number of Homo sapiens tau subtype 1 is NP_058519.3. AD may be associated with tau protein that has become defective and no longer properly stabilizes microtubules.

[0149] As used herein, the term "AD" generally refers to Alzheimer's disease, a chronic neurodegenerative disease that usually starts slowly and gradually worsens over time. For example, eight intellectual domains are most commonly impaired in AD: memory, language, perceptual skills, attention, motor skills, orientation, problem solving, and executive function abilities. These domains are equivalent to the NINCDS-ADRDA Alzheimer's disease criteria listed in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR) published by the American Psychiatric Association.

[0150] As used herein, the term "apoptosis" generally refers to a gene-driven cell self-destruction process characterized by the fragmentation of nuclear DNA, which can be activated by the presence of a stimulus or the removal of an inhibitor or stimulus. Apoptosis is also known as cell suicide, programmed cell death. Bcl-2 family proteins are the main intracellular regulators of apoptosis. Intracellular proteins of the Bcl-2 family help regulate the activation of procaspases. Some members of the Bcl-2 family promote procaspase activation and cell death. For example, the apoptosis promoter Bad plays a role by binding to and inactivating death-suppressing members of the family, while other promoters such as Bax and Bak stimulate cytochrome c to be released from mitochondria. Bax and Bak themselves are activated by other apoptosis-promoting members of the Bcl-2 family such as Bid. Caspase-3 is a caspase protein that interacts with caspase-8 and caspase-9. It is encoded by the CASP3 gene. The sequential activation of caspases plays a key role in the execution phase of apoptosis. Caspases exist as inactive zymogens that undergo proteolytic processing at a conserved aspartic acid residue to produce two subunits, a large subunit and a small subunit, which dimerize to form an active enzyme. Caspase-3 is the major caspase involved in the cleavage of amyloid-β4A precursor protein (also known as APP), which is associated with neuronal death in Alzheimer's disease. Increased levels of procaspase 3 ("Pro-caspase 3") and its cleaved form ("Cl-caspase 3") are often associated with increased apoptosis.

[0151] As used herein, the term "necrosis" generally refers to a form of cell damage that causes premature death of cells in living tissues by autolysis. The signaling pathways responsible for necrosis or necroptosis are generally known. The production of TNFα during viral infection leads to stimulation of its receptor TNFR1. TNFR-related death protein TRADD signals RIPK1, which recruits RIPK3 to form a necrosome. The phosphorylation of MLKL by the ripoptosome ("pMLKL") drives the oligomerization of MLKL, so that MLKL is inserted into the plasma membrane and organelles and permeabilized. The integration of MLKL leads to the release of inflammatory phenotypes and damage-associated molecular patterns (DAMPs), thereby initiating an immune response. Specifically, necroptosis is a form of programmed necrosis, which is performed by mixed lineage kinase domain-like (MLKL) proteins and triggered by receptor interacting protein kinases (RIPK) 1 and 3. It has been found that in postmortem human AD brain, necroptosis is activated, positively correlated with Braak stage, and negatively correlated with brain weight and cognitive scores. Furthermore, a set of genes regulated by RIPK1 has been found to significantly overlap with multiple independent AD transcriptomic signatures, suggesting that RIPK1 activity may explain a large fraction of transcriptomic changes in AD.

[0152] As used herein, the term "gliosis" generally refers to the fibrous proliferation of glial cells in the central nervous system (CNS) injury area. Gliosis is ubiquitous in gliomas and many other neurological disorders such as Alzheimer's disease, and can be detected by the glial fibrillary acid protein (GFAP) levels raised in postmortem tissue samples using immunohistochemistry. Typically, gliosis is a combination of astrocytic hyperplasia and microglial hyperplasia.

[0153] As used herein, the term "synaptic degeneration" generally refers to the loss or dysfunction of synapses, which can be reflected by the state / expression of synaptic markers and / or postsynaptic density. The postsynaptic density (PSD) is a dense specialization of proteins attached to the postsynaptic membrane. The PSD was initially identified as an electron-dense area on the postsynaptic neuron membrane by electron microscopy. The PSD is tightly attached to the presynaptic active zone and ensures that the receptor is closely adjacent to the presynaptic neurotransmitter release site. For example, a depression or swelling of the PSD may indicate synaptic degeneration. The synaptic state can be detected by examining the expression or level of synaptic vesicle proteins. It is reported that synaptic vesicle proteins are integral membrane glycoproteins found in many types of active neurons and are found in the membrane after stimulating neurons.

[0154] As used herein, the term "donor nucleic acid molecule" generally refers to a nucleic acid molecule that provides a heterologous nucleic acid sequence to a recipient (eg, a recipient nucleic acid molecule).

[0155] As used herein, the term "hybridizes to" or "hybridization" when used in the context of molecular biology generally refers to the process of annealing of a single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecule to a complementary DNA or RNA.

[0156] As used herein, the term "induced pluripotent stem cells" or "iPS cells" or "iPSCs" generally refers to cells taken from any tissue of a child or adult, usually the skin or blood, and genetically modified to behave like embryonic stem cells. As the name implies, these cells are pluripotent, meaning they have the ability to form most, if not all, of the cell types of the adult body.

[0157] As used herein, the term "tracrRNA" generally refers to trans-activating crRNA (tracrRNA), which is a small trans-encoded RNA. TracrRNA is complementary to pre-crRNA and base pairs with pre-crRNA to form an RNA duplex. The RNA duplex is cut by RNA-specific ribonuclease RNase III to form a crRNA / tracrRNA hybrid. The hybrid is used as a guide for the nuclease Cas9, which cuts the invading nucleic acid.

[0158] As used herein, the term "APOE" generally refers to apolipoprotein E, whose main function is to transport lipids in peripheral tissues and the brain. APOE has three common subtypes: APOE2, APOE3 and APOE4. It is reported that APOEε4 is the strongest genetic risk factor for AD, and APOEε2 is associated with a reduced risk of AD relative to the common APOEε3 allele. In the CNS, APOE is expressed in a large number of astrocytes. Once APOE has been secreted from the cell, the cell surface ATP-binding cassette transporters ABCA1 and ABCG1 transfer cholesterol and phospholipids to the nascent APOE to form lipoprotein particles. The APOE4 allele causes the cysteine ​​at position 112 to become arginine, and is associated with a 5-fold increase in the risk of AD in monoallele carriers, and a 20-fold increase in homozygous carriers. APOE2 causes the arginine at position 158 to change to cysteine ​​and has a protective effect against AD, while the APOE3 allele is considered neutral (Corder et al., Nat Genet (7) 180-184, 1994; Hauser et al., Cure Alzheimer Res (10) 808-817, 2013). APOE affects the production, aggregation and clearance of amyloid proteins, which are components of amyloid plaques and exacerbate tau-mediated neurodegeneration. APOE alleles also regulate lipid metabolism and cardiovascular risk. About 5%-10% of APOE2 homozygous individuals develop hyperlipoproteinemia type III (HLPIII), while other rare APOE variants are associated with autosomal dominant HLP III inheritance. HLP III is characterized by elevated plasma cholesterol and triglyceride levels and the appearance of nodular or rhabdomyosarcomas of the palmar xanthomas. APOE3ch (referring to the APOE3 Christchurch (R136S) mutation) is associated with profound resistance to clinical onset of Alzheimer's disease, and the R136S mutation significantly reduces the ability of ApoE to bind heparan sulfate proteoglycan (HSPG) / heparin.

[0159] As used herein, the term "subject" includes living organisms that may develop a disease or condition. The term "subject" includes animals (e.g., mammals (e.g., cats, dogs, horses, pigs, cows, goats, sheep, rodents (e.g., mice or rats), rabbits, squirrels, bears, primates (e.g., chimpanzees, monkeys, gorillas, and humans)) and birds (e.g., chickens, ducks, Peking ducks, geese) and transgenic species thereof. In some cases, the subject is a human or non-human primate (e.g., chimpanzee, monkey, macaque, gorilla). In some cases, the subject is a human.

[0160] As used herein, the term "organoid" generally refers to a miniature form of a tissue generated in vitro and exhibiting endogenous three-dimensional organ architecture.

[0161] In this application, when referring to an amino acid substitution, "XnY" means that the amino acid X at residue n is substituted with the amino acid Y.

[0162] Unless otherwise specified, "a," "an," "the," and "at least one" are used interchangeably and mean one or more than one.

[0163] In the present application, the term "comprising" also encompasses "being", "having" and "consisting of". For example, "a composition comprising X and Y" may be understood to encompass a composition comprising at least X and Y. It should also be understood to disclose a composition comprising only X and Y (i.e., a composition consisting of X and Y).

[0164] As used herein, the term "BDNF" generally refers to brain-derived neurotrophic factor. Human BDNF is also referred to as P23560 in the UniProtKB / Swiss-Prot system nomenclature. The protein encoded by the BDNF gene belongs to a member of the nerve growth factor family. Variable splicing produces multiple transcript variants, at least one of which encodes a preprotein proprotein that is processed by proteolysis to produce a mature protein. The binding of this protein to its cognate receptor promotes the survival of neurons in the adult brain. The expression of this gene is reduced in patients with Alzheimer's disease, Parkinson's disease and Huntington's disease. This gene can play a role in the regulation of stress responses and the biology of mood disorders.

[0165] Engineered human cells and methods of producing such cells

[0166] In one aspect, the present application provides an engineered human cell. The endogenous APP gene of the human cell is mutated to encode a mutant APP protein, and compared with the wild-type human APP protein shown in SEQ ID NO: 1, the mutant APP protein comprises amino acid substitutions at the following residues: K670, M671, I716 and / or E693.

[0167] In another aspect, the present application provides a method for producing engineered human cells. The method comprises: introducing a mutation into an endogenous APP gene of a human cell to produce a mutant APP gene, wherein the mutant APP gene encodes a mutant APP protein. Compared to the wild-type human APP protein shown in SEQ ID NO: 1, the mutant APP protein may include amino acid substitutions at the following residues: K670, M671, I716 and / or E693.

[0168] In some embodiments of the method for producing engineered human cells, introducing the mutation into the APP gene comprises knocking a heterologous nucleic acid sequence into an endogenous APP locus, wherein the knock-in replaces at least a portion of the endogenous APP gene with a heterologous nucleic acid sequence, and the heterologous nucleic acid sequence encodes at least a portion of the mutant APP protein.

[0169] In some embodiments, the mutant APP protein comprises an amino acid substitution at K670. In some embodiments, the mutant APP protein comprises an amino acid substitution at M671. In some embodiments, the mutant APP protein comprises an amino acid substitution at I716. In some embodiments, the mutant APP protein comprises an amino acid substitution at E693. In some embodiments, the mutant APP protein comprises an amino acid substitution at K670 and M671. In some embodiments, the mutant APP protein comprises an amino acid substitution at K670, M671 and I716. In some embodiments, the mutant APP protein comprises an amino acid substitution at K670, M671 and E693. In some embodiments, the mutant APP protein comprises an amino acid substitution at I716 and E693. In some embodiments, the mutant APP protein comprises an amino acid substitution at K670, M671, I716 and E693.

[0170] In some embodiments, compared to the wild-type human APP protein shown in SEQ ID NO: 1, the mutant APP protein is mutated at K670, M671, I716 and / or E693. In some embodiments, the mutant APP protein is mutated at K670. In some embodiments, the mutant APP protein is mutated at M671. In some embodiments, the mutant APP protein is mutated at I716. In some embodiments, the mutant APP protein is mutated at E693. In some embodiments, the mutant APP protein is mutated at K670 and M671. In some embodiments, the mutant APP protein is mutated at K670, M671 and I716. In some embodiments, the mutant APP protein is mutated at K670, M671 and E693. In some embodiments, the mutant APP protein is mutated at I716 and E693. In some embodiments, the mutant APP protein is mutated at K670, M671, I716, and E693.

[0171] In some embodiments, the mutant APP protein comprises a Swedish double mutation. For example, the Swedish double mutation may comprise a K670N substitution and a M671L substitution. In some embodiments, the mutant APP protein comprises a Beyreuther / Iberian mutation. For example, the Beyreuther / Iberian mutation may comprise an I716F substitution. In some embodiments, the mutant APP protein comprises an Arctic mutation. For example, the Arctic mutation may comprise an E693G substitution.

[0172] In some embodiments, for the APP gene mutation, the engineered human cell is homozygous. For example, for a mutation at K670, the engineered human cell may be homozygous. For example, for a mutation at M671, the engineered human cell may be homozygous. For example, for a mutation at I716, the engineered human cell may be homozygous. For example, for a mutation at E693, the engineered human cell may be homozygous. For example, for a mutation at K670 and M671, the engineered human cell may be homozygous. For example, for a mutation at K670, M671, and I716, the engineered human cell may be homozygous. For example, for a mutation at K670, M671, and E693, the engineered human cell may be homozygous. For example, for a mutation at I716 and E693, the engineered human cell may be homozygous. For example, the engineered human cell can be homozygous for mutations at K670, M671, I716, and E693.

[0173] For example, for the Swedish double mutation K670N and M671L, the engineered human cell may be homozygous. For example, for the Beyreuther / Iberian mutation I716F, the engineered human cell may be homozygous. For example, for the Arctic mutation E693G, the engineered human cell may be homozygous. For example, for the mutations K670N, M671L and I716F, the engineered human cell may be homozygous. For example, for the mutations K670N, M671L and E693G, the engineered human cell may be homozygous. For example, for the mutations I716F and E693G, the engineered human cell may be homozygous. For example, for the mutations K670N, M671L, I716F and E693G, the engineered human cell may be homozygous.

[0174] The mutant APP protein may also include one or more additional mutations that can affect the amount of Aβ produced, the amount of Aβ fragments produced and / or the ratio of Aβ42 / Aβ40 produced. For example, the mutant APP protein may also include one or more additional or alternative mutations selected from the following: I716V and V717I.

