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WO2025210040A1 - Lignée cellulaire exprimant la protéine tau - Google Patents

Lignée cellulaire exprimant la protéine tau

Info

Publication number
WO2025210040A1
WO2025210040A1 PCT/EP2025/058890 EP2025058890W WO2025210040A1 WO 2025210040 A1 WO2025210040 A1 WO 2025210040A1 EP 2025058890 W EP2025058890 W EP 2025058890W WO 2025210040 A1 WO2025210040 A1 WO 2025210040A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell line
tau
tau protein
protein
4rdak
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/EP2025/058890
Other languages
English (en)
Inventor
Senthilvelrajan KANIYAPPAN
Beate POLKE
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Hoffmann La Roche Inc
Original Assignee
F Hoffmann La Roche AG
Hoffmann La Roche Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG, Hoffmann La Roche Inc filed Critical F Hoffmann La Roche AG
Publication of WO2025210040A1 publication Critical patent/WO2025210040A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0619—Neurons
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/46—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from vertebrates
    • G01N2333/47—Assays involving proteins of known structure or function as defined in the subgroups
    • G01N2333/4701—Details
    • G01N2333/4709—Amyloid plaque core protein
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/28—Neurological disorders
    • G01N2800/2814—Dementia; Cognitive disorders
    • G01N2800/2821—Alzheimer

Definitions

  • the present invention provides cell lines expressing specific Tan proteins.
  • exogenous Tau aggregation inducers such as exogenous Tau.
  • exogenous Tau induced intracellular Tau aggregation is not a validated representation of the pathophysiology of the disease. Furthermore, it requires additional steps, making the model less attractive for high(er) throughput research approaches.
  • the present invention provides a cell line comprising a transgene coding for 4RDAK Tau protein (referred to as H4-Tau4RDAK) or a transgene coding for 1N4R P301L/S320F (referred to as H4-TaulN4R-DM) Tau protein, wherein the Tau protein expression is inducible.
  • H4-Tau4RDAK a transgene coding for 4RDAK Tau protein
  • H4-Tau4RDAK a transgene coding for 1N4R P301L/S320F
  • H4-TaulN4R-DM transgene coding for 1N4R P301L/S320F
  • the cell line is a stable cell line.
  • the cell line is a human H4 epithelial cell line.
  • the transgene expression is under control of a Tet promoter.
  • the transgene codes for 4RDAK Tau protein.
  • the transgene codes for 1N4R P301L/S320F Tau protein.
  • the Tau protein is a human Tau protein.
  • the transgene coding for 1N4R P301L/S320F Tau protein has the nucleotide sequence set forth in Seq. Id. No. 1.
  • the 1N4R P301L/S320F Tau protein of the present invention has the amino acid sequence set forth in Seq. ID. No. 3.
  • the present invention provides the use of the cell lines of the present invention in a method for the identification of molecules modulating Tau protein expressions and aggregation.
  • the molecule modulating Tau protein expression and aggregation is a Tau protein aggregation inhibitor or modulator.
  • Fig. 1 H4 T-REx cells overexpressing 4RDAK Tau (Molecular weight ⁇ 14 kDa) (H4- Tau4RDAK). Upon induction with doxycycline, Tau expression was observed starting from day 1 and appearing to stay expressed until day 5 of induction (tested until day 5). Tau expression seems to reach saturation after 24 hours. T-REx cells express the tetracycline repressor protein, repressing Tau protein expression in the absence of doxycycline.
  • Fig. 2 H4 T-REx cells overexpressing 1N4R Tan isoform with P301L and S320F mutations (Molecular weight ⁇ 58-60kDa) (H4-TaulN4R-DM).
  • T-REx cells express the tetracycline repressor protein, repressing Tau protein expression in the absence of doxycycline .
  • Fig. 3 Sarkosyl extraction separates insoluble Tau (expected to contain bigger aggregates and filaments) from soluble Tau (monomers and small oligomers). H4-Tau4RDAK cells form sarkosyl insoluble Tau aggregates starting on day 1, stable until 5 days of induction (5 days tested).
  • 4RDAK tau protein refers to a Tau protein comprising only the four repeat domains of the Tau protein with a deletion of lysine (K) at 280 position.
  • the 4RDAK Tau protein of the present invention is encoded by the nucleic acid sequence set forth in Seq. Id. No. 2. In short: TauRDAK.
  • H4-Tau4RDAK is used for the cell line overexpressing 4RDAK tau.
  • 1N4R P301L/S320F tau protein as used herein refers to a Tau protein comprising 1N4R Tau isoform (one N-terminal domain and the four repeat domains) carrying the mutations P301L and S320F.
  • the 1N4R P301L/S320F Tau protein of the present invention is encoded by the nucleic acid sequence set forth in Seq. Id. No. 1. In short: TaulN4R-DM (double mutant).
  • the term “H4-TaulN4R-DM” is used for the cell line overexpressing 1N4R P301L/S320F Tau.
  • Tau protein isoforms with GenBank Accession No. P10636-7 (Tau E, 1N4R) and P10636-8 (Tau F, 2N4R) are the naturally occuring Tau protein isoforms used for the generation of the Tau proteins 4RDAK tau and 1N4R P301L/S320F Tau of the present invention.
  • the cell model of the present invention uses an inducible system to overexpress 1N4R P301L/S320F Tau (further referred to as Tau-1N4R-DM) or 4 RD (repeat domain) Tau with a deletion of K280 (further referred to as Tau-4RDAK) in H4 cells, an epithelial cell line isolated form a 37 year old male with neuroglioma.

