Human induced pluripotent stem cell lines for modeling Alzheimer's disease and usage thereof
AssigneeShenzhen Cell Inspire Biotechnology Co., Ltd.
InventorsYubo Yan, Jian Fu, Min Zhou, Yuqing Liu, Lixiang Jiang, Bo Yang, Jiayin Yang
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Abstract
A method of generating a cellular model of Alzheimer's disease (AD) comprises integrating AD related gene to hiPSC to induce increased beta secretase and/or Abeta 42 peptides, and the cellular model of Alzheimer's disease (AD) is prepared by the method.
Claims13
A method of generating a cellular model of Alzheimer's disease (AD), comprising integrating an AD-related gene into human induced pluripotent stem cells hiPSCs to induce increased expression of beta-secretase and/or Abeta-42 peptides as compared to the expression level of beta-secretase and/or Abeta-42 peptides in isogenic hiPSCs without integration of the AD-related gene,
- wherein the AD-related gene is integrated into the hiPSC at the adeno-associated virus integration site 1 (AAVS1) site;
- wherein the AD-related gene is integrated into the hiPSCs by a mammalian expression vector comprising a nucleotide acid sequence encoding an AD-related protein and a reporter, wherein the nucleotide acid sequence is operably linked with a promoter for driving a high level of gene expression in the mammalian expression vector, wherein the promoter is CAG promoter;
- and
- wherein the AD-related gene is beta-secretase 1 (BACE1) gene and is constitutively overexpressed in hiPSCs.
A high throughput method for screening a therapeutic agent for treatment of AD, comprising:
- i) Preparing a cellular model of Alzheimer's disease (AD) from a hiPSC by introducing an expression vector or a genetic construct to the hiPSC, wherein the expression vector comprises a nucleic acid sequence encoding an AD-related protein selected from the group consisting of mutant APP attributing to the onset of AD, PS1dE9 and BACE1 and a reporter, wherein the nucleic acid sequence is operably linked to a promoter for driving high level of gene expression in a mammalian expression vector, and
- wherein the genetic construct comprises a nucleic acid sequence coding for:
- a first promoter;
- a drug selection gene controlled by the first promoter;
- a second promoter;
- an AD-related gene linked to a reporter gene controlled by the second promoter;
- and sequence homologous to human AAVS1 site, wherein all said elements are in a cis order, wherein the AD-related gene is selected from the group consisting of mutant APP attributing to the onset of AD, PS1dE9 gene and BACE1 gene;
- ii) Culturing the candidate compounds with the cellular model for two days to two weeks, and
- iii) measuring beta- secretase level, Abeta-42 concentration, and Abeta42/Abeta-40 ratio before and after adding the candidate compounds;
- wherein reduction of one or more measurements selected from beta-secretase level, Abeta-42 concentration and Abeta42/Abeta-40 ratio indicates the candidate compound is a potential therapeutic agent for treatment of AD.
A drug screening process for screening beta-secretase or Abeta-42 inhibitor, comprising
- i) modifying hiPSC line by constitutively over-expressing BACE1 gene, wherein the hiPSC line is produced by introducing a mammalian expression vector to the hiPSC, wherein the expression vector comprises a nucleic acid sequence encoding an AD-related protein and a reporter, wherein the nucleotide acid sequence is operably linked with a promoter for driving a high level of gene expression in the mammalian expression vector, wherein the promoter is CAG promoter;
- and wherein the AD-related gene is integrate into the hiPSC at AAVS1 site,
- ii) re-differentiating hiPSC line into functional neurons,
- iii) culturing the functional neurons in presence of candidate drug compounds, and
- iv) measuring beta-secretase level, Abeta-42/Abeta-40 ratio, and/or Abeta-42 concentration and selecting compounds that can reduce beta-secretase level, Abeta-42/Abeta-40 ratio, and/or Abeta-42 concentration.
Dependencies are inferred from the claim text.