Gene expression in mammalian cells

Details Gene expression in mammalian cells Gene expression in mammalian cells

1997-10-24

2001-03-27

Schwartzman, Robert A. (Department: 1636)

Drug, bio-affecting and body treating compositions

Lymphokine

Interferon

C435S069400, C435S320100, C435S325000, C435S360000, C530S350000, C536S023520, C536S024100

Reexamination Certificate

active

06207146

ABSTRACT:

This invention relates to the expression of genes in mammalian cells, particularly genes responsible for proteins whose biological activity in vivo is affected by a diversity of factors including specific glycosylation. Examples of such genes are the human &bgr;-interferon (IFN&bgr;), human erythropoietin (EPO), human chorionic gonadotropin, various other cytokines and growth factors as well as specific viral antigens such as Dengue viral proteins whose structure may be relevant for the development of vaccines.
Previously, genes have been extensively expressed in mammalian cell lines, particularly in mutant Chinese Hamster Ovary (CHO) cells deficient in the dihydrofolate reductase gene (dhfr) as devised by the method of Urlaub et al, PNAS U.S.A. 77, 4216-4220, 1980. A variety of expression systems have been used. Many vectors for the expression of genes in such cells are therefore available. Typically, the selection procedures used to isolate cells transformed with the expression vectors rely on using methotrexate to select for transformants in which both the dhfr and the target genes are coamplified.
The dhfr gene, which enables cells to withstand methotrexate, is usually incorporated in the vector with the gene whose expression is desired. Selection of cells under increasing concentrations of methotrexate is then performed. This leads to amplification of the number of dhfr genes present in each cell of the population, as cells with higher copy numbers withstand greater concentrations of methotrexate. As the dhfr gene is amplified, the copy number of the gene of interest increases concomitantly with the copy number of the dhfr gene, so that increased expression of the gene of interest is achieved.
Unfortunately, these amplified genes have been reported to be variably unstable in the absence of continued selection (Schimke, J. Biol. Chem. 263, 5989-5992, 1988). This instability is inherent to the presently available expression systems of CHO dhfr
−
cells.
For many years, several promoters have been used to drive the expression of the target genes such as the SV40 early promoter, the CMV early promoter and the SR&agr; promoter. The CMV and SR&agr; promoters are claimed to be the strongest (Wenger et al, Anal. Biochem. 221, 416-418, 1994).
In one report, the &bgr;-interferon promoter has also been used to drive the expression of the &bgr;-interferon gene in the mutant CHO dhfr
−
cells (U.S. Pat. No. 5,376,567). In this system, however, the selected CHO dhfr
−
cells had to be superinduced by the method of Tan et al (Tan et al, PNAS U.S.A. 67, 464-471, 1970; Tan et al, U.S. Pat. No. 3,773,924) to effect a higher level of &bgr;-interferon production. In this system a significant percentage of the superinduced &bgr;-interferon produced by the CHO dhfr
−
cells was not glycosylated.
The mouse metallothionein gene (mMT1) promoter has also been used for the expression of &bgr;-interferon genes in CHO cells, BHK and LTK
−
mouse cells (Reiser et al 1987 Drug Res. 37, 4, 482-485). However, the expression of &bgr;-interferon with this promoter was not as good as the SV40 early promoter in CHO cells. Further, &bgr;-interferon expression from these cells mediated by the mMT1 promoter was inducible by heavy metals. Heavy metals are however extremely toxic to the cells and this system was therefore abandoned. Instead, Reiser et al used the CHO dhfr
−
expression system in conjunction with the SV40 early promoter (Reiser et al, Drug Res. 37,4, 482-485 (1987) and EP-A-0529300) to produce &bgr;-interferon in CHO dhfr
−
cells as derived by the method of Urlaub et al (1980).
We have now expressed &bgr;-interferon in wild-type CHO cells. Wild-type CHO cells were transfected with a vector comprising a &bgr;-interferon gene under the control of a mouse sarcoma viral enhancer and mouse metallothionein promoter (MSV-mMT1), a neo gene under the control of promoter capable of driving expression of the neo gene in both
E. coli
and mammalian cells and a human metallothionein gene having its own promoter. Transfected cells capable of expressing &bgr;-interferon were selected by first exposing cells to geneticin (antiobiotic G418) and thus eliminating cells lacking the neo gene and then exposing the surviving cells to increasing concentrations of a heavy metal ion.
The heavy metal ion enhanced the MSV-mMT1 promoter for the &bgr;-interferon gene, thus increasing &bgr;-interferon expression. The heavy metal ion also induced the human metallothionein gene promoter, causing expression of human metallothionein. The human metallothionein protected the cells against the toxic effect of the heavy metal ion. The presence of the heavy metal ion ensured that there was continual selection of cells which had the transfecting vector, or at least the &bgr;-interferon gene and the human metallothionein gene and their respective promoters, integrated into their genome.
The selected cells that had been successfully transfected expressed &bgr;-interferon. Expression was surprisingly improved when the cells were cultured in the presence of Zn
2+
. The &bgr;-interferon had improved properties, in particular a higher bioavailability, than prior &bgr;-interferons.
These findings have general applicability. Accordingly, the present invention provides:
a nucleic acid vector comprising:
(i)
a coding sequence which encodes a protein of
interest and which is operably linked to a
promoter capable of directing expression of the
coding sequence in a mammalian cell in the
presence of a heavy metal ion;
(ii)
a first selectable marker sequence which
comprises a metallothionein gene and which is
operably linked to a promoter capable of
directing expression of the metallothionein gene
in a mammalian cell in the presence of a heavy
metal ion; and
(iii)
a second selectable marker sequence which
comprises a neo gene and which is operably linked
to a promoter capable of directing expression of
the neo gene in a mammalian cell;
mammalian cells which harbour a nucleic acid sequence comprising:
(i)
a coding sequence which encodes a protein of
interest and which is operably linked to a
promoter capable of directing expression of
the coding sequence in a mammalian cell in
the presence of a heavy metal ion;
(ii)
a first selectable marker sequence which
comprises a metallothionein gene and which
is operably linked to a promoter capable of
directing expression of the metallothionein
gene in a mammalian cell in the presence of
a heavy metal ion; and optionally
(iii)
a second selectable marker sequence which
comprises a neo gene and which is operably
linked to a promoter capable of directing
expression of the neo gene in a mammalian
cell;
a process for producing such cells, which process comprises
(a)
transfecting mammalian cells with a vector of the
invention;
(b)
exposing the transfected cells to geneticin to
eliminate thereby cells lacking the neo gene; and
(c)
exposing the cells that survive step (a) to
progressively increasing concentrations of a
heavy metal ion to select thereby the desired
cells.
use of a neo gene and a metallothionein gene as selectable marker genes in a single vector; and a process for the preparation of a protein of interest, which process comprising culturing mammalian cells of the invention under conditions allowing expression of the desired protein and recovering the desired protein thus expressed.
By using both a neo gene and a metallothionein gene as selectable markers in a single vector, it is possible to select for transformed mammalian cells, such as wild-type CHO cells, which have multiple copies of the expression vector stably integrated into their genomes. This selection system therefore facilitates the preparation and identification of stably transformed mammalian cells such as the wild-type CHO cells and avoids the need for dhfr
−
cells. The transformed cells enable the stable expression of genes such as the human &bgr;-interferon gene because they have multiple copies, typically at least 20-100 copies or more, of these genes integrated into their genomes.
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