WO2023246838A1

WO2023246838A1 - Disease model and use thereof

Info

Publication number: WO2023246838A1
Application number: PCT/CN2023/101589
Authority: WO
Inventors: Bai Lu; Hang Liu; Xiaoming Guan
Original assignee: Tsinghua University; 4B Technologies (Beijing) Co., Limited
Priority date: 2022-06-22
Filing date: 2023-06-21
Publication date: 2023-12-28
Also published as: WO2023246838A9

Abstract

Provided is a disease model and use thereof. In particular, provided is an engineered human cell with a mutated APP gene, a cellular model derived from the engineered human cell and use thereof.

Description

DISEASE MODEL AND USE THEREOF

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) is the main cause of dementia and is quickly becoming one of the most expensive, lethal, and burdening diseases worldwide. AD is now defined biologically by the presence of senile plaques and neurofibrillary tangles, progressive neuronal death and neuroinflammation. There is a dire need for disease modifying therapies that may prevent or slow the rate of disease progression, but unfortunately none are currently available and numerous phase 3 clinical trials have failed to demonstrate benefits. The potential reasons include 1) translational gaps among the animal species involved in proceeding from initial discoveries in rodent models to human studies, and 2) an unsatisfying patient stratification, meaning heterogenous subgrouping patients based on the disease severity due to the lack of phenotypic and genetic markers.

Many factors may have contributed to this but one of the major hurdles appears to be the shortage of disease models that fully recapitulate the disease pathogenesis. Animal models have played a major role in defining critical disease-related mechanisms and have been at the forefront of evaluating novel therapeutic approaches, with many treatments currently in clinical trial owing their origins to studies initially performed in rodents. Nevertheless, there are significant translational issues that have been raised lately, as there has been some potential discordance between preclinical drug studies and human clinical trials. Fundamental species-specific differences in genome and protein composition between humans and rodents, such as the difference in the number of tau isoforms, have precluded an accurate recapitulation of AD pathology.

On the other hand, in presently available human-cell derived models, ectopic expression or overexpression of APP protein has been employed. However, APP overexpression was observed to perturb axonal transport due to an 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-a and AICD) are overproduced, which may affect normal physiological functions. The use of artificial promoters often results in transgene expression in cells not necessarily identical to those expressing endogenous APP and artificial promoters may compete with endogenous promoters for common transcription factors. Sometimes, the transgene is inserted into a gene locus of the host cell, often in multi-copy manner, which may destroy the functions of endogenous genes. Accordingly, these models can hardly reveal the AD pathology in human patients.

Accordingly, there is an urgent need to develop tools and models that could more accurately recapitulate AD pathogenesis closely mimic in humans.

SUMMARY OF THE INVENTION

The inventors of the present disclosure used gene editing technology to introduce familial mutations (Swedish, Beyreuther/Iberian and/or Arctic) into APP gene of human embryonic stem cells (hESCs) which subsequently differentiated them into brain organoids to build a fully human-derived AD model. Even though the brain organoids only contain certain types of cells comprised by the human brain (for example, the brain organoids do not contain blood vessels, nor do they contain microglial cells, which are often considered important for neuronal functions) . Surprisingly, the cellular model of the present disclosure successfully recapitulated multiple AD-related phenotypes including β-amyloid accumulation, tau protein hyperphosphorylation and aggregation, glial cell proliferation, chronic neuroinflammation, synaptic loss and neuronal death. Furthermore, the inventors introduced APOE3 Christchurch (APOE3ch) mutations into the AD model of the present disclosure and found that APOE3ch APP^NL-G-F organoids (with the Swedish double mutation K670N and M671L (i.e., NL) , the Beyreuther/Iberian mutation I716F (i.e., F) and the Arctic mutation E693G (i.e., G) ) resist multiple AD related pathologies, even in the presence of a high Aβ burden. The mechanisms of such resistance were studied, and it was found that APOE3ch enhanced cellular phagocytosis and lysosomal degradation of neurotoxic substances such as tau aggregates and apoptotic cell by reducing lysosomal cholesterol burden. In conclusion, the cellular models of the present disclosure provide a novel platform for the understanding of AD and drug development. Also, the protective APOE3ch mutation was identified as a promising novel therapeutic target for AD treatment.

In diseases such as AD that are characterized by toxic protein aggregation, the presence of a true interstitial compartment is important for modeling pathology. Previous three-dimensional (3D) tissue engineering approaches have embedded neural progenitors or cell types of interest in a matrix or a scaffold. While these ingenious approaches can model AD phenotypes, they do not recapitulate spontaneous pathology resulting from endogenous cellular characteristics, but rather necessitate the overexpression of familiar AD genes. Several other studies used iPSCs derived from familial AD patients to generate brain organoids. These patients usually carry only one AD mutation or risk gene, which takes a relatively long time to develop pathological phenotype in vivo. Currently, iPSC-derived brain organoids could only be maintained in vitro for a relatively short period of 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. The pathological aggregation of Tau protein has not yet been reported in AD brain organoid model. What’s more, pathological phenotypes, such as abnormal gliosis and neuronal cell death, which appear only in the middle to late stages of Alzheimer's disease, have not been observed in any of these reports, which limits the understanding of the mechanisms of AD.

The present inventors generated knock-in hESCs that harbor Swedish、Beyreuther/Iberian and/or Arctic mutation in the APP gene to help the establishment of AD cerebral organoids model. The model successfully recapitulated several AD-related phenotypes including β-amyloid accumulation, the hyperphosphorylation and aggregation of Tau protein, glial cell proliferation, chronic neuroinflammation, synaptic loss and neuronal death, providing a promising novel tool for the investigation of AD disease mechanisms and high throughput drug screening.

A patient with familial AD Colombia mutations and Christchurch (APOE3ch) mutations was accidentally found to be able to resist the onset of AD. To investigate the potential protective role of APOE3ch, the mutations were introduced into APP^NL-G-F hESCs and they were differentiated into brain organoids. The APOE3ch APP^NL-G-F brain organoids were found to resist multiple AD related pathologies, including alleviating Tau aggregation and neuronal cell death. Mechanistically, APOE3ch can alleviate endolysosomal dysfunctions and then enhance cellular phagocytosis and degradation of neurotoxic substances such as Tau aggregates and apoptotic cell debris, which partially explains its disease modifying effects.

APOE is well known for its role in regulating cellular cholesterol metabolism. It was found that APOE3ch significantly reduced the cholesterol level in neuronal cells. Cholesterol accumulation has reported to cause damage to the endolysosomal functions in neuronal cells, especially in a lysosomal storage disease Niemann-Pick disease, type C1 (NPC1) , in which the mutations in cholesterol transporter lead to the accumulation of cholesterol in lysosomes. It was also demonstrated that the use of cyclodextrin could reduce intracellular cholesterol and in turn partially restored the acidity of lysosomes. Therefore, the present inventors also identified reducing intracellular cholesterol as a potential therapeutic strategy for the treatment of AD.

In one aspect, the present disclosure provides an engineered human cell, wherein the APP gene is mutated to encode for a mutant APP protein, comparing to a wildtype human APP protein as set forth in SEQ ID NO: 1, the mutant APP protein comprises an amino acid substitution at the following residues: K670, M671, I716 and/or E693.

In some embodiments, the mutant APP protein comprises an amino acid substitution at the following residues: K670, M671, I716 and E693.

In some embodiments, the mutant APP protein further comprises one or more additional mutations capable of affecting the amount of Aβ generated, the amount of an Aβ fragment generated, and/or the ratio of Aβ42/Aβ40 generated.

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.

In some embodiments, the engineered human cell is derived from a subject that had, is having or at the risk of developing a disease or disorder.

In some embodiments, the engineered human cell is a human stem cell.

In some embodiments, the engineered human cell comprises a pluripotent stem cell, an embryonic stem cell, an induced pluripotent stem cell, and/or a neuronal stem cell. In some embodiments, the embryonic stem cell is from an established cell line. In some embodiments, the embryonic stem cell is from the human embryonic stem cell (hESC) line H1. In some embodiments, the embryonic stem cell is not obtained via a process in which human embryos are destroyed.

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.

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

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

In some embodiments, the mutant APP comprises an Arctic mutation. In some embodiments, the Arctic mutation comprises a E693G substitution.

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

In some embodiments, the expression level of CTF-α derived from the engineered human cell is not significantly different from that of a corresponding wildtype human cell.

In some embodiments, the expression level of CTF-β derived from the engineered human cell is increased comparing to that of a corresponding wildtype human cell.

In some embodiments, the expression level of Aβ42 derived from the engineered human cell is increased comparing to that of a corresponding wildtype human cell.

In some embodiments, the ratio of Aβ42/Aβ40 derived from the engineered human cell is increased comparing to that of a corresponding wildtype human cell.

In some embodiments of the engineered human cell, hyper-phosphorylation of Tau is detectable.

In some embodiments of the engineered human cell, the hyper-phosphorylation of Tau comprises increased phosphorylation of Ser396, Thr181, Thr205 and/or Ser202 of Tau, comparing to that in a corresponding wildtype human cell.

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 part of an endogenous APP gene is substituted by a heterologous nucleic acid sequence encoding at least a part of the mutant APP protein.

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

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

In some embodiments of the engineered human cell, the mutant APP protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 4-10.

In some embodiments of the engineered human cell, the APOE gene is mutated to encode for a mutant APOE3 protein, comparing to a wildtype human APOE3 protein as set forth in SEQ ID NO: 11, residue R136 is substituted in the mutant APOE3 protein. In some embodiments, the residue R136 in the mutant APOE3 protein is substituted with S.

In some embodiments of the engineered human cell, the mutant APOE3 protein comprises an amino acid sequence as set forth in SEQ ID NO: 12.

In some embodiments, the engineered human cell, is homozygous for the mutant APOE gene.

In some embodiments, 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.

In another aspect, the present disclosure provides a cellular model of a dementia-related neurological disease, comprising an in vitro culture of a plurality of cells derived from the engineered human cell of the present disclosure.

In some embodiments of the cellular model, the dementia-related neurological disease is Alzheimer’s disease.

In some embodiments of the cellular model, the plurality of cell are cultured in two dimensions.

In some embodiments of the cellular model, the plurality of cell are cultured in three dimensions.

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

In some embodiments, the cellular model generates amyloid plaques and Tau-tangles.

In some embodiments of the cellular model, the cells are cultured in a 3D matrigel matrix.

In some embodiments, the cellular model is a brain organoid. In some embodiments, the cellular model is a forebrain organoid. In some embodiments, the cellular model comprises astrocytes, neurons, neural progenitor cells and/or oligodendrocytes. For example, in some cases, the cellular model comprises astrocytes and neurons.

In another aspect, the present disclosure provides a method for generating an engineered human cell. The method comprises introducing a mutation into an endogenous APP gene of a human cell to generate a mutated APP gene, the mutated APP gene encodes for a mutant APP protein, comparing to a wildtype human APP protein as set forth in SEQ ID NO: 1, the mutant APP protein comprises an amino acid substitution at the following residues: K670, M671, I716 and/or E693.

In some embodiments of the method, the mutant APP protein comprises an amino acid substitution at the following residues: K670, M671, I716 and E693.

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

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

In some embodiments of the method, the knocking-in further comprises maintaining the cell under conditions in which 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.

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

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

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.

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.

In some embodiments of the method, the one or more ribonucleic acid (RNA) sequences comprise a nucleic acid sequence as set forth in any one of SEQ ID NO: 13-16.

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

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.

In some embodiments of the method, mutations in the mutated exon 16 and/or the mutated exon 17 results in one or more amino acid substitutions in their encoded polypeptides and wherein the one or more amino acid substitutions comprise an amino acid substitution at the following residues: K670, M671, I716 and/or E693.

In some embodiments of the method, the one or more amino acid substitutions comprise the substitution K670N, M671L, I716F and/or E693G.

In some embodiments of the method, the mutant APP protein further comprises one or more additional mutations capable of affecting the amount of Aβ generated, the amount of an Aβ fragment generated, and/or the ratio of Aβ42/Aβ40 generated.

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 and E693G.

In some embodiments of the method, the human cell is derived from a subject that had, is having or at the risk of developing a disease or disorder.

In some embodiments of the method, the human cell is a human stem cell.

In some embodiments of the method, the human cell is a pluripotent stem cell, an embryonic stem cell, an induced pluripotent stem cell, and/or a neuronal stem cell.

In some embodiments of the method, the embryonic stem cell is from an established cell line.

In some embodiments of the method, the embryonic stem cell is from the human embryonic stem cell (hESC) line H1.

In some embodiments of the method, the embryonic stem cell is not obtained via a process in which human embryos are destroyed.

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

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

In some embodiments of the method, the mutant APP protein comprises a Beyreuther/Iberian mutation. In some embodiments of the method, the Beyreuther/Iberian mutation comprises an I716F substitution.

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

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

In some embodiments of the method, the mutant APP protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 4-10.

In some embodiments, the method further comprises introducing a mutation in the APOE gene to generate a mutant APOE3 protein, comparing to a wildtype human APOE3 protein as set forth in SEQ ID NO: 11, residue R136 is substituted in the mutant APOE3 protein.

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

In some embodiments of the method, the mutant APOE3 protein comprises an amino acid sequence as set forth in SEQ ID NO: 12.

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.

In another aspect, the present disclosure provides an engineered human cell, generated by the method of the present disclosure.

In another aspect, the present disclosure provides a method for generating a cellular model, comprising contacting the engineered human cell of the present disclosure with a differentiation medium to obtain the cellular model.

In some embodiments of the method, the cellular model is a model of a dementia-related neurological disease.

In some embodiments of the method, the dementia-related neurological disease is Alzheimer’s disease.

In some embodiments of the method, the engineered human cell is cultured in a two-dimensional in vitro culture. In some embodiments of the method, the engineered human cell is cultured in a three-dimensional in vitro culture.

In some embodiments of the method, the engineered human cell is cultured in a gel, in a bioreactor, under ultra-low adhesion conditions or on a microchip.

In some embodiments of the method, the engineered human cell is 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 glasses and/or carbon nanotubes.

In some embodiments of the method, the cellular model is a brain organoid. In some embodiments of the method, the cellular model is a forebrain organoid.

In another aspect, the present disclosure provides a cellular model, generated by the method of the present disclosure.

In another aspect, the present disclosure provides a composition, comprising the engineered human cell, or the cellular model of the present disclosure.

In another aspect, the present disclosure provides a kit for screening a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease, comprising the engineered human cell or the cellular model of the present disclosure.

In some embodiments of the kit, the dementia-related neurological disease is Alzheimer’s disease.

In some embodiments of the kit, the substance, device and/or composition comprises a molecule, a membrane-bound vesicle, and/or a cell.

In some embodiments, the kit further comprises one or more additional components selected from the group consisting of: an assay buffer, a control, a substrate, a standard, a detection material, a laboratory supply, a device, a machine, a cell, an organ, a tissue, and a user manual or instruction.

In another aspect, the present disclosure provides a method for screening a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease. The method comprises: exposing the engineered human cell or the cellular model of the present disclosure to a candidate substance, device, and/or composition; assessing the engineered human cell or the cellular model for one or more feature of the dementia-related neurological disease in the presence of the candidate substance, device, and/or composition; and selecting a candidate substance, device, and/or composition capable of preventing, inhibiting and/or reducing one or more the feature of the dementia-related neurological disease.

