Chemistry: molecular biology and microbiology – Animal cell – per se ; composition thereof; process of... – Rodent cell – per se
Reexamination Certificate
1999-11-05
2001-11-06
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Animal cell, per se ; composition thereof; process of...
Rodent cell, per se
C435S320100, C536S024100
Reexamination Certificate
active
06312951
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention relates to DNA sequence elements that augment the expression of recombinant proteins in eukaryotic cells.
BACKGROUND OF THE INVENTION
The development of expression systems for production of recombinant proteins is important for developing a source of a given protein for research or therapeutic use. Expression systems have been developed for both prokaryotic cells, such as
E. coli
, and for eukaryotic cells, which includes both yeast (i.e., Saccharonmices, Pichia and Kluyveromyces spp) and mammalian cells. Expression in mammalian cells is often preferred for manufacturing of therapeutic proteins, since post-translational modifications in such expression systems are more likely to resemble those found in a mammal than the type of post-translational modifications that occur in microbial (prokaryotic) expression systems.
Transcription of eukaryotic genes is regulated by a variety of cis- and trans-acting regulatory elements (reviewed by Dillon and Grosveld,
Trends Genet.
9:134; 1993). Two of the best characterized cis elements are promoters and enhancers. Promoters are DNA sequences immediately 5′ to the coding sequence of the gene and encompass multiple binding sites for trans-acting transcription factors, forming the basal transcription apparatus. Enhancers are also composed of multiple binding sites for trans-acting transcription factors but can be found far up stream or down stream of coding sequences or even within introns. These elements can also act in an orientation independent manner. The activities of promoters and enhancers can be detected in transient expression systems and contain elements which may or may not be tissue specific; they are vulnerable to position effects when studied in stable cell lines or transgenic animals.
Another category of cis- regulatory elements are ones which are believed to regulate the chromatin structure including, locus control regions (LCR) (Grosveld F., et al.,
Cell
51:975, 1987), matrix attachment regions (MAR; Phi-Van et al.,
Mol Cell Biol
10:2302; 1980), scaffold attachment regions (SAR; Gasser and Laemmli,
Trends Genet
3:16, 1987), and insulator elements (Kellum and Schedl,
Cell
64:941, 1991). These elements are similar to enhancers in that they are able to act over long distances, but are unique in that their effects are only detectable in stably transformed cell lines or transgenic animals. LCRs are also dissimilar to enhancers in that they are position and orientation dependent, and are active in a tissue specific manner. In addition, LCR and SAR sequences are characterized by A boxes, T boxes and topoisomerase II sites, which are not typically found in enhancer or promoter sequences. (Gasser and Laemmli, supra; Klehr D., et al.,
Biochemistry
30:1264, 1991).
Internal ribosome entry sites (IRES) are another type of regulatory element that can be found in several viruses and cellular RNAs (reviewed in McBratney et. al.
Current Opinion in Cell Biology
5:961, 1993). IRES are useful in enhancing translation of a second gene product in a bicistronic eukaryotic expression cassette (Kaufman R. J., et al.,
Nucleic Acids Res
19:4485, 1991).
Several vectors are available for expression in mammalian hosts, each containing various combinations of cis- and in some cases trans- regulatory elements to achieve high levels of recombinant protein in a minimal time frame. However, despite the availability of numerous such vectors, the level of expression of a recombinant protein achieved in mammalian systems is often lower than that obtained with a microbial expression system. Moreover, developing a transformed cell line that expresses high levels of a desired protein often requires time consuming cloning and amplification. Accordingly, there is a need in the art to refine and improve expression in mammalian cells, and to identify elements that can augment expression of recombinant proteins and facilitate the use of mammalian cells in recombinant protein production.
SUMMARY OF THE INVENTION
Novel transcription regulatory sequences, expression augmenting sequence elements (EASE), that facilitate high expression of recombinant proteins in mammalian host cells in a short time period, are disclosed. One embodiment of the invention is an expression augmenting sequence element (EASE), that facilitates high expression of recombinant proteins in mammalian host cells in a short time period, which is not active in transient expression systems, does not exhibit characteristics of DNAs that encode a protein, and does not exhibit nucleotide sequence characteristics found in LCR, MAR or SAR. A preferred embodiment of the invention is an EASE that was obtained from Chinese hamster ovary (CHO) cell genomic DNA, proximal to a unique integration site for a recombinant mammalian protein.
In a most preferred embodiment of the invention, the EASE is selected from the group consisting of DNAs comprising nucleotides 1 through 14507, nucleotides 5980 through 14507, nucleotides 8671 through 14507, nucleotides 8671 through 10515, nucleotides 9277 through 10515, nucleotides 8672 through 12273, nucleotides 10100 through 14923 of SEQ ID NO:1, fragments of the foregoing DNAs that have expression augmenting activity, DNAs complementary to the foregoing DNAs, DNAs that are at least about 80% identical in nucleotide sequence to the foregoing DNAs and that have expression augmenting activity, and combinations of the foregoing DNAs that have expression augmenting activity. In one embodiment, the EASE DNA is ligated to a DNA comprising nucleotides 14290 through 14507 of SEQ ID NO:1; alternatively, the EASE DNA is ligated to a DNA comprising nucleotides 12592 through 14507 of SEQ ID NO:1.
Expression vectors comprising the novel EASE are able to transform CHO cells to high expression of recombinant proteins. Thus, another embodiment of the invention is an expression vector comprising an EASE. In a preferred embodiment, the expression vector further comprises a eukaryotic promoter/enhancer driving the expression of a protein of interest. In a most preferred embodiment, the expression vector consists of a bicistronic plasmid wherein a first exon encodes the gene of interest and a second exon encodes an amplifyable dominant selectable marker. A preferred marker is dihydrofolate reductase (DHFR); other amplifyable markers are also suitable for use in the inventive expression vectors. The expression vector may further comprise an IRES sequence between the two exons.
Mammalian host cells can be transformed with the inventive expression vectors, and will produce high levels of recombinant protein in a short period of time. Accordingly, another embodiment of the invention provides a mammalian host cell transformed with the inventive expression vector. In a most preferred embodiment, the host cells are CHO cells.
The invention also provides a method for obtaining a recombinant protein, comprising transforming a host cell with an inventive expression vector, culturing the transformed host cell under conditions promoting expression of the protein, and recovering the protein. In a preferred application of this invention, transformed host cell lines are selected with two selection steps, the first to select for cells expressing the dominant amplifyable marker, and the second step for high expression levels and/or amplification of the marker gene as well as the gene of interest. In a most preferred embodiment, the selection or amplification agent is methotrexate, an inhibitor of DHFR that has been shown to cause amplification of endogenous DHFR genes and transfectcd DHFR sequences.
Moreover, the invention provides a method of identifying additional expression augmenting sequence elements, for example, from other transformed cell lines. Such cell lines will exhibit high levels of expression that are not attributable to high gene copy number. The inventive techniques will be useful in identifying and isolating such EASE, as well as EASE present in non-transformed cells (for example, by hybridization studies or sequence analys
Lee Chi-Chang
Morris Arvia E.
Thomas James N.
Henry Janis C.
Immunex Corporation
Jones Simone L.
Ketter James
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