[0175] The human cells to be engineered can be taken from or can be derived from a subject. The subject can be a patient suffering from a disease or condition. In some cases, the subject has previously suffered from a disease or condition. In some cases, the subject is at risk of developing a disease or condition. The disease or condition can be a neurological disease or condition. In some cases, the disease or condition is not a neurological disease or condition, but a metabolic disease or condition (e.g., diabetes).

[0176] The human cell can be a human stem cell. For example, the human cell can be or can include a pluripotent stem cell, an embryonic stem cell, an induced pluripotent stem cell and / or a neuronal stem cell. The embryonic stem cell can be from a cell line (such as human embryonic stem cell (hESC) line H1) established. For example, the embryonic stem cell is not obtained by a process that destroys a human embryo.

[0177] In the engineered human cells of the present application, the expression level of CTF-α may not be significantly different from the expression level of CTF-α of the corresponding wild-type human cells. In some cases, the expression level of CTF-β in the engineered human cells of the present application may be increased (e.g., may be increased by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, 2 times, 3 times, 4 times, 5 times or more) compared to the expression level of CTF-β of the corresponding wild-type human cells. In some cases, the Aβ42 expression level in the engineered human cells of the present application may be increased (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, 2 times, 3 times, 4 times, 5 times or more) compared to the Aβ42 expression level of corresponding wild-type human cells. In some cases, the ratio of Aβ42 / Aβ40 in the engineered human cells of the present application may be increased (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, 2 times, 3 times, 4 times, 5 times or more) compared to the ratio of Aβ42 / Aβ40 in the corresponding wild-type human cells. In some embodiments, in the engineered human cells of the present application, hyperphosphorylation of Tau is detectable. In some embodiments, the hyperphosphorylation of Tau includes an increase in the phosphorylation level of Thr205 and / or Ser202 in Tau compared to the hyperphosphorylation of Tau in the corresponding wild-type human cells.

[0178] The engineered human cells of the present application may be knock-in cells or may be derived from knock-in cells.

[0179] In some embodiments, at least a portion of the endogenous APP gene can be replaced by a heterologous nucleic acid sequence encoding at least a portion of the mutant APP protein of the present application. In some cases, at least a portion of the endogenous APP gene includes at least a portion of exon 16 of the endogenous APP gene. In some cases, at least a portion of the endogenous APP gene includes at least a portion of exon 17 of the endogenous APP gene. In some cases, the at least a portion of the endogenous APP gene includes at least a portion of exon 16 and at least a portion of exon 17 of the endogenous APP gene. In some cases, the heterologous nucleic acid sequence encodes mutant exon 16 of APP. In some cases, the heterologous nucleic acid sequence includes a sequence of mutant exon 16 of APP. In some cases, the heterologous nucleic acid sequence encodes mutant exon 17 of APP. In some cases, the heterologous nucleic acid sequence includes a sequence of mutant exon 17 of APP. In some cases, the heterologous nucleic acid sequence encodes mutant exon 16 and mutant exon 17 of APP. In some cases, the heterologous nucleic acid sequence comprises a sequence of mutant exon 16 and a sequence of mutant exon 17 of APP. The sequence contained in the heterologous nucleic acid may comprise a partial sequence of mutant exon 16 (e.g., a portion comprising a mutation) and / or may comprise a partial sequence of mutant exon 17 (e.g., a portion comprising a mutation). In some embodiments, the mutation in the mutant exon 16 and / or the mutant exon 17 of APP results in one or more amino acid substitutions in the polypeptide encoded thereby, and wherein the one or more amino acid substitutions comprise amino acid substitutions at the following residues: K670, M671, I716, and / or E693. In some embodiments, the one or more amino acid substitutions comprise substitutions K670N, M671L, I716F, and / or E693G.

[0180] For example, the heterologous nucleic acid may comprise the sequence shown in any one of SEQ ID NOs: 2-3.

[0181] In some cases, the mutant APP protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-10.

[0182] In some cases, in the engineered human cells of the present application, the APOE gene is mutated to encode a mutant APOE3 protein. Compared with the wild-type human APOE3 protein shown in SEQ ID NO: 11, the residue R136 in the mutant APOE3 protein is replaced. In some cases, in the mutant APOE3 protein, the residue R136 is replaced by S. In some cases, the mutant APOE3 protein comprises the amino acid sequence shown in SEQ ID NO: 12. In some cases, the mutant APOE gene encodes APOE3ch.

[0183] In some cases, the engineered human cell is homozygous for the mutant APOE gene. In some cases, the engineered human cell is homozygous for the APOE3ch allele.

[0184] In some cases, the engineered human cells of the present application have altered Tau, APOE2, APOE4 and / or BDNF expression. In some embodiments, the engineered human cells increase Tau, APOE2, APOE4 and / or BDNF expression. In some embodiments, the engineered human cells reduce Tau, APOE2, APOE4 and / or BDNF expression. In some embodiments, the engineered human cells express altered or mutant forms of Tau, APOE2, APOE4 and / or BDNF.

[0185] In some cases, the engineered human cells may contain mutations in one or more additional genes selected from the group consisting of Tau, APOE2, APOE4, and BDNF. For example, such mutations increase or decrease Tau, APOE2, APOE4, and / or BDNF expression. In some cases, such mutations may express mutant forms of Tau, APOE2, APOE4, and / or BDNF.

[0186] In some embodiments of the method, the knock-in includes contacting the genome of the human cell with a gene editing component in the presence of a donor nucleic acid molecule comprising the heterologous nucleic acid sequence. The gene editing component may include: 1) CRISPR-associated (Cas) protein; and 2) one or more ribonucleic acid (RNA) sequences, the sequence comprising i) a sequence complementary to a target portion of an endogenous APP gene, and ii) a binding site for the Cas protein. In some embodiments, the binding site of the Cas protein comprises a tracrRNA sequence.

[0187] In some embodiments, the one or more ribonucleic acid (RNA) sequences comprise the sequence shown in any one of SEQ ID NOs: 13-16.

[0188] In some embodiments, the knock-in further comprises maintaining the cell under conditions wherein the one or more RNA sequences hybridize to the target portion of the endogenous APP gene and the Cas protein cleaves the endogenous APP gene nucleic acid sequence upon the hybridization of the one or more RNA sequences.

[0189] The Cas protein may be a type I Cas protein. In some embodiments, the Cas protein may be a type II Cas protein. In some embodiments, the type II Cas protein may be Cas9. In some embodiments, the Cas protein is Cas9.

[0190] In some embodiments, the Cas protein is introduced into the cell in the form of a protein, a messenger RNA (mRNA) encoding the Cas protein, or a DNA encoding the Cas protein.

[0191] In some embodiments, the one or more RNA sequences are introduced into the cell in the form of one or more RNA molecules or one or more DNA molecules encoding the RNA sequences.

[0192] In some embodiments, the method may further include introducing a mutation into the APOE gene to produce a mutant APOE3 protein of the present application. For example, introducing the mutation into the APOE gene may include knocking a heterologous nucleic acid sequence into an endogenous APOE locus, wherein the knock-in replaces at least a portion of the endogenous APOE gene with a heterologous nucleic acid sequence encoding at least a portion of the mutant APOE3 protein. In some embodiments of the method, the knock-in includes contacting the genome of the human cell with a gene editing component in the presence of a donor nucleic acid molecule comprising the heterologous nucleic acid sequence. The gene editing component may include: 1) CRISPR-related (Cas) protein; and 2) one or more ribonucleic acid (RNA) sequences, the sequence comprising i) a sequence complementary to the target portion of the endogenous APOE gene, and ii) a binding site for the Cas protein. In some embodiments, the binding site of the Cas protein comprises a tracrRNA sequence. In some embodiments, the one or more ribonucleic acid (RNA) sequences comprise a sequence shown in any one of SEQ ID NO: 17-18.

[0193] In another aspect, the present application provides engineered human cells produced by the method of the present application.

[0194] In the method for producing the engineered cell of the present application, a nuclease agent can be used to help modify the target APP locus and / or the APOE locus. Such a nuclease agent can promote homologous recombination between a donor nucleic acid molecule and a target genomic locus. In some embodiments, the nuclease agent includes an endonuclease agent.

[0195] As used herein, the term "recognition site of a nuclease agent" generally refers to a DNA sequence at which a nuclease agent can induce a nick or double-strand break. The recognition site of a nuclease agent can be endogenous (or natural) to the cell, or the recognition site can be exogenous to the cell. In some embodiments, the recognition site can be exogenous to the cell, and therefore is not naturally present in the genome of the cell. In a further embodiment, the exogenous or endogenous recognition site can exist only once in the genome of the host cell. In a specific embodiment, an endogenous or natural site that only appears once in the genome can be identified. Then, such a site can be used to design a nuclease agent that will produce a nick or double-strand break at the endogenous recognition site.

[0196] The length of the recognition site can vary, and includes, for example, a recognition site of at least 4, 6, 8, 10, 12, 14, 16, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides in length. In one embodiment, each monomer of the nuclease agent can recognize a recognition site of at least 9 nucleotides. In other embodiments, the length of the recognition site can be about 9 to about 12 nucleotides, about 12 to about 15 nucleotides, about 15 to about 18 nucleotides, or about 18 to about 21 nucleotides, and any combination of these sub-ranges (e.g., 9-18 nucleotides). The recognition site can be palindromic, that is, the sequence on one strand is the same when read in the opposite direction on the complementary strand. It is recognized that a given nuclease agent can bind to the recognition site and cut the binding site, or alternatively, the nuclease agent can bind to a sequence different from the recognition site. In addition, the term "recognition site" can include both the nuclease agent binding site and the nick / cut site, regardless of whether the nick / cut site is inside or outside the nuclease agent binding site. In another variation, the cutting of the nuclease agent can occur at nucleotide positions directly opposite each other to produce a blunt end cut, or in other cases, the nicks can be staggered to produce single-stranded overhangs, also known as "sticky ends", which can be 5' overhangs or 3' overhangs.

[0197] Any nuclease reagent that induces nicks or double-strand breaks in the desired recognition site can be used in the method of the present application. Naturally occurring or natural nuclease reagents can be used, as long as the nuclease reagent induces nicks or double-strand breaks in the desired recognition site. Alternatively, modified or engineered nuclease reagents can be used. "Engineering nuclease reagents" include nucleases that are engineered (modified or derived) from their native forms to specifically recognize and induce nicks or double-strand breaks in the desired recognition site. Therefore, engineered nuclease reagents can be derived from natural, naturally occurring nuclease reagents, or they can be artificially produced or synthesized. The modification of the nuclease reagent can be as little as one amino acid in a protein cutting agent or one nucleotide in a nucleic acid cutting agent. In some embodiments, the engineered nuclease can include nicks or double-strand breaks in the recognition site, wherein the recognition site is not a sequence recognized by a natural (non-engineered or non-modified) nuclease reagent. Producing nicks or double-strand breaks in a recognition site or other DNA can be referred to as "cutting" or "cutting" the recognition site or other DNA in this article.

[0198] In some embodiments, the nuclease agent can be a transcription activator-like effector nuclease (TALEN). TAL effector nucleases are a class of sequence-specific nucleases that can be used to produce double-strand breaks at specific target sequences in the genome of prokaryotic or eukaryotic organisms. TAL effector nucleases can be produced by fusing natural or engineered transcription activator-like (TAL) effectors or their functional parts to the catalytic domain of a nuclease (e.g., FokI). The unique modular TAL effector DNA binding domain allows the design of proteins with potential recognition specificity for any given DNA. Therefore, the DNA binding domain of the TAL effector nuclease can be engineered to recognize specific DNA target sites and, therefore, used to produce double-strand breaks at the desired target sequence. See WO 2010 / 079430; Morbitzer et al., (2010) PNAS 10.1073 / pnas.1013133107; Scholze & Boch (2010) Virulence 1:428-432; Christian et al., Genetics (2010) 186:757-761; Li et al., (2010) Nuc. Acids Res., (2010) doi:10.1093 / nar / gkq704; and Miller et al., (2011) Nature Biotechnology 29:143-148; all of which are incorporated herein by reference.

[0199] In some embodiments, the nuclease agent may be a zinc finger nuclease (ZFN). For example, each monomer of the ZFN may include 3 or more zinc finger-based DNA binding domains, wherein each zinc finger-based DNA binding domain may bind to a 3bp subsite. In other embodiments, the ZFN may be a chimeric protein comprising a zinc finger-based DNA binding domain operably connected to an independent nuclease. In some embodiments, the independent endonuclease may be a FokI endonuclease. In some embodiments, the nuclease agent may include a first ZFN and a second ZFN, wherein each of the first ZFN and the second ZFN is operably connected to a FokI nuclease, wherein the first ZFN and the second ZFN recognize two continuous target DNA sequences in each chain of the target DNA sequence, the two continuous target DNA sequences being separated by a cleavage site of about 6bp to about 40bp or a cleavage site of about 5bp to about 6bp, and wherein the FokI nuclease dimerizes and produces a double-strand break.

[0200] In some embodiments, the nuclease agent can be a meganuclease. Meganucleases have been divided into four families based on conserved sequence motifs. These motifs are involved in the coordination of metal ions and the hydrolysis of phosphodiester bonds. HEase is known for its long recognition site and tolerance of some sequence polymorphisms in its DNA substrate. Meganuclease domains, structures and functions are known, see, for example, Guhan and Muniyappa (2003) Crit. Rev Biochem Mol Biol 38: 199-248; Lucas et al., (2001) Nucleic Acids Res 29: 960-9; Jurica and Stoddard, (1999) Cell Mol Life Sci 55: 1304-26; Stoddard, (2006) Q Rev Biophys 38: 49-95; and Moure et al., (2002) Nat Struct Biol 9: 764.

