Microorganisms genetically engineered to have modified N-glycosylation activity

• US 9,222,083 B2
• Filed: 04/27/2011
• Issued: 12/29/2015
• 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): Hama, Joanne
• Assistant Examiner(s): HAMUD, FOZIA M
• Application Number:
  US13/095,532
• Publication Number:
  US 20110207676A1
• Time in Patent Office: 1,707 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 a protein that promotes phosphorylation of mannosyl moieties of N-glycans and to comprise a deficiency in Outer CHain elongation (OCH1) activity; 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 relative to the N-glycosylation form of the target protein produced in a corresponding non-genetically engineered cell, and

wherein the protein that promotes phosphorylation of mannosyl moieties is;

(a)(i) a wild-type MNN4 polypeptide;

(a)(ii) a deletion variant that is the polypeptide of (a)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;

(a)(iii) a deletion variant that is the polypeptide of (a)(i) or the variant of (a)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (a)(i) or the variant of (a)(ii);

or

(a)(iv) a substitution variant that is the polypeptide of (a)(i), the variant of (a)(ii), or the variant of (a)(iii), but with no more than 10 conservative substitutions,

wherein the variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (a)(i),

(b)(i) a wild-type PNO1 polypeptide;

(b)(ii) a deletion variant that is the polypeptide of (b)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;

(b)(iii) a deletion variant that is the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (b)(i) or the variant of (b)(ii);

or

(b)(iv) a substitution variant that is the polypeptide of (b)(i), the deletion variant of (b)(ii), or the deletion variant of (b)(iii), but with no more than 10 conservative substitutions,

wherein the variants of (b)(ii), (b)(iii), and (b)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (b)(i), or

(c) 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 human 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 claims 7, wherein the target protein is glucocerebrosidase 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, wherein the altered N-glycosylation form comprises one or more N-glycan structures selected from group consisting of Mans₅G1cNAc₂, Man₈G1cNAc₂, and Man₉G1cNAc₂.

13. The method of claim 1, wherein the altered glycosylation is Mans₅G1cNAc₂, Man₈G1cNAc₂, Man₉G1cNAc₂.

14. The method of claim 1, wherein the cell further comprises a nucleic acid encoding an alpha-mannosidase.

15. The method of claim 14, wherein the alpha-mannosidase is an alpha 1,2mannosidase.

16. The method of claim 14, wherein the alpha-mannosidase is mannosidase 1 (MNS1).

17. The method of claim 14, wherein the alpha-mannosidase is targeted to the endoplasmic reticulum.

18. The method of claim 17, wherein the alpha-mannosidase is a fusion protein comprising an alpha-mannosidase polypeptide and an HDEL endoplasmic reticulum retention peptide.

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

20. The method of claim 19, wherein the additional processing comprises enzymatic or chemical treatment of the altered N-glycosylation form of the target protein.

21. An isolated Yarrowia lipolytica or Arxula adeninivorans cell genetically engineered to express a protein that promotes phosphorylation of mannosyl moieties of N-glycans and to comprise a deficiency in Outer CHain elongation (OCH1) activity,.

wherein the protein that promotes phosphorylation of mannosyl moieties is:

(a)(i) a wild-type MNN4 polypeptide;

(a)(ii) a deletion variant that is the polypeptide of (a)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;

(a)(iii) a deletion variant that is the polypeptide of (a)(i) or the variant of (a)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (a)(i) or the variant of (a)(ii);

or

(a)(iv) a substitution variant that is the polypeptide of (a)(i), the variant of (a)(ii), or the variant of (a)(iii), but with no more than 10 conservative substitutions,

wherein the variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (a)(i),

(b)(i) a wild-type PNO1polypeptide;

(b)(ii) a deletion variant that is the polypeptide of (b)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;

(b)(iii) a deletion variant that is the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (b)(i) or the variant of (b)(ii);

or

(b)(iv) a substitution variant that is the polypeptide of (b)(i), the deletion variant of (b)(ii), or the deletion variant of (b)(iii), but with no more than 10 conservative substitutions,

wherein the variants of (b)(ii), (b)(iii), and (b)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (b)(i), or

(c) a wild-type MNN6 polypeptide.

