WO1993021311A1

WO1993021311A1 - Immortalisation of human cells by gene silencing

Info

Publication number: WO1993021311A1
Application number: PCT/AU1993/000170
Authority: WO
Inventors: Robin Holliday
Original assignee: Commonwealth Scientific And Industrial Research Organisation
Priority date: 1992-04-15
Filing date: 1993-04-15
Publication date: 1993-10-28

Abstract

The present invention relates to a method of silencing a gene(s) in normal human diploid cells. The method involves permeabilising the cells in the presence of 5-methyl dCPT. The cells are then allowed to multiply and the cells in which the gene(s) has been silenced are recovered. In preferred forms of the present invention the histocompatibility genes of the cells are silenced. The present invention also relates to cells in which genes have been silenced by the method of the present invention. The method of the present invention of silencing genes is reversible.

Description

IMMORTALISATION OF HUMAN CELLS BY GENE SILENCING Field of the Invention

The present invention relates to a method for gene silencing and immortalisation of human cells. The method involves the permeabilisation of the cells by electroporation, or by other means in the presence of 5 methyl deoxycytidine triphosphate. Background of the Invention

Human cells grown in culture have finite lifespan. They can be transformed with difficulty to immortal cell lines. Cells isolated from human tumours are also transformed and sometimes grow as an immortal cell line in culture.

The genetic code in DNA consists of four bases - adenine, thymine, guanine and cytosine.. A fifth base, 5 methyl cytosine (5mC), can be produced by the enzymic modification of cytosine after DNA replication. In many biological contexts it is known that 5mC in regulatory regions of genes prevents or silences their activity. There is evidence that in the formation of immortal transformed tumour cells, specific genes become inactive ("tumour suppressor" genes). Usually it is thought that this occurs by mutation, that is, a change in the sequence of bases in DNA. It is also possible that these genes can be inactivated by methylation of cytosine.

A new method was recently developed by Nyce (1991, Gene silencing in mammalian cells by direct incorporation of electroporated 5-methyl-2'-deoxycytidine-5'- triphosphate. Somatic Cell and Molecular Genetics 17.543-550) and Holliday and Ho (1991, Gene silencing in mammalian cells by uptake of 5-methyl deoxycytidine-5 '- triphosphate, Somatic Cell and Molecular Genetics 17:537-542) in which genes are silenced in cultured hamster cells by the direct incorporation of 5mC into DNA. In DNA synthesis the immediate precursors have bases attached to deoxyribose triphosphate (i.e. dATP- dTTP, dGRP and dCTP) . The methylated derivative of cytosine, 5 methyl deoxycytidine triphosphate (5 methyl dCTP) is never synthesised in the cell, nor is it taken up by cells from the medium. The new method depends on the permeabilisation of cells by electroporation, or by other procedures in the presence of 5 methyl dCTP. (Usually electroporation is used to facilitate uptake of DNA itself into cells) . Under these conditions 5 methyl dCTP is incorporated directly into DNA and it has been shown that specific genes of hamster cells then becomes silenced. It is known that once cytosine in DNA is methylated, it can be perpetuated _r or inherited, by a maintenance methylase. Thus the silenced genes are stably inherited. Independent evidence shows that they are partly methylated.

Human fibroblasts grow to 50-70 PDs before they become senescent. Transformed cells never appear in such populations, although they would be strongly selected if they arose. Mutagens and chromosome damaging agents which readily transform rodent cells _r do not induce human fibroblast transformation in vitro. The only method available for obtaining transformation depends on the infection of the cells with an oncogenic virus such as SV40 (or with DNA from the virus). This is believed to induce transformation by inhibiting tumour suppressor genes, but it is well known that these transformed cells very rarely give rise to immortalised cell lines.

The present inventors have now found that genes in human cells may be silenced by permeablisation of the cells in the presence of 5-methyl dCTP.

Accordingly, in a first aspect the present invention consists in a method of silencing a gene(s) in human cells, the method comprising permeabilising the cells in the presence of 5-methyl dCTP, allowing the cells to multiply and recovering the cells in which the gene(s) has been silenced.

While a number of methods of permeabilising the cells may be used including, streptolysin treatment or commercially available reagents known under the trade names of " ransfecton", "TranfectACE" and "Dotap", however, it is presently preferred that the cells are permeabilised by electroporation.