[0201] In some embodiments, the nuclease reagent used in the method of the present application may adopt the CRISPR / Cas system. Such a system may adopt, for example, a Cas9 nuclease, and in some cases, the Cas9 nuclease may be codon optimized for the cell type to be expressed. The system may also adopt a fusion crRNA-tracrRNA construct that acts together with the codon-optimized Cas9. This single RNA may generally be referred to as a small guide RNA or sgRNA. In the sgRNA, the crRNA portion may be identified as a "nucleotide sequence that hybridizes with a target sequence of an endogenous APP gene" (or "targeting sequence"), and tracrRNA may generally be referred to as a "scaffold". In short, a short DNA fragment containing the targeting sequence may be inserted into an sgRNA expression plasmid. The sgRNA expression plasmid may include a targeting sequence (about 20 nucleotides in some embodiments), a form of a tracrRNA sequence (scaffold), and a suitable promoter active in a cell and necessary elements for proper processing in a eukaryotic cell (such as a cell or a cell model). The sgRNA expression cassette and the Cas9 expression cassette may then be introduced into the cell. See, e.g., Mali P et al., (2013) Science 2013 Feb 15;339(6121):823-6; Jinek M et al., Science 2012 Aug 17;337(6096):816-21; Hwang WY et al., Nat Biotechnol 2013 Mar;31(3):227-9; Jiang W et al., Nat Biotechnol 2013 Mar;31(3):233-9; and Cong L et al., Science 2013 Feb 15;339(6121):819-23, each of which is incorporated herein by reference.

[0202] In some embodiments, the method can further include discovering a genetically modified cell comprising the knocked-in heterologous nucleic acid sequence.

[0203] The donor nucleic acid molecule comprising the heterologous nucleic acid sequence may also include a 5' homology arm and a 3' homology arm. The 5' homology arm and the 3' homology arm may flank the heterologous nucleic acid sequence encoding at least a portion of the mutant APP. The homology arm (e.g., 5' homology arm or 3' homology arm) in the donor nucleic acid molecule may have any length sufficient to promote homologous recombination events with the corresponding region in the endogenous APP locus and / or the APOE locus, e.g., at least 5bp, at least 50bp, at least 100bp, at least 150bp, at least 200bp, at least 300bp, at least 400bp, at least 500bp, at least 600bp, at least 700bp, at least 750bp, at least 800bp, at least 850bp, at least 900bp, at least 1kb, at least 1.5kb, at least 5kb in length or longer. In some embodiments, the donor nucleic acid molecule comprises a 5' homology arm that may be about 800 bp to 900 bp in length and a 3' homology arm that may be about 800 bp to 950 bp in length.

[0204] As used herein, when homology arms and target sites (i.e., homologous genomic regions, or corresponding regions within endogenous APP loci and / or APOE loci) have sufficient levels of sequence identity to each other to serve as substrates for homologous recombination reactions, the two regions match or correspond to each other. So-called "homology" refers to that the DNA sequence is identical to or has certain sequence identity with the corresponding sequence or matching sequence. The sequence identity between the corresponding homology arms found in a given target site and the donor nucleic acid molecule can be any degree of sequence identity that allows homologous recombination to occur. For example, the amount of sequence identity that the homology arms of the donor nucleic acid molecule (or a fragment thereof) have with the target site (or a fragment thereof) can be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%, such that the sequences undergo homologous recombination.

[0205] For example, in the donor nucleic acid molecule, the heterologous nucleic acid sequence encoding at least a portion of the mutant APP or APOE may be flanked by: 1) a first homology arm (or 5' homology arm) corresponding to a first region of the endogenous APP locus upstream of exon 16; and 2) a second homology arm (or 3' homology arm) corresponding to a second region of the endogenous APP locus downstream of exon 17. Thus, the donor nucleic acid molecule facilitates integration of the heterologous nucleic acid sequence into the endogenous APP locus of the genome of the cell or cell model through a homologous recombination event that occurs between the homology arms and their corresponding regions within the endogenous APP locus.

[0206] When a nuclease reagent (e.g., Cas protein) is used, the homologous genomic regions corresponding to the 5' and 3' homologous arms of the donor nucleic acid molecule are located close enough to the nuclease target site to promote homologous recombination events between the homologous genomic regions and the homologous arms when a nick or double-strand break at the recognition site occurs. For example, the nuclease target site can be located anywhere between the homologous genomic regions corresponding to the 5' and 3' homologous arms. In some embodiments, the recognition site is adjacent to at least one or two of the corresponding homologous genomic regions.

[0207] The donor nucleic acid molecule may also include a selection box or a reporter gene. The selection box may include a nucleic acid sequence encoding a selection marker, wherein the nucleic acid sequence is operably connected to a promoter. The promoter may be active in desired prokaryotic cells and / or active in desired eukaryotic cells. Such a promoter may be an inducible promoter, an endogenous promoter of a reporter gene or a cell, a heterologous promoter of a reporter gene or a cell, a cell-specific promoter, a tissue-specific promoter, or a developmental stage-specific promoter. In some embodiments, the selection marker may be selected from the following: ampicillin resistance gene (Ampr), neomycin phosphotransferase (neor), hygromycin B phosphotransferase (hygr), puromycin-N-acetyltransferase (puror), blasticidin S deaminase (bsrr), xanthine / guanine phosphoribosyltransferase (gpt) and herpes simplex virus thymidine kinase (HSV-k) and combinations thereof. The selection marker of the donor nucleic acid molecule may be flanked by 5' and 3' homology arms, or may be present at the 5' or 3' end of the homology arms.

[0208] In some embodiments, the Cas protein and one or more RNA sequences can be introduced into human-derived cells as a protein-RNA complex (e.g., RNP).

[0209] In some embodiments, the DNA encoding the Cas protein can be in the form of a first expression construct comprising a first promoter operably connected to a nucleic acid encoding the Cas protein; the DNA encoding the RNA molecule can be in the form of a second expression construct comprising a second promoter operably connected to a nucleic acid sequence encoding the RNA molecule. The first promoter and the second promoter can be active in human cells. In some cases, the first expression construct and the second expression construct are in a single nucleic acid molecule.

[0210] In some cases, the human-derived cell may be an embryonic stem (ES) cell. For example, the cell may be derived from a DA strain or an ACI strain. In some embodiments, the cell may be characterized by the expression of at least one pluripotency marker selected from the following: Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1, and combinations thereof. In some embodiments, the cell may be characterized by one or more of the following features: (a) lack of expression of one or more pluripotency markers selected from the following: c-Myc, Ecat1, and Rexo1; (b) lack of expression of one or more mesoderm markers selected from Brachyury and Bmpr2; (c) lack of expression of one or more endoderm markers selected from Gata6, Sox17, and Sox7; and (d) lack of expression of one or more neural markers selected from Nestin and Pax6.

[0211] In some embodiments, knock-in may include injecting a donor nucleic acid molecule comprising a heterologous nucleic acid sequence of the present application into a human-derived cell.

[0212] Cell models and methods for generating such models

[0213] In one aspect, the present application provides a cell model of a dementia-related neurological disease. The cell model comprises an in vitro culture of multiple cells derived from the engineered human cells of the present application. The dementia-related neurological disease may be Alzheimer's disease.

[0214] The cell model can be produced by culturing the engineered human cells of the present application in two dimensions. In some embodiments, the cell model can be produced by culturing the engineered human cells of the present application in three dimensions.

[0215] For example, the cell model can be produced by contacting the engineered human cells of the present application with a differentiation medium. The engineered human cells can be cultured in a gel, in a bioreactor, under ultra-low adhesion conditions, and / or on a microchip.

[0216] In some embodiments, the engineered human cells are cultured in a matrix. The matrix may be an extracellular matrix. In some cases, the matrix may include one or more of natural molecules, synthetic polymers, bio-synthetic hybrids, metals, ceramics, bioactive glasses, and / or carbon nanotubes.

[0217] For example, the engineered human cells may be engineered human embryonic stem cells, and the method may include culturing the engineered human embryonic stem cells in Matrigel with differentiation medium.

[0218] Technicians can determine the time sufficient to induce differentiation by examining the morphological changes associated with differentiation. In one embodiment, the time sufficient to cultivate the stem cells and induce differentiation is about 5 days to about 180 days. In another embodiment, the time sufficient to induce differentiation is about 7 days to about 15 days. Technicians can determine the time sufficient to induce organoid formation by examining the morphological changes associated with organoid formation. In one embodiment, the time sufficient to induce organoid formation is about 5 days to about 28 days. In another embodiment, the time sufficient to induce organoid formation is about 14 days.

[0219] In some embodiments, the differentiation medium includes mTeSR TM 1 culture medium. In some embodiments, the culture medium may include a neural stem cell induction medium, such as Advanced DMEM / F12, Neurobasal, N2, B27, Glutmax (e.g., about 0.5% to about 5%, such as about 1%), BSA (e.g., about 0.5 μg / mL to about 10 μg / mL, such as about 5 μg / mL), hLIF (e.g., about 0.1 ng / mL to about 50 ng / mL, such as about 10 ng / mL), CHIR99021 (e.g., about 0.3 μM to about 10 μM, such as 3 μM), SB431542 (e.g., about 0.2 μM to about 20 μM, such as about 2 μM), compound E (e.g., about 0.01 μM to about 10 μM, such as about 0.1 μM).

[0220] In some embodiments, the differentiation medium includes DMEM / F12, N2, B27, cAMP (e.g., about 50 ng / mL to about 600 ng / mL, such as 300 ng / mL), vitamin C (e.g., about 0.02 mM to about 20 mM, such as about 0.2 mM), BDNF (e.g., about 1 ng / mL to about 100 ng / mL, such as 10 ng / mL), and GDNF (e.g., about 1 ng / mL to about 50 ng / mL, such as about 10 ng / mL).

[0221] In some embodiments, the differentiation medium comprises a B27 supplement. The B27 supplement can be used at 1x. However, the concentration range of the B27 supplement present in the differentiation medium can be about 0.5x to about 5x. In some embodiments, the differentiation medium comprises the following components: Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12 (DMEM / F12) (ThermoFisher Scientific) (about 1x); B27 supplement (about 1x); bFGF (about 10 mg / mL); EGF (about 20 mg / mL); Penicillin (about 1000 units / mL); and streptomycin (about 100 μg / mL). The differentiation medium may also include or be replaced with other synthetic or natural supplements, growth factors, antibiotics, vitamin metabolites and / or hormones with similar properties known in the art.

[0222] In some embodiments, after forming organoids in a multi-well plate, a bioreactor (e.g., a rotary bioreactor) is used to culture the cells in the differentiation medium. Experiments are conducted to increase the size of the organoids formed. After 3D culture for 4-6 days in the organoid chamber droplets of a multi-well plate, the droplets are transferred to a rotary bioreactor. After 30-60 days of cultivation, the size of the organoids cultured in the rotary bioreactor is 3 times -10 times the size of the organoids cultured in the multi-well plate. Therefore, a rotary bioreactor can be used in some embodiments.

[0223] In some embodiments, the cells are cultured in poly-ornithine coated plates for a time sufficient to induce differentiation. In some embodiments, the method uses commercially available extracellular matrices such as Matrigel. TM Other natural or synthetic extracellular matrices are known in the art for culturing cells. Typically, the extracellular matrix includes laminin, entactin, and collagen. In one embodiment, the method is performed using a 3-dimensional culture device (chamber) that simulates the in vivo environment for culturing the cells, for example, the extracellular matrix can be formed in a plate that can induce stem cells to proliferate under hypoxic conditions. Such 3-dimensional devices are known in the art.

[0224] The cell model of the present application may have one or more of the following features: 1) it is a fully humanized cell model; 2) it exhibits accumulation of β-amyloid protein; 3) it exhibits hyperphosphorylation of Tau protein; 4) it exhibits aggregation of Tau protein; 5) it exhibits glial cell proliferation; 6) it exhibits chronic neuroinflammation; and 7) it exhibits synaptic loss and neuronal death. In some embodiments, the cell model may produce amyloid plaques and Tau tangles.

[0225] In the present application, the cell model can be a brain organoid. In some cases, the cell organoid is a forebrain organoid.

[0226] For example, the cell model of the present application may include the following cells: astrocytes, neurons, neural progenitor cells and / or oligodendrocyte astrocytes.

[0227] Compositions and kits

[0228] In one aspect, the present application provides a composition, which may include the engineered human cells of the present application or the cell model of the present application.

[0229] In another aspect, the present application provides a kit for screening substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of dementia-related neurological diseases. The kit comprises the engineered human cells or cell models of the present application.

[0230] In some embodiments, the dementia-related neurological disease is Alzheimer's disease.

[0231] In some embodiments, the substance, device and / or composition includes molecules, membrane-bound vesicles and / or cells. For example, the substance may include molecules (e.g., small molecules, macromolecules or biological substances). For example, the substance may include membrane-bound vesicles such as exosomes, organelles that can be engineered. For example, the substance may include cells, which may be human cells, and in some cases, the cells may be genetically engineered or otherwise modified.

[0232] The device may be an electronic device and / or a mechanical device. The device may comprise the substance and / or the composition. The device may comprise a medical device, such as a medical device purportedly effective in the treatment, diagnosis, prevention, monitoring and / or prognosis of Alzheimer's disease.

[0233] The candidate substance can be a synthetic compound, a peptide, a protein, a DNA library or a nucleic acid molecule in the library, a tissue extract or a cell culture supernatant of an animal (e.g., a mammal such as mouse, rat, pig, cow, sheep, monkey or human), an extract or a culture product from a plant or microorganism, or any mixture thereof.

[0234] In the present application, the composition may include a mixture derived from one or more organisms. The organism may be a plant, an animal and / or a microorganism. For example, the composition may include a tissue homogenate and / or a blood sample. In some embodiments, the composition may include an extract from one or more plants. For example, the composition may include a candidate Chinese medicine.

[0235] The kit of the present application may also include one or more additional components selected from the following: assay buffer, control, substrate, standard, detection material, laboratory supplies, device, machine, cell, organ, tissue and user manual or instructions. For example, the detection material may include antibodies, derivatives of fluorescein labels, luminescent substrates, detection solutions, scintillation counting fluids, etc. Laboratory supplies may include desalting columns, reaction tubes or microplates (e.g., 96 or 384 well plates).

