Targeted glycosaminoglycan polymers by polymer grafting and methods of making and using same

• US 7,223,571 B2
• Filed: 08/15/2003
• Issued: 05/29/2007
• Est. Priority Date: 04/02/1998
• Status: Expired due to Fees

• Current Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Original Assignee: The Board of Regents of the University of Colorado (University of Colorado System)
• Inventors: DeAngelis, Paul L., Jing, Wei
• Primary Examiner(s): Nashed; Nashaat T.
• Application Number:
  US10/642,248
• Publication Number:
  US 20040132143A1
• Time in Patent Office: 1,383 Days
• Field of Search: None
• US Class Current: 435/97
• CPC Class Codes: A61K 39/102 Pasteurellales, e.g. Actinobacillus, Pasteurella; Haemophilus
• CPC Class Codes: A61K 47/36 Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
• CPC Class Codes: A61K 9/006 Oral mucosa, e.g. mucoadhesive forms, sublingual droplets; Buccal patches or films; Buccal sprays
• CPC Class Codes: A61L 24/08 Polysaccharides A61L24/043 takes precedence
• CPC Class Codes: A61L 27/20 Polysaccharides
• CPC Class Codes: A61L 29/085 Macromolecular materials
• CPC Class Codes: C07H 3/06 Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
• CPC Class Codes: C07K 14/285 from Pasteurellaceae (F), e.g. Haemophilus influenza
• CPC Class Codes: C07K 14/705 Receptors; Cell surface antigens; Cell surface determinants tumour specific antigens C07K14/4748
• CPC Class Codes: C07K 16/28 against receptors, cell surface antigens or cell surface determinants
• CPC Class Codes: C07K 2317/34 Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
• CPC Class Codes: C08B 37/0063 Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
• CPC Class Codes: C08B 37/0069 Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
• CPC Class Codes: C08B 37/0072 Hyaluronic acid, i.e. HA or hyaluronan; Derivatives thereof, e.g. crosslinked hyaluronic acid (hylan) or hyaluronates
• CPC Class Codes: C08B 37/0075 Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
• CPC Class Codes: C08L 5/08 Chitin; Chondroitin sulfate; Hyaluronic acid; Derivatives thereof
• CPC Class Codes: C12N 9/1048 Glycosyltransferases (2.4)
• CPC Class Codes: C12N 9/1051 Hexosyltransferases (2.4.1)
• CPC Class Codes: C12P 19/04 Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• CPC Class Codes: C12P 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: C12P 19/28 : N-glycosides
• CPC Class Codes: C12Q 1/689 : for bacteria

Abstract

The present invention relates to methodology for polymer grafting by a polysaccharide synthase and, more particularly, polymer grafting using the hyaluronate or chondroitin or heparin/heparosan synthases from Pasteurella, in order to create a variety of glycosaminoglycan oligosaccharides having a natural or chimeric or hybrid sugar structure with a targeted size that are substantially monodisperse in size.

Claims

1. A method for enzymatically producing defined glycosaminoglycan polymers comprising the steps of:

providing at least one functional acceptor, wherein the functional acceptor has at least two sugar units selected from the group consisting of uronic acid and hexosamine;

providing at least one recombinant glycosaminoglycan transferase having an empty acceptor site and being capable of elongating the at least one functional acceptor in a controlled fashion to form extended glycosaminoglycan molecules, the at least one recombinant glycosaminoglycan transferase selected from the group consisting of;

(a) a recombinant glycosaminoglycan transferase having an amino acid sequence as set forth in SEQ ID NO;

2;

(b) a recombinant glycosaminoglycan transferase encoded by a nucleotide sequence as set forth in SEQ ID NO;

1;

(c) a truncated form of (a) encoded by a nucleotide sequence as set forth in any of SEQ ID NOS;

10, 20, 27-32 and 71;

(d) a mutated form of (a) encoded by a nucleotide sequence as set forth in any of SEQ ID NOS;

11, 12, 16-19, 33-50;

(e) a recombinant glycosaminoglycan transferase encoded by a nucleotide sequence capable of hybridizing to the nucleotide sequence of SEQ ID NO;

1 under hybridization conditions comprising hybridization at a temperature of 68°

C. in 5×

SSC/5×

Denhardt'"'"'s solution/1.0% SDS, followed with washing in 3×

SSC at 42°

C.; and

providing at least one UDP-sugar selected from the group consisting of UDP-GlcUA, UDP-GlcNAc, and UDP-GlcN in a stoichiometric ratio to the at least one functional acceptor such that the at least one recombinant glycosaminoglycan transferase elongates the at least one functional acceptor to provide glycosaminoglycan polymers wherein the glycosaminoglycan polymers have a desired size distribution such that the glycosaminoglycan polymers are substantially monodisperse in size such that the glycosaminoglycan polymers have a polydispersity value in a range of from 1.0 to 1.2, and wherein the desired size distribution is obtained by controlling the stoichiometric ratio of UDP-sugar to functional acceptor.

