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WO2001066781A1 - Immortalite conditionnelle de cellules - Google Patents

Immortalite conditionnelle de cellules Download PDF

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Publication number
WO2001066781A1
WO2001066781A1 PCT/GB2001/001029 GB0101029W WO0166781A1 WO 2001066781 A1 WO2001066781 A1 WO 2001066781A1 GB 0101029 W GB0101029 W GB 0101029W WO 0166781 A1 WO0166781 A1 WO 0166781A1
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WO
WIPO (PCT)
Prior art keywords
cell
oncogene
cells
myc
conditionally
Prior art date
Application number
PCT/GB2001/001029
Other languages
English (en)
Inventor
John Sinden
Ziping Dong
Original Assignee
Reneuron Limited
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 Reneuron Limited filed Critical Reneuron Limited
Priority to AU37610/01A priority Critical patent/AU3761001A/en
Publication of WO2001066781A1 publication Critical patent/WO2001066781A1/fr

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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/13011—Gammaretrovirus, e.g. murine leukeamia virus
    • C12N2740/13041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/13043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2840/00—Vectors comprising a special translation-regulating system
    • C12N2840/20—Vectors comprising a special translation-regulating system translation of more than one cistron
    • C12N2840/203—Vectors comprising a special translation-regulating system translation of more than one cistron having an IRES