[0236] Screening methods

[0237] In one aspect, the present application provides a method for screening substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of dementia-related neurological diseases. The method may include: exposing the engineered human cells of the present application or the cell model of the present application to candidate substances, devices and / or compositions; evaluating the engineered cells or cell models for one or more characteristics of the dementia-related neurological diseases in the presence of the candidate substances, devices and / or compositions; and selecting candidate substances, devices and / or compositions that can prevent, inhibit and / or alleviate one or more characteristics of the dementia-related neurological diseases.

[0238] In one aspect, the present application provides a method for identifying potential substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of dementia-related neurological diseases. The method may include (i) contacting the engineered human cells of the present application or the cell model of the present application with a candidate substance, device and / or composition to be tested; and (ii) evaluating the activity of the candidate substance, device and / or composition for one or more characteristics of the dementia-related neurological disease.

[0239] In one aspect, the present application provides a method for designing a substance, device and / or composition useful in the treatment, prevention and / or prognosis of a dementia-related neurological disease. The method may include the following steps: (i) exposing the engineered human cells of the present application or the cell model of the present application to a candidate substance, device and / or composition; (ii) evaluating the engineered human cells or the cell model for one or more characteristics of the dementia-related neurological disease; (iii) selecting a candidate substance, device and / or composition that can prevent, inhibit and / or alleviate one or more characteristics of the dementia-related neurological disease; and (iv) modifying the structure and / or composition of the candidate substance, device and / or composition of step (iii) to obtain a modified substance, device and / or composition having improved activity in the treatment, prevention and / or prognosis of the dementia-related neurological disease.

[0240] In some embodiments of the methods, the dementia-related neurological disease is Alzheimer's disease.

[0241] In some embodiments, the features of the dementia-related neurological disease may include: accumulation of β-amyloid protein; hyperphosphorylation of Tau protein; aggregation of Tau protein; glial cell proliferation; chronic neuroinflammation; synaptic loss; neuronal death; the appearance of amyloid plaques; the appearance of Tau tangles; increased expression level of CTF-β; increased expression level of Aβ42; increased ratio of Aβ42 / Aβ40; axonal swelling, axonal fragmentation, accumulation of β-amyloid protein, accumulation of C99 fragment of APP, accumulation of p-Tau, synaptic atrophy, formation of neuronal apoptotic F-actin bundles using phalloidin staining; mitochondrial dysfunction and oxidative damage; autophagy defects; neurotransmitter imbalance; and / or glucose metabolism dysfunction.

[0242] In some embodiments, the hyperphosphorylation of the Tau protein comprises increased phosphorylation of Ser396, Thr181, Thr205 and / or Ser202 in Tau compared to the hyperphosphorylation of the Tau protein in a corresponding wild-type human cell.

[0243] In one aspect, the present application provides use of the engineered human-derived cells of the present application or the cell model of the present application as a model of dementia-related neurological diseases.

[0244] In one aspect, the present application provides use of the engineered human cells of the present application or the cell model of the present application in preparing a model of a dementia-related neurological disease.

[0245] In some embodiments of the use, the dementia-related neurological disease is Alzheimer's disease.

[0246] On the one hand, the present application provides a method for identifying potential biological targets and / or biomarkers of substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of dementia-related neurological diseases. The method may include the following steps: (i) performing quantitative proteomic comparative analysis, lipidomic comparative analysis and / or genomic comparative analysis on the engineered human cells of the present application or the cell model of the present application and control cells or control cell models; (ii) identifying genes, proteins and / or lipids with altered sequences, quantities, expression levels, modifications and / or activities; (iii) wherein the genes, proteins and / or lipids identified in step (ii) are potential biological targets and / or biomarkers of the substances, devices and / or compositions for the treatment, prevention and / or prognosis of the dementia-related neurological diseases.

[0247] In some embodiments of the methods, the dementia-related neurological disease is Alzheimer's disease.

[0248] On the one hand, the present application provides a method for screening biological targets and / or biomarkers useful in diagnosing and / or monitoring dementia-related neurological diseases. The method may include determining the presence and / or level of a substance in a sample obtained from an engineered human cell of the present application or a cell model of the present application before and after detecting the characteristics of the dementia-related neurological disease, and identifying a substance that exhibits a change in presence and / or level before and after the detection.

[0249] In some embodiments of the methods, the dementia-related neurological disease is Alzheimer's disease.

[0250] The characteristics of the dementia-related neurological disease may include: accumulation of β-amyloid protein; hyperphosphorylation of Tau protein; aggregation of Tau protein; glial cell proliferation; chronic neuroinflammation; synaptic loss; neuronal death; the appearance of amyloid plaques; the appearance of Tau tangles; increased expression level of CTF-β; increased expression level of Aβ42 and / or increased ratio of Aβ42 / Aβ40; mitochondrial dysfunction and oxidative damage; autophagy defect; neurotransmitter imbalance; and / or glucose metabolism dysfunction.

[0251] In one aspect, the present application provides the use of the engineered human cells of the present application or the cell model of the present application in preparing a system for screening substances, devices, compositions, biological targets and / or biomarkers useful in the treatment, diagnosis, prevention, monitoring and / or prognosis of dementia-related neurological diseases.

[0252] In some embodiments of the use, the dementia-related neurological disease is Alzheimer's disease.

[0253] In one aspect, the present application provides the engineered human cells or the cell model for screening substances, devices, compositions, biological targets and / or biomarkers useful in the treatment, diagnosis, prevention, monitoring and / or prognosis of dementia-related neurological diseases. The dementia-related neurological disease may be Alzheimer's disease.

[0254] For example, samples (e.g., cells, tissues, or other samples containing DNA or RNA, samples containing proteins, and / or samples containing metabolites) can be obtained from the cells or cell models of the present application before and after the appearance of disease (e.g., Alzheimer's disease) related features (e.g., Aβ accumulation, neurofibrillary tangles, abnormal synaptic morphology or function (atrophy), or neuronal cell death). Then, gene transcription products (transcriptome), gene translation products (proteome), lipids (lipidome), or metabolites (metabolome) derived from the sample can be comprehensively determined, and substances that change after the appearance of disease (e.g., Alzheimer's disease) related features can be identified.

[0255] Nucleic acid microarrays such as DNA microarrays can be used to analyze gene transcription products (e.g., transcriptome). Gene translation products (e.g., proteome) can be analyzed using gel electrophoresis (such as two-dimensional gel electrophoresis) or mass spectrometry (such as time-of-flight mass spectrometry, electrospray ionization mass spectrometry, capillary HPLC / MS and LC / MS / MS). Metabolites (metabolome) can be analyzed using NMR, capillary electrophoresis, LC / MS and / or LC / MS / MS. Mass spectrometry-based technology can be used for lipidomics analysis.

[0256] When the presence / amount of a substance shows a significant difference after the appearance of disease (e.g., Alzheimer's disease)-related features, such a substance can be considered a biological target and / or biomarker of the disease, and can therefore be used for early diagnosis (particularly preclinical diagnosis) of the disease (such as AD). The identified biological target and / or biomarker can be further detected with specific reagents or detection methods. For example, when the biological target and / or biomarker is a protein or peptide, it can be detected by immunoassay using a specific antibody. When the biological target and / or biomarker is a nucleic acid molecule (such as a transcription product), it can be detected by Northern blot analysis using a specific probe or by RT-PCR using a specific primer.

[0257] The method of the present application can be an in vitro method, an ex vivo method or an in vivo method.

[0258] In the method, after treating the cells or cell models of the present application with candidate substances, devices and / or compositions, the cells or organoids can be homogenized using a suitable buffer (such as phosphate-buffered saline) to obtain a homogenized solution. A soluble fraction and / or an insoluble fraction can then be separated from the homogenized solution. The separated soluble fraction and / or insoluble fraction can then be examined by immunoassay, for example, using anti-Aβ antibodies and / or anti-APP antibodies. In some embodiments, the amounts of Aβ42 and Aβ40 can be measured. In some embodiments, the ratio of Aβ42 / Aβ40 can also be calculated.

[0259] In some cases, after separating the cells or organoids, frozen sections or paraffin-embedded sections of the cells / organoids may be prepared. Then, APP / Aβ deposition may be assessed, for example, by immunostaining the cell / organoid sections with anti-APP antibodies and / or anti-Aβ antibodies. In addition, synaptic abnormalities may be assessed by immunostaining sample sections with antibodies to presynaptic or dendritic marker proteins. Morphological abnormalities of cytoskeletal proteins may be assessed by immunostaining brain sections with antibodies to phosphorylated tau. Neuronal cell death may be assessed with Nissl body staining or HE staining (e.g., as described in Am. J. Pathol., Vol. 165, pp. 1289-1300, (2004)). The results may be compared with those of a control group (e.g., in which a candidate substance, device, and / or composition is not applied, or a blank control buffer has been applied instead of a candidate substance, device, and / or composition).

[0260] If the total amount of Aβ is reduced, the amount of Aβ42 is reduced and / or the ratio of Aβ42 / Aβ40 is reduced after application of the candidate substance, device and / or composition, compared to the results obtained from the control group, the candidate substance, device and / or composition can be selected for further study (e.g., as a potential therapeutic agent / device / composition for treating AD or for inhibiting Aβ accumulation).

[0261] If, after application of the candidate substance, device and / or composition, amyloid deposition is reduced, neurofibrillary tangles are reduced, synaptic abnormalities (atrophy) are reduced, neuronal cell death is reduced, and / or inflammatory response is reduced, compared to the results obtained from the control group, the candidate substance, device and / or composition can be selected for further study (e.g., as a potential therapeutic agent / device / composition for treating AD, for inhibiting Aβ accumulation, for inhibiting neurofibrillary tangles, for inhibiting brain damage (e.g., neurodegeneration) or inflammatory response).

[0262] On the other hand, the present application provides a method for screening a substance having affinity for APP or Aβ. For example, a candidate substance can be applied to the cells or cell models of the present application, and then the presence of the candidate substance in the Aβ accumulation area can be checked. Candidate substances with specific affinity for APP or Aβ can be used for early diagnosis of AD.

[0263] Example

[0264] The following examples are set forth in order to provide a complete application and description of how to prepare and use the invention to one of ordinary skill in the art, but not to limit the scope of what the inventors regard as their invention, nor to indicate that the following experiments are all or the only experiments performed. Efforts have been made to ensure the accuracy of the numbers used (e.g., quantities, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature units are degrees Celsius, and pressure is atmospheric pressure or near atmospheric pressure. Standard abbreviations can be used, for example, pl represents picoliters, s or sec represents seconds, min represents minutes, h or hr represents hours, aa represents amino acids, nt represents nucleotides, im represents intramuscular (d), ip represents intraperitoneal (d), sc represents subcutaneous (d), rt represents room temperature, etc.

[0265] Materials and methods

[0266] CRISPR-mediated genome editing

[0267] The CRISPR / Cas9 expression vector (pX459, #48139) was obtained through Addgene (https: / / www.addgene.org / ). The design of sgRNA and the method of inserting sgRNA complementary oligonucleotide DNA into pX458 were the same as previously reported (Ran et al., 2013). The HDRDNA donor vector was constructed using the In-Fusion HD Cloning Kit (Clontech, 639648). One million hESCs were electroporated with 15 μg of CRISPR / Cas9 expression vector and 15 μg of donor vector at a voltage of 1300 V for 10 ms, three times, and then seeded on a 6-well plate. After selection by puromycin, the puromycin resistance gene was removed using Cre recombinase (Clontech, #631449) according to the instructions. After reaching near confluence, cells were replated to form single-cell colonies and genotyped based on previous reports (Li et al., 2016).

[0268] In vitro differentiation of human neuronal cells

[0269] Wild-type (WT) and APP knock-in hESCs were cultured in mTeSRTM1 medium without feeder layers. In vitro differentiation of hNSCs and human neuronal cells was as described previously. In brief, hESCs were passaged at a ratio of 1:5 at confluence. The next day, neural stem cell induction medium (Advanced DMEM / F12:Neurobasal (1:1), 1xN2, 1xB27, 1% Glutmax, 5μg / mL BSA and 10ng / mL hLIF (Millipore), 3μM CHIR99021 (Selleck), 2μM SB431542 (Selleck), 0.1μM compound E (EMD Chemicals)) was added for 7 days to induce human neural stem cells (hNSC). Spontaneous differentiation of human neurons was performed on Matrigel-coated surfaces for 14 days in DMEM / F12, 1xN2, 1xB27, 300 ng / mL cAMP (Sigma-Aldrich), 0.2 mM Vitamin C (Sigma-Aldrich), 10 ng / mL BDNF (Peprotech), and 10 ng / mL GDNF (Peprotech).

[0270] Generation of forebrain organoids

[0271] Generation of forebrain organoids from hESCs was performed as described previously (Qian et al., 2018; Qian et al., 2016). First, hESC colonies were detached with collagenase type IV after 7 days of passaging and washed with fresh stem cell medium in 15 mL conical tubes. On day 0, detached hESC colonies were transferred to ultra-low attachment 6-well plates (Corning Costar) containing 3 mL stem cell medium (without FGF-2) plus 2 μM Dorsomorphine and 2 μM A83–01. On days 5 and 6, half of the medium was replaced with induction medium consisting of DMEM:F12, 1X N2 supplement, 1X penicillin / streptomycin, 1X non-essential amino acids, 1X GlutaMax, 1 μM CHIR99021, and 1 μM SB-431542. On day 7, organoids were embedded in Matrigel and cultured in induction medium for another 7 days. On day 14, embedded organoids were mechanically dissociated from Matrigel by manual pipetting in 5 mL pipette tips. Typically, 20 organoids were transferred to each well of a 12-well spin bioreactor (SpinU) (Qian et al., 2016) containing differentiation medium consisting of DMEM:F12, 1X N2 and B27 supplements, 1X penicillin / streptomycin, 1X 2-mercaptoheptanol, 1X non-essential amino acids, 2.5 μg / ml human insulin.

