Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Recombinant virus encoding one or more heterologous proteins...
Reexamination Certificate
1998-12-10
2001-01-30
Salimi, Ali R. (Department: 1648)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Recombinant virus encoding one or more heterologous proteins...
C424S204100, C435S320100, C435S235100, C435S239000, C435S091100, C435S091330
Reexamination Certificate
active
06180108
ABSTRACT:
RELATED APPLICATIONS
The application to Cho designated MSB-7241, “Human hybrid host cell for mammalian gene expression,” and the application to Cho et al. designated MSB-7255, “Expression system for factor VIII,” contain related subject matter. Both applications were filed on the same day as the current application and are incorporated herein by reference.
BACKGROUND OF THE INVENTION
1. Field
This invention relates generally to the production of biologically active proteins from genetically engineered mammalian cell lines. Specifically, the invention is concerned with a novel expression vector containing an Epstein-Barr virus terminal repeat sequence which enhances integration of expression vectors into the genomic DNA in host mammalian cell lines.
2. Background
Many attempts have been made to increase the stable integration efficiency of expression vectors into genomic DNA by site specific integration.
Random, nonhomologous integration of input DNA into the host cell genome occurs more than 100 times more frequently than targeted homologous recombination (Thomas et al., 1987, Cell 51: 503-512). However, homologous recombination using hotspot, e.g. hypervariable minisatellite DNA, was shown to occur more frequently than random recombination between two defective plasmids in mammalian cells (Wahls et al., 1990, Cell 60: 95-103).
Autoantigenic cellular protein was isolated by Sun et al. (1994, Proc Natl Acad Sci USA 91: 8646-8650). This protein was identified as terminal repeat binding protein, or TRBP. Two terminal repeat binding sites (TRBS1 and TRBS2) for terminal repeat binding protein were also identified by Sun et al. They observed that TRBP binds sequences present in repetitive cellular DNA, e.g. variable-number tandem repeats (VNTR) and immunoglobulin heavy chain class switch regions.
The terminal repeat binding protein binds to G-rich regions of terminal repeats of Epstein-Barr virus (EBV-TR). EBV-TR takes part in processing and packaging of virion DNA (Zimmermann et al., 1995, J Virol 69: 3147-3155). The EBV-TRs are involved in the integration into chromosomal DNA (Matsuo et al., 1984, Science 226: 1322-1325) and in the circularization event of the genome after infection. These sequences are the essential elements for cleavage and packaging of the EBV virion DNA (Hammerschmidt et al., 1989, Nature (London) 340: 393-397; Zimmermann et al. J Virol, 1995, 69: 3147-3155). These data indicate the important role of the EBV-TR sequence in the recombination events. Therefore, we tested EBV-TR for integration events in deriving clones from the transfected cells.
SUMMARY OF THE INVENTION
We have now discovered that cells transfected with an expression vector containing a selectable marker and an EBV-TR sequence show a five to ten fold increase in the number of cells resistant to the selection agent as compared to cells transfected with the same expression vector without an EBV-TR sequence under the same selection conditions. The higher survival ratios under drug selection indicate that the vectors with EBV-TR may enhance the integration frequency of vectors into genomic DNA.
The expression vectors of this invention include an EBV-TR sequence and a selectable marker, such as dihydrofolate reductase (dhfr). The preferred EBV-TR sequence is a 402 bp sequence (given in
FIG. 1
) which includes the core part of the TRBP-binding region from an immortalized lymphoblastoid cell line 6F 11. In a preferred embodiment, the mammalian gene expression vector comprises a CMV enhancer and promoter, an intronic sequence (MIS, as described in U.S. Pat. No. 5,854,021 to Cho et al.) derived from Epstein-Barr virus, a unique restriction enzyme site Hpal to allow for insertion of a protein coding sequence, and a poly A region plus the plasmid backbone with a drug selection marker and the EBV-TR sequence indicated in FIG.
1
. This vector is denoted pSH131 (see FIG.
2
). This vector is used to introduce the appropriate DNA coding sequence of the protein of interest into mammalian cells to stabilize the protein expression in a long term culture in a serum-free medium. In one preferred embodiment, the sequence for an IL-4 mutein was cloned into pSH131 and the resulting vector is pSH135. The EBV-TR sequence was also directly linked to pCIS25D (vector for expressing B-domain deleted rFVIII, designated BDD-FVIII) and resulting vector is pCIS25DTR.
A preferred amplifiable marker is dihydrofolate reductase (dhfr) although other markers such as glutamine synthetase (gs) and multidrug-resistance gene (mdr) can be substituted. These amplifiable markers (dhfr, gs, and mdr) are also selectable markers. A preferred selectable marker is neo (aminoglycoside phosphotransferase, for neomycin resistance); still other preferred markers such as hph (hygromycin B phosphotransferase) and hisD (histidinol dehydroganase) can be substituted.
The cell host to be transfected can be any mammalian cells. Cell lines that are known to accept the integration of selection genes into their chromosomal DNA are optimal; for example, human embryonic kidney cells (e.g. 293S cells), human hybrid of 293S and B-cell origin (e.g. HKB11; ATCC deposit no. CRL 12568, see U.S. Patent application to Cho designated MSB-7241, “Human hybrid host cell for mammalian gene expression,” filed on the same day as the current application and incorporated herein by reference), chinese hamster ovary (CHO), baby hamster kidney (BHK-21), mouse myeloma, and human B-cells.
As one working example, we show that CHO (dhfr-) cells transfected with an expression vector containing dhfr and an EBV-TR sequence showed about a five to ten fold increase in the number of methotrexate (MTX) resistant cells as compared to cells transfected with the same expression vector without EBV-TR under the same selection conditions.
As used herein, serum-free conditions means conditions in which cell growth occurs in media lacking any added serum.
REFERENCES:
Sadler et al. Journal of Virology, Jul. 1995, vol. 69, No. 7, pp. 4577-4581.
Chan Sham-Yuen
Cho Myung-Sam
Bayer Corporation
Salimi Ali R.
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