In a preferred embodiment of the present invention the cells are exposed to a plurality of cycles of electroporation in the presence of 5-methyl dCTP. Preferably, the cells are exposed to 3-8 cycles.

In a further preferred embodiment of the present invention the electroporation is conducted at less than 300V and preferably at 250V. It is also preferred that the electroporation is carried out at 25 μF .

In a preferred embodiment of the present invention the cells when subjected to permeablisation in the presence of 5-methyl dCTP are in S phase.

Typically, the method of the present invention will also include a selection step in which cells containing silenced genes are selected for. This selection procedure will, of course, vary according to the gene(s) which has been silenced.

In a preferred embodiment of the present invention the tumour suppressor genes of the cell are silenced thereby rendering the human cell immortal.

In yet a further preferred embodiment of the present invention the HLA-A histocompatibility gene is silenced. In this embodiment of the present invention the selection procedure involves selection for cells with lacking expression of the HLA-A antigen. Detailed Description of the Invention

In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples. The human foetal lung fibroblast strain, MRC-5, was used and the cells were grown and subcultured in minimal essential medium (MEM) containing 10% serum, with non-essential amino acids, penicillin and streptomycin. Young early passage (approximately 20) cells were harvested after treatment with tryspin-versene. These cells were seeded in flasks with MEM and 10% serum, and incubated for 24 hrs. Subsequently the medium was removed, the cells were washed with phosphate buffered saline, and MEM without serum was added. After a further 24 hrs incubation, 10% serum is added to the medium. 20 hours later the majority of cells are synthesising DNA (S phase) . They are harvested with trypsin versene and washed twice with sucrose phosphate buffer (272 mM sucrose? 7mM NaH₂P0₄; ImM MgCl₂) and 3 x 10 cells were suspended in 0.5 ml buffer containing 20μl of a lOmM solution of 5 methyl dCTP and held for 10 mins on ice before electroporation. Electroporation was carried out with a BioRad Gene Pulser set at 25μF and 250v. After treatment the cells were again held 10 minutes on ice

2 before diluting into 10 ml medium in a 25 cm flask.

The medium was changed after 24 hours and the flask incubated until the cells became confluent.

The effectiveness of the method for silencing genes was demonstrated for the X-linked gene coding for the enzyme hypoxanthine-guanine phosphoribosyl transferase

(HPRT) . After treatment with 5 methyl dCTP, cells were subcultured after 3-4 days, incubated a further 3-4 days and then harvested. Cells were seeded in 10cm dishes with either 2.5 x 10 4 or 5 x 104 cells per plate containing

15 ml medium. After 24 hours 6 thioguanine (6TG) was added at a concentration of 6μg/ml. The medium containing 6TG was replaced after 7 days and again after a further 7 days incubation. Colonies which grew in the presence of 6TG are deficient in HPRT. Cells from individual colonies were isolated and shown to have the following properties. They were unable to grow in a medium containing hypoxanthine, aminopterin and thymidine (standard HAT medium), whereas normal MRC-5 cells grow in this medium. The cells resistant to 6TG were treated for 24 hrs with lμg/ml 5 azacytidine which is a DNA demethylating agent. These cells produced colonies in HAT medium, showing that the silent HPRT gene was reactivated to produce active enzyme. These reactivated cells will not grow in medium containing 6TG. Thus, the procedure outlined has been shown to silence a gene in normal human diploid fibroblasts. The method could also be applied to any gene of interest, where the cells with a silent gene can be selected. A further example is the silencing of the HLA-A histocompatibility gene, where cells lacking expression of the antigen can be selected with the appropriate specific antiserum and complement.

To select immortalised cells two possibilities are available. First, the treatment repeated several times can result in the appearance of cells with altered morphology. These cells lack "contact inhibition", which is a characteristic of transformed cells, so they continue to grow when normal diploid cells cease growth at confluence. Thus, during the subculture of the mixed population the transformed cells are selected, and an immortal line can be obtained. Second, when normal cells become senescent they do not form a confluent monolayer. However, any immortal cells which are present continue to grow to form small foci. These are isolated and constitute an immortal cell line.

As well as the silencing of tumour suppressor genes and standard genes for metabolic activity (such as HPRT), the method is applicable to genes coding for histocompatibility antigens (HLA) , or other genes with specialised functions. Dominant inherited conditions which are caused by over-expression of a gene, or the production of an abnormal gene product,- could be eliminated in appropriate cells (such as cells of the haematopoietic system, or other appropriate stem cells) by the gene silencing procedure described.

