Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
2000-09-15
2002-06-04
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S368000, C536S023500
Reexamination Certificate
active
06399384
ABSTRACT:
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 practiced, 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 vitro, prior to transplantation.
Reynolds and Weiss, Science, 1992;255:1707, disclose the use of epidermal growth factor (EGF) to induce the in vitro proliferation of adult mouse brain cells. Under suitable conditions it was thought that the cells could be induced to differentiate into astrocytes and neurons.
International Patent Application No. WO-A-94/16059 discloses a technique for maintaining a primary neuronal cell culture in vitro by culturing the cells in a serum-free media supplemented with at least one trophic factor.
International Patent Application No. 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 in the undifferentiated state. Upon transplantation the oncogene is switched off due to the higher temperature of the human body (37° 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 in the undifferentiated state until transplantation, at which point the cells differentiate, in response to the specific damage, into the phenotype of the damaged or lost cells. U.S. Pat. No. 5,688,692 also discloses cells expressing a non-DNA binding, temperature-sensitive T antigen.
However, it is recognised that although human cells expressing oncogenes can have an extended life, they still stop dividing and eventually undergo crisis (cell death).
It has also been proposed that human cells can be immortalised by reconstituting telomerase activity BY incorporating an exogenous copy of the catalytic subunit of human telomerase (Bodnar et al, Science, 1998; 279:249-252). Telomerase acts to maintain telomerase found at the ends of chromosomes, and it is believed that the gradual shortening of telomeres during cell duplication contributes to senescence and that therefore reconstituting telomerase immortalises cells. Human telomerase has now been used to immortalise many different cell types.
Counter et al., PNAS (USA), 1998; 95(25):4723-14728, also discloses that ectopic expression of the telomerase catalytic subunit (hTERT) can allow post senescent cells to proliferate beyond crisis to cellular immortality. The cells studied were transformed with an oncogene expressing SV40 T-antigen. However, the authors conclude that hTERT expression alone may suffice to immortalise cells, and that activation of hTERT may be a critical step in tumour progression. Therefore, the general teaching of this publication is that hTERT transformed cells would be immortal. This means that the cells would be unsuitable for use in transplantation therapy.
Therefore, while many of the techniques disclosed above may be useful, there is still a need for 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-inducible oncogene and at least the catalytic subunit of the telomerase complex 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-inducible or temperature-sensitive oncogene, and an exogenous polynucleotide encoding at least the catalytic sub-unit of the telomerase complex.
According to a second aspect of the invention, a recombinant polynucleotide construct comprises a gene that encodes at least the catalytic sub-unit of the telomerase complex, and a conditionally-inducible 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-inducible oncogene and an exogenous polynucleotide encoding the catalytic sub-unit of the telomerase gene.
According to a fourth aspect, the cells of the present invention may be used in therapy, in particular in the manufacture of a medicament for the treatment of a disease associated with cell loss or damage. For example, neuroepithelial stem cells may be used for the treatment of disorders associated with brain damage e.g. Alzheimers.
It has been found that cells according to the present invention retain a high level of stability and at non-permissive temperatures are not immortal.
This is a surprising and important finding as it would be expected, based on the prior art, for the cells to remain immortal, due to the reconstitution of the telomerase activity. However, it appears that, although the gene encoding the telomerase is constitutive, the telomerase does not act to retain immortality. 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.
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Mark F. Mehler et al, Progenitor Cell Biology, Implications for Neural Regeneration, Section Editor: David E. Pleasure, MD Arch Neurol/vol. 56, Jul. 1999.*
Kiyono, Tohru, Scott A. Foster, Jenn I. Koop et al. (Nov. 5, 1998) “Both Rb/p16INK4ainactivation and telomerase activity are required to immortalize human epithelial cells”Nature396:84-88.
Stamps, Alasdair C., susan C. Davies, Jacqueline Burman and Michael J. O'Hare (1994) “Analysis Of Proviral Integration In Human Mammary Epithelial Cell Lines Immortalized By Retroviral Infection With A Temperature-Sensitive SV40 T-Antigen Construct”Int. J. Cancer57:865-874.
Hahn, William C., Christopher M. Counter, Ante S. Lundberg et al. (Jul. 29, 1999) “Creation of human tumour cells with defined genetic elements”Nature400:464-468.
Zhu, Jiyue, He Wang, J. Michael Bishop and Elizabeth H. Blackburn (Mar. 1999) “Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening”Proc. Natl. Acad. Sci. USA96:3723-3728.
Bodnar, Andrea G., Michel Quellette, Maria Frolkis et al. (Jan. 1998) “Extension of Life-Span by Introduction of Telomerase into Normal Human Cells”Science279:349-252.
Counter, Christopher M. et al. (Dec. 1998) “Dissociation among in vitro telomerase activity, telomere maintenance, and cellular immortalization”PNAS(USA) 95(25):14723-14728.
Reynolds, Brent A. and Samuel Weiss (Mar. 1992) “Generation of Neuro
Ketter James
Reneuron Limited
Saliwanchik Lloyd & Saliwanchik
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