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WO2001011030A2 - Procede et moyen permettant d'induire la multiplication cellulaire de cellules au repos - Google Patents

Procede et moyen permettant d'induire la multiplication cellulaire de cellules au repos Download PDF

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Publication number
WO2001011030A2
WO2001011030A2 PCT/DE2000/002725 DE0002725W WO0111030A2 WO 2001011030 A2 WO2001011030 A2 WO 2001011030A2 DE 0002725 W DE0002725 W DE 0002725W WO 0111030 A2 WO0111030 A2 WO 0111030A2
Authority
WO
WIPO (PCT)
Prior art keywords
cells
cell
proliferation
gene
multiplication
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.)
Ceased
Application number
PCT/DE2000/002725
Other languages
German (de)
English (en)
Other versions
WO2001011030A3 (fr
Inventor
Gary S. Jennings
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.)
HepaVec AG fur Gentherapie
Develogen AG
Original Assignee
HepaVec AG fur Gentherapie
Develogen AG
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 HepaVec AG fur Gentherapie, Develogen AG filed Critical HepaVec AG fur Gentherapie
Priority to AU77697/00A priority Critical patent/AU7769700A/en
Publication of WO2001011030A2 publication Critical patent/WO2001011030A2/fr
Publication of WO2001011030A3 publication Critical patent/WO2001011030A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C12N5/067—Hepatocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • 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
    • C12N2510/02—Cells for production
    • 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
    • C12N2510/04—Immortalised cells
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the invention relates to a method and means for the induction of cell propagation in the few cell types of human and animal type which normally no longer divide or which show a very limited propagation capacity in vitro.
  • cell multiplication is to be achieved without general changes in the combination of the cellular genome and thus reversible properties occurring in the cells.
  • the invention preferably relates to novel hepatocytes with an extended lifespan
  • immortalized cell lines can be established directly from tumor riaterals or through different ones Treatments of primary cells can be isolated from normal tissue.
  • an immortalizing gene was introduced into the genome of the target cell, specifically a gene from DNA tumor viruses (such as "Large T antigen from Simian V ⁇ rus-40").
  • DNA tumor viruses such as "Large T antigen from Simian V ⁇ rus-40"
  • the continuous expression of the immortalizing gene led to undesired, also transformed and tumorigenic properties of the cells, which can also lead to a decrease in the cell-typical properties up to the switching off or even loss of the specific cellular tasks.
  • the object of the invention was therefore to achieve a method for inducing a non-immortalizing and non-malignant proliferation of resting cells from an organism to a homogeneous cell population.
  • a time-limited expression of an exogenous, immortalizing gene is used to stimulate cell mitosis.
  • the invention is implemented according to the claims.
  • the invention relates to novel hepatocytes with an extended lifespan, which are particularly suitable for extracorporeal assumption of liver functions, for pharmacological and toxicological investigation of drugs in vitro, for the production of recombinant proteins and for the production of glycosylated proteins.
  • the invention presented here is based on the surprising finding that the removal of an immortalizing gene after the beginning of cell division produces a cell population which shows a defined proliferation capacity in the presence of growth factors.
  • the subject of the presented invention is therefore the ⁇
  • T antigen in a population of target cells from a primary culture.
  • the gene construct which contains the immortalizing gene is also another genetic Un construct, which also has the special property that the expression of the gene in the target cell can be switched off at a later freely selectable point in time.
  • plasmids which do not contain a recognition sequence for the start of replication for plasmid-related replication in the target cell (FIG. 1 a) or a replication-deficient retrovirus is used in which the immortalizing gene, in addition to other foreign sequences from loxP Sequences is framed.
  • loxP sequences allow the framed gene sequences to be eliminated using the enzyme cre recombinase (FIG. 1b).
  • the Cre recombinase is introduced into the target cell by means of a non-integrating, replication-deficient adenovirus (FIG. 1b).
  • the cells produced according to the invention with an extended lifespan are preferably used for the pharmacological and toxicological investigation of medicinal substances in vitro, for the production of recombinant proteins and for the production of glycosylated proteins.
  • Plasmid pCMV-Tagori (Figure la) is transduced into resting primary mouse hepatocytes. After six weeks, proliferating colonies were spiked, about a third of which did not express Large T antigen. These cells could continue to be subcultured for 15-20 passages at a division rate of 1: 3 to 1: 5. After further passages, the cell division decreased until the cell division completely ceased.
  • hepatocytes Resting primary mouse hepatocytes were infected with loxP-HyTK-large-T retrovirus ( Figure lb), which integrated into the Zeil genome. Immortalized hepatocyte clones were isolated and established as cell lines expressing Large T antigen. Such proliferating cell lines were infected with a special adenovirus construct which expresses cre recombinase. The enzyme cre recombinase cuts out the sequences framed by loxP sequences, in this case Large T antigen together with the CMV promoter and HyTK. The resulting cells were treated with ganciclovir, which is activated intracellularly by HyTK to form a cell toxin. This kills those cells in which the cre construct has not been excised correctly.
  • the cell lines with finite lifespan described here remain genetically identical to the primary cells from which they were derived, but they have a wide range prolonged proliferation span compared to primary cells of approximately 50-100 cell divisions.
  • ELS cells Process for the production of cells with an extended lifespan (ELS cells) by means of a time-limited expression of an exogenous, immortalizing gene which is introduced into the respective cell, or by injection of a proliferation-promoting protein.
  • the method according to claim 10 characterized in that it is nucleic acid sequences and / or genes which influence the retinoblastoma cell cycle regulation point, the p53 cell cycle regulation point and / or the telomerase activity.
  • Novel hepatocytes with extended lifespan made from primary hepatocytes.
  • Cells according to claim 12 characterized in that the cells proliferate 15-20 passages stably.
  • Cells according to claim 12 and 13 characterized in that the cells have a euploid (diploid, tetraploid, polyploid) set of chromosomes. 15. Cells according to claim 12 to 14, characterized in that the cells are not immortalized and have no tumorigenic properties.
  • Cells according to claim 12 to 16 characterized in that the cells express cytochrome P450.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Genetics & Genomics (AREA)
  • Zoology (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Chemical & Material Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé et un moyen permettant d'induire la multiplication cellulaire des types de cellules humaines et animales qui normalement ne se divisent plus ou affichent une capacité de multiplication in-vitro extrêmement limitée. On obtient notamment une multiplication cellulaire sans modification générale de la composition du génome cellulaire et donc des propriétés réversibles des cellules. L'invention concerne, de préférence, de nouveaux hépatocytes ayant une dure de vie allongée.
PCT/DE2000/002725 1999-08-10 2000-08-10 Procede et moyen permettant d'induire la multiplication cellulaire de cellules au repos Ceased WO2001011030A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU77697/00A AU7769700A (en) 1999-08-10 2000-08-10 Method and means for inducing cell-multiplication of non-active cells