In another aspect, the present disclosure provides a method for identifying a potential substance, device, and/or composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease, comprising: (i) contacting the engineered human cell or the cellular model of the present disclosure with a candidate substance, device, and/or composition to be tested; and (ii) assessing the activity of the candidate substance, device, and/or composition on one or more feature of the dementia-related neurological disease.

In another aspect, the present disclosure provides a method for designing a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease. The method comprises the steps of: (i) exposing the engineered human cell or the cellular model to a candidate substance, device, and/or composition; (ii) assessing the engineered human cell or the cellular model for one or more feature of the dementia-related neurological disease; (iii) selecting a candidate substance, device, and/or composition capable of preventing, inhibiting and/or reducing one or more the feature 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 with improved activity in the treatment, prevention and/or prognosis of the dementia-related neurological disease.

In some embodiments of the method, the feature of the dementia-related neurological disease comprises: β-amyloid accumulation; hyperphosphorylation of Tau protein; aggregation of Tau protein; glial cell proliferation; chronic neuroinflammation; synaptic loss; neuronal death; appearance of amyloid plaques; 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 deficit; neurotransmitter imbalance; and/or dysfunctional glucose metabolism.

In some embodiments of the method, the hyper-phosphorylation of Tau protein comprises increased phosphorylation of Ser396, Thr181, Thr205 and/or Ser202 of Tau, comparing to that in a corresponding wildtype human cell.

In another aspect, the present disclosure provides use of the engineered human cell or the cellular model of the present disclosure as model of a dementia-related neurological disease.

In another aspect, the present disclosure provides use of the engineered human cell or the cellular model of the present disclosure in the preparation of a model of a dementia-related neurological disease.

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

In another aspect, the present disclosure provides a method for identifying a potential biological target and/or biomarker of a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease. The method comprises the steps of: (i) making a quantitative proteomic, lipidomic and/or genomic comparative analysis of the engineered human cell or the cellular model of the present disclosure with a control human cell or a control cellular model; (ii) identifying a gene, a protein and/or a lipid with an altered sequence, quantity, 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.

In some embodiments of the method, the dementia-related neurological disease is Alzheimer's disease.

In another aspect, the present disclosure provides a method of screening for a biological target and/or biomarker useful in the diagnosis and/or monitoring of a dementia-related neurological disease, comprising determining a presence and/or a level of a substance in a sample obtained from the engineered human cell or from the cellular model of the present disclosure both before and after detection of a feature of the dementia-related neurological disease and identifying a substance showing a change of the presence and/or level before and after the detection.

In another aspect, the present disclosure provides use of the engineered human cell or the cellular model of the present disclosure in the preparation of a system for screening a substance, a device, a composition, a biological target and/or a biomarker useful in the treatment, diagnosis, prevention, monitoring and/or prognosis of a dementia-related neurological disease. In some embodiments of the use, the dementia-related neurological disease is Alzheimer's disease.

In another aspect, the present disclosure provides the engineered human cell or the cellular model of the present disclosure, for use in the screening for a substance, a device, a composition, a biological target and/or a biomarker useful in the treatment, diagnosis, prevention, monitoring and/or prognosis of a dementia-related neurological disease. In some embodiments, the dementia-related neurological disease is Alzheimer's disease.

Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure. Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.

INCORPORATION BY REFERENCE

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 DRAWING

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are employed, and the accompanying drawings (also “FIG. ” , “Fig. ” and “FIG. ” herein) , of which:

FIGs. 1A–1G illustrate increased accumulation of extracellular amyloid-β deposits in APP^NL-G-F Knock-in cerebral organoids.

FIGs. 2A-2J illustrate Tau pathology in APP^NL-G-F cerebral organoids.

FIGs. 3A-3E illustrate enhanced gliosis and neuroinflammation APP^NL-G-F cerebral organoids.

FIGs. 4A-4H illustrate increased synaptic loss, apoptosis and necrosis in APP^NL-G-F cerebral organoids.

FIGs. 5A-5H illustrate that APOE3ch alleviates multiple AD-related phenotypes in APP^NL-G-F cerebral organoids.

FIGs. 6A-6H illustrate that APOE3ch activates endolysosomal functions in APP^NL-G-F cerebral organoids.

FIGs. 7A-7D illustrate the effects of the TrkB antibody using the APP^NL-G-F cerebral organoids.

FIG. 8 illustrates the knock-in targeting strategy.

FIG. 9 illustrates the differentially expressed proteins in the AD cerebral organoid at 3 months. Red (right) , increased proteins in the AD group; blue (left) , decreased proteins in the AD group. -log10 (p. adj) >0.5, Fold change>1.

FIG. 10 illustrates the GSEA analyses using KEGG database enriched biological processes. NES>0 stands for the enriched pathways in the AD group; NES<0 stands for the enriched pathways in the control group.

FIG. 11 illustrates the GSEA analyses using GO database enriched biological processes. NES>0 stands for the enriched pathways in the AD group; NES<0 stands for the enriched pathways in the control group.

DETAILED DESCRIPTION

While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

As used herein, the term “CRISPR” generally refers to Clustered Regularly Interspaced Short Palindromic Repeats. The CRISPR loci usually differs from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) . Generally, the repeats are short elements that appear in regularly spaced clusters with unique intervening sequences of a substantially constant length. The repeat sequences are highly conserved between strains, but the number of interspersed repeats and the sequences of the spacer regions usually differ from strain to strain.

As used herein, the terms “sgRNA” , “guide RNA” , “single guide RNA” and “synthetic guide RNA” are interchangeable and generally refer to the polynucleotide sequence comprising the guide sequence. The guide sequence is about 20 bp and is within the guide RNA that specifies the target site.

As used herein, the term “heterologous nucleic acid sequence” generally refers to a nucleic acid sequence derived from a foreign source and/or present in a non-endogenous form. For example, a heterologous nucleic acid sequence may originate from a foreign subject, may originate from a foreign species, may be artificially synthesized, may be positioned in a foreign locus and/or may be substantially modified.

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 molecules of DNA known as homologous sequences or homologous arms.

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

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 immunity system found in certain bacteria, such as Streptococcus pyogenes and other bacteria. For example, a Cas9 protein may comprise not only the wildtype Cas9 found in Streptococcus pyogenes, but also its various variants, such as those described in WO2013/176772A1. In some embodiments, a Cas9 protein may comprise a Cas9 sequence from S. pyogenes, N. meningitidis, S. thermophilus and T. denticola, as described in Esvelt et al., Nature Methods, 10 (11) : 1116-1121, 2013.

As used herein, the term “Cas9 coding sequence” generally refers to a polynucleotide sequence capable of being transcribed and/or translated, according to a genetic code functional in a host cell/host animal, to produce a Cas9 protein. The Cas9 coding sequence may be a DNA (such as a plasmid) or an RNA (such as an mRNA) .

As used herein, the term “Cas9 riboprotein” generally refers to a protein/RNA complex consisting of Cas9 protein and an associated guide RNA.

As used herein, the term “CRISPR/Cas9 system” generally refers to a tool for site-specific genomic targeting in an organism. For example, it may be a type II CRISPR/Cas system, which is a prokaryotic adaptive immune response system that uses non-coding RNAs to guide the Cas9 nuclease to induce site-specific DNA cleavage. This DNA damage is repaired by cellular DNA repair mechanisms, either via the non-homologous end joining DNA repair pathway (NHEJ) or the homology directed repair (HDR) pathway. The CRISPR/Cas9 system may be harnessed to create a simple, RNA- programmable method to mediate genome editing in mammalian cells and may be used to generate gene knockouts (via insertion/deletion) or knock-ins (via HDR) .

As used herein, the term “knocking-in” or “knock in” generally refers to a genetic engineering process that involves the one-for-one substitution of DNA sequence information in a genetic locus or the insertion of sequence information not found within the endogenous locus. Knocking-in may involve a gene inserted into a specific locus and may thus be a “targeted” insertion.

As used herein, the term “vector” generally refers to a DNA molecule used as a vehicle to artificially carry foreign genetic material into another cell or host, where it can be replicated and/or expressed.

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 for substituting an endogenous DNA fragment.

As used herein, the term “embryonic stem cell” or “ES cell” generally refers to a pluripotent stem cell derived from the inner cell mass (ICM) of a blastocyst (an early-stage preimplantation embryo of a mammal) , that can be cultured after an extended period in vitro, before it is inserted/injected into the cavity of a normal blastocyst and be induced to resume a normal program of embryonic development to differentiate into various cell types of an adult mammal, including germ cells.

As used herein, the term “homozygote” or “homozygous” is used with respect to a particular 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 both homologous chromosomes have the same alleles or copies of the gene/DNA.

As used herein, the term “heterozygote” or “heterozygous” is used with respect to a particular 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 the two homologous chromosomes have different alleles/copies/versions of the gene or DNA.

As used herein, the term “mutated APP protein” or “mutant APP protein” generally refers to an amyloid precursor protein that has an addition, a deletion and/or a substitution of at least one amino acid residue.

As used herein, the term “Swedish double mutation” or “Swedish mutation” generally refers to a double mutation in the APP gene originally found in a Swedish family, which is located before the amyloid β-peptide (Aβ) region of APP and results in an increased production and secretion of Aβ, as described in Nat Genet. 1992 Aug; 1 (5) : 345-7. The Swedish double mutation is located in exon 16 of the human APP gene and is the only known mutation immediately adjacent to the β-secretase site in APP. In some cases, the Swedish mutation results in a substitution of two amino acids, lysine (K) 670 and methionine (M) 671. The Swedish double mutation has been shown to increase total Aβ levels. Specifically, there is increased production and secretion of Aβ40 and Aβ42, but the ratio of Aβ40/Aβ42 is generally not affected. In some embodiments, the Swedish double mutation comprises the amino acid substitutions K670N and M671L.

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 may affect γ-secretase cleavage specificity and cause a dramatic increase in the Aβ42/Aβ40 ratio. In some embodiments, the Beyreuther/Iberian mutation comprises the amino acid substitution I716F.

As used herein, the term “Arctic mutation” generally refers to a mutation in the APP gene (e.g., at residue E693) that leads to an increased propensity and faster rate of Aβ40 protofibril formation. It is also known as “E22G” , because it affects the twenty-second amino acid of Aβ peptides. The Arctic mutation was one of several pathogenic APP mutations found to confer resistance to neprilysin-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.

As used herein, the term “not significantly different” generally refers to that the difference between two values or two objects are not substantial. For example, when two values are compared, a difference of 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 may be regarded as not significant different.

As used herein, the term “Aβ oligomers” generally refers to soluble amyloid β (Aβ) peptide aggregates, which normally form small clumps. An Aβ oligomer may be a dimer, a trimer, or other multimers of the Aβ peptide.

As used herein, the term “Aβ plaque” or “Amyloid plaque” generally refers to fibrillar aggregates of Aβ peptides (e.g., Aβ42 and/or Aβ40) , wherein many copies of the Aβ peptides stick together to form fibrils or fibrous deposits (e.g., plaques) .

As used herein, the term “substantial accumulation of Aβ peptide” generally refers to that formation of Aβ oligomers or Amyloid plaques may be detected using commonly employed detection methods or tools, such as specific Aβ antibody staining.

As used herein, the term “Aβ” or “Amyloid-β” generally refers to Amyloid-β peptides produced from the regulated intramembrane proteolysis of the amyloid precursor protein (APP) . Sequential proteolytic cleavage events by β-and γ-secretase generate Aβ peptides of varying lengths, including Aβ40 and Aβ42. Its two extra hydrophobic residues give Aβ42 a higher propensity to aggregate into soluble oligomers and insoluble deposits than Aβ40 or the range of shorter peptides that have been observed in recent years by mass spectrometry analysis of cerebral spinal fluid (CSF) . Multiple aggregated forms of Aβ exist, from dimers to β-pleated sheet fibrils in compact neuritic plaques. Excess amounts of Aβ can induce a variety of pathologic processes. Aβ can impair neuronal and glial function, synaptic physiology, neurotransmission and cognition. Evidence points to transcellular spread and templated seeding and the resulting deposition of aggregated Aβ into extraneuronal amyloid plaques is a pathological hallmark of AD.

As used herein, the term “hyper-phosphorylated” or “hyper-phosphorylation” refers to a state of being abnormally phosphorylated at one or more additional sites. For example, phosphorylation of the protein tau was found to negatively regulate its activity in promoting microtubule assembly, and abnormally hyperphosphorylated tau has been considered to be the major component of PHFs in AD. Normal brain tau contains 2–3 moles of phosphate per mole tau. Studies on human brain biopsy tissue indicated that several serine and threonine residues of tau are normally phosphorylated at low substoichiometrical levels. The phosphorylation level of tau isolated from autopsied AD brain is 3-to 4-fold higher than that of normal human brains. Tau phosphorylation at different sites has a different impact on its biological function and on its pathogenic role. Studies of the binding between hyperphosphorylated tau and normal tau suggest that Ser199/Ser202/Thr205, Thr212, Thr231/Ser235, Ser262/Ser356, and Ser422 are among the critical phosphorylation sites that convert tau to an inhibitory molecule that sequesters normal microtubule-associated proteins from microtubules.

As used herein, the term “neuronal loss” generally refers to a reduction in the amount or function of neuron cells in an organism. Neuronal loss may be revealed as death of neuron cells.

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

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 functional abilities. These domains are equivalent to the NINCDS-ADRDA Alzheimer's Criteria as listed in the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV-TR) published by the American Psychiatric Association.

As used herein, the term “apoptosis” generally refers to a genetically directed process of cell self-destruction that is marked by the fragmentation of nuclear DNA, which may be activated either by the presence of a stimulus or removal of a suppressing agent or stimulus. Apoptosis is also known as cell suicide, programmed cell death. Bcl-2 Family Proteins are among the main intracellular regulators of apoptosis. The Bcl-2 family of intracellular proteins helps regulate the activation of procaspases. Some members of the Bcl-2 family promote procaspase activation and cell death. For example, the apoptosis promoter Bad functions by binding to and inactivating the death-inhibiting members of the family, whereas others, like Bax and Bak, stimulate the release of cytochrome c from mitochondria. Bax and Bak are themselves 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. Sequential activation of caspases plays a central role in the execution-phase of cell apoptosis. Caspases exist as inactive proenzymes that undergo proteolytic processing at conserved aspartic residues to produce two subunits, large and small, that dimerize to form the active enzyme. Caspase-3 is the predominant caspase involved in the cleavage of amyloid-beta 4A precursor protein (also known as APP) , which is associated with neuronal death in Alzheimer's disease. Increased level of procaspase 3 ( “Pro-caspase3” ) and its cleaved form ( “Cl-caspase3” ) is often associated with increased apoptosis.

As used herein, the term “necrosis” generally refers to a form of cell injury which results in the premature death of cells in living tissue by autolysis. The signaling pathway responsible for carrying out necrosis or necroptosis is generally understood. Production of TNFα during viral infection leads to stimulation of its receptor TNFR1. The TNFR-associated death protein TRADD signals to RIPK1 which recruits RIPK3 forming the necrosome. Phosphorylation of MLKL ( “pMLKL” ) by the ripoptosome drives oligomerization of MLKL, allowing MLKL to insert into and permeabilize plasma membranes and organelles. Integration of MLKL leads to the inflammatory phenotype and release of damage-associated molecular patterns (DAMPs) , which elicit immune responses. Specifically, necroptosis, a programmed form of necrosis, is executed by the mixed lineage kinase domain-like (MLKL) protein, which is triggered by receptor-interactive protein kinases (RIPK) 1 and 3. It has been found that necroptosis was activated in postmortem human AD brains, positively correlated with Braak stage, and inversely correlated with brain weight and cognitive scores. In addition, it has been found that the set of genes regulated by RIPK1 overlapped significantly with multiple independent AD transcriptomic signatures, indicating that RIPK1 activity could explain a substantial portion of transcriptomic changes in AD.

