Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-09-12
2001-10-30
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S069100, C435S358000, C435S320100, C536S024100
Reexamination Certificate
active
06309841
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., Saccharomyces, 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).
Another type of regulatory element is the HMG-I(Y) family. The HMG-I(Y) family of “high mobility group” nonhistone chromatin proteins are founding members of a new category of mammalian gene trans-regulatory proteins called “architectural transcription factors” (Grosschedl, et al.,
Trends Genet
. 10:94-100 (1994); Bustin and Reeves,
Prog. Nucleic Acid Res. Mol. Biol
. 54:35-100 (1996)). In contrast to most transcription factors that bind to specific nucleotide recognition sites in the major groove, architectural transcription factors are characterized by their ability to recognize and modulate DNA and chromatin structure and typically bind to the minor groove of DNA substrates. The HMIG-I(Y) family consists of three closely related proteins, HMG-I, HMG-Y and HMG-IC. Each possess three independent DNA-binding domains called “A.T-hooks” because of their ability to recognize and bind to the narrow minor groove of stretches of A.T-rich nucleotides. A.T-hooks also recognize distorted DNA structures such as those present on synthetic four-way junctions (Hill and Reeves,
Nucleic Acids Res
. 25:3523-31 (1997)), Hill et al.,
Nucleic Acids Res
. 27:2135-44 (1999)), supercoiled plasmids (Nissen and Reeves,
J. Biol. Chem
. 270:4344-4360 (1995)), and the surface of nucleosome core particles (Reeves and Wolffe,
Biochemistry
35:5063-74 (1996)).
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 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 such as clusters of A and T boxes and topoisomerase II sites. The instant invention may contain certain putative MAR as defined by Singh et al. (
Nucleic Acids Res
. 25:1419-25(1997). 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 46 through 14507 of a nucleotide sequence set forth in SEQ ID NO:1, nucleotides 5980 through 14507 of a nucleotide sequence set forth in SEQ ID NO:1, nucleotides 8671 through 14507 of the nucleotide sequence set forth in SEQ ID NO:1, nucleotides 8671 through 10516 ligated to nucleotides 12592 through 14507 of the nucleotide sequence set forth in SEQ ID NO:1, nucleotides 8671 through 10516 ligated to nucleotides 14291 through 14507 of the nucleotide sequence set forth in SEQ ID NO:1, nucleotides 9277 through 10516 ligated to nucleotides 14291 through 14507 of the nucleotide sequence set forth in SEQ ID NO: 1, fragments of the foregoing DNAs that have expression augmenting activity, DNAs complementary to the forgoing DNAs, and combinations of the foregoing DNAs that have expression augmenting activity.
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 an eukaryotic promoter/enhaficer 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
Morris Arvia E.
Thomas James N.
Henry Janis C.
Immunex Corporation
Jones Simone L.
Ketter James
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