Definitions

  • the present invention relates to the immortalisation of mammalian cells for therapeutic application.
  • transplantation of suitable cells into a damaged brain may improve or correct any sensory, motor, behavioural or psychological deficits caused by the damage.
  • WO-A-94/16059 discloses a technique for maintaining a primary neuronal cell culture in vi tro by culturing the cells m a serum-free media supplemented with at least one trophic factor.
  • WO-A-97/10329 discloses an alternative technique, using a conditionally- immortalised cell line.
  • This cell line comprises an immortalising temperature-sensitive oncogene which, under permissive conditions, maintains neuroepithelial stem cells m the undifferentiated state.
  • the oncogene Upon transplantation the oncogene is switched off due to the higher temperature of the human body (37 C C) and the cells differentiate into the cell types required to repair damage.
  • the advantage of using the oncogene is that the cells are maintained m the undifferentiated state until transplantation, at which point the cells differentiate, m response to the specific damage, into the phenotype of the damaged or lost cells.
  • US-A-5688692 discloses cells expressing a non-DNA binding, temperature-sensitive T antigen.
  • a recombinant, or genetically engineered, mammalian cell comprises a conditionally-mducible or temperature- sensitive oncogene and, separately, an exogenous polynucleotide encoding a member of the myc oncogene family.
  • a recombinant polynucleotide construct comprises a gene that encodes a member of the myc family of oncogenes and a conditionally-mducible or temperature-sensitive oncogene.
  • a method for immortalising a mammalian cell comprises incorporating, within a proliferating mammalian cell, a conditionally- mducible oncogene and an exogenous polynucleotide encoding a member of the myc family of oncogenes.
  • Cells of the present invention may be used m therapy, m particular m the manufacture of a medicament for the treatment of a disease associated with cell loss or damage.
  • Figure 1 is a schematic illustration of a polynucleotide construct containing both the temperature- sensitive oncogene encoding the SV40 large T-antigen and the C-myc oncogene;
  • FIG. 2 is a schematic illustration of an alternative construct with the temperature-sensitive oncogene and the C-myc oncogene m a different order. Description of the Invention
  • the present invention discloses methods for preparing cells which are suitable for transplantation therapy and which are immortal up to the time of transplantation.
  • the cells require a conditionally-mducible oncogene to be present.
  • conditionally-mducible is used herein to refer to oncogenes, the expression of which can be regulated under certain conditions.
  • the oncogene will undergo expression when so-called permissive conditions are applied.
  • some oncogenes are temperature- sensitive and are only expressed when the temperature of their environment is below a certain value.
  • the oncogene that is used is a non-DNA binding, temperature- sensitive, mutant of the SV- 40 large T-antigen gene, e.g.
  • U19tsA58 (Almazon and McKay, Brain Res., 1992;579:234-245). Suitable alternatives are also known and include the oncogene of the polyoma T- antigen adenovirus EIA and HPV16 or 18E7. Suitable cDNA variants may also be used. The cells also require an exogenous polynucleotide that encodes a member of the myc oncogene family.
  • the myc proto-oncogene family includes C- yc, N-myc, -myc, B-myc, V-myc and Gag-.myc.
  • a review of the myc family is provided by Alt et al . , Cold Spring Harbour Sy p . Quant. Biol . , 1986;51:931-941.
  • At least C-myc, N-myc, L- myc and B-myc have a similar gene structure and encode nuclear phosphoprotems with homologous ammo acid sequences (Legouy et al . , EMBO J, 1987;6:3359-3366; and Ingvarsson et al . , Mol . Cell Biol . , 1988;8:3168-3174).
  • Each of these myc oncogenes is suitable for use m the present invention, although C- yc is preferred..
  • exogenous is used herein m its normal context to refer to the polynucleotide introduced into the cell.
  • the conditionally-mducible oncogene and the polynucleotide encoding the myc gene may be comprised m a recombinant DNA or retroviral vector or construct to transduce/ infect the cells.
  • the two components may be incorporated into one vector or each may be comprised a separate vector.
  • the vectors or constructs of the invention may further comprise a suitable promoter region to initiate transcription of DNA and a selectable marker which may be used to identify those cells that have undergone transduction/mfection . Regulation of expression may be carried out by methods known to the skilled person.
  • regulation may be effected using the long terminal repeat (LTR) promoter.
  • LTR long terminal repeat
  • Alternative promoters will be apparent to the skilled person.
  • regulation may be effected using the cytomegalovirus (CMV) promoter.
  • CMV cytomegalovirus
  • the CMV promoter is a very strong promoter, and may be preferred when the cells are neural cells, e.g. neuroepithelial stem cells.
  • the cell may be an endothelial cell, and may be used for the revascularisation of the leg, heart and other organs.
  • the cell is a human somatic cell, e.g. human epithelial stem cell, which is capable of differentiation into a specific cell type.
  • a particularly preferred cell is a human neuroepithelial stem cell which may be used m neural transplantation to repair cell loss or damage and correct behavioural or psychological deficits.
  • neuroepithelial cells may be used in the treatment of Alzheimer's disease, Parkinson's disease, stroke and other forms of cerebral ischaemia, cerebral palsy, multiple sclerosis, Huntingdon's disease and Creuzfeld-Jacob ' s disease.
  • the cell may be a differentiated cell, e.g. the ⁇ cells of Islets of Langerhans .
  • Additional cells may include but are not limited to those obtainable from the endocrine glands, retinal cells, cochlear cells, liver cells, kidney cells, pancreatic cells, osteoblast and osteoclasts, haemopoietic cells, myoblasts and keratmocytes .
  • the oncogene and the myc gene are incorporated into the cell during the early culture phase, usually within the first 10 cell divisions.
  • the order of incorporating the oncogene and myc gene is not critical to the success of the method, although it is preferred that the myc gene is introduced first. This is because it has been found, surprisingly, that introducing the myc gene first provides better assurance for achieving a dipoid cell line.
  • the transduced or infected cells may be cultured under conditions known to those skilled m the art. It is preferable that the cells are cultured under non-stressed conditions. A skilled person will appreciate the conditions suitable for each particular cell type, based on conventional culture techniques.
  • the constructs may be used to transduce suitable cells to produce conditionally-immortalised cells that have improved stability during passaging.
  • the myc gene may activate directly the catalytic subunit of telomerase (Wu et al . , Nature Genetics, 1999; 21:220-224). This may maintain the chromosomes during cell replication.
  • the recombinant cells of the invention may have use m therapy.
  • Methods for the preparation of formulations for delivery to a patient will be apparent to the skilled person. Suitable excipients, diluents etc, will again be apparent based on current practice m preparing cell -based therapies.
  • the amount of cells required for delivery will vary depending on the form of treatment, the severity of the disease/damage, and the need for applying multiple doses over a treatment period. The skilled person can readily determine the appropriate treatment based on existing cell transplantation therapies.
  • the cells may be administered using conventional techniques, for example, for neuroepithelial cells, intracerebral injection.
  • thermolabile T-antigen derived from the non-DNA mutant of the SV40 early region (U19tsA58) ; the human c-myc gene; and a dominantly-acting selectable neomycin phosphotransferase resistant marker (Neo) which encodes resistance to G418 (Clontech) .
  • the final construct is assembled in the Moloney murine leukemia virus (MoMuLV) and Moloney murine sarcoma virus (MoMuSV) based retroviral vector, pLNCX (Clontech) (Miller and Buttimore, Mol. Cell Biol. 1986; 6:2895-2902).
  • MoMuLV LTR is used to drive neo
  • the CMV promoter is used to drive U19tsA58.
  • An internal ribosome entry site (IRES) is integrated between the U19tsA58 and c-myc genes (fused in frame to the c-myc gene) to induce reinitiation of translation by eukaryotic ribosomes .
  • the resulting construct is shown in Figure 1.
  • Construct 2 MoMuLV LTR is used to drive neo
  • the CMV promoter is used to drive c -myc .
  • constructs are packaged into a retroviral producer system using the human origin TEFLY cell system and methods disclosed in US 6165715, and clonal producers are generated that produced virus at a titer of 10 4 - 10 5 colony forming units/ml.
  • Cultures of human fetal central nervous system, liver, kidney and pancreas from a hospital source using ethical protocols are digested, and freshly isolated single dividing precursor cell suspensions are placed into tissue culture using standard techniques (such as described m WO- A-97/10329) and infected with 3-5 rounds of retrovirus derived m growth media. Following selection for several days under either permissive temperature culture (i.e. at 33°C) and/or G418 treatment, colonies of cells gradually form. These can be transferred as single isolated clones or polyclonal populations and expanded under permissive temperature conditions.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Oncology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