As used herein, the term “gliosis” generally refers to a fibrous proliferation of glial cells in injured areas of the central nervous system (CNS) . Gliosis is prevalent in glioma as well as in many other neurological disorders, such as Alzheimer’s disease, and may be detected by elevated glial fibrillary acidic protein (GFAP) levels in postmortem tissue samples using immunohistochemistry. Normally, gliosis is a combination of astrocytosis and microgliosis.

As used herein, the term “synaptic degeneration” generally refers to loss or dysfunction of synapses, it may be reflected by the state/expression of synaptic marker and/or postsynaptic densities. The postsynaptic density (PSD) is a protein dense specialization attached to the postsynaptic membrane. PSDs were originally identified by electron microscopy as an electron-dense region at the membrane of a postsynaptic neuron. The PSD is in close apposition to the presynaptic active zone and ensures that receptors are in close proximity to presynaptic neurotransmitter release sites. For example, hollowing or swelling of PSDs may indicate synaptic degeneration. Synaptic state may be detected by examining the expression or level of Synaptophysin. Synaptophysin has been reported to be an integral membrane glycoprotein found in many types of active neurons and has been found in the membrane after stimulation of the neurons.

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 (e.g., a receiving nucleic acid molecule) .

As used herein, the term “hybridize to” or “hybridization” , when used in the context of molecular biology, generally refers to a process in which single-stranded deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) molecules anneal to complementary DNA or RNA.

As used herein, the term “induced pluripotent stem cell” or “iPS cell” or “iPSC” generally refers to a cell taken from any tissue (usually skin or blood) from a child or adult and is genetically modified to behave like an embryonic stem cell. As the name implies, these cells are pluripotent, which means that they have the ability to form most, if not all, adult cell types.

As used herein, the term “tracrRNA” generally refers to trans-activating crRNA (tracrRNA) , which is a small trans-encoded RNA. TracrRNA is complementary to, base pairs with a pre-crRNA forming an RNA duplex. This is cleaved by RNase III, an RNA-specific ribonuclease, to form a crRNA/tracrRNA hybrid. This hybrid acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid.

As used herein, the term “APOE” generally refers to Apolipoprotein E, whose primary function is to transport lipids in peripheral tissues and in the brain. APOE has three common isoforms: APOE2, APOE3 and APOE4. APOE ε4 has been reported to be the strongest genetic risk factor for AD and APOE ε2 is associated with a decreased risk of AD relative to the common APOE ε3 allele. In the CNS, APOE is abundantly expressed in astrocytes. Once APOE has been secreted from the cells, the cell-surface ATP-binding cassette transporters ABCA1 and ABCG1 transfer cholesterol and phospholipids to nascent APOE to form lipoprotein particles. APOE4 allele leads to a change of cysteine to arginine at position 112 and is associated with a 5-fold increase in AD risk in single allele carriers, reaching a 20-fold increase in homozygote carriers. APOE2 leads to an amino acid change of arginine to cysteine at position 158 and is protective for AD whereas the APOE3 allele is thought to be neutral (Corder et al, Nat Genet (7) 180-184, 1994; Hauser et al, Cure Alzheimer Res (10) ; 808-817, 2013) . APOE impacts amyloid production, aggregation, and clearance, is a component of amyloid plaques, and exacerbates tau-mediated neurodegeneration. APOE alleles also regulate lipid metabolism and cardiovascular risk. About 5-10%of APOE2 homozygote individuals develop hyperlipoproteinemia type III (HLP III) , whereas other APOE rare variants are linked to autosomal dominant HLP III. HLP III is characterized by increased plasma cholesterol and triglycerides levels and by the presence of tuberous or striated palmar xanthomas. APOE3ch (which refers to the APOE3 Christchurch (R136S) mutation) is associated with a profound resistance to the clinical onset of Alzheimer’s disease, and that the R136S mutation significantly diminishes the ability of ApoE to bind heparan sulfate proteoglycans (HSPG) /heparin.

As used herein, the term “subject” includes living organisms in which a disease or disorder may occur. 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) ) , as well as avian (e.g. chickens, ducks, Peking ducks, geese) , and transgenic species thereof. In some cases, the subject is a human or a non-human primate (e.g., chimpanzee, monkey, macaque, gorilla) . In certain cases, the subject is a human being.

As used herein, the term “organoid” generally refers to a miniature form of a tissue that is generated in vitro and exhibits endogenous three-dimensional organ architecture.

In the present disclosure, when referring to an amino acid substitution, “XnY” means that the amino acid X at residue n is substituted by the amino acid Y.

Unless otherwise specified, “a” , “an” , “the” and “at least one” are used interchangeably and refer to one or more than one.

In the present disclosure, the term “comprise” also encompasses “is” , “has” and “consist of” . For example, “a composition comprising X and Y” may be understood to encompass a composition that comprises at least X and Y. It shall also be understood to disclose a composition that only comprises X and Y (i.e., a composition consisting of X and Y) .

As used herein, the term “BDNF” generally refers to Brain Derived Neurotrophic Factor. Human BDNF is also referred to as P23560 in UniProtKB/Swiss-Prot. The BDNF gene encodes a member of the nerve growth factor family of proteins. Alternative splicing results in multiple transcript variants, at least one of which encodes a preproprotein that is proteolytically processed to generate the mature protein. Binding of this protein to its cognate receptor promotes neuronal survival in the adult brain. Expression of this gene is reduced in Alzheimer's , Parkinson's , and Huntington's disease patients. This gene may play a role in the regulation of the stress response and in the biology of mood disorders.

Engineered human cell and methods for generating such cells

In one aspect, the present disclosure provides an engineered human cell. The endogenous APP gene of the human cell is mutated to encode for a mutant APP protein, comparing to a wildtype human APP protein as set forth in SEQ ID NO: 1, said mutant APP protein comprises an amino acid substitution at the following residues: K670, M671, I716 and/or E693.

In another aspect, the present disclosure provides a method for generating an engineered human cell. The method comprises: introducing a mutation into an endogenous APP gene of a human cell to generate a mutated APP gene, the mutated APP gene encodes for a mutant APP protein. Comparing to a wildtype human APP protein as set forth in SEQ ID NO: 1, the mutant APP protein may comprise an amino acid substitution at the following residues: K670, M671, I716 and/or E693.

In some embodiments of the method for generating the engineered human cell, introducing the mutation into the APP gene comprises knocking-in a heterologous nucleic acid sequence into an endogenous APP gene locus, wherein said knocking-in substitutes at least a part of the endogenous APP gene with a heterologous nucleic acid sequence encoding at least a part of said mutant APP protein.

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.

In some embodiments, comparing to the wildtype human APP protein as set forth 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.

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 a E693G substitution.

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

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

The mutant APP protein may further comprise one or more additional mutations capable of affecting the amount of Aβ generated, the amount of an Aβ fragment generated, and/or the ratio of Aβ42/Aβ40 generated. For example, the mutant APP protein may further comprise one or more additional or alternative mutations selected from the group consisting of: I716V and V717I.

The human cell to be engineered may be taken from or may be derived from a subject. The subject may be a patient having a disease or disorder. In some cases, the subject had a disease or disorder before. In some cases, the subject is at the risk of developing a disease or disorder. The disease or disorder may be a neurological disease or disorder. In some cases, the disease or disorder is not a neurological disease or disorder, but a metabolic disease or disorder (e.g., diabetes) .

The human cell may be a human stem cell. For example, the human cell may be or may comprise a pluripotent stem cell, an embryonic stem cell, an induced pluripotent stem cell, and/or a neuronal stem cell. The embryonic stem cell may be from an established cell line (such as the human embryonic stem cell (hESC) line H1) . For example, the embryonic stem cell is not obtained via a process in which human embryos are destroyed.

In the engineered human cell of the present disclosure, the expression level of CTF-α may be not significantly different from that of a corresponding wildtype human cell. In some cases, in the engineered human cell of the present disclosure, the expression level of CTF-β may be increased (e.g., may be increased by about 5%, by about 10%, by about 15%, by about 20%, by about 25%, by about 30%, by about 35%, by about 40%, by about 45%, by about 50%, by about 55%, by about 60%, by about 65%, by about 70%, by about 75%, by about 80%, by about 85%, by about 90%, by about 95%, by about 100%, by 2 folds, by 3 folds, by 4 folds, by 5 folds, or more) comparing to that of a corresponding wildtype human cell. In some cases, in the engineered human cell of the present disclosure, the expression level of Aβ42 may be increased (e.g., by about 5%, by about 10%, by about 15%, by about 20%, by about 25%, by about 30%, by about 35%, by about 40%, by about 45%, by about 50%, by about 55%, by about 60%, by about 65%, by about 70%, by about 75%, by about 80%, by about 85%, by about 90%, by about 95%, by about 100%, by 2 folds, by 3 folds, by 4 folds, by 5 folds, or more) comparing to that of a corresponding wildtype human cell. In some cases, in the engineered human cell of the present disclosure, the ratio of Aβ42/Aβ40 may be increased (e.g., by about 5%, by about 10%, by about 15%, by about 20%, by about 25%, by about 30%, by about 35%, by about 40%, by about 45%, by about 50%, by about 55%, by about 60%, by about 65%, by about 70%, by about 75%, by about 80%, by about 85%, by about 90%, by about 95%, by about 100%, by 2 folds, by 3 folds, by 4 folds, by 5 folds, or more) comparing to that of a corresponding wildtype human cell. In some embodiments, in the engineered human cell of the present disclosure, hyper-phosphorylation of Tau is detectable. In some embodiments, the hyper-phosphorylation of Tau comprises increased phosphorylation of Thr205 and/or Ser202 of Tau, comparing to that in a corresponding wildtype human cell.

The engineered human cell of the present disclosure may be a knock-in cell or may be derived from a knock-in cell.

In some embodiments, at least a part of the endogenous APP gene may be substituted by a heterologous nucleic acid sequence encoding at least a part of the mutant APP protein of the present disclosure. In some cases, the at least part of the endogenous APP gene comprises at least a part of exon 16 of the endogenous APP gene. In some cases, the at least part of the endogenous APP gene comprises at least a part of exon 17 of the endogenous APP gene. In some cases, the at least part of the endogenous APP gene comprises at least a part of exon 16 and at least a part of exon 17 of the endogenous APP gene. In some cases, the heterologous nucleic acid sequence encodes for a mutated exon 16 of APP. In some cases, the heterologous nucleic acid sequence comprises sequences of a mutated exon 16 of APP. In some cases, the heterologous nucleic acid sequence encodes for a mutated exon 17 of APP. In some cases, the heterologous nucleic acid sequence comprises sequences of a mutated exon 17 of APP. In some cases, the heterologous nucleic acid sequence encodes for a mutated exon 16 and a mutated exon 17 of APP. In some cases, the heterologous nucleic acid sequence comprises sequences of a mutated exon 16 and sequences of a mutated exon 17 of APP. The sequences comprised in the heterologous nucleic acid may contain part of the sequences of the mutated exon 16 (e.g., the mutation containing portion) and/or may contain part of the sequences of the mutated exon 17 (e.g., the mutation containing portion) . In some embodiments, mutations in the mutated exon 16 and/or the mutated exon 17 of APP result in one or more amino acid substitutions in their encoded polypeptides and wherein said one or more amino acid substitutions comprise an amino acid substitution at the following residues: K670, M671, I716 and/or E693. In some embodiments, the one or more amino acid substitutions comprise the substitution K670N, M671L, I716F and/or E693G.

For example, the heterologous nucleic acid may comprise a sequence as set forth in any one of SEQ ID NO: 2-3.

In some cases, the mutant APP protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 4-10.

In some cases, in the engineered human cell of the present disclosure, the APOE gene is mutated to encode for a mutant APOE3 protein. Comparing to a wildtype human APOE3 protein as set forth in SEQ ID NO: 11, residue R136 is substituted in the mutant APOE3 protein. In some cases, in the mutant APOE3 protein, the residue R136 is substituted with S. In some cases, the mutant APOE3 protein comprises an amino acid sequence as set forth in SEQ ID NO: 12. In some cases, the mutant APOE gene encodes for APOE3ch.

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.

In some cases, the engineered human cell of the present disclosure may have altered expression of Tau, APOE2, APOE4 and/or BDNF. In some embodiments, the engineered human cell has increased expression of Tau, APOE2, APOE4 and/or BDNF. In some embodiments, the engineered human cell has decreased expression of Tau, APOE2, APOE4 and/or BDNF. In some embodiments, the engineered human cell expresses an altered form or a mutated form of Tau, APOE2, APOE4 and/or BDNF.

In some cases, the engineered human cell may comprise a mutation in one or more additional genes selected from the group consisting of: Tau, APOE2, APOE4 and BDNF. For example, such a mutation may lead to increased expression or decreased expression of Tau, APOE2, APOE4 and/or BDNF. In some cases, such a mutation may lead to expression of a mutated form of Tau, APOE2, APOE4 and/or BDNF.

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

In some embodiments, said one or more ribonucleic acid (RNA) sequences comprise a sequence as set forth in any one of SEQ ID NO: 13-16.

In some embodiments, said knocking-in further comprises maintaining the cell under conditions in which 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 said hybridization of said one or more RNA sequences.

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.

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.

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 said RNA sequences.

In some embodiments, the method may further comprise introducing a mutation in the APOE gene to generate the mutant APOE3 protein of the present disclosure. For example, introducing the mutation into the APOE gene may comprise knocking-in a heterologous nucleic acid sequence into an endogenous APOE gene locus, wherein said knocking-in substitutes at least a part of the endogenous APOE gene with a heterologous nucleic acid sequence encoding at least a part of said mutant APOE3 protein. In some embodiments of the method, said knocking-in comprises contacting the genome of the human cell with gene-editing components in the presence of a donor nucleic acid molecule comprising said heterologous nucleic acid sequence. The gene-editing components may comprise: 1) a CRISPR associated (Cas) protein; and 2) one or more ribonucleic acid (RNA) sequences that comprise i) a sequence complementary to a target portion of the endogenous APOE gene, and ii) a binding site for the Cas protein. In some embodiments, the binding site for the Cas protein comprises a tracrRNA sequence. In some embodiments, said one or more ribonucleic acid (RNA) sequences comprise a sequence as set forth in any one of SEQ ID NO: 17-18.

In another aspect, the present disclosure provides an engineered human cell that is generated by the method of the present disclosure.

In the method of the present disclosure for generating the engineered cell, nuclease agents may be utilized to aid in the modification of the target APP gene locus and/or the APOE gene locus. Such a nuclease agent may promote homologous recombination between the donor nucleic acid molecule and the target genomic locus. In some embodiments, the nuclease agent comprises an endonuclease agent.

As used herein, the term “recognition site for a nuclease agent” generally refers to a DNA sequence at which a nick or double-strand break may be induced by a nuclease agent. The recognition site for a nuclease agent can be endogenous (or native) to the cell or the recognition site can be exogenous to the cell. In some embodiments, the recognition site may be exogenous to the cell and thereby is not naturally occurring in the genome of the cell. In further embodiments, the exogenous or endogenous recognition site may be present only once in the genome of the host cell. In specific embodiments, an endogenous or native site that occurs only once within the genome may be identified. Such a site can then be used to design nuclease agents that will produce a nick or double-strand break at the endogenous recognition site.

