Chemistry: molecular biology and microbiology – Process of mutation – cell fusion – or genetic modification – Introduction of a polynucleotide molecule into or...
Reexamination Certificate
1999-02-11
2002-07-09
Clark, Deborah J. R. (Department: 1632)
Chemistry: molecular biology and microbiology
Process of mutation, cell fusion, or genetic modification
Introduction of a polynucleotide molecule into or...
C435S006120, C435S320100, C435S325000, C435S369000, C435S467000, C530S350000
Reexamination Certificate
active
06417002
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods which allow for the stable maintenance and selection of at least one episome in eukaryotic cells. The invention can be used to maintain and select episomes in cultured cells as well as in cells that are the subject of gene therapy without the need for exogenous selection factors, such as antibiotics.
BACKGROUND OF THE INVENTION
In the field of molecular biology it is often desirable to express exogenous proteins in eukaryotic cells. This can be achieved through the stable maintenance of exogenous DNA encoding the proteins of interest in the desired cells. One method for producing stably transfected eukaryotic cells which express a gene of interest requires integration of multiple genes into one or more chromosomal loci. However, integration of exogenous genes into chromosomes is unpredictable and it is normally necessary to screen many clonal cell populations to obtain a cell line in which all of the desired genes are expressed at an appropriate level. This process is time consuming.
Recently, non-integrating, autonomously replicating episomal vectors have been used to circumvent many of these difficulties. The Epstein Barr Virus (EBV) Nuclear Antigen 1 protein (EBNA 1) has been used to stably maintain plasmids containing the EBV origin of replication (oriP) in primate cells (Reisman, D. et al,
Mol. Cell. Biol.
5: 1822-1832, 1985; Yates, J. L. et al.,
Nature
313:812-815, 1985). EBNA1 is the only protein that is required in primate and canine cells to maintain plasmids that contain an EBV origin of replication in an episomal state.
Transfection of cell lines that already express EBNA1 can be extremely advantageous, as the ability of such cells to stably maintain episomal constructs can be enhanced by several orders of magnitude and stable cell lines can be generated in as little as two to three weeks (Horlick et al.,
Prot. Exp. And Purific.
9:301-308, 1997). These methods, however, require the additional step of producing a cell line which constitutively expresses EBNA1 from an integrated gene. Alternately, a single plasmid which contains the EBV oriP and the EBNA1 gene and the gene or genes encoding a protein of interest can be used to transfect cells. For this technique to succeed, all of these genes must be driven by strong promoters, which can cause “promoter occlusion” (Greger, I. H. et al.,
Nuc. Acid Res.
26(5) 1214-1301, 1998; Kadesch, T. et al.,
Mol. Cell. Biol.
6(7): 2593-2601, 1986).
One solution to the problem of promoter occlusion, described in U.S. patent application Ser. No. 09/130,114, filed on Aug. 6, 1998, uses two episomes, one of which contains an EBNA1-encoding gene and the other of which contains a gene encoding a protein of interest. In this approach, the episome containing the gene which encodes the protein of interest is maintained as an autonomously replicating plasmid in the presence of the episome containing the EBNA1 gene.
In all of the methods described above, however, conventional selection markers are employed in order to select for cells that have been successfully transfected with an episome encoding the desired sequences. Such selection normally involves exposing transfected cells to antibiotics or other substances that initiate the relevant selection process. Where transfected genes integrate into the cellular chromosome, a single marker is required. Where the continued maintenance of multiple episomes is desired, each episome generally carries its own resistance marker. It would be advantageous to select and maintain transfected cells, both in conventional cell-culture and in gene therapy methods, without the need for an exogenous selection agent, such as an antibiotic. It would also be advantageous to do so quickly, reliably, and without positional effects associated with certain prior art methods.
Accordingly, there is a need in the art for novel methods which allow for the stable maintenance of one or more episomes in eukaryotic cells. Furthermore, there is a need for methods and compositions that allow for the stable maintenance of exogenous DNA in cells of a mammalian organism, i.e., that can be used in gene therapy in vivo or ex vivo.
SUMMARY OF THE INVENTION
In one embodiment, the present invention relates to a method for transfecting eukaryotic cells with exogenous nucleic acid encoding a protein or RNA the expression of which is desired in the eukaryotic cells. According to the invention, the eukaryotic cell is transfected with the exogenous nucleic acid encoding the protein or RNA the expression of which is desired, and with nucleic acid encoding a kill antagonist protein whose expression prohibits the occurrence of cell death resulting from expression of a kill agonist protein by the cell. Successfully transfected eukaryotic cells express the exogenous nucleic acid encoding the protein or RNA the expression of which is desired, and also both the kill agonist protein and the kill antagonist protein, allowing the eukaryotic cell to live. Unsuccessfully transfected eukaryotic cells, i.e., wherein the exogenous nucleic acid encoding the desired protein or RNA is not expressed, express the kill agonist protein, but not the kill antagonist protein. Therefore, the unsuccessfully transfected eukaryotic cells do not live. Preferably, the nucleic acid encoding the protein or RNA the expression of which is desired, and the nucleic acid encoding the kill antagonist protein, are contained in an episome transfected into the cell.
The present invention also provides, in another aspect, methods for obtaining a eukaryotic cell stably transformed with at least one episome, which methods can be used in cell culture or in intact organisms. These methods are carried out by the steps of:
(a) transfecting a eukaryotic cell with:
(i) a first episome which comprises (a) an EBV origin of replication (oriP); (b) a gene encoding a first protein whose expression results in cell death; and (c) a selectable marker for eukaryotic cells; and
(ii) a second episome comprising (a) an EBV(oriP); and (b) a gene encoding a second protein whose expression prohibits the occurrence of cell death resulting from expression of the first protein, to produce doubly transfected cells, wherein said cells express an EBNA-1 protein, preferably from nucleic acid contained in at least one of the first and second episomes.
(b) maintaining the doubly transfected cells under conditions in which (i) the first and second proteins and the selectable marker are expressed and (ii) the selective pressure specified by the marker is maintained. Under these conditions, only cells containing both episomes live. The selectable marker on the first episome can be a conventional marker, such as DNA encoding antibiotic resistance, or can encode a second kill antagonist protein that antagonizes a second kill agonist protein expressed by the cell.
Preferably, either or both episomes additionally comprise nucleic acid that encodes a third protein whose expression is desired, in particular a protein that is not normally a selectable marker such as a therapeutic protein. Where only one of the episomes encodes an EBNA1 protein, the protein of interest is preferably expressed from the other episome.
It is also possible, according to the invention, to transfect cells with episomes that do not encode a particular protein or RNA desired to be expressed by the cells. One or both of the episomes may, for example, instead contain a nucleic acid sequence useful as a tag to identify the transfected cells. Other uses of the invention will be apparent to those skilled in this art.
In another series of embodiments, the first episome comprises (a) an EBV origin of replication and (b) a gene encoding a first protein whose expression results in cell death; the second episome comprises (a) an EBV origin of replication and (b) a gene encoding a second protein whose expression prohibits the occurrence of cell death resulting from expression of the first protein; and at least one of the episomes preferably comprises a gene encoding an EBNA1 protein. In
Chelsky Daniel
Horlick Robert A.
Clark Deborah J. R.
Heslin Rothenberg Farley & & Mesiti P.C.
Paras, Jr. Peter
Pharmacopeia Inc.
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