WO2001066781A1

WO2001066781A1 - Conditional immortalisation of cells

Info

Publication number: WO2001066781A1
Application number: PCT/GB2001/001029
Authority: WO
Inventors: John Sinden; Ziping Dong
Original assignee: Reneuron Limited
Priority date: 2000-03-10
Filing date: 2001-03-09
Publication date: 2001-09-13
Also published as: AU3761001A; GB0005856D0

Abstract

A recombinant mammalian cell comprises a conditionally-inducible oncogene and an exogenous polynucleotide encoding a member of the myc oncogene family.

Description

CONDITIONAL IMMORTALISATION OF CELLS

Field of the Invention

The present invention relates to the immortalisation of mammalian cells for therapeutic application. Background to the Invention

There is a growing awareness and understanding of the importance of transplantation therapy to treat damage to tissues and organs. While organ transplantation is widely practised, therapies based on the transplantation of individual cells are still in a relatively early phase of clinical development.

For example, there is growing recognition that the transplantation of suitable cells into a damaged brain may improve or correct any sensory, motor, behavioural or psychological deficits caused by the damage.

For cell-based therapies to be useful, it must be possible to obtain sufficient cells for transplantation. One means for ensuring this is to culture undifferentiated cells under conditions which allow repeated cell division and growth. One difficulty with using undifferentiated cells is that unregulated cell division must be switched off either prior to or on transplantation into the patient, to prevent uncontrolled growth at the site of transplantation . Many different techniques have been developed to provide suitable cells for transplantation. With regard to neural transplantation, one approach has been to maintain undifferentiated foetal cells under culture conditions that permit cell division to occur, and to subsequently induce differentiation in vi tro, prior to transplantation.

Reynolds and Weiss, Science, 1992 ; 255 : 1707 , disclose the use of epidermal growth factor (EGF) to induce the in vi tro proliferation of adult mouse brain cells. Under suitable conditions it was thought that the cells could be induced to differentiate into astrocytes and neurons.

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. Upon transplantation the oncogene is switched off due to the higher temperature of the human body (37^CC) 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.

Bernard et al . , Journal of Neurosc ence Research, 1989;24:9-20, discloses the use of c-myc and N-rayc to immortalise neural precursor cells.

It is recognised that, although cells immortalised with oncogenes have an extended life, they have decreased genetic stability and eventually succumb to crisis (cell death) . Although many of the techniques disclosed above may be useful, there is still a need for improved methods to obtain cells which retain immortality prior to transplantation, but which lose immortality on transplantation . Summary of the Invention

The present invention is based on the realisation that cells transduced with a conditionally-mducible oncogene and a member of the myc family of oncogenes have increased genetic stability and are immortal under permissive conditions but lose immortality under non-permissive conditions . According to one aspect of the present invention, 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.

According to a second aspect of the invention, 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.

According to a third aspect, 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.

It has been found that cells according to the present invention retain a high level of stability, and at non- permissive conditions are not immortal. This is a surprising and important finding as it would be expected for the cells to remain immortal, due to the activity of the myc gene. The retention of conditionality and increased stability, makes the cells of the present invention suitable to be passaged serially to derive a cell line for transplantation. Description of the Drawings

The accompanying drawings illustrate the invention. 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; and

Figure 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. The term "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. For example, some oncogenes are temperature- sensitive and are only expressed when the temperature of their environment is below a certain value. In one embodiment of the invention, 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..

The term "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. For example, regulation may be effected using the long terminal repeat (LTR) promoter. Alternative promoters will be apparent to the skilled person. For example, regulation may be effected using the cytomegalovirus (CMV) promoter. The CMV promoter is a very strong promoter, and may be preferred when the cells are neural cells, e.g. neuroepithelial stem cells.

Methods for constructing myc constructs for transduction into cells can be found m Bernard et al . , supra . Retroviral vectors, including the Zen vectors

(Haπlaran et al . , Oncogene Research, 1988;3:387-399) may be utilised.

Methods for introducing suitable constructs into cells, are known to the skilled person.

Any mammalian cell may be used m the present invention. For example, the cell may be an endothelial cell, and may be used for the revascularisation of the leg, heart and other organs. Preferably, 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. For example, 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. Alternatively, the cell may be a differentiated cell, e.g. the β 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 .

Preferably, 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.

The following Example illustrates the invention with reference to the accompanying drawings. Example

Production of suitable expression vectors is achieved by co-expressing 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). Construct 1

MoMuLV LTR is used to drive neo, and 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, and the CMV promoter is used to drive c -myc . An internal ribosome entry site

(IRES) is integrated between the c -myc and U19tsA58 genes

(fused in frame to the U19tsA58 gene) to induce reinitiation of translation by eukaryotic ribosomes. The resulting construct is shown in Figure 2.

The 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 10⁴ - 10⁵ 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.

From the pure populations of clones of precursor cells, transduced with the combined tsA58Ul9-c-myc construct, senescence is overcome and continued proliferation for over 100 doublings is observed without overt crisis or change m phenotype . Transferral to non- permissive temperature culture (37-39°C) results m growth arrest and the appearance of mature, differentiated phenotypes m the cells. The presence of the c-myc gene in the cells does not alter the conditionally immortal phenotype of the cells conferred by the tsA58U19 gene, but preserves the stability of the diploid karyotype of the cells at later passage. Cell lines derived using transduction with the tsA58U19 gene only, demonstrate frequent instability and polyploidy at later passage. Thus, c-myc introduced into the cell's genome at an early stage is able to protect the subsequent cell line from karyotypic instability, and this is a requirement for the eventual safe use of cell line transplants into a diseased or injured patient.

Claims

I. A mammalian cell comprising a conditionally-mducible oncogene and an exogenous polynucleotide encoding a member of the myc oncogene family.

2. A cell according to claim 1, wherein the conditionally-mducible oncogene and the exogenous polynucleotide are comprised m a recombinant vector.

3. A cell according to claim 1 or claim 2, wherein the conditionally-mducible oncogene is temperature-sensitive.

4. A cell according to any preceding claim, which is a human somatic cell .

5. A cell according to any preceding claim, which is a mammalian stromal fibroblast cell or a mammalian microvascular cell.

6. A cell according to any of claims 1 to 4, which is a human stem cell .

7. A cell according to claim 6, which is a neuroepithelial stem cell.

8. A cell according to any preceding claim, wherein the conditionally-mducible oncogene comprises the temperature- sensitive mutant of the SV-40 T-antigen gene.

9. A cell according to claim 8, wherein the mutant is tsA58-U19.

10. A cell according to any preceding claim, wherein the myc oncogene is C-myc or V-myc .

II. A recombinant polynucleotide that encodes a member of the myc oncogene family and a conditionally-mducible oncogene .

12. A polynucleotide according to claim 11, further comprising a selectable marker gene and a promoter region.

13. A method for immortalising a mammalian cell, comprising incorporating withm a proliferating cell a conditionally-mducible oncogene and an exogenous polynucleotide encoding a member of the myc oncogene family.

14. A method according to claim 13, wherein the oncogene and exogenous polynucleotide are incorporated into the cell within the first 10 cell divisions.

15. A method according to claim 13 or claim 14, wherein the exogenous polynucleotide is introduced into the cell before the oncogene.

16. Use of a cell according to any of claims 1 to 10, in the manufacture of a medicament for the treatment of a disease associated with cell loss or damage.

17. Use according to claim 16, wherein the cell is a neuroepithelial cell.