Claims for Patent: 5,578,461

Summary for Patent: 5,578,461

Title:	Gene manipulation and expression using genomic elements
Abstract:	Expression of mammalian target genes is achieved by employing chromosomal target DNA, either native primary cells or YACs in a yeast host, where the YACs include a fragment of a mammalian chromosome, the fragment comprising the target gene. Employing homologous recombination, an amplifiable gene is integrated into the mammalian fragment at a site to allow for amplification. In the same step, or one or more steps, as desired, the mammalian gene and/or the transcriptional system may be modified by in vivo mutagenesis. The resulting construct from homologous recombination may then be transformed into a mammalian expression host and integrated into the host genome, either randomly or by homologous recombination. The amplifiable gene may then be amplified by an appropriate agent providing for multiple copies of the target gene and the expression host grown to provide for high yields of the desired wild-type or modified protein.
Inventor(s):	Sherwin; Stephen (San Francisco, CA), Klapholz; Sue (Stanford, CA), Skoultchi; Arthur (Larchmont, NY)
Assignee:	Cell Genesys, Inc. (Foster City, CA)
Application Number:	08/102,567
Patent Claims:	1. A method for production of a mammalian gene product in cell culture, comprising culturing a mammalian continuous cell line which expresses an exogenous mammalian target gene controlled by a nucleotide regulatory sequence heterologous to said mammalian target gene, and recovering the mammalian target gene product from said cell culture, in which said mammalian continuous cell line was prepared by: (a) integrating, via targeted homologous recombination, said nucleotide heterologous to said mammalian target gene said target gene contained in a yeast artificial chromosome maintained in a yeast host cell, so that said integrated nucleotide regulatory sequence is operably associated with said mammalian target gene contained in said yeast artificial chromosome to form a recombined mammalian target gene; in said yeast host cell and (b) transferring said recombined mammalian target gene to a mammalian continuous cell line capable of expressing said target gene product under the control of said integrated nucleotide regulatory sequence so that the mammalian target gene product is expressed by the mammalian continuous cell line in culture. 2. The method of claim 1 in which said mammalian continuous cell line further contains an amplifiable gene operatively associated with said exogenous mammalian target gene controlled by said heterologous nucleotide regulatory sequence, wherein: (a) said amplifiable gene is integrating, via targeted homologous recombination, proximal to said mammalian target gene contained in said yeast artificial chromosome maintained in said yeast host cell, so that the coding sequence of said mammalian target gene product is not disrupted and said heterologous regulatory sequence is operatively associated with said mammalian target gene; and (b) transferring said recombined mammalian target gene into a mammalian continuous cell line, and wherein the expression of said mammalian target gene controlled by said heterologous regulatory sequence and amplified when said mammalian continuous cell line is cultured under conditions that amplify said amplifiable gene. 3. A method for producing a mammalian continuous cell line used for protein production in culture, comprising: (a) integrating, via targeted homologous recombination, a nucleotide regulatory sequence heterologous to a mammalian target gene contained in a yeast artificial chromosome maintained in a yeast host cell, so that said integrated nucleotide regulatory sequence is operably associated with said mammalian target gene to form a recombined mammalian target gene; and (b) transferring said recombined mammalian target gene into a mammalian continuous cell line capable of expressing said target gene product under the control of said integrated regulatory sequence, so that said mammalian target gene product is expressed by said mammalian continuous cell line in culture. 4. The method for producing the mammalian continuous cell line of claim 3, which further comprises: (a) integrating, via targeted homologous recombination, an amplifiable gene proximal to said mammalian target gene contained in said yeast artificial chromosome maintained in said yeast host cell, so that the coding sequence of said mammalian target gene product is not disrupted, and said heterologous regulatory sequence is operably associated with said mammalian target gene; and (b) transferring the recombined mammalian target gene to a mammalian continuous cell line, and wherein the expression of said mammalian target gene controlled by said heterologous regulatory sequence is amplified when said mammalian continuous cell line is cultured under conditions that amplify said amplifiable gene. 5. The method of claim 1, 2, 3 or 4 in which said mammalian target gene is a human gene. 6. The method of claim 1, 2, 3 or 4 in which said heterologous regulatory sequence is a viral promoter or a promoter/enhancer. 7. The method of claim 6 in which said promoter/enhancer is a cytomegalovirus promoter/enhancer. 8. The method of claim 2 or 4 in which said amplifiable gene is dihydrofolate reductase, metallothionein-I, metallothionein-II, adenosine deaminase, ornithine decarboxylase, or glutamine synthetase. 9. The method of claim 1, 2, 3 or 4 in which said mammalian continuous cell line is a Chinese hamster ovary cell line, a monkey kidney cell line, a C127 mouse fibroblast cell line, a 3T3 mouse cell line, a Vero cell line or a 293 cell line. 10. The method of claim 1, 2, 3 or 4 in which the mammalian target gene further contains a mutation located in said amino acid coding region, an intron, the 5' untranslated region, or the 3' untranslated region of the gene.

Details for Patent 5,578,461

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		2013-11-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	For Injection	103132			2013-11-26
Merck Sharp & Dohme Corp.	INTRON A	interferon alfa-2b	Injection	103132			2013-11-26