The present disclosure is the first example of immortalisation of human fibroblasts in vitro not involving the infection of cells with an oncogenic virus such as SV40. This is a major advance over the prior art. Immortal human cell lines are important for several reasons. Inherited defects can be most readily studied at the biochemical and molecular level using cells obtained from affected individuals. Such cells, however, have only finite growth in culture, so many biochemical and genetic studies are hindered or are impossible. Laboratories frequently try to obtain SV40 transformed derivatives which still carry the inherited defect but grow indefinitely. Permanent cell lines, however, have only sometimes been obtained by this procedure. Human hybridomas are difficult to work with because as they are often unstable and have a finite lifespan. Immortalisation of human hybridomas would make it possible to obtain continuous production of antibody.

Human diploid fibroblasts are also routinely used for the growth of viruses for the production of vaccines. It is necessary, however, to continually renew populations by defrosting a frozen ampoule of young cells. It is possible that immortalised cells which were not transformed with an oncogenic virus, such as SV40, would be more suitable for vaccine production.

It is often the case that cells with specialised functions in vivo cannot grown in culture, or only for a very small number of divisions. This is true, for example, for cells of the islets of Langerhans in the pancreas which synthesise and secrete insulin. If such cells could be transformed and grown indefinitely in culture, a major advance would have been achieved. There are many types of specialised cells, including stem line cells which could be advantageously used for in vitro organ culture. Such cells could be studied far more easily than in vivo, or they may be important because they synthesis a specific hormone, growth factor or other product of pharmaceutical significance.

It is important to emphasise that the gene silencing procedure which is described can also be reversed by demethylating the DNA. This is an advantage over other procedures which may depend 1) on the incorporation on additional DNA sequences, 2) the integration of viral genomes, or 3) mutation of the gene of interest, as none of these 3 types of event can be readily reversed.

As will be recognised by those skilled in the art the present invention has a wide number of applications. By silencing the histocompatibility genes "univeral" donor cells for use in transplantation can be produced. Indeed, it is foreseeable that a wide range of cells which do not express antigens typically involved in transplantation rejection can be produced. This opens up the possibility of universal donor cells.

As mentioned above, the present invention can be used to produce immortalised human cells. Such immortalised cells have a wide range of uses not the least of which industrial production of valuable products include virus production.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS : -

1. A method of silencing a gene(s) in normal diploid human cells, the method comprising permeabilising the cells in the presence of 5-methyl dCTP, allowing the cells to multiply and recovering the cells in which the gene(s) has been silenced.

2. A method as claimed in claim 1 in which the cells are exposed to a plurality of cycles of electroporation in the presence of 5-methyl dCTP.

3. A method as claimed in claim 2 in which the cells are exposed to 3-8 cycles of electroporation.

4. A method as claimed in claim 2 or claim 3 in which the electroporation is conducted at less than 300V and at

25 μF. 5. A method as claimed in claim 4 in which the electroporation is conducted at 250V.

6. A method as claimed in any one of claims 1 to 5 in which the method includes a selection step in which cells containing the silence gene(s) are selected for.

7. A method as claimed in any one of claims 1 to 6 in which the cells when subjected to permeablisation in the presence of 5-methyl dCTP are in S phase.

8. A method as claimed in any one of claims 1 to 7 in which tumour suppressor genes of the human cell are silenced thereby rendering the human cell immortal.

9. A method as claimed in any one of claims 1 to 7 in which the histocompatibility genes are silenced.

10. A method as claimed in claim 9 in which the HLA-A histocompatibility gene is silenced.

11. A human cell in which a gene(s) has been silenced by the method as claimed in any one of claims 1 to 7.

12. A human cell which does not express the histocompatibility antigens, the cell being characterised in that the histocompatibility genes have been silenced by permeabilising the cell in the presence of 5-methyl dCTP.

13. A human cell which does not express the HLA-A antigen, the cell being characterised in that the HLA-A gene has been silenced by permeabilising the cell in the presence of 5-methyl dCTP.

14. A transformed, immortal human cell, the cell being characterised in that the cell has been transformed by permeabilising the cell in the presence of 5-methyl dCTP.

15. A human cell in which a gene(s) has been silenced by the method as claimed in any one of claims 1 to 7 and in which the silenced gene(s) has subsequently been reactivated.