The length of the recognition site can vary, and includes, for example, recognition sites that are 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 may recognize a recognition site of at least 9 nucleotides. In other embodiments, the recognition site may be from about 9 to about 12 nucleotides in length, from about 12 to about 15 nucleotides in length, from about 15 to about 18 nucleotides in length, or from about 18 to about 21 nucleotides in length, and any combination of such subranges (e.g., 9-18 nucleotides) . The recognition site could be palindromic, that is, the sequence on one strand reads the same in the opposite direction on the complementary strand. It is recognized that a given nuclease agent can bind the recognition site and cleave that binding site or alternatively, the nuclease agent can bind to a sequence that is the different from the recognition site. Moreover, the term recognition site may comprise both the nuclease agent binding site and the nick/cleavage site irrespective whether the nick/cleavage site is within or outside the nuclease agent binding site. In another variation, the cleavage by the nuclease agent can occur at nucleotide positions immediately opposite each other to produce a blunt end cut or, in other cases, the incisions can be staggered to produce single-stranded overhangs, also called “sticky ends” , which can be either 5’ overhangs, or 3’ overhangs.

Any nuclease agent that induces a nick or double-strand break into a desired recognition site can be used in the methods of the present disclosure. A naturally-occurring or native nuclease agent can be employed so long as the nuclease agent induces a nick or double-strand break in a desired recognition site. Alternatively, a modified or engineered nuclease agent can be employed. An “engineered nuclease agent” comprises a nuclease that is engineered (modified or derived) from its native form to specifically recognize and induce a nick or double-strand break in the desired recognition site. Thus, an engineered nuclease agent can be derived from a native, naturally-occurring nuclease agent or it can be artificially created or synthesized. The modification of the nuclease agent can be as little as one amino acid in a protein cleavage agent or one nucleotide in a nucleic acid cleavage agent. In some embodiments, the engineered nuclease may comprise a nick or double-strand break in a recognition site, wherein the recognition site was not a sequence that would have been recognized by a native (non-engineered or non-modified) nuclease agent. Producing a nick or double-strand break in a recognition site or other DNA can be referred to herein as “cutting” or “cleaving” the recognition site or other DNA.

In some embodiments, the nuclease agent may be a Transcription Activator-Like Effector Nuclease (TALEN) . TAL effector nucleases are a class of sequence-specific nucleases that can be used to make double-strand breaks at specific target sequences in the genome of a prokaryotic or eukaryotic organism. TAL effector nucleases may be created by fusing a native or engineered transcription activator-like (TAL) effector, or functional part thereof, to the catalytic domain of an endonuclease, such as, for example, FokI. The unique, modular TAL effector DNA binding domain allows for the design of proteins with potentially any given DNA recognition specificity. Thus, the DNA binding domains of the TAL effector nucleases can be engineered to recognize specific DNA target sites and thus, used to make double-strand breaks at desired target sequences. 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 herein incorporated by reference.

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

In some embodiments, the nuclease agent may be a meganuclease. Meganucleases have been classified into four families based on conserved sequence motifs. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites, and for tolerating some sequence polymorphisms in their DNA substrates. Meganuclease domains, structure and function are known, see for example, Guhan and Muniyappa (2003) Crit. Rev Biochem Mol Biol 38: 199-248; Lucas et al., (2001) Nucleic Acids Res29: 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.

In some embodiments, the nuclease agent employed in the methods of the present disclosure may employ a CRISPR/Cas system. Such systems can employ, for example, a Cas9 nuclease, which in some instances, may be codon-optimized for the desired cell type in which it is to be expressed. The system may further employ a fused crRNA-tracrRNA construct that functions with the codon-optimized Cas9. This single RNA may be often referred to as a small guide RNA or sgRNA. Within an sgRNA, the crRNA portion may be identified as the “nucleotide sequence hybridizing to the target sequence of the endogenous APP gene” (or a “targeting sequence” ) and the tracrRNA may be often referred to as the “scaffold” . Briefly, a short DNA fragment containing the targeting sequence may be inserted into an sgRNA expression plasmid. The sgRNA expression plasmid may comprise the targeting sequence (in some embodiments around 20 nucleotides) , a form of the tracrRNA sequence (the scaffold) as well as a suitable promoter that is active in the cell and necessary elements for proper processing in eukaryotic cells (such as cell or cellular model) . The sgRNA expression cassette and the Cas9 expression cassette may then be introduced into the cell. See, for example, 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 W Y et al. Nat Biotechnol 2013 March; 31 (3) : 227-9; Jiang W et al. Nat Biotechnol 2013 March; 31 (3) : 233-9; and, Cong L et al. Science 2013 Feb. 15; 339 (6121) : 819-23, each of which is herein incorporated by reference.

In some embodiments, the method may further comprise identifying a genetically modified cell comprising the knocked-in heterologous nucleic acid sequence.

The donor nucleic acid molecule comprising the heterologous nucleic acid sequence may also comprise a 5’ homologous arm and a 3’ homologous arm. The 5’ homologous arm and the 3’ homologous arm may flank the heterologous nucleic acid sequence encoding at least a part of the mutant APP. A homologous arm in the donor nucleic acid molecule (e.g., the 5’ homologous arm or the 3’ homologous arm) may be of any length that is sufficient to promote a homologous recombination event with a corresponding region in the endogenous APP gene locus and/or the APOE gene locus, for example, at least 5 bps, at least 50 bps, at least 100 bps, at least 150 bps, at least 200 bps, at least 300 bps, at least 400 bps, at least 500 bps, at least 600 bps, at least 700 bps, at least 750 bps, at least 800 bps, at least 850 bps, at least 900 bps, at least 1kb, at least 1.5kb, at least 5kb in length or greater. In some embodiments, the donor nucleic acid molecule comprises a 5’ homologous arm that may be about 800-900 bps in length and a 3’ homologous arm that may be about 800-950 bps in length.

As used herein, a homologous arm and a target site (i.e., a cognate genomic region, or a corresponding region within the endogenous APP gene locus and/or the APOE gene locus) match or correspond to one another when the two regions share a sufficient level of sequence identity to one another to act as substrates for a homologous recombination reaction. By “homology” , it is meant that the DNA sequences either are identical or share a certain sequence identity to a corresponding or matching sequence. The sequence identity between a given target site and the corresponding homologous arm found in the donor nucleic acid molecule can be any degree of sequence identity that allows for homologous recombination to occur. For example, the amount of sequence identity shared by the homologous arm of the donor nucleic acid molecule (or a fragment thereof) and 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.

For example, in the donor nucleic acid molecule, the heterologous nucleic acid sequence encoding at least a part of the mutant APP or APOE may be flanked by: 1) a first homologous arm (or a 5’ homologous arm) corresponding to a first region of the endogenous APP gene locus upstream of exon 16; and 2) a second homologous arm (or a 3’ homologous arm) corresponding to a second region of the endogenous APP gene locus downstream of exon 17. As such, the donor nucleic acid molecule thereby aids in the integration of the heterologous nucleic acid sequence into the endogenous APP gene locus of genome of the cell or cellular model through a homologous recombination event that occurs between the homologous arms and their corresponding regions within the endogenous APP gene locus.

When nuclease agents (e.g., the Cas protein) are employed, the cognate genomic regions corresponding to the 5’ and 3’ homologous arms of a donor nucleic acid molecule are located in sufficient proximity to the nuclease target sites so as to promote the occurrence of a homologous recombination event between the cognate genomic regions and the homologous arms upon a nick or double-strand break at the recognition site. For example, the nuclease target sites can be located anywhere between the cognate genomic regions corresponding to the 5’ and 3’ homologous arms. In some embodiments, the recognition site is immediately adjacent to at least one or both of the corresponding cognate genomic regions.

The donor nucleic acid molecule may also comprise a selection cassette or a reporter gene. The selection cassette may comprise a nucleic acid sequence encoding a selection marker, wherein the nucleic acid sequence is operably linked to a promoter. The promoter may be active in a prokaryotic cell of interest and/or active in a eukaryotic cell of interest. Such promoters may be an inducible promoter, a promoter that is endogenous to the reporter gene or the cell, a promoter that is heterologous to the reporter gene or to the 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 group consisting of: Ampicillin resistance gene (Ampr) , neomycin phosphotransferase (neor) , hygromycin B phosphotransferase (hygr) , puromycin-N-acetyltransferase (puror) , blasticidin S deaminase (bsrr) , xanthine/guanine phosphoribosyl transferase (gpt) and herpes simplex virus thymidine kinase (HSV-k) and a combination thereof. The selection marker of the donor nucleic acid molecule may be flanked by the 5’ and 3’ homologous arms or found either 5’ or 3’ to the homologous arms.

In some embodiments, the Cas protein and the one or more RNA sequences may be introduced into the human cell as a protein-RNA complex (e.g., RNP) .

In some embodiments, the DNA encoding the Cas protein may be in the form of a first expression construct comprising a first promoter operably linked to a nucleic acid encoding the Cas protein; the DNA encoding the RNA molecules may be in the form of a second expression construct comprising a second promoter operably linked to a nucleic acid sequence encoding the RNA molecules. The first and the second promoters may be active in the human cell. In some cases, the first and the second expression constructs are in a single nucleic acid molecule.

In some cases, the human 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 group consisting of: Dnmt3L, Eras, Err-beta, Fbxo15, Fgf4, Gdf3, Klf4, Lef1, LIF receptor, Lin28, Nanog, Oct4, Sox15, Sox2, Utf1 and a combination 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 group consisting of: c-Myc, Ecat1 and Rexo1; (b) lack of expression of one or more mesodermal markers selected from the group consisting of Brachyury and Bmpr2; (c) lack of expression of one or more endodermal markers selected from the group consisting of Gata6, Sox17 and Sox7; and (d) lack of expression of one or more neural markers selected from the group consisting of Nestin and Pax6.

In some embodiments, the knocking-in may comprise injecting the donor nucleic acid molecule comprising the heterologous nucleic acid sequence of the present disclosure into the human cell.

Cellular model and methods for generating such a model

In one aspect, the present disclosure provides a cellular model of a dementia-related neurological disease. The cellular model comprises an in vitro culture of a plurality of cells derived from the engineered human cell of the present disclosure. The dementia-related neurological disease may be Alzheimer’s disease.

The cellular model may be generated by culturing the engineered human cell of the present disclosure in two dimensions. In some embodiments, the cellular model may be generated by culturing the engineered human cell of the present disclosure in three dimensions.

For example, the cellular model may be generated by contacting the engineered human cell of the present disclosure with a differentiation medium. The engineered human cell may be cultured in a gel, in a bioreactor, under ultra-low adhesion conditions and/or on a microchip.

In some embodiments, the engineered human cell is cultured in a matrix. The matrix may be an extracellular matrix. In some cases, the matrix may comprise one or more of natural molecules, synthetic polymers, biological-synthetic hybrids, metals, ceramics, bioactive glasses and/or carbon nanotubes.

For example, the engineered human cell may be an engineered human embryonic stem cell, and the method may comprise culturing the engineered human embryonic stem cell in matrigel with the differentiation medium.

A skilled person may determine the time sufficient to induce differentiation by examining morphological changes associated with differentiation. In one embodiment, the time sufficient to culture the stem cells and induce differentiation is from about 5 days to about 180 days. In another embodiment, the time sufficient to induce differentiation is about 7 days to about 15 days. A skilled person can determine the time sufficient to induce organoid formation by examining morphological changes associated with organoid formation. In one embodiment, the time sufficient to induce organoid formation is from about 5 days to about 28 days. In another embodiment, the time sufficient to induce organoid formation is about 14 days.

In some embodiments, the differentiation medium comprises a mTeSR^TM1 medium. In some embodiments, the medium may comprise 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 0.5μg/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) .

In some embodiments, the differentiation medium comprises 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 0.02mM to about 20mM, 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) .

In some embodiments, the differentiation medium comprises B27 supplement. The B27 supplement may be used at 1x. However, the concentration of B27 supplement present in the differentiation medium may range from 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 further comprise or be substituted with other supplements, growth factors, antibiotics, vitamins metabolites, and/or hormones, synthetic or natural with similar properties as known in the art.

In some embodiments, the cells are cultured in the differentiation medium using a bioreactor (e.g., a spinning bioreactor) after the organoids are formed in a multi-well plate (s) . An experiment was performed to increase the size of the formed organoids. After 4-6 days of 3D culture in organoid chamber droplets in a multi-well plate, the droplets were transferred to a spinning bioreactor. The organoids cultured in the spinning bioreactor became 3-10 fold larger in size than those cultured in multi-well plates at 30-60 days after culturing. Thus, a spinning bioreactor may be used in some embodiments.

In some embodiments, the cells are cultured in poly-ornithine coated plates for the time sufficient to induce differentiation. In some embodiments, the method is performed with a commercially available extracellular matrix such as Matrigel^TM. Other natural or synthetic extracellular matrices are known in the art for culturing cells. In general, an extracellular matrix comprises laminin, entactin, and collagen. In an embodiment, the method is performed using a 3-dimensional culture device (chamber) that mimics an in vivo environment for the culturing of the cells, e.g., the extracellular matrix may be formed inside a plate that is capable of inducing the proliferatio of stem cells under hypoxic conditions. Such 3-dimensional devices are known in the art.

The cellular model of the present disclosure may have one or more of the following features: 1) is a fully human-derived cellular model; 2) shows β-amyloid accumulation; 3) shows hyperphosphorylation of Tau protein; 4) shows aggregation of Tau protein; 5) shows glial cell proliferation; 6) shows chronic neuroinflammation; and 7) shows synaptic loss and neuronal death. In some embodiments, the cellular model may generate amyloid plaques and Tau-tangles.

In the present disclosure, the cellular model may be a brain organoid. In some cases, the cellular organoid is a forebrain organoid.

For example, the cellular model of the present disclosure may comprise the following cells: astrocytes, neurons, neural progenitor cells and/or oligodendrocytes astrocytes.

Compositions and Kits

In one aspect, the present disclosure provides a composition. The composition may comprise the engineered human cell of the present disclosure, or the cellular model of the present disclosure.

In another aspect, the present disclosure provides a kit for screening a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease. The kit comprises the engineered human cell or the cellular model of the present disclosure.

In some embodiments, the dementia-related neurological disease is Alzheimer’s disease.

In some embodiments, the substance, device and/or composition comprises a molecule, a membrane-bound vesicle, and/or a cell. For example, the substance may comprise a molecule (e.g., a small molecule, a macromolecule, or a biological substance) . For example, the substance may comprise a membrane-bound vesicle, such as an exosome, an organelle, which may be engineered. For example, the substance may comprise a cell, which may be a human cell, in some cases, the cell may be genetically engineered or otherwise modified.

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 alleged to be effective in the treatment, diagnosis, prevention, monitoring and/or prognosis of Alzheimer’s disease.

A candidate substance may be a synthetic compound, a peptide, a protein, a DNA library or a nucleic acid molecule in the library, a tissue extract or cell culture supernatant of an animal (e.g., a mammal, such as a mouse, a rat, a pig, cattle, a sheep, a monkey, or a human being) , an extract or a cultured product from a plant or a microorganism, or any mixtures thereof.

In the present disclosure, the composition may comprise 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 comprise a tissue homogenate and/or a blood sample. In some embodiments, the composition may comprise extracts from one or more plants. For example, the composition may comprise a candidate traditional Chinese medicine.

