Compositions and methods for improved glycoprotein sialylation
View Patent ↗The present invention provides compositions and methods for the production of glycoproteins with enhanced sialylation. In particular, the invention provides cell lines comprising disrupted sialidase expression and methods of using the cell lines to produce glycoproteins with enhanced sialylation.
1. A cell line, the cell line comprising disrupted expression of at least one chromosomally integrated nucleic acid sequence encoding a non-cytoplasmic sialidase, the amino acid sequence of the non-cytoplasmic sialidase is (a) at least about 95% identical to SEQ ID NO:2, and/or (b) at least about 80% identical to SEQ ID NO:4.
2. The cell line of claim 1 , wherein the amino acid sequence of the non-cytoplasmic sialidase is (a) at least about 97% identical to SEQ ID NO:2, and/or (b) at least about 90% identical to SEQ ID NO:4.
3. The cell line of claim 1 , wherein the amino acid sequence of the non-cytoplasmic sialidase consists of SEQ ID NO:2 and/or SEQ ID NO:4.
4. The cell line of claim 1 , wherein the expression of the nucleic acid sequence encoding the non-cytoplasmic sialidase is disrupted by a technique selected from the group consisting of RNA interference, homologous recombination, and site-specific recombination.
5. The cell line of claim 4 , wherein RNA interference (RNAi) is mediated by an RNAi agent selected from the group consisting of short interfering RNA, a short hairpin RNA, and an RNAi expression vector.
6. The cell line of claim 1 , wherein the cell line is selected from the group consisting of an insect cell line, a mammalian cell line, a rodent cell line, a non-human primate cell line, and a human cell line.
7. The cell line of claim 1 , wherein the cell line is a Chinese hamster ovary (CHO) cell line.
8. The cell line of claim 7 , wherein the amino acid sequence of the non-cytoplasmic sialidase consists of SEQ ID NO:2 and/or SEQ ID NO:4.
9. The cell line of claim 1 , further comprising disrupted expression of a chromosomally integrated nucleic acid sequence encoding a cytoplasmic sialidase, the amino acid sequence of the cytoplasmic sialidase is at least about 85% identical to SEQ ID NO:5.
10. The cell line of claim 9 , wherein the amino acid sequence of the cytoplasmic sialidase consists of SEQ ID NO:5.
11. The cell line of claim 9 , wherein the expression of the nucleic acid sequence encoding the cytoplasmic sialidase is disrupted by a technique selected from the group consisting of RNA interference, homologous recombination, and site-specific recombination.
12. The cell line of claim 11 , wherein RNA interference (RNAi) is mediated by an RNAi agent selected from the group consisting of short interfering RNA, a short hairpin RNA, and an RNAi expression vector.
13. The cell line of claim 9 , wherein the cell line is selected from the group consisting of an insect cell line, a mammalian cell line, a rodent cell line, a non-human primate cell line, and a human cell line.
14. The cell line of claim 9 , wherein the cell line is a Chinese hamster ovary (CHO) cell line.
15. The cell line of claim 14 , wherein the amino acid sequence of the non-cytoplasmic sialidase consists of SEQ ID NO:2 and/or SEQ ID NO:4.
16. A method for producing a glycoprotein, the method comprising expressing a nucleic acid sequence encoding the glycoprotein in a cell comprising disrupted expression of at least one chromosomally integrated nucleic acid sequence encoding a non-cytoplasmic sialidase, the amino acid sequence of the non-cytoplasmic sialidase is (a) at least about 95% identical to SEQ ID NO:2, and/or (b) at least about 80% identical to SEQ ID NO:4, wherein the glycoprotein has increased sialylation relative to a suitable control.
17. The method of claim 16 , wherein the amino acid sequence of the non-cytoplasmic sialidase is (a) at least about 97% identical to SEQ ID NO:2, and/or (b) at least about 90% identical to SEQ ID NO:4.
18. The method of claim 16 , wherein the amino acid sequence of the non-cytoplasmic sialidase consists of SEQ ID NO:2 and/or SEQ ID NO:4.
19. The method of claim 16 , wherein the expression of the nucleic acid sequence encoding the non-cytoplasmic sialidase is disrupted by a technique selected from the group consisting of RNA interference, homologous recombination, and site-specific recombination.
20. The method of claim 19 , wherein RNA interference (RNAi) is mediated by an RNAi agent selected from the group consisting of short interfering RNA, a short hairpin RNA, and an RNAi expression vector.
21. The method of claim 16 , wherein the nucleic acid sequence encoding the glycoprotein is chromosomally integrated or extrachromosomal.
22. The method of claim 16 , wherein the cell is selected from the group consisting of an insect cell, a mammalian cell, a rodent cell, a non-human primate cell, and a human cell.
23. The method of claim 16 , wherein the cell is a Chinese hamster ovary (CHO) cell.
24. The method of claim 23 , wherein the amino acid sequence of the non-cytoplasmic sialidase consists of SEQ ID NO:2 and/or SEQ ID NO:4.
25. The method of claim 16 , wherein the cell further comprises disrupted expression of a chromosomally integrated nucleic acid sequence encoding a cytoplasmic sialidase, the amino acid sequence of the cytoplasmic sialidase is at least about 85% identical to SEQ ID NO:5.
26. The method of claim 25 , wherein the amino acid sequence of the cytoplasmic sialidase consists of SEQ ID NO:5.
27. The method of claim 25 , wherein the expression of the nucleic acid sequence encoding the cytoplasmic sialidase is disrupted by a technique selected from the group consisting of RNA interference, homologous recombination, and site-specific recombination.
28. The method of claim 27 , wherein RNA interference (RNAi) is mediated by an RNAi agent selected from the group consisting of short interfering RNA, a short hairpin RNA, and an RNAi expression vector.
29. The method of claim 25 , wherein the cell is selected from the group consisting of an insect cell, a mammalian cell, a rodent cell, a non-human primate cell, and a human cell.
30. The method of claim 25 , wherein the cell is a Chinese hamster ovary (CHO) cell.
31. The method of claim 30 , wherein the amino acid sequence of the non-cytoplasmic sialidase consists of SEQ ID NO:2 and/or SEQ ID NO:4.
32. An isolated nucleic acid encoding a polypeptide whose amino acid sequence consists of SEQ ID NO:2 or SEQ ID NO:4.