[0272] Tissue preparation

[0273] Whole organoids were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) for 30 min at room temperature. Organoids were washed three times with PBS and then immersed in 30% sucrose solution overnight. Unless otherwise stated, organoids were embedded in tissue freezing medium and sectioned at 8 μm thickness using a cryostat (Leica).

[0274] Immunohistochemistry and microscopy techniques

[0275] For immunostaining, frozen sections were washed with PBS and then permeabilized with 0.5% Triton-X in PBS for 1 hour. Tissues were blocked with blocking medium for 30 minutes, and the blocking medium consisted of 10% donkey serum in PBS (PBST) containing 0.05% Triton-X. The primary antibody diluted in blocking solution was applied to the sections and left overnight at 4°C. After washing with PBST for at least 5 times, the secondary antibody diluted in blocking solution was applied to the sections and continued for 1 hour at room temperature or left overnight at 4°C. Finally, the sections were washed with PBST for at least 5 times before sealing. The secondary antibody was: AlexaFluor 488-coupled antibody, AlexaFluor 546-coupled antibody (Invitrogen) used at a dilution of 1:500. Images were captured by confocal microscopy (Zeiss LSM 880). Sample images were prepared in ImageJ (NIH). The information of the primary antibodies used in this study and their dilutions are as follows: anti-Aβ antibody (1:1000, Cell Signaling Technology, 8243), cleaved caspase-3 (Cell Signaling Technology, 9661, 1:1000), PSD95 (Abcam, ab2723, 1:1000), synaptophysin (Abcam, ab8049, 1:1000), AT8 (Thermo Fisher Scientific, MN1020, 1:1000), Tau5 (Millipore, 577801, 1:1000), MC1 (1:20; gift from P. Davies, Albert Einstein College of Medicine, New York, USA), anti-human Aβ1–12 (6E10 1:1000, Covance, 39320), anti-APP-CTF (1:1,000, Sigma, A8717), APP (Thermo Fisher Scientific, 14-9749-82, 1:1000), GFAP (Millipore, MAB360, 1:1000), EEA1 (Cell Signaling Technology, 2411, 1:1000), LAMP1 (Cell Signaling Technology, 9091, 1:1000), anti-RIPK1 (1:1,000, BDBiosciences, 610459), anti-RIPK3 (1:500, Stata Crus, 374639), anti-MLKL (1:1000, EnoGene, E11-11361C)

[0276] Hypoxia analysis

[0277] Hypoxic cells in organoids were detected using the Hypoxia Probe Kit (HPI). Pimonidazole hydrochloride was added to the culture medium at a working concentration of 200 μM and incubated with the organoids for 2 hours before being fixed for analysis. Immunochemical detection of hypoxic cells containing pimonidazole was performed according to the manufacturer's instructions. Images were captured by confocal microscopy (Zeiss LSM 880) using the same acquisition parameters (laser intensity and gain), and the "tiling" / "stitching" function in Zen software (Zeiss) was used when necessary. In order to quantify the percentage of hypoxic area in sliced ​​and unsliced ​​organoids, the area marked by the hypoxia probe was measured using ImageJ software and divided by the total organoid area marked by DAPI.

[0278] Western blotting

[0279] Samples from the soluble fraction were loaded onto 4%–20% sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF Immobilon FL membranes (Millipore). After blocking with 5% skim milk in PBS, the membranes were blotted overnight with primary antibodies in 5% skim milk containing 0.01% Tween-20 and then probed with horseradish peroxidase-conjugated secondary antibodies and detected with SuperSignal West Femto chemiluminescent substrate (Pierce). The information of the primary antibodies and their dilutions used in this study are as follows: cleaved caspase-3 (Cell Signaling Technology, 9661, 1:1000), caspase-3 (Cell Signaling Technology, 9662, 1:1000), PSD95 (Abcam, ab2723, 1:1000), synaptophysin (Abcam, ab8049, 1:1000), AT8 (Thermo Fisher Scientific, MN1020, 1:1000), Tau5 (Millipore, 577801, 1:1000), APP (Thermo Fisher Scientific, 14-9749-82, 1:1000), GFAP (Millipore, MAB360, 1:1000), EEA1 (Cell Signaling Technology, 2411, 1:1000), LAMP1 (Cell Signaling Technology, 9091, 1:1000), anti-RIPK1 (1:1,000, BD Biosciences, 610459), anti-RIPK3 (1:500, Stata Crus, 374639), anti-MLKL (1:1000, EnoGene, E11-11361C), anti-pMLKL (1:1000, Abcam, ab196436).

[0280] ELISA quantification

[0281] Aβ40 and Aβ42 in the culture medium were measured using human β-amyloid (1–40) ELISA kit (ThermoFisher, KHB3481) and human β-amyloid (1–42) ELISA kit (Thermo Fisher, KHB3441), respectively, according to the manufacturer’s instructions. The reaction was stopped and read at 450 nm using a microplate reader (Biotek).

[0282] Culture of various major cell types

[0283] Human embryonic stem (ES) cells and induced pluripotent stem (iPS) cells. H1 human ES cells were obtained from WiCell Research Resources (Wicell, WI), maintained in feeder-free conditions using mTeSR1 medium (Stem Cell Technologies), and used to generate induced human neuronal (iN) cells at intermediate (about 50) passages (Zhang et al., 2013). Cells were cultured as described (Zhang et al., 2013).

[0284] Mouse embryonic fibroblasts (MEFs) were isolated from mouse embryos (Harlan Laboratories, Inc.) of the CF-1 strain harvested at 12.5-13.5 pc. Briefly, embryos were dissected from terminally anesthetized mice. The head and viscera were removed, the remaining carcasses were minced, digested with trypsin into single cell suspensions and plated on T75 flasks. Before use in experiments, cultured MEFs (P0) were frozen or expanded up to 3 times (P3).

[0285] Lysotracker staining

[0286] Neuronal cells were incubated with 500 nM Lysotracker Red dye (Invitrogen) for 30 minutes at 37° C. The cells were then washed with PBS and the fluorescence intensity was measured by flow cytometry or Opera Phenix high-content confocal microscopy.

[0287] Cholesterol staining

[0288] Accumulation of free cholesterol in macrophages was revealed by fillipin staining at 50 μg / ml for 1 hour at room temperature. The cells were then washed with PBS and the fluorescence intensity was measured by flow cytometry or Opera Phenix high-content confocal microscopy.

[0289] TMT-MS-based quantitative proteomics

[0290] AD brain organoids and controls at three months were homogenized in 8M urea lysis buffer (8M urea, 10mM Tris, 1mM PMSF, 1mM protease inhibitor cocktail). Protein digestion was performed on 100μg of each sample. The samples were reduced by alkylation with dithiothreitol and iodoacetamide. After centrifugation at 2000g for 10 minutes, 0.4% trifluoroacetic acid (TFA) was added to the supernatant and desalted with a Sep-Pak column. Each sample was dissolved with 100mM TEAB buffer, labeled with a TMT 10-plex kit, and incubated at room temperature for 1 hour. The reaction was quenched with 5% hydroxylamine. All samples were combined and dried with SpeedVac. The labeled peptides were desalted with a Sep-Pak column. The samples were separated by HPLC and analyzed by mass spectrometry.

[0291] Example 1 APP NL-G-F Generation of hESCs and cell models derived from them

[0292] To better recapitulate key aspects of AD in human cells, Swedish (K670M / N671L), Beyreuther / Iberian (I716F), and Arctic (E693G) mutations of APP were knocked into human H1 embryonic stem cells (hESCs).

[0293] The targeted scheme for gene editing is shown in Figure 8 Containing all three homozygous APP mutations (APP NL-G-F ) of human ESCs were verified by PCR and Sanger sequencing of colonies from single putative edited cells. Several protocols have been developed to generate neural organoids from human pluripotent stem cells. The protocol reported by Qian, X et al. (2016) was used to generate neural organoids from APP NL-G-F hESCs and WT controls generated forebrain organoids and obtained complex, dense 3D neural organoids.

[0294] To characterize AD-related phenotypes in the generated brain organoids, the levels of 40-amino acid and 42-amino acid amyloid-β isoforms (Aβ40 and Aβ42) were measured in the conditioned medium after 6 weeks of differentiation. NL-G-F Conditioned medium from brain organoids showed a significant decrease in Aβ40 (approximately 3-fold) and an increase in Aβ42 levels (approximately 1.5-fold) ( Figures 1A to 1D ). APP NL-G-F The Aβ42 / Aβ40 ratio in brain organoids was also increased (approximately 4-fold). Figure 1A Representative immunocytochemistry images of neurons (MAP2) and astrocytes (GFAP) in brain organoids at day 90 of differentiation are shown. Figure 1B to Figure 1CShown are the protein levels of Aβ40 and Aβ42 in the culture medium measured by ELISA. Protein levels were normalized to the total protein concentration of the corresponding samples (n=4). Figure 1D The ratio of Aβ42 / Aβ40 is shown (n=4).

[0295] To investigate the state of Aβ accumulation and deposition, brain organoids were analyzed at different time points (2 and 3 months, respectively). Analysis at 60 days (2 months) and 90 days (3 months) of culture determined the presence of APP NL-G-F The area of ​​Aβ aggregates in organoids gradually increased compared with the control ( Figure 1E to Figure 1G ), which is barely detectable in conventional 2D cultures, where secreted amyloid-β diffuses into the bulk of the culture medium. These data suggest that APP NL-G-F Strong Aβ aggregation present in organoids that appears to develop in a time-dependent manner. Figure 1E Provided are representative immunocytochemistry images of Aβ and MAP2 in brain organoids at day 60. Scale, 50 μm. Figure 1F Provides representation Figure 1E Bar graph of the number of Aβ spots in .gamma.-Throughput PCR (n=4). Figure 1G Provided are representative immunocytochemistry images of Aβ and MAP2 in brain organoids at day 90. Scale, 50 μm. Figure 1H Provides representation Figure 1G Bar graph of the number of Aβ spots in .gamma.-Throughput PCR (n=4).

[0296] Example 2 APP NL-G-F Tau pathology in forebrain organoids

[0297] Abnormal phosphorylation, mislocalization, and aggregation of tau protein have long been considered key features of AD. NL-G-F Tau pathology in organoids. Of note, since Tau hyperphosphorylation is known to precede tau aggregation, first, phosphorylated Tau (p-Tau) levels were analyzed by Western blotting using antibodies against p-Tau (phosphorylation at Ser202 / Thr205, Ser396, and Thr181). It has been reported that the increase of p-Tau at specific sites occurs in a relative order. Interestingly, when APP NL-G-F When Aβ aggregates were already observed in organoids, APP NL-G-F There were no significant differences in p-Tau levels between organoids and control organoids. At 3 months, in the RIPA soluble fraction, APP NL-G-F Organoids showed significantly higher AT8 / t-Tau levels than controls ( FIG. 2A to FIG. 2B). Ser396 / t-Tau and Thr181 / t-Tau began to increase significantly in the RIPA soluble fraction or the Sarcosyl sodium insoluble fraction near 4 months, but not in the control, while AT8 / t-Tau levels continued to increase. Figure 2A Provided on the 90th day in the APP NL-G-F Representative Western blots of total tau and P-tau S202 / T205 (AT8) in organoids and WT organoids. The upper and lower bands in the AT8 blot represent different tau isoforms. Figure 2B Shows Figure 2A Densitometry quantification of P-tau / total tau levels in WT mice (n=5).

[0298] Next, multiple antibodies were used to detect different stages of conformational changes in Tau. First, APN-mab005, a monoclonal antibody that recognizes the early stages of aggregated Tau, was used. NL-G-F Brain organoids began to show more aggregated APN-mab005-positive Tau than WT organoids at 3 months ( Figure 2C to Figure 2F ). MC1 immunoreactivity (indicative of intermediate conformational / pathological changes of Tau) was then assessed in organoids at 4 months and in APP NL-G-F Significant increases were found in organoids ( Figure 2G to Figure 2H ). Moreover, at 4 months of differentiation, in APP NL-G-F A significant increase in AT8-positive p-Tau levels was also found in a subset of cells ( Figures 2I to 2J ). As a result, in the organoid model, hyperphosphorylated Tau appeared after amyloid-β accumulation, and the increase of p-Tau at specific sites appeared in a relative order, which is consistent with previous reports on AD pathology in human patients.

[0299] Figure 2C Representative immunocytochemistry images of tau aggregation using APN-mab005, a specific antibody against aggregated tau with early pathological phenotypes in day 60 brain organoids are provided. Scale, 50 μm. Figure 2D Provides representation Figure 2C Bar graph of the number of aggregated tau spots in the WT mice (n=4). Figure 2E Provided are representative immunocytochemistry images of tau aggregation using APN-mAb005 in brain organoids at day 90. Scale, 50 μm. Figure 2F Provides representation Figure 2E Bar graph of the number of aggregated tau spots in the WT mice (n=4).

[0300] Figure 2GRepresentative immunocytochemistry images of disease-specific conformational changes of tau using MC1 antibody in brain organoids at day 120 are provided. Scale, 20 μm. Figure 2H Provides representation Figure 2G Bar graph of the number of aggregated tau spots in the WT mice (n=8).

[0301] Fig.2I Provided are representative immunocytochemistry images of P-tau S202 / T205 (AT8) antibody in brain organoids at day 120. Scale, 50 μm. Figure 2J Provides representation Fig.2I Bar graph of the number of aggregated tau spots in the WT mice (n=8).

