Microorganisms genetically engineered to have modified N-glycosylation activity

• US 10,023,854 B2
• Filed: 09/14/2012
• Issued: 07/17/2018
• Est. Priority Date: 04/03/2007
• Status: Active Grant

• Current Assignee: Universiteit Gent Vzw, VIB VZW
• Original Assignee: Oxyrane UK Limited, Universiteit Gent Vzw, VIB VZW
• Inventors: Callewaert, Nico Luc Marc, Vervecken, Wouter, De Pourcq, Karen Jacqueline Marcel, Geysens, Steven Christian Jozef, Guerfal, Mouna
• Primary Examiner(s): Bunner, Bridget E
• Assistant Examiner(s): HAMUD, FOZIA M
• Application Number:
  US13/620,259
• Publication Number:
  US 20130195835A1
• Time in Patent Office: 2,132 Days
• 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 a protein capable of promoting mannosyl phosphorylation of N-glycans; 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, and

wherein the protein capable of promoting mannosyl phosphorylation of N-glycans is selected from the group consisting of;

a wild-type MNN4 polypeptide or a biologically active variant of a wild-type MNN4 polypeptide;

a wild-type PNO1 polypeptide or a biologically active variant of a wild-type PNO1 polypeptide; and

a wild-type MNN6 polypeptide.

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 glucocerebrosidase, alpha galactosidase, or galactocerebrosidase.

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

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

12. The method of claim 1, further comprising additional processing of the glycoprotein.

13. The method of claim 12, wherein the additional processing comprises enzymatic or chemical treatment of the altered N-glycosylation form of the target protein.

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

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

16. The method of claim 15, wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.

17. The method of claim 16, wherein the deficiency in an N-glycosylation activity is a deficiency in Outer CHain elongation (OCH1) activity.

18. The method of claim 15, 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 18, wherein the expressed protein having N-glycosylation activity is an alpha-mannosidase, or a biologically active variant thereof.

20. The method of claim 18, wherein the alpha-mannosidase is an alpha 1,2 mannosidase.

21. The method of claim 18, wherein the alpha-mannosidase is MNS1.

22. The method of claim 18, wherein the alpha-mannosidase is targeted to the endoplasmic reticulum.

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

36. The method of claim 29, wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.

37. The method of claim 36, wherein the deficiency in an N-glycosylation activity is a deficiency in Outer CHain elongation (OCH1) activity.

38. The method of claim 29, wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.

39. The method of claim 38, wherein the expressed protein having N-glycosylation activity is an alpha-mannosidase, or a biologically active variant thereof.

23. An isolated Yarrowia lipolytica or Arxula adeninivorans cell genetically engineered to express a protein capable of promoting mannosyl phosphorylation of N-glycans, wherein the cell is capable of producing a target polypeptide in an altered glycosylation form when a nucleic acid encoding the target polypeptide is introduced into the cell, and

wherein the protein capable of promoting mannosyl phosphorylation of N-glycans is selected from the group consisting of:

a wild-type MNN4 polypeptide or a biologically active variant of a wild-type MNN4 polypeptide;

a wild-type PNO1 polypeptide or a biologically active variant of a wild-type PNO1 polypeptide; and

a wild-type MNN6 polypeptide.

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

25. The cell of claim 24, wherein the target polypeptide is a human polypeptide.

26. The cell of claim 24, wherein the target polypeptide is a polypeptide associated with a lysosomal storage disorder (LSD).

27. The cell of claim 26, wherein the LSD is selected from the group consisting of Gaucher disease, Tay-Sachs disease, Pompe disease, Niemann-Pick disease, and Fabry disease.

28. The cell of claim 26, wherein the target polypeptide is selected from the group consisting of glucocerebrosidase, alpha galactosidase, galactocerebrosidase, alpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase, beta-hexosaminidase, beta-D-mannosidase, alpha-L-fucosidase, arylsulfatase B, arylsulfatase A, alpha-N-acteylgalactosaminidase, aspartylglucosaminidase, iduronate-2-sulfatase, alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase, alpha- neuraminidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin K, lipoprotein lipase, and human alpha- galactosidase A.