The kit of the present disclosure may also comprise one or more additional components selected from the group consisting of: an assay buffer, a control, a substrate, a standard, a detection material, a laboratory supply, a device, a machine, a cell, an organ, a tissue, and a user manual or instruction. For example, the detection material may comprise antibodies, fluorescein-labelled derivatives, luminogenic substrates, detection solutions, scintillation counting fluid, etc. Laboratory supplies may comprise desalting column, reaction tubes or microplates (e.g. 96-or 384-well plates) .

Screening Methods

In one aspect, the present disclosure provides a method for screening a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease. The method may comprise: exposing the engineered human cell of the present disclosure or the cellular model of the present disclosure to a candidate substance, device, and/or composition; assessing the engineered cell or cellular model for one or more feature of the dementia-related neurological disease in the presence of the candidate substance, device, and/or composition; and selecting a candidate substance, device, and/or composition capable of preventing, inhibiting and/or reducing one or more feature of the dementia-related neurological disease.

In one aspect, the present disclosure provides a method for identifying a potential substance, device, and/or composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease. The method may comprise (i) contacting the engineered human cell of the present disclosure or the cellular model of the present disclosure with a candidate substance, device, and/or composition to be tested; and (ii) assessing the activity of the candidate substance, device, and/or composition on one or more feature of the dementia-related neurological disease.

In one aspect, the present disclosure provides a method for designing a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease. The method may comprises the steps of: (i) exposing the engineered human cell of the present disclosure or the cellular model of the present disclosure to a candidate substance, device, and/or composition; (ii) assessing the engineered human cell or the cellular model for one or more feature of the dementia-related neurological disease; (iii) selecting a candidate substance, device, and/or composition capable of preventing, inhibiting and/or reducing one or more feature 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 with improved activity in the treatment, prevention and/or prognosis of the dementia-related neurological disease.

In some embodiments, the feature of the dementia-related neurological disease may comprise: β-amyloid accumulation; hyperphosphorylation of Tau protein; aggregation of Tau protein; glial cell proliferation; chronic neuroinflammation; synaptic loss; neuronal death; appearance of amyloid plaques; appearance of Tau-tangles; increased expression level of CTF-β; increased expression level of Aβ42; increased ratio of Aβ42/Aβ40; axonal swelling, axonal segment breaks, beta-amyloid accumulation, C99 fragment of APP accumulation, p-Tau accumulation, synaptic atrophy, neuronal apoptosis F-Actin bundles formation using Phalloidin staining; mitochondrial dysfunction and oxidative damage; autophagy deficit; neurotransmitter imbalance; and/or dysfunctional glucose metabolism.

In some embodiments, the hyper-phosphorylation of Tau protein comprises increased phosphorylation of Ser396, Thr181, Thr205 and/or Ser202 of Tau, comparing to that in a corresponding wildtype human cell.

In one aspect, the present disclosure provides use of the engineered human cell of the present disclosure or the cellular model of the present disclosure as model of a dementia-related neurological disease.

In one aspect, the present disclosure provides use of the engineered human cell of the present disclosure or the cellular model of the present disclosure in the preparation of a model of a dementia-related neurological disease.

In one aspect, the present disclosure provides a method for identifying a potential biological target and/or biomarker of a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease. The method may comprise the steps of: (i) making a quantitative proteomic, lipidomic and/or genomic comparative analysis of the engineered human cell of the present disclosure or the cellular model of the present disclosure with a control cell or a control cellular model; (ii) identifying a gene, a protein and/or a lipid with an altered sequence, quantity, 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.

In one aspect, the present disclosure provides a method of screening for a biological target and/or biomarker useful in the diagnosis and/or monitoring of a dementia-related neurological disease. The method may comprise determining a presence and/or a level of a substance in a sample obtained from the engineered human cell of the present disclosure or from the cellular model of the present disclosure both before and after detection of a feature of the dementia-related neurological disease and identifying a substance showing a change of the presence and/or level before and after the detection.

The feature of the dementia-related neurological disease may comprise: β-amyloid accumulation; hyperphosphorylation of Tau protein; aggregation of Tau protein; glial cell proliferation; chronic neuroinflammation; synaptic loss; neuronal death; appearance of amyloid plaques; 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 deficit; neurotransmitter imbalance; and/or dysfunctional glucose metabolism.

In one aspect, the present disclosure provides use of the engineered human cell of the present disclosure or the cellular model of the present disclosure in the preparation of a system of screening for a substance, a device, a composition, a biological target and/or a biomarker useful in the treatment, diagnosis, prevention, monitoring and/or prognosis of a dementia-related neurological disease.

In one aspect, the present disclosure provides the engineered human cell or the cellular model, which is for use in the screening for a substance, a device, a composition, a biological target and/or a biomarker useful in the treatment, diagnosis, prevention, monitoring and/or prognosis of a dementia-related neurological disease. The dementia-related neurological disease may be Alzheimer's disease.

For example, a sample (e.g., cells, tissues, or other DNA-or RNA-containing sample, protein-containing sample and/or metabolite-containing sample) may be taken from the cell or the cellular model of the present disclosure before and after the appearance of a disease (e.g., Alzheimer's disease) related feature (e.g., Aβ accumulation, neurofibrillary tangle, morphologically or functionally abnormal (collapsed) synapse, or neuronal cell death) . Then, a gene transcription product (transcriptome) , a gene translation product (proteome) , a lipid (lipidome) or a metabolite (metabolome) derived from the sample may be comprehensively assayed and a substance that changes after the appearance of a disease (e.g., Alzheimer's disease) related feature may be identified.

Gene transcription products (e.g., transcriptome) may be analyzed using nucleic acid microarray, such as a DNA microarray. Gene translation products (e.g., proteome) may be analyzed using gel electrophoresis (such as a two-dimensional gel electrophoresis) , or mass spectrometry (such as time-of-flight mass spectrometry, electronspray ionization mass spectrometry, capillary HPLC/MS and LC/MS/MS) . Metabolites (metabolome) may be analyzed using NMR, capillary electrophoresis, LC/MS and/or LC/MS/MS. Mass spectrometry-based techniques may be used in lipidomic analysis.

When the presence/amount of a substance shows a significant difference after the appearance of a disease (e.g., Alzheimer's disease) related feature, such a substance may be considered as a biological target and/or biomarker of the disease, which may then be used in early diagnosis (particularly a preclinical diagnosis) of the disease (such as AD) . The identified biological target and/or biomarker may be further detected with a specific agent or a detection method. For example, when the biological target and/or biomarker is a protein or a peptide, it may be detected with an immunoassay using a specific antibody. When the biological target and/or biomarker is a nucleic acid molecule (such as a transcription product) , it may be detected with Northern blot analysis using a specific probe, or with RT-PCR using specific primers.

The method of the present disclosure may be an in vitro method, an ex vivo method, or an in vivo method.

In the method, after treating the cell or the cellular model of the present disclosure with the candidate substance, device and/or composition, the cell or organoid may be homogenized using a suitable buffer (such as a phosphate-buffered saline) to obtain a homogenized solution. A soluble fraction and/or an insoluble fraction may then be isolated from the homogenized solution. Afterwards, the isolated soluble fraction and/or insoluble fraction may be examined with an immunoassay, e.g., using an anti-Aβ antibody and/or an anti-APP antibody. In some embodiments, the amount of Aβ42 and Aβ40 can be measured. In some embodiments, the ratio of Aβ42/Aβ40 can be calculated as well.

In some cases, after isolating the cell or organoid, a frozen section or a paraffin-embedded section of the cell/organoid may be prepared. Then, APP/Aβ deposition may be evaluated, e.g., by immunostaining the cell/organoid section with an anti-APP antibody and/or an anti-Aβ antibody. In addition, synapse abnormality may be evaluated by immunostaining the sample section with an antibody against a marker protein of the presynapse or the dendrite. Morphological abnormality of cell skeleton proteins may be evaluated by immunostaining the brain section with an antibody against the phosphorylated tau. Neuronal cell death may be evaluated with Nissl body staining or HE staining (e.g., as described in Am. J. Pathol., vol. 165, pages 1289-1300, (2004) ) . The results may be compared with that of a control group (e.g., wherein the candidate substance, device and/or composition has not been applied, or a blank control buffer has been applied instead of the candidate substance, device and/or composition) .

Comparing to the results obtained from the control group, after applying the candidate substance, device and/or composition, if the total amount of Aβ decreases, the amount of Aβ42 decreases and/or the ratio of Aβ42/Aβ40 decreases, then, the candidate substance, device and/or composition may be selected for further study (e.g., as a potential therapeutic agent/device/composition for treating AD, or for suppressing the accumulation of Aβ) .

Comparing to the results obtained from the control group, after applying the candidate substance, device and/or composition, if amyloid deposition decreases, neurofibrillary tangle decreases, synapse abnormality (collapse) decreases, neuronal cell death decreases and/or inflammatory reaction decreases, then, the candidate substance, device and/or composition may be selected for further study (e.g., as a potential therapeutic agent/device/composition for treating AD, for suppressing the accumulation of Aβ, for suppressing neurofibrillary tangle, for suppressing brain lesion (e.g., neurodegeneration) or inflammatory reaction) .

In another aspect, the present disclosure provides a screening method for a substance having an affinity for APP or Aβ. For example, a candidate substance may be applied to the cell or cellular model of the present disclosure, then, a presence of the candidate substance in an area of Aβ accumulation may be examined. A candidate substance having a specific affinity for APP or Aβ may be used in early diagnosis of AD.

Examples

The following examples are set forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc. ) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., pl, picoliter (s) ; s or sec, second (s) ; min, minute (s) ; h or hr, hour (s) ; aa, amino acid (s) ; nt, nucleotide (s) ; i. m., intramuscular (ly) ; i. p., intraperitoneal (ly) ; s. c., subcutaneous (ly) ; r. t., room temperature; and the like.

Materials and Methods

CRISPR-mediated genome editing

CRISPR/Cas9 expression vector (pX459, #48139) was obtained through Addgene (https: //www. addgene. org/) . sgRNA design and insertion of sgRNA-complementary oligo DNA into pX458 was conducted as previously reported (Ran et al., 2013) . HDR DNA donor vector was built 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 by using Neon Transfection System (Invitrogen, #MPK5000) at 1300 V, 10 ms, 3 times, then seeded on a 6-well plate. After the puromycin selection, the puromycin resistance gene was removed by Cre recombinase (Clontech, #631449) according to the instruction. After reaching sub confluency, cells were re-plated to form single-cell colonies, and genotyping was performed based on the previous report (Li et al., 2016) .

In Vitro differentiation of human neuronal cells

Wildtype (WT) and APP knock-in hESCs were cultured in the mTeSRTM1 medium in a feeder-free way. The in vitro differentiation of hNSCs and human neuronal cells was as described previously. Briefly, the hESCs were passaged at a ratio of 1: 5 when confluent. On the next day, neural stem cell induction medium (Advanced DMEM/F12: Neurobasal (1: 1) , 1xN2, 1xB27, 1%Glutmax, 5 μg/mL BSA and 10 ng/mL hLIF (Millipore) , 3 μMCHIR99021 (Selleck) , 2 μM SB431542 (Selleck) , 0.1 μM Compound E (EMD Chemicals) ) was added for 7 days for the induction of human neural stem cell (hNSC) . Spontaneous differentiation of human neurons was performed 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) for 14 days on Matrigel coated surface.

Generation of forebrain organoids

Generation of forebrain organoids from hESCs was performed as previously described (Qian et al., 2018; Qian et al., 2016) . First, hESC colonies were detached with Collagenase Type IV 7 days after passage and washed with fresh stem cell medium in a 15 mL conical tube. On Day 0, detached hESC colonies were transferred to an ultra-Low attachment 6-well plate (Corning Costar) , containing 3 mL of stem cell medium (without FGF-2) , plus 2 μM Dorsomorphine and 2 μM A83–01. On Day 5 and Day 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, 1μM CHIR99021, and 1 μM SB-431542. On Day 7, organoids were embedded in Matrigel and cultured in the induction medium for 7 more days. On Day 14, embedded organoids were mechanically dissociated from Matrigel by manual pipetting in a 5 mL pipette tip. Typically, 20 organoids were transferred to each well of a 12-well spinning bioreactor (SpinU) (Qian et al., 2016) containing differentiation medium consisting of DMEM: F12, 1X N2 and B27 Supplements, 1X Penicillin/Streptomycin, 1X 2-Mercaptoenthanol, 1X Non-essential Amino Acids, 2.5 μg/ml human Insulin.

Tissue preparation

Whole organoids were fixed in 4%Paraformaldehyde in Phosphate Buffered Saline (PBS) for 30 mins at room temperature. Organoids were washed 3 times with PBS and then immersed in 30%sucrose solution overnight. Organoids were embedded in tissue freezing medium and sectioned with a cryostat (Leica) at 8 μm thickness unless otherwise specified.

Immunohistochemistry and microscopy

For immunostaining, cryosectioned slides were washed with PBS before permeabilization with 0.5%Triton-X in PBS for 1 hr. Tissues were blocked with blocking medium consisting of 10%donkey serum in PBS with 0.05%Triton-X (PBST) for 30 mins. Primary antibodies diluted in blocking solution were applied to the sections overnight at 4℃. After washing with PBST for a minimum of 5 times, secondary antibodies diluted in blocking solution were applied to the sections for 1hr at room temperature or overnight at 4℃. Finally, sections were washed with PBST for a minimum of 5 times before mounting. Secondary antibodies were: AlexaFluor 488, 546-conjugated antibodies (Invitrogen) used at 1: 500 dilution. Images were captured by a confocal microscope (Zeiss LSM 880) . Sample images were prepared in ImageJ (NIH) . The information of primary antibodies and their dilutions used in this study 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; a 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, BD Biosciences, 610459) , anti-RIPK3 (1: 500, Stata Crus, 374639) , anti-MLKL (1: 1000, EnoGene, E11-11361C)

Analysis of hypoxia

Detection of hypoxic cells in organoids was performed using Hypoxyprobe Kit (HPI) . Pimonidazole HCL was added to culture medium at 200 μM working concentration, and incubated with organoids for 2 hr before fixation for analysis. Immunochemical detection of hypoxic cells containing pimonidazole was performed following the manufacturer’s instructions. Images were captured by a confocal microscope (Zeiss LSM 880) using the same acquisition parameters (laser intensity and gain) , and “tile” / “stitching” functions in the Zen software (Zeiss) were used when necessary. To quantify the percentage of hypoxic area in sliced and unsliced organoids, the area labeled by the hypoxyprobe was measured using ImageJ software and divided by the total organoid area marked by DAPI.

Western blotting

Samples in soluble fraction were loaded into a 4–20%sodium dodecyl sulfate-polyacrylamide gel, and transferred to PVDF Immobilon FL membranes (Millipore) . After blocking with 5%non-fat milk in PBS, membranes were blotted overnight with primary antibodies in 5%non-fat milk containing 0.01%Tween-20, and then probed with horseradish peroxidase-conjugated secondary antibody, detected by SuperSignal West Femto Chemiluminescent Substrate (Pierce) . The information of 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) , .

ELISA quantification

The Aβ40 and Aβ42 in medium were measured using the Human β-Amyloid (1–40) ELISA Kit (Thermo Fisher, KHB3481) and Human β-Amyloid (1–42) ELISA Kit (Thermo Fisher, KHB3441) respectively, according to the manufacturers’ instructions. The reaction was stopped and read at 450 nm with a microplate reader (Biotek) .

Culture of various principal types of cells

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 condition using mTeSR1 medium (Stem Cell Technologies) , and used at intermediate (～50) passage numbers to generate human induced neuronal (iN) cells (Zhang et al., 2013) . Cells were cultured as described (Zhang et al., 2013) .