[0302] Example 3 APP NL-G-F Increased gliosis and neuroinflammation in forebrain organoids

[0303] Both genetic epidemiological and experimental data suggest a key role for gliosis and neuroinflammation in the progression of AD pathology. Microglia and astrocytes are key regulators of inflammatory responses in the central nervous system. Unlike neurons and astrocytes, which are derived from the ectoderm, microglia migrate early in embryonic development from the yolk sac, a structure derived from the mesoderm. Organoids generated according to the present application are primarily populated by neurons and astrocytes. In order to study APP NL-G-F Gliosis in forebrain organoids, measuring APP NL-G-F Protein levels of GFAP (astrocyte marker) in forebrain organoids. Western blot analysis showed that APP NL-G-F Moderate increase in GFAP levels in forebrain organoids ( FIG. 3A to FIG. 3B ), while APP was observed at 4 months NL-G-F Significant increase in GFAP in forebrain organoids ( FIG. 3C to FIG. 3D ). Figure 3A Provided on the 90th day in the APP NL-G-F Representative western blot of GFAP in WT organoids and p38 organoids. Figure 3B Provided Figure 3A Densitometry quantification of GFAP levels in WT mice (n=8). Figure 3C Provided on the 120th day in the APP NL-G-F Representative western blot of GFAP in WT organoids and p38 organoids. Figure 3D Provided Figure 3C Densitometry quantification of GFAP levels in WT mice (n=4).

[0304] Then, test the app NL-G-FThe secretion of chemokines and cytokines in the culture medium of forebrain organoids showed a significant increase in the secretion of proinflammatory factors interleukin 6 (IL6) and interleukin 8 (IL8), while the secretion of neuroprotective macrophage migration inhibitory factor (MIF) and anti-apoptotic serine protease inhibitor E1 was significantly reduced. NL-G-F A slight increase in the chemokine CXCL1 and a significant decrease in CCL2 and CXCL12 were also observed in the forebrain organoid culture medium. TNFα and IL-1β, which are mainly secreted by microglia, were almost undetectable in the organoid culture medium. Figure 3E ). Figure 3E It was shown that the APP from day 90 was measured by human cytokine array kit NL-G-F Chemokine and cytokine secretion in conditioned media of organoids or WT organoids (n=2).

[0305] Example 4 APP NL-G-F Increased synaptic degeneration and cell death in forebrain organoids

[0306] Synaptic degeneration is now considered an intermediate step and a key pathophysiological feature of AD. Extensive evidence suggests that in AD, synaptic number is reduced and synaptic function is impaired, which occurs after Aβ accumulation and is associated with disease progression. NL-G-F Synaptic loss in forebrain organoids, with Western blot analysis of presynaptic synaptophysin and postsynaptic PSD95 at 3 and 4 months of age. NL-G-F A slight decrease in the presynaptic membrane protein synaptophysin was observed in forebrain organoids, but an increase in the postsynaptic membrane protein PSD95 was observed ( FIG. 4A to FIG. 4B ), which may be due to compensatory enhancement of the postsynaptic membrane when the presynaptic membrane is damaged in the early stages of the disease. NL-G-F A significant reduction in the presynaptic membrane protein synaptophysin was observed in forebrain organoids, accompanied by a slight reduction in PSD95 ( FIG. 4C to FIG. 4D ), indicating the progression of AD-related synaptic loss. Figure 4A Provided on the 90th day in the APP NL-G-F Representative western blots of synaptophysin and PSD95 in WT organoids and WT organoids. Figure 4B Provided Figure 4A Densitometry quantification of synaptophysin and PSD95 levels in WT mice (n=4). Figure 4C Provided on the 120th day in the APP NL-G-F Representative western blots of synaptophysin and PSD95 in WT organoids and WT organoids. Figure 4D Provided Figure 4CDensitometry quantification of synaptophysin and PSD95 levels in WT mice (n=4).

[0307] Neuronal death is another prominent pathological feature of AD. Cell death in the AD brain has been attributed to at least two mechanisms: apoptosis and necrosis. To assess neuronal apoptosis in brain organoids, cleaved caspase-3 was analyzed by immunostaining at 3 months. It was noted that brain organoids grown as spheroids in suspension culture could expand to 3 mm to 4 mm in diameter. Due to the lack of a functional circulatory system, the viability of cells within large cortical organoids is limited by the limited supply of oxygen and nutrients delivered by surface diffusion. To avoid the possible impact of hypoxia on apoptosis in the core of the organoids, the organoids were co-stained with a pimonidazole-based hypoxia probe. The results showed that in WT and APP NL-G-F However, in WT forebrain organoids, cleaved caspase 3 signals largely colocalized with the hypoxia dye, whereas in APP NL-G-F In forebrain organoids, cleaved caspase 3 signals were detected in areas without hypoxia dye staining, indicating that apoptosis occurs independently of hypoxia ( Figure 4E ). Figure 4E Representative immunocytochemical images of apoptosis marker cleaved CASP3 and pimonidazole-based hypoxia probe (HPOX) in brain organoids at differentiation day 90 are provided. Scale, 80 μm. Bars represent the area (%) of cleaved CASP3 spots in immunocytochemical images (n=8).

[0308] Necrosis is the uncontrolled lysis of cells, which also leads to neuronal cell death in AD. Necroptosis, a form of programmed necrosis, has recently been discovered in postmortem AD brains. Three key proteins, RIPK1, RIPK3, and their substrate MLKL, are involved in the initiation of necroptosis. Phosphorylated MLKL can trigger MLKL aggregates to form homodimers, which induces membrane damage and leads to cell death. Similarly, MLKL co-stained with the hypoxia dye HOPX. In WT forebrain organoids, MLKL was not observed in either HOPX-positive or -negative areas, while in APP NL-G-F In forebrain organoids, the amount of aggregated MLKL signaling was significantly increased ( Figure 4F ). It shows that necroptosis in forebrain organoids is not induced by hypoxia. Programmed necrosis complex-related proteins were analyzed by Western blot. Consistent with the observations in AD patients, in APP NL-G-F Phosphorylated forms of MLKL and RIPK1 were significantly increased in forebrain organoids, whereas RIPK3 and total MLKL remained unchanged ( Figure 4G-4H). Taken together, these observations suggest that both apoptosis and necroptosis may contribute to APP NL-G-F Neuronal loss in forebrain organoids.

[0309] Figure 4F Provided are representative immunocytochemical images of the necroptosis marker MLKL in brain organoids at day 90 of differentiation. Scale, 80 μm. Bars represent the area (%) of MLKL spots in the immunocytochemical images (n=8). Figure 4G Provided on the 90th day in the APP NL-G-F Representative Western blots of RIPK1, RIPK3, MLKL, and phosphorylated MLKL levels in organoids and WT organoids. Figure 4H Provided Figure 4G Densitometry quantification of RIPK1, RIPK3, MLKL and phosphorylated MLKL levels in WT mice (n=4).

[0310] Example 5 APOE3-Christchurch (R136S) in APP NL-G-F Neuroprotection in forebrain organoids

[0311] A rare APOE variant, designated APOE3 Christchurch (APOEch), was found in some members of a large Colombian family with autosomal dominant Alzheimer's disease (ADAD), generating a missense mutation from arginine to serine at amino acid position 136 (corresponding to codon 154), due to the E280A mutation in PSEN1. A woman homozygous for APOEch was described who did not develop mild cognitive impairment (MCI) until her 70s, almost 30 years after the typical age of onset associated with PSEN1 mutations. The APOEch mutation was thought to account for her resilience to the highly penetrant familial PSEN1 mutation of AD. This individual was found to have an abnormally high brain amyloid burden compared with other PSEN1-E280A carriers, but only limited amounts of Tau tangles or other signs of neurodegeneration detectable by brain imaging. These suggest that the AD-protective effect of the APOEch mutation may be achieved through mechanisms that limit Tau pathology and neurodegeneration. However, only one case of homozygous APOEch mutation has been reported. More clinical cases and / or experimental animal studies are needed to confirm these observations and explore the underlying molecular and cellular mechanisms.

[0312] Then study the APP NL-G-F Whether the conversion of APOE3 to APOE3ch in forebrain organoids is sufficient to ameliorate AD-related pathology. NL-G-FThe contribution of Aβ accumulation in forebrain organoids was found to be NL-G-F Forebrain organoids and age-matched APOE3 APP NL-G-F Forebrain organoids showed fewer Aβ spots ( Figure 5A-5B ). NL-G-F Tau pathology was assessed in forebrain organoids. Western blotting using anti-phosphorylated Tau (p-Tau) AT8 antibody at 3 months showed that APP with APOE3ch NL-G-F p-Tau and p-Tau / total Tau ratio were significantly reduced in forebrain organoids ( Figure 5C-5D ). Consistent with the results, APP NL-G-F Forebrain organoids were stained with AT8 at 4 months and less conformationally altered Tau was found in organoids with APOE3ch ( Figure 5E-5F ). Figure 5A Provides APOE3 APP at day 90 of differentiation NL-G-F and APOE3ch APP NL-G-F Representative immunocytochemistry images of Aβ in brain organoids. Scale, 80 μm. Figure 5B Provides representation Figure 5A Bar graph of the number of Aβ spots in . Figure 5C APOE3 APP provided at 90 days NL-G-F and APOE3ch APP NL-G-F Representative Western blots of total tau and P-tau S202 / T205 (AT8) levels in brain organoids. The upper and lower bands in the AT8 blot represent different tau isoforms. Figure 5D Provided Figure 5C Densitometry quantification of total tau and P-tau S202 / T205 (AT8) levels in p347 cells (n=5). Figure 5E Provides APOE3 APP at day 120 of differentiation NL-G-F and APOE3ch APP NL-G-F Representative immunocytochemistry images of P-tau S202 / T205 (AT8) antibody in brain organoids. Scale, 80 μm. Fig. 5F Provides representation Figure 5E Bar graph of the number of aggregated tau spots in the .gif (n=5).

[0313] Next, we will study whether APOE3ch improves APP NL-G-F Elevated neuronal death in forebrain organoids. Immunocytochemistry results showed that APP NL-G-FSignificant reduction in cleaved CASP3 immunoreactivity in forebrain organoids with APOE3ch ( Figure 5G-5H ). In summary, these results suggest that NL-G-F AD-associated phenotypes observed in forebrain organoids can be greatly ameliorated by converting APOE3 to APOE3ch. Figure 5G Provided on day 120 of differentiation in APOE3 APP NL-G-F and APOE3chAPP NL-G-F Representative immunocytochemistry images of cleaved CASP3 antibody in brain organoids. Scale, 50 μm. Figure 5H Provides representation Figure 5G Bar graph of the area (%) of cleaved CASP3 spots in Figure 5 (n=6).

[0314] Example 6 APOE3ch enhances lysosomal function to clear neurotoxic proteins

[0315] The dominant theory in AD research is that increased Aβ levels, Aβ aggregates, or amyloid plaques lead to Tau pathology, which subsequently causes AD-related cognitive decline. Clearly, the new findings in patients with APOE3ch do not support this widely discussed hypothesis. In fact, this study provides direct evidence that Aβ / amyloid accumulation alone is not sufficient to cause AD, and Aβ may induce Tau pathology and cognitive decline only in the presence of normal functional APOE. Further understanding of this causal chain is critical to better understand AD pathogenesis and improve drug development.

[0316] As mentioned above, APOE3ch leads to APP at day 120 NL-G-F Cleaved caspase 3 was significantly reduced in forebrain organoids. NL-G-F When the protein levels of cleaved caspase 3 were examined at the early stage (day 80) of forebrain organoids, the expression of cleaved caspase 3 in APOE3 and APOE3ch APP NL-G-F No differences were observed between forebrain organoids. NL-G-F The protein levels of cleaved caspase 3 in forebrain organoids were higher at day 80 than at day 120 ( Fig. 6A ). Fig. 6A Provided on days 80 and 120 of differentiation in APOE3 APP NL-G-F and APOE3ch APP NL-G-F Representative immunocytochemistry images of cleaved CASP3 antibody in brain organoids. Scale, 50 μm. Bars represent the area (%) of cleaved CASP3 spots (n=6).

[0317] In the periphery, cells undergoing apoptosis expose and release signals that attract phagocytes, which engulf cells undergoing apoptosis and maintain homeostasis, while in the central nervous system, microglia and astrocytes are responsible for engulfing the debris of apoptotic cells, thereby maintaining tissue. In the early stages of forebrain organoid differentiation, GFAP-positive astrocytes were almost undetectable, while at day 120, astrocytes appeared. The engulfment of apoptotic cells by astrocytes may explain why in APP NL-G-F The levels of cleaved caspase 3 were reduced in forebrain organoids at day 120. This suggests that APOE3ch APP NL-G-F Astrocytes in forebrain organoids may be able to more efficiently engulf and digest apoptotic debris. To investigate this, fluorescently labeled Tau was added to differentiated APOE3 and APOE3chAPP NL-G-F In cells, APOE3ch APP was observed NL-G-F Increased Tau protein uptake in cells ( Figure 6B Moreover, fluorescently labeled Tau is mostly co-localized with lysosomes after being taken into cells ( Figure 6C ). Figure 6B Figure 3 shows the addition of fluorescently labeled tau to differentiated APOE3 APP NL-G-F and APOE3ch APP NL-G-F The cells were incubated with tau proteins and the uptake of tau protein was measured by FACS analysis. The bar graph represents the fluorescence intensity of tau protein in the cells (n=9). Figure 6C APOE3 APP is available NL-G-F and APOE3ch APP NL-G-F Representative images of cells stained with fluorescently labeled tau and lysotracker dye. Scale, 20 μm.

[0318] Then test whether APOE3ch can enhance APP NL-G-F Endocytosis and lysosomal degradation in forebrain organoids. First, early endosomes were investigated by immunocytochemistry of EEA1 protein and APOE3 APP NL-G-F Forebrain organoids have more APOE3ch than APP NL-G-F Forebrain organoids have significantly larger early endosomes ( Fig.6D ), which is consistent with the results observed in AD patients, where larger early endosome volume and abnormal endosomal function ultimately lead to impaired endocytosis and degradation function. Lysosomal function was then studied using lysotracker dye staining. NL-G-F The cells had a higher number of lysosomes, but the acidity of these lysosomes was defective compared with WT cells, which could be expressed in APOE3chAPPNL-G-F Regeneration in cells Fig.6D ). The function of lysosomal hydrolases is highly dependent on the acidic environment of the lysosomal cavity. Therefore, APOE3ch can increase APP NL-G-F The degradation capacity of lysosomes in cells and help clear neurotoxic proteins. Fig.6D Provided on day 120 of differentiation in APOE3 APP NL-G-F and APOE3ch APP NL-G-F Representative immunocytochemistry images of the early endosomal marker EEA1 in brain organoids. Scale, 50 μm. Bars represent the number of EEA1 puncta (n=6).