30. 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.

31. The cell of claim 30, wherein the deficiency in an N-glycosylation activity is a deficiency in Outer CHain elongation (OCH1) activity.

32. 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.

33. The cell of claim 32, wherein the expressed protein having N-glycosylation activity is an alpha-mannosidase, or a biologically active variant thereof.

34. The substantially pure culture of claim 33, wherein said cell further comprises a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form.

a wild-type MNN6 polypeptide.

35. The method of claim 28, wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.

40. The method of claim 34, wherein the target protein is a human protein.

41. The method of claim 34, wherein the target protein is a protein associated with a lysosomal storage disorder (LSD).

42. The method of claim 41, wherein the LSD is selected from the group consisting of Gaucher disease, Tay-Sachs disease, Pompe disease, Niemann-Pick disease, and Fabry disease.

43. The method of claim 41, 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-D-mannosidase, alpha-L-fucosidase, arylsulfatase B, arylsulfatase A, alpha-N-acteylgalactosaminidase, aspartylglucosaminidase, iduronate-2-sulfatase, alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase, alpha-neuraminidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin K, lipoprotein lipase, and human alpha-galactosidase A.

50. The culture of claim 33, wherein the cell is further genetically engineered to comprise at least one additional modification of an N-glycosylation activity.

51. The culture of claim 35, wherein the at least one additional modification of an N-glycosylation activity comprises a deficiency in an N-glycosylation activity.

52. The culture of claim 51, wherein the deficiency in an N-glycosylation activity is a deficiency in Outer CHain elongation (OCH1) activity.

53. The culture of claim 35, wherein the at least one additional modification of an N-glycosylation activity comprises expression of a protein having N-glycosylation activity.

54. The substantially pure culture of claim 53, wherein the expressed protein having N-glycosylation activity is an alpha-mannosidase, or a biologically active variant thereof.

44. A substantially pure culture of Yarrowia lipolytica or Arxula adeninivorans cells, wherein the culture comprises a cell genetically engineered to express a protein capable of promoting mannosyl phosphorylation of N-glycans, wherein the cell is capable of producing a target polypeptide in an altered glycosylation form when a nucleic acid encoding the target polypeptide is introduced into the cell, and

wherein the protein capable of promoting mannosyl phosphorylation of N-glycans is selected from the group consisting of:

a wild-type MNN4 polypeptide or a biologically active variant of a wild-type MNN4 polypeptide;

a wild-type PNO1polypeptide or a biologically active variant of a wild-type PNO1polypeptide; and

a wild-type MNN6 polypeptide.

45. The substantially pure culture of claim 44, wherein said cell further comprises a nucleic acid encoding a target polypeptide, wherein the cell procedures the target polypeptide in an altered N-glycosylation form.

46. The culture of claim 45, wherein the target polypeptide is a human polypeptide.

47. The culture of claim 45, wherein the target polypeptide is a protein associated with a lysosomal storage disorder (LSD).

48. The culture of claim 44, wherein the LSD is selected from the group consisting of Gaucher disease, Tay-Sachs disease, Pompe disease, Niemann-Pick disease, and Fabry disease.

49. The culture of claim 47, wherein the target polypeptide is selected from the group consisting of glucocerebrosidase, alpha galactosidase, galactocerebrosidase, alpha-L-iduronidase, beta-D-galactosidase, beta-glucosidase, beta-hexosaminidase, beta-D-mannosidase, alpha-L-fucosidase, arylsulfatase B, arylsulfatase A, alpha-N-acteylgalactosaminidase, aspartylglucosaminidase, iduronate-2-sulfatase, alpha-glucosaminide-N-acetyltransferase, beta-D-glucoronidase, hyaluronidase, alpha-L-mannosidase, alpha- neuraminidase, phosphotransferase, acid lipase, acid ceramidase, sphinogmyelinase, thioesterase, cathepsin K, lipoprotein lipase, and human alpha-galactosidase A.