Murine embryonic fibroblasts (MEFs) were isolated from mouse embryos of CF-1 strain (Harlan Laboratories, Inc. ) harvested at 12.5-13.5 postcoitum (p.c. ) . Briefly, embryos were dissected out of terminally anesthetized mice. The head and internal organs were removed, and the remaining carcasses were finely minced, trypsinized into single-cell suspensions, and plated onto T75 flasks. The cultured MEFs (P0) were frozen or expanded for up to 3 times (P3) before they were used for experiments.

Lysotracker staining

Neuronal cells were incubated with 500 nM Lysotracker Red dye (Invitrogen) at 37℃ for 30min. Then cells were washed with PBS and the fluorescent intensity was measures by flow cytometry or Opera Phenix high-content confocal microscope.

Cholesterol staining

The buildup of free cholesterol in macrophages was disclosed by filipin staining at 50 μg/ml at room temperature for 1 hours. Then cells were washed with PBS and the fluorescent intensity was measures by flow cytometry or Opera Phenix high-content confocal microscope.

TMT-MS-based quantitative proteomics

The AD cerebral organoid and control at three months were homogenized in 8M urea lysis buffer (8M urea, 10mM Tris, 1mM PMSF, 1mM protease inhibitor cocktail) . 100 μg of each sample was subjected to protein digestion. Samples were reduced with dithiothreitol and iodoacetamide alkylation. After centrifuge at 2000 g for 10 min, the supernatant was added with 0.4%trifluoroacetic acid (TFA) and de-salted with Sep-Pak column. Each sample was resolved with 100 mM TEAB buffer, and labeled with TMT 10-plex kit and allowed to incubate for 1h at room temperature. The reaction was quenched with 5%hydroxylamine. All samples were combined and dried with SpeedVac. Labeled peptides were de-salted with Sep-Pak column. The samples were subjected to HPLC fractionation and mass spectrometry.

Example 1 Generation of APP^NL-G-F hESCs and cellular models derived from such cells

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

The targeting scheme of the gene editing is illustrated in FIG. 8. Human ESCs harboring all three homozygous APP mutations (APP^NL-G-F) were verified by PCR and Sanger sequencing of colonies derived from single putatively edited cells. Several protocols have been developed to create neural organoids from human pluripotent stem cells. The protocols reported by Qian, X. et al. (2016) was employed to generate the forebrain organoids from the APP^NL-G-F hESCs and WT controls, and complex dense 3D neural organoids were obtained.

To characterize AD related phenotypes in the brain organoids generated, the levels of the 40-amino-acid and 42-amino-acid amyloid-β isoforms (Aβ40 and Aβ42) in conditional media after 6-week of differentiation were measured. Conditional media from APP^NL-G-F cerebral organoids revealed dramatic decrease in Aβ40 (～3-fold) while the Aβ42 (～1.5-fold) level increased as compared to the control (FIGs. 1A-1D) . The Aβ42/Aβ40 ratio was also increased (～4-fold) in APP^NL-G-F cerebral organoids. FIG. 1A illustrates representative immunocytochemistry images of the neurons (MAP2) and astrocytes (GFAP) in cerebral organoids at Day 90 of differentiation. FIGs. 1B–1C illustrate protein levels of Aβ40 and Aβ42 in the culture medium measured by ELISA. Protein level were normalized to the total protein concentration of the respective sample (n=4) . FIG. 1D illustrates the ratio of Aβ42/Aβ40 (n=4) .

To investigate the status of Aβ accumulation and deposition, cerebral organoids were utilized for the analyses at different time points (2 and 3 months, respectively) . Analysis at 60 days (2 month) and 90 days (3 month) of culture identified a progressive increase in the area of Aβ aggregates in the APP^NL-G-F organoids compared to controls (FIGs. 1E-1G) which could barely be detected in conventional 2D cultures, where secreted amyloid-β diffuses into a large volume of media. These data demonstrate the presence of robust Aβ aggregation in APP^NL-G-F organoids that appears to develop in a time-dependent manner. FIG. 1E provides representative immunocytochemistry image of Aβ and MAP2 in cerebral organoids at Day 60. Scale, 50 μm. FIG. 1F provides bar graphs representing the number of Aβ puncta in FIG. 1E (n = 4) . FIG. 1G provides representative immunocytochemistry image of Aβ and MAP2 in cerebral organoids at Day 90. Scale, 50 μm. FIG. 1H provides bar graphs representing the number of Aβ puncta in FIG. 1G (n=4) .

Example 2 Tau pathology in APP^NL-G-F Forebrain Organoids

The abnormal phosphorylation, mislocalization, and aggregation of the Tau protein have long been recognized as key hallmarks of AD. So, Tau pathology in APP^NL-G-F organoids was also examined over time. Notably, as the Tau hyperphosphorylation is known to precede tau aggregation, first, phosphorylated Tau (p-Tau) levels were analyzed with western blots using antibodies against p-Tau (phosphorylated at Ser202/Thr205, Ser396 and Thr181) . It was reported that the increases in p-Tau at specific sites occurred in a relative order. Interestingly, there was no significant difference in p-Tau levels between APP^NL-G-F and control organoids at 2-month, when Aβ aggregates had already been observed in the APP^NL-G-F organoids. At 3-month, the APP^NL-G-F organoids exhibit significantly greater level of AT8/t-Tau than controls in RIPA-soluble fractions (FIGs. 2A-2B) . Ser396/t-Tau and Thr181/t-Tau began to increase significantly near the time of 4-month in RIPA-soluble fractions or in sarkosyl insoluble fractions but not in controls while AT8/t-Tau levels continued to increase. FIG. 2A provides representative Western blots of total tau and P-tau S202/T205 (AT8) in APP^NL-G-F and WT organoids at Day 90. The upper and lower bands in the AT8 blots represent different tau isoforms. FIG. 2B illustrates densitometry quantification of P-tau/total tau level in FIG. 2A (n = 5) .

Next, multiple antibodies were used to detect different stage of conformationally altered Tau. First, APN-mab005, a monoclonal antibody that recognizes early stage of aggregated Tau, was used. APP^NL-G-F cerebral organoids at 3-month started to exhibit more APN-mab005-positive Tau aggregated than WT organoids (FIGs. 2C-2F) . Then, the MC1 immunoreactivities (which is indicative of intermediate state conformational/pathological change of Tau) were evaluated in 4-month organoids and a significant increase was found in APP^NL-G-F organoids (FIGs. 2G-2H) . What’s more, dramatic increases in AT8-positive p-Tau levels were also found in a portion of APP^NL-G-F cells at 4-month of differentiation (FIGs. 2I-2J) . As a result, in the organoid model, the hyperphosphorylated Tau occurred following the appearance of amyloid-β accumulation and the increases in p-Tau at specific sites occurred in a relative order, consistent with previous reports for the AD pathology in human patients.

FIG. 2C provides representative immunocytochemistry image of tau aggregation using APN-mab005, a specific antibody for early pathological phenotype aggregated tau in cerebral organoids at Day 60. Scale, 50 μm. FIG. 2D provides bar graphs representing the number of aggregated tau puncta in FIG. 2C (n = 4) . FIG. 2E provides representative immunocytochemistry image of tau aggregation using APN-mab005 in cerebral organoids at Day 90. Scale, 50 μm. FIG. 2F provides bar graphs representing the number of aggregated tau puncta in FIG. 2E (n = 4) .

FIG. 2G provides representative immunocytochemistry image of disease-specific conformational change of tau using MC1 antibody in cerebral organoids at Day 120. Scale, 20 μm. FIG. 2H provides bar graphs representing the number of aggregated tau puncta in FIG. 2G (n = 8) .

FIG. 2I provides representative immunocytochemistry image of P-tau S202/T205 (AT8) antibodies in cerebral organoids at Day 120. Scale, 50 μm. FIG. 2J provides bar graphs representing the number of aggregated tau puncta in FIG. 2I (n = 8) .

Example 3 Gliosis and neuroinflammation increased in APP^NL-G-F forebrain organoids

Genetic epidemiological and experimental data all suggested a critical role of 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 derive from ectoderm, microglia migrate early during embryo development from the yolk-sac, a mesodermal-derived structure. The organoids generated according to the present disclosure are mainly populated with neurons and astrocytes. To investigate the gliosis in APP^NL-G-F forebrain organoids, the protein levels of GFAP (an astrocyte marker) in APP^NL-G-F forebrain organoids were measured. Western blot analysis revealed a moderate increase in the levels of GFAP in APP^NL-G-F forebrain organoids at 3-month (FIGs. 3A-3B) , while a significant increase of GFAP in APP^NL-G-F forebrain organoids was observed at 4-month (FIGs. 3C-3D) . FIG. 3A provides representative Western blots of of GFAP in APP^NL-G-F and WT organoids at Day 90. FIG. 3B provides densitometry quantification of GFAP levels in FIG. 3A (n = 8) . FIG. 3C provides representative Western blots of GFAP in APP^NL-G-F and WT organoids at Day 120. FIG. 3D provides densitometry quantification of GFAP levels in FIG. 3C (n = 4) .

Then, the chemokine and cytokine secretion in APP^NL-G-F forebrain organoids culture medium were tested and significant increase in the secretion of the pro-inflammatory factors interleukin 6 (IL6) and interleukin 8 (IL8) were observed, while the secretion of the neuroprotective macrophage migration inhibitory factor (MIF) and the anti-apoptotic Serpin E1 were significantly decreased. What’s more, a mild increase of the chemokine CXCL1 and significant decreased of CCL2 and CXCL12 were also observed in APP^NL-G-F forebrain organoids culture medium. TNFα and IL-1β, which were mainly secreted by microglia, were barely detected in the organoid culture medium (FIG. 3E) . FIG. 3E demonstrates that chemokine and cytokine secretion in conditioned media from APP^NL- ^G-F or WT organoids at Day 90 were determined by Human Cytokine Array kits (n = 2) .

Example 4 Synaptic degeneration and cell death increased in APP^NL-G-F forebrain organoids

Synapse degeneration is now regarded as an intermediate step and critical pathophysiological hallmark of AD. Substantial evidence indicates that in AD, there is a decrease in the number of synapses as well as impairments in synaptic functions, which occurs later than Aβ accumulation and correlates with disease progression. To evaluate the synaptic loss in APP^NL-G-F forebrain organoids, presynaptic synaptophysin and postsynaptic PSD95 were analyzed by western blots at 3-month and 4-month. A mild decrease of the presynaptic membrane protein Synaptophysin was observed but an increase of the postsynaptic membrane protein PSD95 was observed in APP^NL-G-F forebrain organoids at 3-month (FIGs. 4A-4B) , which might be due to the compensatory enhancement of the postsynaptic membrane in the early stage of the disease when the presynaptic membrane underwent damage. At 4-month of differentiation, a significant decrease in the presynaptic membrane protein Synaptophysin was observed accompanied by a mild decrease in PSD95 in APP^NL-G-F forebrain organoids (FIGs. 4C-4D) , indicating the progression of AD related synapse loss. FIG. 4A provides representative Western blots of Synaptophysin and PSD95 in APP^NL-G-F and WT organoids at day 90. FIG. 4B provides densitometry quantification of Synaptophysin and PSD95 levels in FIG. 4A (n=4) . FIG. 4C provides representative Western blots of Synaptophysin and PSD95 in APP^NL-G-F and WT organoids at day 120. FIG. 4D provides densitometry quantification of Synaptophysin and PSD95 levels in FIG. 4C (n = 4) .

Neuronal death is another prominent pathological feature of AD. Cell death in AD brain has been attributed by at least two mechanisms: apoptosis and necrosis. To evaluate the neuronal apoptosis in the cerebral organoids, cleaved caspase-3 was analyzed by immunostaining at 3 months. It was noted that brain organoids grown as spheres in suspension culture can expand up to 3 to 4 mm in diameter. Due to the lack of a functional circulation system, the viability of cells within the large cortical organoids is restricted by the limited supply of oxygen and nutrients delivered via surface diffusion. To avoid the possible influences of hypoxia on apoptosis in the core portion of organoids, the organoids were co-stained by a pimonidazole-based hypoxia probe. The results showed that apoptosis could be observed in both the WT and APP^NL-G-F forebrain organoids. However, in WT forebrain organoids, the cleaved caspase 3 signal mostly co-localized with the hypoxic dye while in APP^NL-G-F forebrain organoids, the cleaved caspase 3 signal could be detected in regions with no hypoxic dye staining, indicating the occurrence of apoptosis independent of hypoxia (FIG. 4E) . FIG. 4E provides representative immunocytochemistry images of cellular apoptosis marker cleaved CASP3 and a pimonidazole-based hypoxia probe (HPOX) in cerebral organoids at Day 90 of differentiation. Scale, 80 μm. Bar graphs represent the area (%) of cleaved CASP3 puncta in immunocytochemistry images (n = 8) .

Necrosis is the uncontrolled lysis of cells that also contributes to the neuronal cell death in AD. Necroptosis, a programmed form of necrosis was recently identified in postmortem AD brains. Three critical proteins, RIPK1, RIPK3 and its substrate MLKL, are involved in the initiation of necroptosis. Phosphorylated MLKL can trigger MLKL aggregates to form homodimers, which induce membrane damage that leads to cell death. Similarly, MLKL was co-stained with the hypoxic dye HOPX. In WT forebrain organoids, MLKL was observed in neither HOPX positive nor negative regions, while in APP^NL-G-F forebrain organoids, there was a significant increase in the amount of aggregated MLKL signal (FIG. 4F) . It indicates that necroptosis in forebrain organoids was not induced by hypoxia. Programmed necrosis complex-associated proteins were analyzed by western blots. In agreement with the observations in AD patients, phosphorylated form of MLKL and RIPK1 was found to be significantly elevated in APP^NL-G-F forebrain organoids, while RIPK3 and total MLKL remained unchanged (FIGs. 4G-4H) . Taken together, these observations suggested that both apoptosis and necroptosis may contribute to the neuronal loss in APP^NL-G-F forebrain organoids.

FIG. 4F provides representative immunocytochemistry images of cellular necroptosis marker MLKL in cerebral organoids at Day 90 of differentiation. Scale, 80 μm. Bar graphs represent the area (%) of MLKL puncta in immunocytochemistry images (n = 8) . FIG. 4G provides representative Western blots of RIPK1、RIPK3、MLKL and phosphorylated MLKL levels in APP^NL-G-F and WT organoids at day 90. FIG. 4H provides densitometry quantification of RIPK1、RIPK3、MLKL and phosphorylated MLKL levels in FIG. 4G (n = 4) .