[0319] APOE functions as a lipid chaperone and promotes the cellular uptake of cholesterol and lipoproteins through receptor-mediated endocytosis. It has been reported that cholesterol accumulation can inhibit lysosomal function by activating the mTORC1 signaling pathway. APP was then tested NL-G-F The lipid and cholesterol composition in cells was analyzed and it was found that APOE3ch reduced the amount of intracellular lipid and cholesterol ( Figure 6E-6H ). This may explain how APOE3ch increases APP NL-G-F Lysosomal function in cells. Fig. 6E Provides WT, APOE3APP NL-G-F and APOE3ch APP NL-G-F Representative images of lysotracker dye staining in cells. Scale, 30 μm. Bar graph represents the fluorescence intensity of lysotracker dye in cells (n=7). Fig. 6F APOE3 APP is available NL-G-F and APOE3ch APP NL-G-F Representative images of filipin staining in cells. Scale, 80 μm. Bars represent the fluorescence intensity of filipin dye in cells (n=6). Figure 6G APOE3 APP treated with or without HPβCD was provided NL-G-F Representative images of filipin staining in cells. Scale, 80 μm. Bars represent the fluorescence intensity of filipin dye in cells (n=6). Figure 6H APOE3 APP treated with or without HPβCD was provided NL-G-F Representative images of lysotracker dye staining in cells. Scale, 30 μm. Bar graph represents the fluorescence intensity of lysotracker dye in cells (n=6).

[0320] Example 7 TrkB antibody rescues synaptic loss and cell death in AD organoids

[0321] To further evaluate the APP NL-G-F The function of organoids, the effect of TrkB antibody on organoids was studied.

[0322] In order to investigate the therapeutic effects of TrkB antibodies on AD, it is important to determine whether TrkB antibodies can bind to their target TrkB and its downstream signaling events and ultimately exhibit positive cellular functions. NL-G-F Organoids were treated with BDNF (5 nM), TrkB antibody (15 nM) or vehicle for 30 minutes or 2 hours, and TrkB downstream signaling events were analyzed by Western blot. The amino acid sequence of the TrkB antibody is shown in SEQ ID NOs: 19-26. The HCDR1 of the TrkB antibody is shown in SEQ ID NO: 19. The HCDR2 of the TrkB antibody is shown in SEQ ID NO: 20. The HCDR3 of the TrkB antibody is shown in SEQ ID NO: 21. The LCDR1 of the TrkB antibody is shown in SEQ ID NO: 22. The LCDR2 of the TrkB antibody is shown in SEQ ID NO: 23. The LCDR3 of the TrkB antibody is shown in SEQ ID NO: 24. The VH of the TrkB antibody is shown in SEQ ID NO: 25. The VL of the TrkB antibody is shown in SEQ ID NO: 26.

[0323] WT and APP NL-G-F Organoids (day 120) were treated with vehicle (lanes 1-4) or TrkB antibody (15 nM, lanes 5-10) for 12 days and subjected to Western blotting ( FIG. 7A to FIG. 7B WT organoids (day 100) were treated with TrkB antibody (15 nM, lanes 1-3) or BDNF (5 nM, lanes 4-6) for 0, 0.5, and 2 hours and subjected to Western blotting ( FIG. 7C to FIG. 7D ).

[0324] Western blot showed that TrkB antibody rescued APP NL-G-F Synapse loss and cell death in organoids. And similar to BDNF, TrkB antibodies actively triggered all major signaling pathways.

[0325] Therefore, the APP of this application NL-G-F Organoids can be successfully used to identify substances useful in AD treatment.

[0326] Example 8 Proteomic Study of Human Brain Organoid Model of Alzheimer's Disease (AD)

[0327] Human brain organoids with AD at 3 months showed Aβ aggregation and tau tangles, which are pathological features of AD. To support the molecular mechanisms of AD pathophysiology, we performed APPNL-G-F (AD) Organoids and age-matched APP WT (Control) organoids were subjected to isobaric multiple tandem mass tag (TMT) labeling mass spectrometry (MS) analysis. To reduce heterogeneity between samples, the two organoids were combined into one sample. We used three samples in each group. Organoid samples were homogenized, digested and labeled with TMT, followed by HPLC and MS. Proteomics revealed the expression levels of 6600 proteins. Bioinformatics analysis supported the differentially expressed proteins and enriched for statistically significant biological processes. The results are shown in Figures 9 to 11 middle. Fig. 9 Shown are proteins differentially expressed in AD brain organoids at 3 months. This experiment sought to enrich for relevant biological processes based on statistically significant differentially expressed proteins. Fig.10 GSEA analysis of biological processes enriched using the KEGG database is shown. The data suggest that AD is associated with the following pathways: Alzheimer's disease, Huntington's disease, Parkinson's disease, oxidative phosphorylation, tight junctions, RNA degradation, regulation of ribosomes and actin cytoskeleton. In contrast, the ERBB signaling pathway and ubiquitin-mediated proteolysis were enriched in the control group. Fig.11 GSEA analysis of biological processes enriched using the GO database is shown. The analysis showed that AD was associated with the following pathways: protein folding, metabolic processes of carbohydrate derivatives, cellular amino acid biosynthesis processes, mitochondrial organization, oxidative phosphorylation, and biological processes involved in interaction with the host. In contrast, dendrite development and protein aggregation were enriched in the control group.

[0328] In conclusion, quantitative proteomics was performed to determine the molecular characteristics of the AD brain organoid model. A total of 6600 proteins were analyzed for GSEA analysis of differentially expressed proteins and biological processes. Based on the GSEA analysis using the KEGG annotation database, the following experimental results were obtained. First, AD organoids showed comprehensive changes similar to Alzheimer's disease and other neurodegenerative diseases, such as Huntington's disease and Parkinson's disease in protein expression profiles. Secondly, the regulation of oxidative phosphorylation, tight junctions, RNA degradation, ribosomes and actin cytoskeleton was enriched in AD organoids. Third, ERBB signaling pathways and ubiquitin-mediated proteolysis were enriched in the control group, which means that such proteolysis was downregulated in AD brain organoids. Consistent with the above results, enrichment based on GO annotations showed an upregulation of signaling pathways, which involved protein folding, carbohydrate derivative metabolic processes, cellular amino acid biosynthesis processes, mitochondrial organization, oxidative phosphorylation and biological processes involved in interacting with the host. However, dendritic development and protein aggregation were downregulated in AD brain organoids. These enriched biological processes are consistent with the results of proteomic studies using postmortem human AD brains. These data confirm that AD brain organoids reproduce the pathophysiological changes of human AD. Due to the extensive individual heterogeneity between human brain samples, the limited number of cases, and the differences in AD brain libraries, it is more difficult for the data to achieve statistical significance or maintain consistency between proteomic studies. In contrast, the AD brain organoid model exhibits higher uniformity than different individual human brain samples. In addition, AD human brain organoids reveal disease-related signaling pathways that are not captured in conventional AD mouse models. All of these features support that the molecular trajectory of AD in brain organoid models is similar to that of AD patients. Brain organoids exhibit advantages over AD mouse models because they capture stronger phenotypes in a shorter time frame. Therefore, this AD brain organoid model will allow meaningful mechanistic studies and effective drug testing and screening.

[0329] Although exemplary embodiments of the present invention have been shown and described herein, it is obvious to those skilled in the art that these embodiments are provided by way of example only. The present invention is not limited to the specific embodiments provided in this specification. Although the present invention has been described with reference to the above description, the description and explanation of the embodiments herein should not be understood in a restrictive manner. Without departing from the present invention, many changes, modifications and substitutions will occur to those skilled in the art. In addition, it should be understood that all aspects of the present invention are not limited to the specific description, configuration or relative proportion described herein, which depend on various conditions and variables. It should be understood that various alternatives of the embodiments of the present invention described herein can be adopted when implementing the present invention. Therefore, it can be expected that the present invention will also cover any such alternatives, modifications, variations or equivalents. The following claims are intended to define the scope of the present invention, and thus cover methods and structures and their equivalents within the scope of these claims.

Claims

1. An engineered human cell, wherein the APP gene is mutated to encode a mutant APP protein, and compared with the wild-type human APP protein shown in SEQ ID NO: 1, the mutant APP protein comprises amino acid substitutions at the following residues: K670, M671, I716 and / or E693.

2. The engineered human cell of claim 1, wherein the mutant APP protein comprises amino acid substitutions at the following residues: K670, M671, I716, and E693.

3. An engineered human cell according to any one of claims 1-2, wherein the mutant APP protein further comprises one or more additional mutations, and the one or more additional mutations can affect the amount of Aβ produced, the amount of Aβ fragments produced and / or the ratio of Aβ42 / Aβ40 produced.

4. The engineered human cell according to any one of claims 1-3, wherein the mutant APP protein further comprises one or more additional or alternative mutations selected from the group consisting of: I716V and V717I.

5. The engineered human cell according to any one of claims 1-4, wherein the engineered human cell is derived from a subject who has suffered from a disease or condition, is suffering from a disease or condition, or is at risk of suffering from a disease or condition.

6. The engineered human cell according to any one of claims 1 to 5, wherein the engineered human cell is a human stem cell.

7. The engineered human cells according to any one of claims 1-6, wherein the engineered human cells comprise pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells and / or neuronal stem cells.

8. The engineered human-derived cell of claim 7, wherein the embryonic stem cell is from an established cell line.

9. The engineered human cell according to any one of claims 7-8, wherein the embryonic stem cell is from the human embryonic stem cell (hESC) line H1.

10. The engineered human-derived cell according to any one of claims 7 to 9, wherein the embryonic stem cell is not obtained by a process of destroying a human embryo.

11. The engineered human cell according to any one of claims 1-10, wherein the mutant APP protein comprises a Swedish double mutation.

12. The engineered human cell of claim 11, wherein the Swedish double mutation comprises a K670N substitution and a M671L substitution.

13. The engineered human cell according to any one of claims 1-12, wherein the mutant APP protein comprises a Beyreuther / Iberian mutation.

14. The engineered human cell of claim 13, wherein the Beyreuther / Iberian mutation comprises an I716F substitution.

15. The engineered human cell according to any one of claims 1-14, wherein the mutant APP comprises an Arctic mutation.

16. The engineered human cell of claim 15, wherein the Arctic mutation comprises an E693G substitution.

17. The engineered human cell according to any one of claims 1-16, which is homozygous for one or more mutations at the following residues of the APP protein: K670, M671, I716 and / or E693.

18. The engineered human cell according to any one of claims 1 to 17, wherein the expression level of CTF-α of the engineered human cell is not significantly different from the expression level of CTF-α of the corresponding wild-type human cell.

19. The engineered human cell of any one of claims 1-18, wherein the engineered human cell has an increased expression level of CTF-β compared to the expression level of CTF-β of a corresponding wild-type human cell.

20. The engineered human cell according to any one of claims 1-19, wherein the engineered human cell has an increased expression level of Aβ42 compared to the expression level of Aβ42 of a corresponding wild-type human cell.

21. The engineered human cell according to any one of claims 1-20, wherein the ratio of Aβ42 / Aβ40 of the engineered human cell is increased compared to the ratio of Aβ42 / Aβ40 of a corresponding wild-type human cell.

22. The engineered human cell of any one of claims 1-21, wherein hyperphosphorylation of Tau is detectable.

23. The engineered human cell of claim 22, wherein the hyperphosphorylation of Tau in the engineered human cell comprises an increased phosphorylation level of Thr205 and / or Ser202 in Tau compared to the hyperphosphorylation of Tau in the corresponding wild-type human cell.

24. An engineered human cell according to any one of claims 1-23, wherein the cell is a knock-in cell or is derived from a knock-in cell, and wherein at least a portion of the endogenous APP gene is replaced by a heterologous nucleic acid sequence, which encodes at least a portion of the mutant APP protein.

25. The engineered human cell of claim 24, wherein the at least a portion of the endogenous APP gene comprises at least a portion of exon 16 and / or at least a portion of exon 17 of the endogenous APP gene.

26. The engineered human cell according to any one of claims 24-25, wherein the heterologous nucleic acid sequence encodes or comprises a mutated exon 16 and / or a mutated exon 17.

27. The engineered human cell according to any one of claims 1-26, wherein the mutant APP protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-10.

28. The engineered human cell according to any one of claims 1-27, wherein the APOE gene is mutated to encode a mutant APOE3 protein, and residue R136 in the mutant APOE3 protein is substituted compared to the wild-type human APOE3 protein shown in SEQ ID NO:

11.

29. The engineered human cell of claim 28, wherein the residue R136 in the mutant APOE3 protein is substituted by S.

30. The engineered human cell according to any one of claims 28-29, wherein the mutant APOE3 protein comprises the amino acid sequence shown in SEQ ID NO:

12.

31. The engineered human cell according to any one of claims 28-30, which is homozygous for the mutated APOE gene.

32. The engineered human cell of any one of claims 1-31, further comprising a mutation in one or more additional genes selected from the group consisting of: Tau, APOE2, APOE4, and BDNF.

33. A cell model of a dementia-related neurological disease, the cell model comprising an in vitro culture of a plurality of cells derived from the engineered human cells described in any one of claims 1-32.

34. The cell model of claim 33, wherein the dementia-related neurological disease is Alzheimer's disease.

35. The cell model according to any one of claims 33-34, wherein the plurality of cells are cultured in two dimensions.

36. The cell model according to any one of claims 33-35, wherein the plurality of cells are cultured in three dimensions.

