Claims for Patent: 10,023,854

Summary for Patent: 10,023,854

Title:	Microorganisms genetically engineered to have modified N-glycosylation activity
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.
Inventor(s):	Callewaert; Nico Luc Marc (Hansbeke-Nevele, BE), Vervecken; Wouter (Landskouter, BE), De Pourcq; Karen Jacqueline Marcel (Ghent, BE), Geysens; Steven Christian Jozef (Wannegem-Lede, BE), Guerfal; Mouna (Veltem-Beisem, BE)
Assignee:	Oxyrane UK Limited (Manchester, GB) VIB vzw (Ghent, BE) Universiteit Gent (Ghent, BE)
Application Number:	13/620,259
Patent 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. 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. 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. 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. 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. 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. 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. 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.

Details for Patent 10,023,854

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Bausch & Lomb Incorporated	VITRASE	hyaluronidase	Injection	021640	May 05, 2004		2032-09-14
Bausch & Lomb Incorporated	VITRASE	hyaluronidase	Injection	021640	December 02, 2004		2032-09-14
Amphastar Pharmaceuticals, Inc.	AMPHADASE	hyaluronidase	Injection	021665	October 26, 2004		2032-09-14
Akorn, Inc.	HYDASE	hyaluronidase	Injection	021716	October 25, 2005		2032-09-14