Selon l'invention, une cellule recombinée de mammifère comprend un oncogène inductible sous condition et un polynucléotide exogène codant pour un élément de la famille oncogène myc.
PCT/GB2001/001029 2000-03-10 2001-03-09 Immortalite conditionnelle de cellules WO2001066781A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU37610/01A AU3761001A (en) 2000-03-10 2001-03-09 Conditional immortalisation of cells

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0005856.0 2000-03-10
GB0005856A GB0005856D0 (en) 2000-03-10 2000-03-10 Genetic constructs

Publications (1)

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WO2001066781A1 true WO2001066781A1 (fr) 2001-09-13

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AU (1) AU3761001A (fr)
GB (1) GB0005856D0 (fr)
WO (1) WO2001066781A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003035879A2 (fr) * 2001-10-26 2003-05-01 Reneuron Limited Promoteurs destines a reguler la differenciation cellulaire
EP1645626A1 (fr) * 2004-09-30 2006-04-12 Reneuron Limited Lignée cellulaire
WO2012059223A1 (fr) * 2010-11-02 2012-05-10 Helmholtz-Zentrum für Infektionsforschung GmbH Procédés et vecteurs pour l'immortalisation de cellules

Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003882A1 (fr) * 1987-10-09 1989-05-05 Immunex Corporation Vecteurs retroviraux de transformation de promoteurs multiples
WO1989003872A1 (fr) * 1987-10-29 1989-05-05 Amrad Corporation Limited Production de lignees cellulaires precurseurs neurales
WO1989009816A1 (fr) * 1988-04-12 1989-10-19 Massachusetts Institute Of Technology Procede de manipulation des types cellulaires d'eucaryotes
WO1996024669A1 (fr) * 1995-02-10 1996-08-15 The Regents Of The University Of California Lignees de cellules pancreatiques humaines: developpements et utilisations
WO1997010329A1 (fr) * 1995-09-12 1997-03-20 Reneuron Limited Transplantation neurale a l'aide de cellules neuroepitheliales pluripotentes
WO1997030168A1 (fr) * 1996-02-20 1997-08-21 The Regents Of The University Of California Systeme retroviral modulable destine a la modification genetique de cellules
WO1997039117A1 (fr) * 1996-04-17 1997-10-23 The University Of Liverpool Lignees de cellules a immortalisation conditionnelle derivant d'animaux transgeniques
WO1999053028A1 (fr) * 1998-04-14 1999-10-21 Signal Pharmaceuticals, Inc. Lignees de cellules du snp et leur methode d'utilisation
WO2000009669A1 (fr) * 1998-08-12 2000-02-24 Signal Pharmaceuticals, Inc. Lignees cellulaires du mesencephale humain et procedes d'utilisation desdites lignees cellulaires
WO2001021790A1 (fr) * 1999-09-17 2001-03-29 Reneuron Limited Immortalisation conditionnelle de cellules