37. The cell model according to any one of claims 33-36, wherein the cell model has one or more of the following features: 1) It is a fully humanized cell model; 2) exhibit β-amyloid accumulation; 3) showing hyperphosphorylation of Tau protein; 4) showing aggregation of Tau protein; 5) exhibited glial cell proliferation; 6) exhibit chronic neuroinflammation; 7) exhibit synaptic loss and neuronal death; 8) exhibit mitochondrial dysfunction and oxidative damage; 9) exhibit autophagy defects; 10) Demonstrates neurotransmitter imbalance; and / or 11) Demonstrates glucose metabolism dysfunction.

38. The cell model according to any one of claims 33-37, which produces amyloid plaques and Tau tangles.

39. The cell model according to any one of claims 33-38, wherein the cells are cultured in a 3D Matrigel matrix.

40. The cell model according to any one of claims 33-39, which is a brain organoid.

41. The cell model according to any one of claims 33-40, which is a forebrain organoid.

42. The cell model according to any one of claims 33-41, comprising astrocytes, neurons, neural progenitor cells and / or oligodendrocytes.

43. A method for producing engineered human cells, the method comprising: Mutations are introduced into the endogenous APP gene of human cells to produce a mutated APP gene, which encodes a mutant APP protein. Compared with the wild-type human APP protein shown in SEQ ID NO: 1, the mutant APP protein comprises amino acid substitutions at the following residues: K670, M671, I716 and / or E693.

44. The method of claim 43, wherein the mutant APP protein comprises amino acid substitutions at the following residues: K670, M671, I716, and E693.

45. according to the method described in any one of claims 43-44, wherein the introduction of mutation into the APP gene comprises knocking a heterologous nucleic acid sequence into an endogenous APP locus, wherein the knock-in replaces at least a portion of the endogenous APP gene with a heterologous nucleic acid sequence encoding at least a portion of the mutant APP protein.

46. ​​The method of claim 45, wherein the knock-in comprises contacting the genome of the human-derived cell with: 1) CRISPR-associated (Cas) proteins; and 2) one or more ribonucleic acid (RNA) sequences, wherein the one or more RNA sequences comprise: i) a sequence complementary to a target portion of the endogenous APP gene; and ii) a binding site for the Cas protein.

47. The method of any one of claims 45-46, wherein the knock-in further comprises maintaining the cell under conditions wherein the one or more RNA sequences hybridize with the target portion of the endogenous APP gene and the Cas protein cuts the endogenous APP gene nucleic acid sequence upon the hybridization of the one or more RNA sequences.

48. The method of any one of claims 46-47, wherein the Cas protein is Cas9.

49. The method of any one of claims 46-48, wherein the binding site of the Cas protein comprises a tracrRNA sequence.

50. The method of any one of claims 46-49, wherein the Cas protein is introduced into the cell in the form of a protein, a messenger RNA (mRNA) encoding the Cas protein, or a DNA encoding the Cas protein.

51. according to the method described in any one of claims 46-50, wherein the one or more RNA sequences are introduced into the cell in the form of one or more RNA molecules or one or more DNA molecules encoding the RNA sequences.

52. The method of any one of claims 46-51, wherein the one or more ribonucleic acid (RNA) sequences comprise the nucleic acid sequence shown in any one of SEQ ID NOs: 13-16.

53. The method according to any one of claims 45-52, wherein the at least a portion of the endogenous APP gene comprises at least a portion of exon 16 and / or at least a portion of exon 17 of the endogenous APP gene.

54. The method according to any one of claims 45-53, wherein the heterologous nucleic acid sequence comprises a mutated exon 16 and / or a mutated exon 17 of the human APP gene.

55. A method according to claim 54, wherein the mutation in the mutated exon 16 and / or the mutated exon 17 results in one or more amino acid substitutions in the polypeptide encoded thereby, and wherein the one or more amino acid substitutions include amino acid substitutions at the following residues: K670, M671, I716 and / or E693.

56. The method of claim 55, wherein the one or more amino acid substitutions comprise substitutions K670N, M671L, I716F and / or E693G.

57. The method according to any one of claims 43-56, wherein the mutant APP protein further comprises one or more additional mutations, which can affect the amount of Aβ produced, the amount of Aβ fragments produced and / or the ratio of Aβ42 / Aβ40 produced.

58. The method of any one of claims 43-57, wherein the mutant APP protein further comprises one or more additional or alternative mutations selected from the group consisting of: I716V and V717I.

59. The method of any one of claims 43-58, wherein the human-derived cell is derived from a subject who has had, is suffering from, or is at risk of developing a disease or disorder.

60. The method of any one of claims 43-59, wherein the human-derived cells are human stem cells.

61. The method according to any one of claims 43-60, wherein the human-derived cells are pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells and / or neuronal stem cells.

62. The method of claim 61, wherein the embryonic stem cells are from an established cell line.

63. The method of any one of claims 61-62, wherein the embryonic stem cells are from the human embryonic stem cell (hESC) line H1.

64. The method of any one of claims 61-63, wherein the embryonic stem cells are not obtained by a process that destroys a human embryo.

65. The method of any one of claims 43-64, wherein the mutant APP protein comprises a Swedish double mutation.

66. The method of claim 65, wherein the Swedish double mutation comprises a K670N substitution and a M671L substitution.

67. The method of any one of claims 43-66, wherein the mutant APP protein comprises a Beyreuther / Iberian mutation.

68. The method of claim 67, wherein the Beyreuther / Iberian mutation comprises an I716F substitution.

69. The method of any one of claims 43-68, wherein the mutant APP comprises an Arctic mutation.

70. The method of claim 69, wherein the Arctic mutation comprises an E693G substitution.

71. The method of any one of claims 43-70, wherein the engineered human cells are homozygous for one or more mutations at the following residues of the APP protein: K670, M671, I716 and / or E693.

72. The method according to any one of claims 43-71, wherein the mutant APP protein comprises the amino acid sequence shown in any one of SEQ ID NOs: 4-10.

73. The method according to any one of claims 43-72, further comprising introducing a mutation into the APOE gene to produce a mutant APOE3 protein, wherein residue R136 in the mutant APOE3 protein is substituted compared to the wild-type human APOE3 protein shown in SEQ ID NO:

11.

74. The method of claim 73, wherein the residue R136 in the mutant APOE3 protein is substituted with S.

75. The method of any one of claims 73-74, wherein the mutant APOE3 protein comprises the amino acid sequence shown in SEQ ID NO:

12.

76. The method of any one of claims 73-75, wherein the engineered human cells are homozygous for the mutated APOE gene.

77. The method of any one of claims 43-76, wherein the engineered human-derived cells further comprise mutations in one or more additional genes selected from the group consisting of: Tau, APOE2, APOE4, and BDNF.

78. An engineered human cell produced by the method of any one of claims 43-77.

79. A method for producing a cell model, the method comprising contacting the engineered human-derived cell of any one of claims 1-32 with a differentiation medium to obtain the cell model.

80. The method of claim 79, wherein the cell model is a model of a dementia-related neurological disease.

81. The method of claim 80, wherein the dementia-related neurological disease is Alzheimer's disease.

82. The method of any one of claims 79-81, wherein the engineered human cells are cultured in a two-dimensional in vitro culture.

83. The method of any one of claims 79-82, wherein the engineered human cells are cultured in a three-dimensional in vitro culture.

84. The method of any one of claims 79-83, wherein the engineered human cells are cultured in a gel, in a bioreactor, under ultra-low adhesion conditions, or on a microchip.

85. The method of any one of claims 79-84, wherein the engineered human cells are cultured in a matrix.

86. The method of claim 85, wherein the matrix is ​​an extracellular matrix and / or wherein the matrix comprises one or more of natural molecules, synthetic polymers, bio-synthetic hybrids, metals, ceramics, bioactive glasses and / or carbon nanotubes.

87. The method of any one of claims 79-86, wherein the cell model is a brain organoid.

88. The method of any one of claims 79-87, wherein the cell model is a forebrain organoid.

89. A cell model produced by the method of any one of claims 79-88.

90. A composition comprising the engineered human cell of any one of claims 1-32 or the cell model of any one of claims 33-42.

91. A kit for screening substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of dementia-related neurological diseases, the kit comprising the engineered human cells described in any one of claims 1-32 or the cell model described in any one of claims 33-42.

92. The kit of claim 91, wherein the dementia-related neurological disease is Alzheimer's disease.

93. The kit of any one of claims 91-92, wherein the substance, device and / or composition comprises a molecule, a membrane-bound vesicle and / or a cell.

94. A kit according to any one of claims 91-93, further comprising one or more additional components selected from the group consisting of assay buffers, controls, substrates, standards, detection materials, laboratory supplies, devices, machines, cells, organs, tissues, and user manuals or instructions.

95. A method for screening for substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of dementia-related neurological diseases, the method comprising: Exposing the engineered human cell of any one of claims 1 to 32 or the cell model of any one of claims 33 to 42 to a candidate substance, device and / or composition; evaluating the engineered human-derived cell or the cell model for one or more characteristics of the dementia-related neurological disease in the presence of the candidate substance, device and / or composition; and Candidate substances, devices and / or compositions are selected that are capable of preventing, inhibiting and / or ameliorating one or more of the characteristics of the dementia-related neurological disease.

96. A method of identifying potential substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of a dementia-related neurological disease, the method comprising: (i) contacting the engineered human cell of any one of claims 1 to 32 or the cell model of any one of claims 33 to 42 with a candidate substance, device and / or composition to be tested; and (ii) evaluating the activity of the candidate substance, device and / or composition on one or more features of the dementia-related neurological disease.

97. A method of designing a substance, device and / or composition useful in the treatment, prevention and / or prognosis of a dementia-related neurological disease, the method comprising the steps of: (i) exposing the engineered human cell of any one of claims 1 to 32 or the cell model of any one of claims 33 to 42 to a candidate substance, device and / or composition; (ii) evaluating the engineered human cell or the cell model for one or more characteristics of the dementia-related neurological disease; (iii) selecting candidate substances, devices and / or compositions that are capable of preventing, inhibiting and / or alleviating one or more of the characteristics of the dementia-related neurological disease; (iv) modifying the structure and / or composition of the candidate substance, device and / or composition of step (iii) to obtain a modified substance, device and / or composition having improved activity in the treatment, prevention and / or prognosis of the dementia-related neurological disease.

98. The method of any one of claims 95-97, wherein the dementia-related neurological disease is Alzheimer's disease.

99. The method of any one of claims 95-98, wherein the features of the dementia-related neurological disease include: β-amyloid accumulation; Hyperphosphorylation of Tau protein; Aggregation of Tau protein; Glial cell proliferation; Chronic neuroinflammation; Synaptic loss; Neuronal death; the appearance of amyloid plaques; The emergence of Tau tangles; Increased expression levels of CTF-β; Aβ42 expression levels increased; The ratio of Aβ42 / Aβ40 increased; mitochondrial dysfunction and oxidative damage; Autophagy defects; Neurotransmitter imbalance; and / or Dysfunction of glucose metabolism.

100. The method of claim 99, wherein the hyperphosphorylation of the Tau protein in the engineered human cells comprises an increased phosphorylation level of Thr205 and / or Ser202 in Tau compared to the hyperphosphorylation of the Tau protein in the corresponding wild-type human cells.

101. Use of the engineered human cell of any one of claims 1-32 or the cell model of any one of claims 33-42 as a model of dementia-related neurological diseases.

102. Use of the engineered human cell of any one of claims 1-32 or the cell model of any one of claims 33-42 in preparing a model of dementia-related neurological diseases.

103. The use according to any one of claims 101-102, wherein the dementia-related neurological disease is Alzheimer's disease.

104. A method of identifying potential biological targets and / or biomarkers of substances, devices and / or compositions useful in the treatment, prevention and / or prognosis of dementia-related neurological diseases, the method comprising the steps of: (i) performing quantitative proteomic, lipidomic and / or genomic comparative analysis on the engineered human cell of any one of claims 1 to 32 or the cell model of any one of claims 33 to 42 and a control human cell or a control cell model; (ii) identifying genes, proteins and / or lipids with altered sequence, amount, expression level, modification and / or activity; (iii) wherein the gene, protein and / or lipid identified in step (ii) is a potential biological target and / or biomarker of the substance, the device and / or the composition useful in the treatment, prevention and / or prognosis of the dementia-related neurological disease.

105. The method of claim 104, wherein the dementia-related neurological disease is Alzheimer's disease.

106. A method for screening biological targets and / or biomarkers useful in diagnosing and / or monitoring dementia-related neurological diseases, the method comprising determining the presence and / or level of a substance obtained from a sample of the engineered human cells described in any one of claims 1-32 or the cell model described in any one of claims 33-42 before and after detecting characteristics of the dementia-related neurological disease, and identifying a substance that exhibits a change in the presence and / or level before and after the detection.

107. The method of claim 106, wherein the dementia-related neurological disease is Alzheimer's disease.

108. The method of any one of claims 106-107, wherein the features of the dementia-related neurological disease include: β-amyloid accumulation; Hyperphosphorylation of Tau protein; Aggregation of Tau protein; Glial cell proliferation; Chronic neuroinflammation; Synaptic loss; Neuronal death; the appearance of amyloid plaques; The emergence of Tau tangles; Increased expression levels of CTF-β; Aβ42 expression levels increased; The ratio of Aβ42 / Aβ40 increased; mitochondrial dysfunction and oxidative damage; Autophagy defects; Neurotransmitter imbalance; and / or Dysfunction of glucose metabolism.

109. Use of the engineered human cells described in any one of claims 1-32 or the cell model described in any one of claims 33-42 in preparing a system for screening substances, devices, compositions, biological targets and / or biomarkers useful in the treatment, diagnosis, prevention, monitoring and / or prognosis of dementia-related neurological diseases.

110. The use according to claim 109, wherein the dementia-related neurological disease is Alzheimer's disease.

111. An engineered human cell according to any one of claims 1-32 or a cell model according to any one of claims 33-42, for screening substances, devices, compositions, biological targets and / or biomarkers useful in the treatment, diagnosis, prevention, monitoring and / or prognosis of dementia-related neurological diseases.

112. The engineered human cell or cell model of claim 111, wherein the dementia-related neurological disease is Alzheimer's disease.

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