Claims for Patent: 5,561,053

Summary for Patent: 5,561,053

Title:	Method for selecting high-expressing host cells
Abstract:	A method for selecting recombinant host cells expressing high levels of a desired protein is described. This method utilizes eukaryotic host cells harboring a DNA construct comprising a selectable gene (preferably an amplifiable gene) and a product gene provided 3\' to the selectable gene. The selectable gene is positioned within an intron defined by a splice donor site and a splice acceptor site and the selectable gene and product gene are under the transcriptional control of a single transcriptional regulatory region. The splice donor site is generally an efficient splice donor site and thereby regulates expression of the product gene using the transcriptional regulatory region. The transfected cells are cultured so as to express the gene encoding the product in a selective medium comprising an amplifying agent for sufficient time to allow amplification to occur, whereupon either the desired product is recovered or cells having multiple copies of the product gene are identified.
Inventor(s):	Crowley; Craig W. (Portola Valley, CA)
Assignee:	Genentech, Inc. (South San Francisco, CA)
Application Number:	08/286,740
Patent Claims:	1. A DNA construct comprising in order from 5' to 3': a) a transcriptional regulatory region; b) a transcriptional initiation site; c) a selectable gene positioned within an intron defined by a 5' splice donor site comprising an efficient splice donor sequence such that the efficiency of splicing a messenger RNA having said splice donor sequence is between about 80% and 99% as determined by quantitative PCR, and a 3' splice acceptor site; d) a product gene encoding a product of interest; and e) a transcriptional termination site; wherein the transcriptional regulatory region regulates transcription of both the selectable gene and the product gene. 2. The DNA construct of claim 1 wherein the splice donor site comprises a consensus splice donor sequence. 3. The DNA construct of claim 1 wherein the splice donor site comprises the sequence GACGTAAGT. 4. The DNA construct of claim 1 wherein the selectable gene is an amplifiable gene. 5. The DNA construct of claim 4 wherein the amplifiable gene is DHFR. 6. The DNA construct of claim 1 wherein the transcriptional regulatory region comprises a promoter and an enhancer. 7. A vector comprising the DNA construct of claim 1. 8. The vector of claim 7 wherein the selectable gene of the DNA construct is an amplifiable gene. 9. The vector of claim 7 that is capable of replication in a eukaryotic host. 10. A eukaryotic host cell comprising the vector of claim 9. 11. A eukaryotic host cell comprising the DNA construct of claim 4. 12. A eukaryotic host cell comprising the DNA construct of claim 1 integrated into a chromosome of the host cell. 13. The host cell of claim 12 that is a mammalian cell. 14. A method for producing a product of interest comprising culturing the host cell of claim 10 so as to express the product gene and recovering the product from the host cell culture. 15. The method of claim 14 further comprising recovering the product from the culture medium. 16. The method of claim 14 wherein the selectable gene is an amplifiable gene and the splice donor site comprises an efficient splice donor sequence. 17. A method for producing a product of interest comprising culturing the host cell of claim 11 so as to express the product gene in a selective medium comprising an amplifying agent for sufficient time to allow amplification to occur, and recovering the product. 18. A method for producing eukaryotic cells having multiple copies of a product gene comprising transforming eukaryotic cells with the DNA construct of claim 4 growing the cells in a selective medium comprising an amplifying agent for a sufficient time for amplification to occur, and selecting cells having multiple copies of the product gene. 19. The method of claim 18 further comprising culturing the selected cell 80 as to express the product gene and recovering from the selected cells the product of interest. 20. The method of claim 18 wherein the DNA construct is introduced into the eukaryotic cells by electroporation.

Details for Patent 5,561,053

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132	06/04/1986		2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2039-02-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2039-02-26