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989003882A1 (fr) * 1987-10-09 1989-05-05 Immunex Corporation Vecteurs retroviraux de transformation de promoteurs multiples
WO1989003872A1 (fr) * 1987-10-29 1989-05-05 Amrad Corporation Limited Production de lignees cellulaires precurseurs neurales
WO1989009816A1 (fr) * 1988-04-12 1989-10-19 Massachusetts Institute Of Technology Procede de manipulation des types cellulaires d'eucaryotes
WO1996024669A1 (fr) * 1995-02-10 1996-08-15 The Regents Of The University Of California Lignees de cellules pancreatiques humaines: developpements et utilisations
WO1997010329A1 (fr) * 1995-09-12 1997-03-20 Reneuron Limited Transplantation neurale a l'aide de cellules neuroepitheliales pluripotentes
WO1997030168A1 (fr) * 1996-02-20 1997-08-21 The Regents Of The University Of California Systeme retroviral modulable destine a la modification genetique de cellules
WO1997039117A1 (fr) * 1996-04-17 1997-10-23 The University Of Liverpool Lignees de cellules a immortalisation conditionnelle derivant d'animaux transgeniques
WO1999053028A1 (fr) * 1998-04-14 1999-10-21 Signal Pharmaceuticals, Inc. Lignees de cellules du snp et leur methode d'utilisation
WO2000009669A1 (fr) * 1998-08-12 2000-02-24 Signal Pharmaceuticals, Inc. Lignees cellulaires du mesencephale humain et procedes d'utilisation desdites lignees cellulaires
WO2001021790A1 (fr) * 1999-09-17 2001-03-29 Reneuron Limited Immortalisation conditionnelle de cellules

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
BERNARD O ET AL: "ROLE OF THE C-MYC AND THE N-MYC PROTO-ONCOGENES IN THE IMMORTALIZATION OF NEURAL PRECURSORS", JOURNAL OF NEUROSCIENCE RESEARCH, vol. 24, no. 1, 1989, pages 9 - 20, XP001010016, ISSN: 0360-4012 *
GRAY JEFFREY A ET AL: "Prospects for the clinical application of neural transplantation with the use of conditionally immortalized neuroepithelial stem cells.", PHILOSOPHICAL TRANSACTIONS OF THE ROYAL SOCIETY OF LONDON B BIOLOGICAL, vol. 354, no. 1388, August 1999 (1999-08-01), Aug., 1999, pages 1407 - 1421, XP001010006, ISSN: 0962-8436 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003035879A2 (fr) * 2001-10-26 2003-05-01 Reneuron Limited Promoteurs destines a reguler la differenciation cellulaire
WO2003035879A3 (fr) * 2001-10-26 2003-09-25 Reneuron Ltd Promoteurs destines a reguler la differenciation cellulaire
EP1645626A1 (fr) * 2004-09-30 2006-04-12 Reneuron Limited Lignée cellulaire
JP2006122045A (ja) * 2004-09-30 2006-05-18 Reneuron Ltd 細胞株
US7416888B2 (en) 2004-09-30 2008-08-26 Reneuron Limited Cell lines
US7419827B2 (en) 2004-09-30 2008-09-02 Reneuron Limited Cell lines
US7666672B2 (en) 2004-09-30 2010-02-23 Reneuron Limited Cell lines
WO2012059223A1 (fr) * 2010-11-02 2012-05-10 Helmholtz-Zentrum für Infektionsforschung GmbH Procédés et vecteurs pour l'immortalisation de cellules
US9453203B2 (en) 2010-11-02 2016-09-27 Helmholtz-Zentrum Fur Infektionsforschung Methods and vectors for cell immortalisation
EP3257943A3 (fr) * 2010-11-02 2018-01-10 Helmholtz-Zentrum für Infektionsforschung GmbH Procédés et vecteurs pour l'immortalisation de cellules

Also Published As

Publication number Publication date
AU3761001A (en) 2001-09-17
GB0005856D0 (en) 2000-05-03

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