Search Images Maps Play YouTube News Gmail Drive More »
Advanced Patent Search | Web History | Sign in

Patents

The present invention involves the production of large quantities of human .alpha.-Gal A by cloning and expressing the .alpha.-Gal A coding sequence in eukaryotic host cell expression systems. The eukaryotic expression systems, and in particular the mammalian host cell expression system described herein provide for the appropriate cotranslational and posttranslational modifications required for proper processing, e.g., glycosylation, phosphorylation, etc. and sorting of the expression product so that an glycosylation, phosphorylation, etc. and sorting of the expression product so that an active enzyme is produced. In addition, the expression of fusion proteins which simplify purification is described.

Inventors: Robert J. Desnick, David F. Bishop, Yiannis A. Ioannou
Original Assignee: Mount Sinai School of Medicine of the City of New York
Primary Examiner: Keith D. Hendricks
Current U.S. Classification: 435/208; 435/252.3; 435/320.1; 435/325; 435/358; 435/365
International Classification: C12N 940; C12N 1500; C12N 500; C12N 120

View patent at USPTO
Search USPTO Assignment Database

Citations

Cited PatentFiling dateIssue dateOriginal AssigneeTitle
US3966555Jan 30, 1975Jun 29, 1976Societe d'Assistance Technique pour Produits Nestle S.A.Alpha-galactosidase production
US3972777Apr 30, 1975Aug 3, 1976Hokkaido Sugar Co., Ltd.Method for recovery of refined .alpha.-galactosidase
US4450238Nov 5, 1982May 22, 1984E. N. I. Ente Nazionale IdrocarburiBiologically pure culture of Saccharomyces cerevisiae

Referenced by

Citing PatentFiling dateIssue dateOriginal AssigneeTitle
US6083725Sep 12, 1997Jul 4, 2000Transkaryotic Therapies, Inc.Tranfected human cells expressing human .alpha.-galactosidase A protein
US6118045Jul 29, 1996Sep 12, 2000Pharming B.V.
The Universiteit Leiden
Academic Hospital
Eramus Universiteit		Lysosomal proteins produced in the milk of transgenic animals
US6210666Oct 21, 1998Apr 3, 2001Orphan Medical, Inc.Truncated .alpha.-galactosidase A to treat fabry disease
US6270989Mar 17, 1995Aug 7, 2001Transkaryotic Therapies, Inc.Protein production and delivery
US6395884Apr 6, 2000May 28, 2002Transkaryotic Therapies, Inc.Therapy for
US6455037Nov 1, 1996Sep 24, 2002Mount Sinai School of Medicine of the City University of New York
The Austin Research Institute		Cells expressing an
US6458574Mar 11, 1999Oct 1, 2002Transkaryotic Therapies, Inc.Treatment of a
US6534300Aug 10, 2000Mar 18, 2003Genzyme Glycobiology Research Institute, Inc.Methods for producing highly phosphorylated lysosomal hydrolases
US6537785Aug 10, 2000Mar 25, 2003Genzyme Glycobiology Research Institute, Inc.Methods of treating lysosomal storage diseases
US6565844May 14, 1999May 20, 2003Transkaryotic Therapies, Inc.Protein production and protein delivery
US6566099Jan 27, 2000May 20, 2003Transkaryotic Therapies, Inc.Nucleic acid encoding a chimeric polypeptide
US6642038Aug 10, 2000Nov 4, 2003Genzyme Glycobiology Research Institute, Inc.GlcNAc phosphotransferase of the lysosomal targeting pathway
US6670165Nov 9, 2001Dec 30, 2003Genzyme Glycobiology Research Institute, Inc.Methods for producing highly phosphorylated lysosomal hydrolases
US6770468Aug 10, 2000Aug 3, 2004Genzyme Glycobiology Research Institute, Inc.Phosphodiester-a-GlcNAcase of the lysosomal targeting pathway
US6800472Dec 21, 2001Oct 5, 2004Genzyme Glycobiology Research Institute, Inc.Expression of lysosomal hydrolase in cells expressing pro-N-acetylglucosamine-1-phosphodiester
US6828135Nov 29, 2002Dec 7, 2004Genzyme Glycobiology Research Institute, Inc.Phosphodiester
US6861242Jul 2, 2001Mar 1, 2005Genzyme Glycobiology Research Institute, Inc.Methods for producing highly phosphorylated lysosomal hydrolases
US6890748Mar 20, 2002May 10, 2005Large Scale Biology CorporationProduction of lysosomal enzymes in plants by transient expression
US6905856Dec 21, 2001Jun 14, 2005Genzyme Glycobiology Research Institute, Inc.Soluble GlcNAc phosphotransferase
US7011831Sep 4, 2002Mar 14, 2006Research Corporation Technologies, Inc.Recombinant α-galactosidase A therapy for Fabry disease
US7067127Sep 9, 2003Jun 27, 2006Genzyme Glycobiology Research Institute Inc.GIcNAc phosphotransferase of the lysosomal targeting pathway
US7122354Dec 12, 2002Oct 17, 2006Transkaryotic Therapies, Inc.Nucleic acid encoding a chimeric polypeptide
US7135322Jul 29, 2004Nov 14, 2006Novazyme Pharmaceuticals, Inc.Expression of lysosomal hydrolase in cells expressing pro-N-acetylglucosamine-1-phosphodiester α-N-acetyl glucosimanidase
US7351410Jun 30, 2003Apr 1, 2008Genzyme Therapeutic Products Limited PartnershipTreatment of Pompe's disease
US7371366Aug 9, 2005May 13, 2008Genzyme CorporationGlcNAc phosphotransferase of the lysosomal targeting pathway
US7396811Apr 30, 2002Jul 8, 2008ZyStor Therapeutics, Inc.Subcellular targeting of therapeutic proteins
US7396811Apr 30, 2002Jul 8, 2008ZyStor Therapeutics, Inc.Subcellular targeting of therapeutic proteins
US7560424Oct 16, 2002Jul 14, 2009ZyStor Therapeutics, Inc.Targeted therapeutic proteins
US7629309Nov 3, 2004Dec 8, 2009Zystor Therapeutics, Inc.Targeted therapeutic proteins
US7655226Jan 30, 2008Feb 2, 2010Genzyme Therapeutic Products Limited PartnershipTreatment of Pompe's disease
US7785856Sep 12, 2007Aug 31, 2010ZyStor Therapeutics, Inc.Acid alpha-glucosidase and fragments thereof
US7833742Apr 13, 2006Nov 16, 2010Shire Human Genetic Therapies, Inc.Treatment of α-galactosidase A deficiency
US7858576May 30, 2007Dec 28, 2010ZyStor Therapeutics, Inc.Method for targeting lysosomal enzymes
US7935336Nov 17, 2006May 3, 2011Tokyo Metropolitan Organization for Medical Research
ALTIF Laboratories		Highly functional enzyme having α-galactosidase activity
US7981864Feb 19, 2008Jul 19, 2011Biomarin Pharmaceutical Inc.Methods and compositions for targeting proteins across the blood brain barrier
US8207114Nov 2, 2009Jun 26, 2012Biomarin Pharmaceutical Inc.Targeted therapeutic proteins