22. The cell of claim 21, said cell further comprising a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form relative to the N-glycosylation form of the target protein produced in a corresponding non-genetically engineered cell.

23. The cell of claim 21, said cell further comprising a nucleic acid encoding an alpha-mannosidase.

24. 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 a protein that promotes phosphorylation of mannosyl moieties of N-glycans and to comprise a deficiency in Outer CHain elongation (OCH1) activity,

wherein the contacting of the target protein with the cell lysate results in an altered N-glycosylation form of the target protein relative to the N-glycosylation form of the target protein produced from the cell lysate of a corresponding non-genetically engineered cell, and

wherein the protein that promotes phosphorylation of mannosyl moieties is:

(a)(i) a wild-type MNN4 polypeptide;

(a)(ii) a deletion variant that is the polypeptide of (a)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;

(a)(iii) a deletion variant that is the polypeptide of (a)(i) or the variant of (a)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (a)(i) or the variant of (a)(ii);

or

(a)(iv) a substitution variant that is the polypeptide of (a)(i), the variant of (a)(ii), or the variant of (a)(iii), but with no more than 10 conservative substitutions,

wherein the variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (a)(i),

(b)(i) a wild-type PNO1 polypeptide;

(b)(ii) a deletion variant that is the polypeptide of (b)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;

(b)(iii) a deletion variant that is the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (b)(i) or the variant of (b)(ii);

or

(b)(iv) a substitution variant that is the polypeptide of (b)(i), the deletion variant of (b)(ii), or the deletion variant of (b)(iii), but with no more than 10 conservative substitutions,

wherein the variants of (b)(ii), (b)(iii), and (b)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (b)(i), or

(c) a wild-type MNN6 polypeptide.

25. The method of claim 24, wherein the cell further comprises a nucleic acid encoding an alpha-mannosidase.

26. A substantially pure culture of Yarrowia lipolytica or Arxula adeninivorans cells, a substantial number of which are genetically engineered to express a protein that promotes phosphorylation of mannosyl moieties of N-glycans and to comprise a deficiency in Outer CHain elongation (OCH1) activity, and

wherein the protein that promotes phosphorylation of mannosyl moieties is:

(a)(i) a wild-type MNN4 polypeptide;

(a)(ii) a deletion variant that is the polypeptide of (a)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;

(a)(iii) a deletion variant that is the polypeptide of (a)(i) or the variant of (a)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (a)(i) or the variant of (a)(ii);

or

(a)(iv) a substitution variant that is the polypeptide of (a)(i), the variant of (a)(ii), or the variant of (a)(iii), but with no more than 10 conservative substitutions,

wherein the variants of (a)(ii), (a)(iii) and (a)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (a)(i),

(b)(i) a wild-type PNO1 polypeptide;

(b)(ii) a deletion variant that is the polypeptide of (b)(i) but lacks up to twenty amino acid segments, each segment consisting of two amino acids;

(b)(iii) a deletion variant that is the polypeptide of (b)(i) or the variant of (b)(ii) but lacks up to twenty single, non-contiguous amino acids of the polypeptide of (b)(i) or the variant of (b)(ii);

or

(b)(iv) a substitution variant that is the polypeptide of (b)(i), the deletion variant of (b)(ii), or the deletion variant of (b)(iii), but with no more than 10 conservative substitutions,

wherein the variants of (b)(ii), (b)(iii), and (b)(iv) have at least 25% of the phosphorylation-promoting activity of the polypeptide of (b)(i), or

(c) a wild-type MNN6 polypeptide.

27. The substantially pure culture of claim 26, said cells further comprising a nucleic acid encoding a target protein, wherein the cell produces the target protein in an altered N-glycosylation form relative to the N-glycosylation form of the target protein produced in a corresponding non-genetically engineered cell.

28. The substantially pure culture of claim 26, said cell further comprising a nucleic acid encoding an alpha-mannosidase.