Landscapes

  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Immunology (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Medicinal Chemistry (AREA)
  • Neurology (AREA)
  • Urology & Nephrology (AREA)
  • Toxicology (AREA)
  • Wood Science & Technology (AREA)
  • Microbiology (AREA)
  • Hematology (AREA)
  • Cell Biology (AREA)
  • Pathology (AREA)
  • Food Science & Technology (AREA)
  • Neurosurgery (AREA)
  • General Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • General Engineering & Computer Science (AREA)
  • Physics & Mathematics (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biophysics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne un modèle cellulaire permettant d'étudier des maladies associées à la protéine Tau.
PCT/EP2025/058890 2024-04-04 2025-04-02 Lignée cellulaire exprimant la protéine tau Pending WO2025210040A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
EP24168462.0 2024-04-04
EP24168462 2024-04-04

Publications (1)

Publication Number Publication Date
WO2025210040A1 true WO2025210040A1 (fr) 2025-10-09

Family

ID=90717679

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP2025/058890 Pending WO2025210040A1 (fr) 2024-04-04 2025-04-02 Lignée cellulaire exprimant la protéine tau

Country Status (1)

Country Link
WO (1) WO2025210040A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006007864A1 (fr) * 2004-07-17 2006-01-26 Max Planck Geselllschaft Zur Förderung Der Wissenschaft Traitement d'etats neurodegeneratifs
WO2025031900A1 (fr) * 2023-08-09 2025-02-13 Ludwig-Maximilians-Universität München Modèle dérivé d'ipsc humain pour tauopathies

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006007864A1 (fr) * 2004-07-17 2006-01-26 Max Planck Geselllschaft Zur Förderung Der Wissenschaft Traitement d'etats neurodegeneratifs
WO2025031900A1 (fr) * 2023-08-09 2025-02-13 Ludwig-Maximilians-Universität München Modèle dérivé d'ipsc humain pour tauopathies

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BUCHHOLZ SARAH ET AL: "The six brain-specific TAU isoforms and their role in Alzheimer's disease and related neurodegenerative dementia syndromes", ALZHEIMER'S & DEMENTIA, vol. 20, no. 5, 31 March 2024 (2024-03-31), US, pages 3606 - 3628, XP093281013, ISSN: 1552-5260, DOI: 10.1002/alz.13784 *
MICHAEL DETURE ET AL: "tau Assembly in Inducible Transfectants Expressing Wild-Type or FTDP-17 tau", THE AMERICAN JOURNAL OF PATHOLOGY, vol. 161, no. 5, 1 November 2002 (2002-11-01), pages 1711 - 1722, XP055053502, ISSN: 0002-9440, DOI: 10.1016/S0002-9440(10)64448-3 *
PIR GHULAM JEELANI ET AL: "Suppressing Tau Aggregation and Toxicity by an Anti-Aggregant Tau Fragment", MOLECULAR NEUROBIOLOGY, SPRINGER US, NEW YORK, vol. 56, no. 5, 8 September 2018 (2018-09-08), pages 3751 - 3767, XP036763055, ISSN: 0893-7648, [retrieved on 20180908], DOI: 10.1007/S12035-018-1326-Z *
REMMLER D. ET AL: "On the way to precision formulation additives: 2D-screening to select solubilizers with tailored host and release capabilities", JOURNAL OF CONTROLLED RELEASE, vol. 285, 1 September 2018 (2018-09-01), NL, pages 96 - 105, XP093281031, ISSN: 0168-3659, DOI: 10.1016/j.jconrel.2018.06.032 *

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