Cells deficient in CMP-N-acetylneuraminic acid hydroxylase and/or glycoprotein alpha-1,3-galactosyltransferase

• US 8,980,583 B2
• Filed: 06/29/2012
• Issued: 03/17/2015
• Est. Priority Date: 06/30/2011
• Status: Active Grant

• Current Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Original Assignee: Sigma-Aldrich Company (Sigma-Aldrich Corporation)
• Inventors: Lin, Nan, George, Henry J., Mascarenhas, Joaquina, Collingwood, Trevor N., Kayser, Kevin J., Achtien, Katherine
• Primary Examiner(s): Gebreyesus, Kagnew H
• Application Number:
  US13/538,825
• Publication Number:
  US 20130004992A1
• Time in Patent Office: 991 Days
• Field of Search: None
• US Class Current: 435/69.4
• CPC Class Codes: C12N 2510/00 Genetically modified cells
• CPC Class Codes: C12N 2510/02 Cells for production
• CPC Class Codes: C12N 2510/04 Immortalised cells
• CPC Class Codes: C12N 9/0071 acting on paired donors with incorporation of molecular oxygen (1.14)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12Y 114/18002 CMP-N-acetylneuraminate monooxygenase (1.14.18.2)

Abstract

The present invention provides non-human mammalian cell lines that are deficient in CMP-Neu5Ac hydroxylase (Cmah) and/or glycoprotein alpha-1,3-galactosyltransferase (Ggta1). Also provided are methods for using the cells disclosed herein for producing recombinant proteins with human-like patterns of glycosylation.

Claims

1. A Chinese hamster ovary (CHO) cell line deficient in glutamine synthetase (GS), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (Cmah), and glycoprotein alpha-1,3-galactosyltransferase-1 (Ggta1).

2. The CHO cell line of claim 1, wherein the cell line comprises an inactivated chromosomal sequence encoding Cmah, and an inactivated chromosomal sequence encoding Ggta1.

3. The CHO cell line of claim 2, wherein the inactivated chromosomal sequences encoding Cmah and Ggta1 comprise a deletion of at least one nucleotide, an insertion of at least one nucleotide, a substitution of at least one nucleotide, or a combination thereof.

4. The CHO cell line of claim 2, wherein the chromosomal sequences are inactivated with a targeting endonuclease.

5. The CHO cell line of claim 4, wherein the targeting endonuclease is a zinc finger nuclease.

6. The CHO cell line of claim 2, wherein the inactivated chromosomal sequences encoding Cmah and Ggta1 comprise no exogenously introduced sequence.

7. The CHO cell line of claim 2, wherein the inactivated chromosomal sequences encoding Cmah and Ggta1 are monoallelic and the cell line produces a reduced amount of Cmah and Ggta1.

8. The CHO cell line of claim 2, wherein the inactivated chromosomal sequences encoding Cmah and Ggta1 are biallelic and the cell line produces no Cmah or Ggta1.

9. The CHO cell line of claim 1, wherein the cell line produces at least one protein having a reduced content of or no N-glycolylneuraminicacid (Neu5Gc) residues.

10. The CHO cell line of claim 2, wherein the inactivated chromosomal sequence encoding Cmah comprises a deletion of 17 or 31 base pairs, and the inactivated chromosomal sequence encoding Ggta1 comprises a deletion of 3, 4, or 23 base pairs.

11. A Chinese hamster ovary (CHO) cell line comprising inactivated chromosomal sequences encoding glutamine synthetase (GS), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (Cmah), and glycoprotein alpha-1,3-galactosyltransferase-1 (Ggta1).

12. The CHO cell line of claim 11, wherein the inactivated chromosomal sequences encoding GS, Cmah, and Ggta1 are biallelic and the cell line produces no GS, Cmah, or Ggta1.