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Generated: August 21, 2019

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Claims for Patent: 5,045,455

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Summary for Patent: 5,045,455
Title: Factor VIII:C cDNA cloning and expression
Abstract:Methods and compositions are provided for recombinant DNA production of Factor VIIIC and truncated derivatives thereof. Based on amino acid sequences, probes are developed for isolating messenger RNA, cDNA and/or chromosomal DNA encoding for Factor VIIIC. The Factor VIIIC gene in its entirety, a fragment thereof, or a cDNA is then used for expression of Factor VIIIC in a host. The bacteriophage .lambda.FVIII23D containing the 14.43kb EcoRI fragment was deposited at the A.T.C.C. on Jan. 4, 1984 and given Accession No. 40094. Also, the vector pSVF8-200 was deposited at the A.T.C.C. on July 17, 1985 and given Accession No. 40190.
Inventor(s): Kuo; George (San Francisco, CA), Masiarz; Frank (San Francisco, CA), Truett; Martha (Oakland, CA), Valenzuela; Pablo (San Francisco, CA), Rasmussen; Mirella E. (Copenhagen, DK), Favaloro; Jennifer M. (Victoria, AU), Caput; Daniel (San Francisco, CA), Burke; Rae L. (San Francisco, CA), Pachl; Carol (Oakland, CA)
Assignee: Chiron Corporation (Emeryville, CA)
Application Number:07/538,691
Patent Claims:1. An introl-free DNA molecule comprising a coding sequence for the 92.5 kd peptide subunit of human Factor VIII:C wherein said peptide subunit is (i) cleavable by thromobin to yield the 52.5 kd and 40 kd peptide subunits of human Factor VIII:C, and (ii) capable of forming a Ca.sup.++ -bridged complex with the 77/80 kd peptide subunit of human Factor VIII:C, said complex having coagulation activity; and wherein said coding sequence is flanked by 5' and 3' sequences which are not derived from human Factor VIII:C.

2. A DNA molecule according to claim 1, wherein said DNA molecule is an extrachromosomal element.

3. A DNA molecule according to claim 1 wherein said extrachromosomal element is pSVF8-200.

4. A composition comprising host cells containing heterologous DNA substantially free of cells that do not comprise said heterologous DNA, wherein said heterologous DNA comprises an intron-free DNA molecule comprising a coding sequence for the 92.5 kd peptide subunit of human Factor VIII:C wherein said peptide subunit is (i) cleavable by thrombin to yield the 52.5 kd and 40 kd peptide subunits of human Factor VIII:C, and (ii) capable of forming a Ca.sup.++ -bridged complex with the 77/80 kd peptide subunit of human Factor VIII:C, said complex having coagulation activity; and wherein said coding sequence is flanked by 5' and 3' sequences which are not derived from human Factor VIII:C.

5. A composition according to claim 4 wherein said cells are mammalian cells.

6. A composition according to claim 5 wherein said mammalian cells are COS cells.

7. A process for preparing a recombinant human Factor VIII:C polypeptide, said process comprising:

(a) providing a population of host cells produced by introducing into a host cell an exogenous DNA molecule comprising (1) an intron-free sequence coding for the 92.5 kd peptide subunit of human Factor VIII:C wherein said peptide subunit is (i) cleavable by thrombin to yield the 52.5 kd and 40 kd peptide subunits of human Factor VIII:C, and (ii) capable of forming a Ca.sup.++ -bridged complex with the 77/80 kd peptide subunit of human Factor VIII:C, said complex having coagulation activity; wherein said intron-free sequence is flanked by 5' and 3' sequences which are not derived from human Factor VIII:C; and (2) transcriptional and translational signals recognized by said host cell that will provide for expression of said intron-free sequence; and

(b) growing said population of host cells under conditions whereby said intron-free sequence is expressed.

8. A process according to claim 7 wherein said host cell is a mammalian cell.

9. A process according to claim 7 wherein said polynucleotide is part of an extrachromosomal element having a plasmid or viral origin of replication and said host cell is a mammalian cell transformed or transfected by said extrachromosomal element.

10. A process according to claim 8 wherein said recombinant human Factor VIII:C polypeptide is glycosylated.

11. A process according to claim 8 wherein said transcriptional and translational signals comprise a mammalian viral promoter sequence.

12. A process according to claim 11 wherein said transcriptional and translational signals comprise an SV40 virus early region promoter and a SV40 virus late region polyadenylation sequence.

13. A process according to claim 7 wherein said polynucleotide sequence further encodes a leader sequence that will provide for secretion of said recombinant human Factor VIII:C polypeptide.

14. A DNA molecule according to claim 1 further comprising transcriptional and translational control signals that are compatible with a selected cellular host and that will provide for the expression of said coding sequence in said selected cellular host, said transcriptional and translational control sequences being heterologous to said coding sequence.

15. A DNA molecule according to claim 14 wherein said selected cellular host is a mammalian cell.

16. A microorganism or mammalian cell comprising the DNA molecule of claim 15.

Details for Patent 5,045,455

Applicant Tradename Biologic Ingredient Dosage Form BLA Number Approval Date Patent No. Assignee Estimated Patent Expiration Status Orphan Source
Schering INTRON A interferon alfa-2b VIAL 103132 001 1986-06-04   Try a Free Trial Chiron Corporation (Emeryville, CA) 2008-09-03 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 002 1986-06-04   Try a Free Trial Chiron Corporation (Emeryville, CA) 2008-09-03 RX search
Schering INTRON A interferon alfa-2b VIAL 103132 003 1986-06-04   Try a Free Trial Chiron Corporation (Emeryville, CA) 2008-09-03 RX search
>Applicant >Tradename >Biologic Ingredient >Dosage Form >BLA >Number >Approval Date >Patent No. >Assignee >Estimated Patent Expiration >Status >Orphan >Source

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