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DE19936793 1999-08-10
DE19936793.0 1999-08-10
DE19957951.2 1999-10-20
DE19957951 1999-10-20

Publications (2)

Publication Number Publication Date
WO2001011030A2 true WO2001011030A2 (fr) 2001-02-15
WO2001011030A3 WO2001011030A3 (fr) 2001-05-31

Family

ID=26054489

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE2000/002725 Ceased WO2001011030A2 (fr) 1999-08-10 2000-08-10 Procede et moyen permettant d'induire la multiplication cellulaire de cellules au repos

Country Status (3)

Country Link
AU (1) AU7769700A (fr)
DE (1) DE10039856A1 (fr)
WO (1) WO2001011030A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1427850A4 (fr) * 2001-08-16 2006-02-08 Phase 1 Molecular Toxicology I Genes et jeux ordonnes d'echantillons humains pertinents sur le plan toxicologique
DE102007041655A1 (de) * 2007-09-03 2009-03-05 Medicyte Gmbh Vermehrung von primären Zellen und deren Verwendung

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6110743A (en) * 1995-02-10 2000-08-29 The Regents Of The University Of California Development and use of human pancreatic cell lines
DE19626830A1 (de) * 1996-07-04 1998-01-08 Max Delbrueck Centrum Konditionales Immortalisationsverfahren für humane Tumorzellen zur Herstellung einer Vakzine

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1427850A4 (fr) * 2001-08-16 2006-02-08 Phase 1 Molecular Toxicology I Genes et jeux ordonnes d'echantillons humains pertinents sur le plan toxicologique
DE102007041655A1 (de) * 2007-09-03 2009-03-05 Medicyte Gmbh Vermehrung von primären Zellen und deren Verwendung

Also Published As

Publication number Publication date
DE10039856A1 (de) 2001-05-03
AU7769700A (en) 2001-03-05
WO2001011030A3 (fr) 2001-05-31

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