Example 5 The neuroprotection role of APOE3-Christchurch (R136S) in APP^NL-G-F forebrain organoids

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

It was then investigated whether converting APOE3 to APOE3ch in APP^NL-G-F forebrain organoids was sufficient to ameliorate the AD related pathologies. The contribution of the APOE3ch variant to Aβ accumulation in APP^NL-G-F forebrain organoids was then investigated and it was found that after 3 months of culture, APOE3ch APP^NL-G-F forebrain organoids showed less Aβ puncta compared to age-matched APOE3 APP^NL-G-F forebrain organoids (FIGs. 5A-5B) . Tau pathology was evaluated in the APP^NL-G-F forebrain organoids. Western blots using anti-phosphorylated Tau (p-Tau) AT8 antibody at 3-month showed that p-Tau and the p-Tau/total Tau ratio decreased markedly in APP^NL-G-F forebrain organoids with APOE3ch (FIGs. 5C-5D) . Consistent with the results, the APP^NL- ^G-F forebrain organoids were stained with AT8 at 4-month and less conformationally altered Tau was found in organoids with APOE3ch (FIGs. 5E-5F) . FIG. 5A provides representative immunocytochemistry images of Aβ in APOE3 APP^NL-G-F and APOE3ch APP^NL-G-F cerebral organoids at Day 90 of differentiation. Scale, 80 μm. FIG. 5B provides bar graphs representing the number of Aβpuncta in FIG. 5A (n = 8) . FIG. 5C provides representative Western blots of total tau and P-tau S202/T205 (AT8) levels in APOE3 APP^NL-G-F and APOE3ch APP^NL-G-F cerebral organoids at Day 90. The upper and lower bands in the AT8 blots represent different tau isoforms. FIG. 5D provides densitometry quantification of total tau and P-tau S202/T205 (AT8) levels in FIG. 5C (n = 5) . FIG. 5E provides representative immunocytochemistry images of P-tau S202/T205 (AT8) antibody in APOE3 APP^NL-G-F and APOE3ch APP^NL-G-F cerebral organoids at Day 120 of differentiation. Scale, 80 μm. FIG. 5F provides bar graphs representing the number of aggregated tau puncta in FIG. 5E (n = 5) .

Next, it was investigated whether APOE3ch ameliorated elevated neuronal death in the APP^NL-G- ^F forebrain organoids. Immunocytochemistry results showed a significant reduction of cleaved CASP3 immunoreactivity in the APP^NL-G-F forebrain organoids with APOE3ch (FIGs. 5G-5H) . Taken together, these results indicate that the AD related phenotypes observed in the APP^NL-G-F forebrain organoids could largely be ameliorated through converting APOE3 to APOE3ch. FIG. 5G provides representative immunocytochemistry images of cleaved CASP3 antibody in APOE3 APP^NL-G-F and APOE3ch APP^NL-G-F cerebral organoids at day 120 of differentiation. Scale, 50 μm. FIG. 5H provides bar graphs representing the area (%) of cleaved CASP3 puncta in FIG. 5G (n = 6) .

Example 6 APOE3ch enhances lysosomal functions to clear neural toxic proteins

A dominant theory in AD research has been that increased Aβ levels, Aβ aggregates or amyloid plaques lead to Tau pathologies and subsequently to AD-related cognitive decline. Clearly, the new findings in the patient with APOE3ch do not support this widely discussed hypothesis. Indeed, the study provides direct evidence that Aβ/amyloid accumulation alone is not sufficient to cause AD, Aβ might induce Tau pathologies and cognitive decline only in the presence of normally functional APOE. Further understanding of this chain of causality will be vital for better understanding AD pathogenesis and improving drug development.

As described above, APOE3ch resulted in a significant reduction in cleaved caspase 3 in the APP^NL-G-F forebrain organoids at Day 120. However, when the protein level of cleaved caspase 3 was tested in earlier stage (day 80) of APP^NL-G-F forebrain organoids, no differences were observed between APOE3 and APOE3ch APP^NL-G-F forebrain organoids. What’s more, the protein level of cleaved caspase 3 in APOE3 APP^NL-G-F forebrain organoids was higher at Day 80 compared to Day 120 (FIG. 6A) . FIG. 6A provides representative immunocytochemistry images of cleaved CASP3 antibody in APOE3 APP^NL-G-F and APOE3ch APP^NL-G-F cerebral organoids at Day 80 and 120 of differentiation. Scale, 50 μm. Bar graphs represent the area (%) of cleaved CASP3 puncta (n = 6) .

In the periphery, cells undergoing apoptosis expose and release signals that attract phagocytes, which engulf the cells undergoing apoptosis and maintain homeostasis in vivo, while in the central nervous system, microglia and astrocytes are responsible to engulf the debris of apoptotic cells, thus maintain the tissue. In the early stage of forebrain organoid differentiation, GFAP positive astrocytes are barely detected while at Day 120, astrocytes have emerged. The engulfment of apoptotic cells by astrocytes might explain why the level of cleaved caspase 3 decreased at Day 120 in APP^NL-G-F forebrain organoids. This suggests that astrocytes in APOE3ch APP^NL-G-F forebrain organoids might be able to engulf and digest apoptotic debris more efficiently. To investigate that, fluorescent labelled Tau was added into differentiated APOE3 and APOE3ch APP^NL-G-F cells and an increase in the uptake of Tau protein in APOE3ch APP^NL-G-F cells was observed (FIG. 6B) . What’s more, the fluorescent labelled Tau mostly colocalized with lysosome after being taken into cells (FIG. 6C) . FIG. 6B shows that fluorescent labelled tau was added into differentiated APOE3 APP^NL-G-F and APOE3ch APP^NL-G- ^F cells and the uptake of tau protein was measured by FACS analysis. Bar graphs represent the fluorescence intensity of tau protein in cells (n = 9) . FIG. 6C provides representative images of fluorescent labelled tau and lysotracker dye staining in APOE3 APP^NL-G-F and APOE3ch APP^NL-G-F cells. Scale, 20 μm.

It was then tested whether APOE3ch could enhance the endocytosis and lysosomal degradation capacity in APP^NL-G-F forebrain organoids. Firstly, the early endosome was studied by immunocytochemistry of EEA1 protein and it was found that the APOE3 APP^NL-G-F forebrain organoids had significantly larger early endosomes than the APOE3ch APP^NL-G-F forebrain organoids (FIG. 6D) , which is consistent with that observed in AD patients, in which the larger early endosomal volume and abnormal endosomal function ultimately lead to impaired endocytosis and degradation functions. Then, the lysosomal functions were investigated with the staining of lysotracker dye. It was observed that APOE3 APP^NL-G-F cells had higher number of lysosomes but the acidity of these lysosomes is defective compared to WT cells, which could be rejuvenated in APOE3ch APP^NL-G-F cells (FIG. 6D) . The functions of the lysosomal hydrolases are highly dependent on the acidic environment in the lysosomal lumen. Thus, APOE3ch might improve the degradation capacity of lysosomes in APP^NL-G-F cells and help the clearance of neural toxic proteins. FIG. 6D provides representative immunocytochemistry images of early endosome marker EEA1 in APOE3 APP^NL-G-F and APOE3ch APP^NL-G-F cerebral organoids at Day 120 of differentiation. Scale, 50 μm. Bar graphs represent the number of EEA1 puncta (n = 6) .

APOE functions as a lipid chaperone and facilitates cellular uptake of cholesterol and lipoproteins through receptor-mediated endocytosis. It has been reported that the accumulation of cholesterol can suppress lysosomal functions by activating mTORC1 signaling pathway. The lipid and cholesterol composition in APP^NL-G-F cells were then tested and it was found that APOE3ch reduced the amount of both intracellular lipids and cholesterol (FIGs. 6E-6H) . This might explain how APOE3ch increased the lysosomal functions in APP^NL-G-F cells. FIG. 6E provides representative images of lysotracker dye staining in WT, APOE3 APP^NL-G-F and APOE3ch APP^NL-G-F cells. Scale, 30 μm. Bar graphs represent the fluorescence intensity of lysotracker dye in cells (n = 7) . FIG. 6F provides representative images of filipin staining in APOE3 APP^NL-G-F and APOE3ch APP^NL-G-F cells. Scale, 80 μm. Bar graphs represent the fluorescence intensity of filipin dye in cells (n = 6) . FIG. 6G provides representative images of filipin staining in APOE3 APP^NL-G-F cells treated with or without HPβCD. Scale, 80 μm. Bar graphs represent the fluorescence intensity of filipin dye in cells (n = 6) . FIG. 6H provides representative images of lysotracker dye staining in APOE3 APP^NL-G-F cells treated with or without HPβCD. Scale, 30 μm. Bar graphs represent the fluorescence intensity of lysotracker dye in cells (n =6) .

Example 7 TrkB antibody rescued synaptic loss and cell death in the AD organoid

To further evaluate the functions of the APP^NL-G-F organoids, effects of TrkB antibodies on the organoids were investigated.

To investigate the therapeutic effects of TrkB antibody on AD, it is important to determine whether TrkB antibody could engage its target TrkB and its downstream signaling events and eventually exhibit positive cellular functions. The APP^NL-G-F organoids were treated with BDNF (5 nM) , TrkB antibody (15 nM) or a vehicle for 30 min or 2h, and the TrkB downstream signaling events were analyzed by western blots. The amino acid sequences of the TrkB antibody are as set forth in SEQ ID NO: 19-26. The HCDR1of the TrkB antibody is as set forth in SEQ ID NO: 19. The HCDR2 of the TrkB antibody is as set forth in SEQ ID NO: 20. The HCDR3 of the TrkB antibody is as set forth in SEQ ID NO: 21. The LCDR1 of the TrkB antibody is as set forth in SEQ ID NO: 22. The LCDR2 of the TrkB antibody is as set forth in SEQ ID NO: 23. The LCDR3 of the TrkB antibody is as set forth in SEQ ID NO: 24. The VH of the TrkB antibody is as set forth in SEQ ID NO: 25. The VL of the TrkB antibody is as set forth in SEQ ID NO: 26.

WT and APP^NL-G-F organoids (Day 120) were treated with vehicle (lanes 1-4) or the TrkB antibody (15 nM, lanes 5-10) for 12 days and subjected to western blotting (FIGs. 7A-7B) . WT organoid (Day 100) was treated with the TrkB antibody (15 nM, lanes 1-3) or BDNF (5 nM, lanes 4-6) for 0, 0.5 and 2 hr and subjected to western blotting (FIGs. 7C-7D) .

The western blot shows that the TrkB antibody rescued synaptic loss and cell death in the APP^NL- ^G-F organoids. And similar to BDNF, the TrkB antibody actively triggered all major signaling pathways.

Thus, the APP^NL-G-F organoids of the present disclosure can be successfully used for identifying substances useful in AD treatment.

Example 8 Proteomic study of human cerebral organoid model of Alzheimer’s disease (AD)

Human cerebral organoid of AD at 3 months exhibited Aβ aggregation and tau tangles, which are the pathological hallmarks of AD. To underpin the molecular mechanism underlying AD pathophysiology, we performed isobaric multiplex tandem mass tags (TMTs) labeling mass spectrometry (MS) analyses on 3-month APP^NL-G-F (AD) organoid and age-matched APP^WT (control) organoid. To reduce the heterogeneity between samples, two organoids were combined into one sample. We employed three samples in each group. The organoid samples were homogenized, digested and labeled with TMT, followed by HPLC and MS. The proteomics revealed the expression levels of 6600 proteins. Bioinformatic analysis underpinned the differentially expressed proteins and enriched biological processes with statistical significance. Results are shown in FIGs. 9-11. FIG. 9 indicated the differentially expressed proteins in the AD cerebral organoid at 3 months. Based on the differentially expressed proteins with statistical significance, the related biological processes were sought to be enriched. FIG. 10 illustrated the GSEA analyses using KEGG database enriched biological processes. The data indicated that AD is associated with the following pathways: Alzheimer’s disease, Huntington’s disease, Parkinson’s disease, oxidative phosphorylation, tight junction, RNA degradation, ribosome and regulation of actin cytoskeleton. On contrast, ERBB signaling pathway and ubiquitin-mediated proteolysis are enriched in the control group. FIG. 11 showed the GSEA analyses using GO database enriched biological processes. The analyses indicate AD is associated with the following pathways: protein folding, carbohydrate derivative metabolic process, cellular amino acid biosynthetic process, mitochondrion organization, oxidative phosphorylation and biological process involved in interaction with host. On contrast, dendrite development and protein polymerization are enriched in the control group.

It was concluded that quantitative proteomics was performed to determine the molecular features of the AD cerebral organoid model. In total, 6600 proteins were analyzed for deferentially expressed proteins and GSEA analysis of biological processes. Based on the GSEA analysis using KEGG annotation database, several findings were found. First, AD organoid showed comprehensive changes in protein expression profile that resembles Alzheimer’s disease and other neurodegenerative diseases, such as Huntington’s disease and Parkinson’s disease. Second, oxidative phosphorylation, tight junction, RNA degradation, ribosome and regulation of actin cytoskeleton were enriched in the AD organoid. Third, ERBB signaling pathway and ubiquitin-mediated proteolysis were enriched in the control group, which means downregulation in the AD cerebral organoid. Consistently, the enrichment based on the GO annotation indicated an upregulation of signaling pathways involving protein folding, carbohydrate derivative metabolic process, cellular amino acid biosynthetic process, mitochondrion organization, oxidative phosphorylation and biological process involved in interaction with host. Whereas, dendrite development and protein polymerization were downregulated in the AD cerebral organoid. These enriched biological processes were in consistent with the proteomic study using human postmortem AD brains. These data substantiate that the AD cerebral organoid recapitulates the pathophysiological changes of human AD. Due to broad heterogeneity between individual human brain samples, limited case numbers and the divergence of AD brain banks, it makes the data harder to reach statistical significance or keep consistence between each proteomic study. On contrast, the AD cerebral organoid model shows higher homogeneity than individual human brain sample. In addition, the AD human cerebral organoid uncovers disease-related signaling pathways that are not captured in the conventional AD mice models. All these features support that the molecular trajectory of AD progression in cerebral organoid model is similar to AD patient. The cerebral organoid shows superiority over AD mice model in that it captures a stronger phenotype with shorter time frame. Thus, this AD cerebral organoid model will allow meaningful mechanistic study and efficient drug testing and screening.