Claims

1. A method for producing human .alpha.-galactosidase A comprising:

(a) culturing a mammalian cell containing a chromosomally integrated nucleotide sequence encoding human .alpha.-galactosidase A controlled by a regulatory sequence that promotes gene expression and a selectable marker controlled by the same or different regulatory sequence, so that the .alpha.-galactosidase A nucleotide sequence is stably overexpressed and an enzymatically active .alpha.-galactosidase A enzyme is secreted by the mammalian cell; and
(b) isolating enzymatically active .alpha.-galactosidase A enzyme from the mammalian cell culture.

2. The method according to claim 1 wherein, in the presence of selection, the chromosomally integrated nucleotide sequences are amplified.

3. The method according to claim 1 in which the nucleotide sequence encoding human .alpha.-galactosidase A encodes the amino acid sequence depicted in FIGS. 1A-1C [SEQ ID No: 2] from amino acid residue number 1 to 430.

4. The method according to claim 1 in which the nucleotide sequence encoding human .alpha.-galactosidase A encodes the amino acid sequence depicted in FIGS. 1A-1C [SEQ. ID No. 1] from amino acid[residue number 31 to 430.

5. The method according to claim 1 in which the regulatory sequence that promotes gene expression is a viral promoter.

6. The method according to claim 1 in which the regulatory sequence that promotes gene expression is an inducible promoter.

7. The method according to claim 1 in which the selectable marker is dihydrofolate reductase.

8. The method according to claim 2 in which the selectable marker is dihydrofolate reductase and the selection is methotrexate.

9. The method according to claim 1 in which the mammalian cell is a Chinese hamster ovary cell line.

10. A mammalian cell comprising a chromosomally integrated nucleotide sequence encoding human .alpha.-galactosidase A controlled by a regulatory sequence that promotes gene expression and a selectable marker controlled by the same or different regulatory sequence, so that the .alpha.-galactosidase A nucleotide sequence is stably overexpressed and an enzymatically active .alpha.-galactosidase A enzyme is secreted by the mammalian cell.

11. The mammalian cell of claim 10 wherein the chromosomally integrated nucleotide sequences are amplified.

12. The mammalian cell according to claim 10 in which the nucleotide sequence encoding human .alpha.-galactosidase A encodes the amino acid sequence depicted in FIGS. 1A-1C [SEQ. ID. No. 2] from amino acid residue number 1 to 430.

13. The mammalian cell according to claim 10 in which the nucleotide sequence encoding human .alpha.-galactosidase A encodes the amino acid sequence depicted in FIG. 1A [SEQ. ID No: 1] from amino acid residue number 31 to 430.

14. The mammalian cell according to claim 10 in which the regulatory sequence that promotes gene expression is a viral promoter.

15. The mammalian cell according to claim 10 in which the regulatory sequence that promotes gene expression is an inducible promoter.

16. The mammalian cell according to claim 10 in which the selectable marker is dihydrofolate reductase.

17. The mammalian cell according to claim 10 in which the mammalian cell is a Chinese hamster ovary cell line.

Drawings