Microorganisms genetically engineered to have modified N-glycosylation activity

• US 9,206,408 B2
• Filed: 09/14/2012
• Issued: 12/08/2015
• Est. Priority Date: 04/03/2007
• Status: Active Grant

• Current Assignee: Oxyrane UK Limited, Universiteit Gent Vzw, VIB VZW
• Original Assignee: Oxyrane UK Limited, Universiteit Gent Vzw, VIB VZW
• Inventors: Callewaert, Nico Luc Marc, Vervecken, Wouter, Geysens, Steven Christian Jozef, Guerfal, Mouna, De Pourcq, Karen Jacqueline Marcel
• Primary Examiner(s): Hama, Joanne
• Assistant Examiner(s): HAMUD, FOZIA M
• Application Number:
  US13/620,306
• Publication Number:
  US 20130190253A1
• Time in Patent Office: 1,180 Days
• Field of Search: None
• US Class Current: 1/1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 25/00 Drugs for disorders of the nervous system
• CPC Class Codes: A61P 29/00 Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• CPC Class Codes: A61P 3/00 Drugs for disorders of the metabolism of the blood or the extracellular fluid A61P7/00
• CPC Class Codes: A61P 3/06 Antihyperlipidemics
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 37/00 Drugs for immunological or allergic disorders
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 7/06 Antianaemics
• CPC Class Codes: C07K 14/00 Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12N 9/24 acting on glycosyl compounds (3.2)
• CPC Class Codes: C12N 9/2402 hydrolysing O- and S- glycosyl compounds (3.2.1)
• CPC Class Codes: C12N 9/2465 acting on alpha-galactose-glycoside bonds, e.g. alpha-galactosidase (3.2.1.22)
• CPC Class Codes: C12N 9/2488 Mannanases
• CPC Class Codes: C12P 21/005 Glycopeptides, glycoproteins
• CPC Class Codes: C12P 21/06 produced by the hydrolysis of a peptide bond, e.g. hydrolysate products preparing foodstuffs by protein hydrolysis A23J3/00
• CPC Class Codes: C12Y 204/01232 Initiation-specific alpha-1,6-mannosyltransferase (2.4.1.232)
• CPC Class Codes: C12Y 302/01024 Alpha-mannosidase (3.2.1.24)
• CPC Class Codes: C12Y 302/01045 : Glucosylceramidase (3.2.1.45), i.e. beta-glucocerebrosidase
• CPC Class Codes: C12Y 302/01046 : Galactosylceramidase (3.2.1.46)
• CPC Class Codes: C12Y 302/01084 : Glucan 1,3-alpha-glucosidase (3.2.1.84), i.e. mutanase
• CPC Class Codes: C12Y 302/01113 : Mannosyl-oligosaccharide 1,2-alpha-mannosidase (3.2.1.113), i.e. alpha-1,2-mannosidase
• CPC Class Codes: Y02P 20/52 : using catalysts, e.g. selective catalysts

Abstract

Described herein are methods and genetically engineered cells useful for producing an altered N-glycosylation form of a target molecule. Also described are methods and molecules with altered N-glycosylation useful for treating a variety of disorders such as metabolic disorders.

Claims

1. A method of producing an altered N-glycosylation form of a target protein, the method comprising:

providing a Yarrowia lipolytica or an Arxula adeninivorans cell genetically engineered to express an α

-1,2-mannosidase polypeptide; and

introducing into the cell a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form.

2. The method of claim 1, further comprising isolating the altered N-glycosylation form of the target protein.

3. The method of claim 1, wherein the target protein is an exogenous protein.

4. The method of claim 1, wherein the target protein is an endogenous protein.

5. The method of claim 1, wherein the target protein is a mammalian protein.

6. The method of claim 1, wherein the target protein is a pathogen protein, a lysosomal protein, a growth factor, a cytokine, a chemokine, or a fusion protein.

7. The method of claim 1, wherein the target protein is a protein associated with a lysosomal storage disorder (LSD).

8. The method of claim 7, wherein the lysosomal storage disorder is Gaucher disease, Tay-Sachs disease, Pompe disease, Niemann-Pick disease, or Fabry disease.

9. The method of claim 7, wherein the target protein is selected from the group consisting of glucocerebrosidase, alpha-galactosidase, galactocerebrosidase, alpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase, beta-hexosaminidase, beta-Dmannosidase, alpha-L-fucosidase, arylsulfatase B, arylsulfatase A, alpha-Nacteylgalactosaminidase, aspartylglucosaminidase, iduronate-2-sulfatase, alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase, alphaneuraminidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin K, and lipoprotein lipase.