While exemplary embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. It is not intended that the invention be limited by the specific examples provided within the specification. While the invention has been described with reference to the aforementioned specification, the descriptions and illustrations of the embodiments herein are not meant to be construed in a limiting sense. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. Furthermore, it shall be understood that all aspects of the invention are not limited to the specific depictions, configurations or relative proportions set forth herein which depend upon a variety of conditions and variables. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is therefore contemplated that the invention shall also cover any such alternatives, modifications, variations or equivalents. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

An engineered human cell, wherein the APP gene is mutated to encode for a mutant APP protein, comparing to a wildtype human APP protein as set forth in SEQ ID NO: 1, said mutant APP protein comprises an amino acid substitution at the following residues: K670, M671, I716 and/or E693.
The engineered human cell of claim 1, wherein said mutant APP protein comprises an amino acid substitution at the following residues: K670, M671, I716 and E693.
The engineered human cell of any one of claims 1-2, wherein said mutant APP protein further comprises one or more additional mutations capable of affecting the amount of Aβgenerated, the amount of an Aβ fragment generated, and/or the ratio of Aβ42/Aβ40 generated.
The engineered human cell of any one of claims 1-3, wherein said mutant APP protein further comprises one or more additional or alternative mutations selected from the group consisting of: I716V and V717I.
The engineered human cell of any one of claims 1-4, which is derived from a subject that had, is having or at the risk of developing a disease or disorder.
The engineered human cell of any one of claims 1-5, which is a human stem cell.
The engineered human cell of any one of claims 1-6, which comprises a pluripotent stem cell, an embryonic stem cell, an induced pluripotent stem cell, and/or a neuronal stem cell.
The engineered human cell of claim 7, wherein said embryonic stem cell is from an established cell line.
The engineered human cell of any one of claims 7-8, wherein said embryonic stem cell is from the human embryonic stem cell (hESC) line H1.
The engineered human cell of any one of claims 7-9, wherein said embryonic stem cell is not obtained via a process in which human embryos are destroyed.
The engineered human cell of any one of claims 1-10, wherein said mutant APP protein comprises a Swedish double mutation.
The engineered human cell of claim 11, wherein said Swedish double mutation comprises a K670N substitution and a M671L substitution.
The engineered human cell of any one of claims 1-12, wherein said mutant APP protein comprises a Beyreuther/Iberian mutation.
The engineered human cell of claim 13, wherein said Beyreuther/Iberian mutation comprises an I716F substitution.
The engineered human cell of any one of claims 1-14, wherein said mutant APP comprises an Arctic mutation.
The engineered human cell of claim 15, wherein said Arctic mutation comprises a E693G substitution.
The engineered human cell of 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.
The engineered human cell of any one of claims 1-17, wherein the expression level of CTF-α is not significantly different from that of a corresponding wildtype human cell.
The engineered human cell of any one of claims 1-18, wherein the expression level of CTF-β is increased comparing to that of a corresponding wildtype human cell.
The engineered human cell of any one of claims 1-19, wherein the expression level of Aβ42 is increased comparing to that of a corresponding wildtype human cell.
The engineered human cell of any one of claims 1-20, wherein the ratio of Aβ42/Aβ40 is increased comparing to that of a corresponding wildtype human cell.
The engineered human cell of any one of claims 1-21, wherein hyper-phosphorylation of Tau is detectable.
The engineered human cell of claim 22, wherein said hyper-phosphorylation of Tau comprises increased phosphorylation of Thr205 and/or Ser202 of Tau, comparing to that in a corresponding wildtype human cell.
The engineered human cell of any one of claims 1-23, wherein said cell is a knock-in cell or is derived from a knock-in cell and wherein at least a part of an endogenous APP gene is substituted by a heterologous nucleic acid sequence encoding at least a part of said mutant APP protein.
The engineered human cell of claim 24, wherein said at least part of the endogenous APP gene comprises at least a part of exon 16 and/or at least a part of exon 17 of the endogenous APP gene.
The engineered human cell of any one of claims 24-25, wherein said heterologous nucleic acid sequence encodes for or comprises a mutated exon 16 and/or a mutated exon 17.
The engineered human cell of any one of claims 1-26, wherein said mutant APP protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 4-10.
The engineered human cell of any one of claims 1-27, wherein the APOE gene is mutated to encode for a mutant APOE3 protein, comparing to a wildtype human APOE3 protein as set forth in SEQ ID NO: 11, residue R136 is substituted in the mutant APOE3 protein.
The engineered human cell of claim 28, wherein the residue R136 in the mutant APOE3 protein is substituted with S.
The engineered human cell of any one of claims 28-29, wherein the mutant APOE3 protein comprises an amino acid sequence as set forth in SEQ ID NO: 12.
The engineered human cell of any one of claims 28-30, which is homozygous for the mutant APOE gene.
The engineered human cell of any one of claims 1-31, which further comprises a mutation in one or more additional genes selected from the group consisting of: Tau, APOE2, APOE4 and BDNF.
A cellular model of a dementia-related neurological disease, comprising an in vitro culture of a plurality of cells derived from the engineered human cell of any one of claims 1-32.
The cellular model of claim 33, wherein said dementia-related neurological disease is Alzheimer’s disease.
The cellular model of any one of claims 33-34, wherein said plurality of cell are cultured in two dimensions.
The cellular model of any one of claims 33-35, wherein said plurality of cell are cultured in three dimensions.
The cellular model of any one of claims 33-36, which has one or more of the following features:

1) is a fully human-derived cellular model;

2) shows β-amyloid accumulation;

3) shows hyperphosphorylation of Tau protein;

4) shows aggregation of Tau protein;

5) shows glial cell proliferation;

6) shows chronic neuroinflammation;

7) shows synaptic loss and neuronal death;

8) shows mitochondrial dysfunction and oxidative damage;

9) shows autophagy deficit;

10) shows neurotransmitter imbalance; and/or

11) shows dysfunctional glucose metabolism.
The cellular model of any one of claims 33-37, which generates amyloid plaques and Tau-tangles.
The cellular model of any one of claims 33-38, wherein said cells are cultured in a 3D matrigel matrix.
The cellular model of any one of claims 33-39, which is a brain organoid.
The cellular model of any one of claims 33-40, which is a forebrain organoid.
The cellular model of any one of claims 33-41, which comprises astrocytes, neurons, neural progenitor cells and/or oligodendrocytes.
A method for generating an engineered human cell, comprising: introducing a mutation into an endogenous APP gene of a human cell to generate a mutated APP gene, said mutated APP gene encodes for a mutant APP protein, comparing to a wildtype human APP protein as set forth in SEQ ID NO: 1, said mutant APP protein comprises an amino acid substitution at the following residues: K670, M671, I716 and/or E693.
The method of claim 43, wherein said mutant APP protein comprises an amino acid substitution at the following residues: K670, M671, I716 and E693.
The method of any one of claims 43-44, wherein said introducing a mutation into the APP gene comprises knocking-in a heterologous nucleic acid sequence into an endogenous APP gene locus, wherein said knocking-in substitutes at least a part of the endogenous APP gene with a heterologous nucleic acid sequence encoding at least a part of said mutant APP protein.
The method of claim 45, wherein said knocking-in comprises contacting the genome of said human cell with the following in the presence of a donor nucleic acid molecule comprising said heterologous nucleic acid sequence:

1) a CRISPR associated (Cas) protein; and

2) one or more ribonucleic acid (RNA) sequences that comprise: i) a sequence complementary to a target portion of the endogenous APP gene; and ii) a binding site for the Cas protein.
The method of any one of claims 45-46, wherein said knocking-in further comprises maintaining the cell under conditions in which 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 said hybridization of said one or more RNA sequences.
The method of any one of claims 46-47, wherein said Cas protein is Cas9.
The method of any one of claims 46-48, wherein said binding site for the Cas protein comprises a tracrRNA sequence.
The method of any one of claims 46-49, wherein said 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.
The method of any one of claims 46-50, wherein said 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 said RNA sequences.
The method of any one of claims 46-51, wherein said one or more ribonucleic acid (RNA) sequences comprise a nucleic acid sequence as set forth in any one of SEQ ID NO: 13-16.
The method of any one of claims 45-52, wherein said at least part of the endogenous APP gene comprises at least a part of exon 16 and/or at least a part of exon 17 of the endogenous APP gene.
The method of any one of claims 45-53, wherein said heterologous nucleic acid sequence comprises a mutated exon 16 and/or a mutated exon 17 of the human APP gene.
The method of claim 54, wherein mutations in said mutated exon 16 and/or said mutated exon 17 results in one or more amino acid substitutions in their encoded polypeptides and wherein said one or more amino acid substitutions comprise an amino acid substitution at the following residues: K670, M671, I716 and/or E693.
The method of claim 55, wherein said one or more amino acid substitutions comprise the substitution K670N, M671L, I716F and/or E693G.
The method of any one of claims 43-56, wherein said mutant APP protein further comprises one or more additional mutations capable of affecting the amount of Aβgenerated, the amount of an Aβ fragment generated, and/or the ratio of Aβ42/Aβ40 generated.
The method of any one of claims 43-57, wherein said mutant APP protein further comprises one or more additional or alternative mutations selected from the group consisting of: I716V and V717I.
The method of any one of claims 43-58, wherein said human cell is derived from a subject that had, is having or at the risk of developing a disease or disorder.
The method of any one of claims 43-59, wherein said human cell is a human stem cell.
The method of any one of claims 43-60, wherein said human cell is a pluripotent stem cell, an embryonic stem cell, an induced pluripotent stem cell, and/or a neuronal stem cell.
The method of claim 61, wherein said embryonic stem cell is from an established cell line.
The method of any one of claims 61-62, wherein said embryonic stem cell is from the human embryonic stem cell (hESC) line H1.
The method of any one of claims 61-63, wherein said embryonic stem cell is not obtained via a process in which human embryos are destroyed.
The method of any one of claims 43-64, wherein said mutant APP protein comprises a Swedish double mutation.
The method of claim 65, wherein said Swedish double mutation comprises a K670N substitution and a M671L substitution.
The method of any one of claims 43-66, wherein said mutant APP protein comprises a Beyreuther/Iberian mutation.
The method of claim 67, wherein said Beyreuther/Iberian mutation comprises an I716F substitution.
The method of any one of claims 43-68, wherein said mutant APP comprises an Arctic mutation.
The method of claim 69, wherein said Arctic mutation comprises a E693G substitution.
The method of any one of claims 43-70, wherein said engineered human cell is homozygous for one or more mutations at the following residues of the APP protein: K670, M671, I716 and/or E693.
The method of any one of claims 43-71, wherein said mutant APP protein comprises an amino acid sequence as set forth in any one of SEQ ID NO: 4-10.
The method of any one of claims 43-72, further comprising introducing a mutation in the APOE gene to generate a mutant APOE3 protein, comparing to a wildtype human APOE3 protein as set forth in SEQ ID NO: 11, residue R136 is substituted in the mutant APOE3 protein.
The method of claim 73, wherein the residue R136 in the mutant APOE3 protein is substituted with S.
The method of any one of claims 73-74, wherein the mutant APOE3 protein comprises an amino acid sequence as set forth in SEQ ID NO: 12.
The method of any one of claims 73-75, wherein said engineered human cell is homozygous for the mutant APOE gene.
The method of any one of claims 43-76, wherein said engineered human cell further comprises a mutation in one or more additional genes selected from the group consisting of: Tau, APOE2, APOE4 and BDNF.
An engineered human cell, generated by the method of any one of claims 43-77.
A method for generating a cellular model, comprising contacting the engineered human cell of any one of claims 1-32 with a differentiation medium to obtain the cellular model.
The method of claim 79, wherein said cellular model is a model of a dementia-related neurological disease.
The method of claim 80, wherein said dementia-related neurological disease is Alzheimer’s disease.
The method of any one of claims 79-81, wherein said engineered human cell is cultured in a two-dimensional in vitro culture.
The method of any one of claims 79-82, wherein said engineered human cell is cultured in a three-dimensional in vitro culture.
The method of any one of claims 79-83, wherein said engineered human cell is cultured in a gel, in a bioreactor, under ultra-low adhesion conditions or on a microchip.
The method of any one of claims 79-84, wherein said engineered human cell is cultured in a matrix.
The method of claim 85, wherein said 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 glasses and/or carbon nanotubes.
The method of any one of claims 79-86, wherein said cellular model is a brain organoid.
The method of any one of claims 79-87, wherein said cellular model is a forebrain organoid.
A cellular model, generated by the method of any one of claims 79-88.
A composition, comprising the engineered human cell of any one of claims 1-32, or the cellular model of any one of claims 33-42.
A kit for screening a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease, comprising the engineered human cell of any one of claims 1-32, or the cellular model of any one of claims 33-42.
The kit of claim 91, wherein the dementia-related neurological disease is Alzheimer’s disease.
The kit of any one of claims 91-92, wherein said substance, device and/or composition comprises a molecule, a membrane-bound vesicle, and/or a cell.
The kit of any one of claims 91-93, further comprising one or more additional components selected from the group consisting of: an assay buffer, a control, a substrate, a standard, a detection material, a laboratory supply, a device, a machine, a cell, an organ, a tissue, and a user manual or instruction.
A method for screening a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease, comprising:

exposing the engineered human cell of any one of claims 1-32 or the cellular model of any one of claims 33-42 to a candidate substance, device, and/or composition;

assessing said engineered human cell or the cellular model for one or more feature of the dementia-related neurological disease in the presence of said candidate substance, device, and/or composition; and

selecting a candidate substance, device, and/or composition capable of preventing, inhibiting and/or reducing one or more said feature of the dementia-related neurological disease.
A method for identifying a potential substance, device, and/or composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease, comprising:

(i) contacting the engineered human cell of any one of claims 1-32 or the cellular model of any one of claims 33-42 with a candidate substance, device, and/or composition to be tested; and

(ii) assessing the activity of said candidate substance, device, and/or composition on one or more feature of the dementia-related neurological disease.
A method for designing a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease, comprising the steps of:

(i) exposing the engineered human cell of any one of claims 1-32 or the cellular model of any one of claims 33-42 to a candidate substance, device, and/or composition;

(ii) assessing said engineered human cell or said cellular model for one or more feature of the dementia-related neurological disease;

(iii) selecting a candidate substance, device, and/or composition capable of preventing, inhibiting and/or reducing one or more said feature 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 with improved activity in the treatment, prevention and/or prognosis of the dementia-related neurological disease.
The method of any one of claims 95-97, wherein said dementia-related neurological disease is Alzheimer’s disease.
The method of any one of claims 95-98, wherein said feature of the dementia-related neurological disease comprises:

β-amyloid accumulation;

hyperphosphorylation of Tau protein;

aggregation of Tau protein;

glial cell proliferation;

chronic neuroinflammation;

synaptic loss;

neuronal death;

appearance of amyloid plaques;

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 deficit;

neurotransmitter imbalance; and/or

dysfunctional glucose metabolism.
The method of claim 99, wherein said hyper-phosphorylation of Tau protein comprises increased phosphorylation of Thr205 and/or Ser202 of Tau, comparing to that in a corresponding wildtype human cell.
Use of the engineered human cell of any one of claims 1-32 or the cellular model of any one of claims 33-42 as model of a dementia-related neurological disease.
Use of the engineered human cell of any one of claims 1-32 or the cellular model of any one of claims 33-42 in the preparation of a model of a dementia-related neurological disease.
The use of any one of claims 101-102, wherein said dementia-related neurological disease is Alzheimer's disease.
A method for identifying a potential biological target and/or biomarker of a substance, a device, and/or a composition useful in the treatment, prevention and/or prognosis of a dementia-related neurological disease, comprising the steps of:

(i) making a quantitative proteomic, lipidomic and/or genomic comparative analysis of the engineered human cell of any one of claims 1-32 or the cellular model of any one of claims 33-42 with a control human cell or a control cellular model;

(ii) identifying a gene, a protein and/or a lipid with an altered sequence, quantity, 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.
The method of claim 104, wherein said dementia-related neurological disease is Alzheimer's disease.
A method of screening for a biological target and/or biomarker useful in the diagnosis and/or monitoring of a dementia-related neurological disease, comprising determining a presence and/or a level of a substance in a sample obtained from the engineered human cell of any one of claims 1-32 or from the cellular model of any one of claims 33-42 both before and after detection of a feature of the dementia-related neurological disease and identifying a substance showing a change of said presence and/or level before and after said detection.
The method of claim 106, wherein said dementia-related neurological disease is Alzheimer's disease.
The method of any one of claims 106-107, wherein said feature of the dementia-related neurological disease comprises:

β-amyloid accumulation;

hyperphosphorylation of Tau protein;

aggregation of Tau protein;

glial cell proliferation;

chronic neuroinflammation;

synaptic loss;

neuronal death;

appearance of amyloid plaques;

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 deficit;

neurotransmitter imbalance; and/or

dysfunctional glucose metabolism.
Use of the engineered human cell of any one of claims 1-32 or the cellular model of any one of claims 33-42 in the preparation of a system of screening for a substance, a device, a composition, a biological target and/or a biomarker useful in the treatment, diagnosis, prevention, monitoring and/or prognosis of a dementia-related neurological disease.
The use of claim 109, wherein the dementia-related neurological disease is Alzheimer's disease.
The engineered human cell of any one of claims 1-32 or the cellular model of any one of claims 33-42, for use in the screening for a substance, a device, a composition, a biological target and/or a biomarker useful in the treatment, diagnosis, prevention, monitoring and/or prognosis of a dementia-related neurological disease.
The engineered human cell or the cellular model of claim 111, wherein the dementia-related neurological disease is Alzheimer's disease.