10. The method of claim 7, wherein the target protein is human alpha-galactosidase A.

11. The method of claim 1, wherein the altered N-glycosylation form comprises one or more N-glycan structures selected from group consisting of Man₅GlcNAc₂, Man₈GlcNAc₂, and Man₉GlcNAc₂.

12. The method of claim 1, wherein the α

-1,2-mannosidase polypeptide is a MNS1 polypeptide.

13. The method of claim 1, wherein the α

-1,2-mannosidase polypeptide is targeted to the endoplasmic reticulum.

14. The method of claim 1, further comprising additional processing of the target protein in its altered N-glycosylation form.

15. The method of claim 14, wherein the additional processing comprises enzymatic or chemical treatment of the altered N-glycosylation form of the target protein.

16. The method of claim 1, wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.

17. The method of claim 16, wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.

18. The method of claim 16, wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.

19. The method of claim 1, wherein the target protein is a human protein.

20. The method of claim 18, wherein the expressed protein is a polypeptide capable of effecting mannosyl phosphorylation of N-glycans.

21. The method of claim 1, wherein the α

-1,2-mannosidase is the Saccharomyces cerevisiae MNS1 polypeptide or the S. cerevisiae MNS1 polypeptide comprising one or more amino substitutions selected from the group consisting of R273L, R273G, R269S, and S272G.

22. An isolated Yarrowia lipolytica or Arxula adeninivorans cell genetically engineered to express an α

-1,2-mannosidase polypeptide, wherein the cell is capable of producing a target protein in an altered glycosylation form if a nucleic acid encoding the target protein is introduced into the cell.

23. The cell of claim 22, said cell further comprising a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form.

24. The cell of claim 22, wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.

25. The cell of claim 24, wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.

26. The cell of claim 24, wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.

27. The cell of claim 22, wherein the α

-1,2-mannosidase polypeptide is a MNS1 polypeptide.

28. The cell of claim 22, wherein the α

-1,2-mannosidase polypeptide is targeted to the endoplasmic reticulum.

29. The cell of claim 26, wherein the expressed protein is a polypeptide capable of effecting mannosyl phosphorylation of N-glycans.

30. The cell of claim 22, wherein the α

-1,2-mannosidase is the Saccharomyces cerevisiae MNS1 polypeptide or the S. cerevisiae MNS1 polypeptide comprising one or more amino substitutions selected from the group consisting of R273L, R273G, R269S, and S272G.

31. A method of producing an altered N-glycosylation form of a target protein, the method comprising contacting a target protein with a cell lysate prepared from a Yarrowia lipolytica or an Arxula adeninivorans cell genetically engineered to express an α

-1,2-mannosidase polypeptide, wherein the contacting of the target protein with the cell lysate results in an altered N-glycosylation form of the target protein.

32. The method of claim 31, wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.

33. The method of claim 32, wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.

34. The method of claim 32, wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.

35. The method of claim 31, wherein the alpha α

-1,2-mannosidase polypeptide is a MNS1 polypeptide.

36. The method of claim 31, wherein the α

-1,2-mannosidase polypeptide is targeted to the endoplasmic reticulum.

37. The method of claim 34, wherein the expressed protein is a polypeptide capable of effecting mannosyl phosphorylation of N-glycans.

38. The method of claim 31, wherein the α

-1,2-mannosidase is the Saccharomyces cerevisiae MNS1 polypeptide or the S. cerevisiae MNS1 polypeptide comprising one or more amino substitutions selected from the group consisting of R273L, R273G, R269S, and S272G.

39. A substantially pure culture of Yarrowia lipolytica or Arxula adeninivorans cells, wherein the culture comprises a cell genetically engineered to express an α

-1,2-mannosidase polypeptide, and wherein the cell is capable of producing a target protein in an altered glycosylation form if a nucleic acid encoding the target protein is introduced into the cell.

40. The substantially pure culture of claim 39, said cell further comprising a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form.

41. The substantially pure culture of claim 39, wherein the α

-1,2-mannosidase polypeptide is a MNS1 polypeptide.

42. The substantially pure culture of claim 39, wherein the α

-1,2-mannosidase polypeptide is targeted to the endoplasmic reticulum.

43. The substantially pure culture of claim 39, wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.

44. The substantially pure culture of claim 43, wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.

45. The substantially pure culture of claim 43, wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.

46. The substantially pure culture of claim 45, wherein the expressed protein is a polypeptide capable of effecting mannosyl phosphorylation of N-glycans.

47. The substantially pure culture of claim 39, wherein the α

-1,2-mannosidase is the Saccharomyces cerevisiae MNS1 polypeptide or the S. cerevisiae MNS1 polypeptide comprising one or more amino substitutions selected from the group consisting of R273L, R273G, R269S, and S272G.