2. The method of claim 1 wherein, in the step of providing at least one functional acceptor, uronic acid is further defined as a uronic acid selected from the group consisting of GlcUA, iduronic acid (IdoUA), and GalUA.

3. The method of claim 1 wherein, in the step of providing at least one functional acceptor, hexosamine is further defined as a hexosamine selected from the group consisting of GlcNAc, GalNAc, GlcN, and GalN.

4. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the functional acceptor is a hyaluronic acid (HA) oligosaccharide having between three sugar units and oligosaccharide polymer having molecular weight of about 4.2 kDa.

5. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the functional acceptor is an HA polymer having a mass in a range of from about 3.5 kDa to about 2 MDa.

6. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the functional acceptor is a chondroitin oligosaccharide comprising at least three sugar units.

7. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the functional acceptor is a chondroitin polymer.

8. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the functional acceptor is a chondroitin sulfate polymer.

9. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the functional acceptor is a heparin, heparan or heparosan polymer.

10. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the functional acceptor is an extended acceptor selected from the group consisting of HA chains, chondroitin chains, heparosan chains, mixed glycosaminoglycan chains, analog containing chains, and combinations thereof.

11. The method of claim 1 wherein, in the step of providing at least one recombinant glycosaminoglycan transferase, the at least one recombinant glycosaminoglycan transferase comprises a recombinant single action glycosyltransferase capable of adding only one sugar unit selected from the group consisting of GlcUA, GlcNAc, and GlcN.

12. The method of claim 1, wherein the at least one recombinant glycosaminoglycan transferase is immobilized and the at least one functional acceptor and the at least one of UDP-GlcUA, UDP-GlcNAc, and UDP-GlcN are in a liquid phase.

13. The method of claim 1, wherein the at least one functional acceptor is immobilized and the at least one UDP-sugar are in a liquid phase.

14. The method of claim 1, further comprising the step of providing a divalent metal ion.

15. The method of claim 14, wherein the divalent metal ion is selected from the group consisting of manganese, magnesium, cobalt, nickel and combinationS thereof.

16. The method of claim 1, wherein the method occurs in a buffer having a pH from about 6 to about 8.

17. The method of claim 1 wherein the substantially monodisperse glycosaminoglycan polymers have a molecular weight in a range of from about 3.5 kDa to about 0.5 MDa.

18. The method of claim 17 wherein the substantially monodisperse glycosaminoglycan polymers have a polydispersity value in a range of from about 1.0 to about 1.1.

19. The method of claim 18 wherein the substantially monodisperse glycosaminoglycan polymers have a polydispersity value in a range of from about 1.0 to about 1.05.

20. The method of claim 1 wherein the substantially monodisperse glycosamirioglycan polymers have a molecular weight in a range of from about 0.5 MDa to about 4.5 MDa.

21. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor comprises a moiety selected from the group consisting of a fluorescent tag, a radioactive tag, an affinity tag, a detection probe, a medicant, and combinations thereof.

22. The method of claim 1 wherein, in the step of providing at least one UDP-sugar, at least one UDP-sugar is radioactively labeled.

23. The method of claim 1 wherein the glycosaminoglycan polymers are chimeric or hybrid glycosaminoglycans comprising more than one type of polymer backbone.

24. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor is a plurality of functional acceptors immobilized on a substrate.

25. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor is a plurality of functional acceptors in a liquid phase.

26. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor is immobilized on a microtiter plate.

27. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor is immobilized on a microarray slide.

28. The method of claim 1 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor is sulfated or is a modified oligosaccharide.

29. The method of claim 1 wherein the ratio of UDP-sugar to functional acceptor is low to produce products having a molecular weight less than about 0.5 MDa.

30. The method of claim 1 wherein the ratio of UDP-sugar to functional acceptor is high to produce products having a molecular weight greater than about 0.5 MDa.

31. The method of claim 1 wherein the substantially monodisperse glycosaminoglycan polymers have a polydispersity value in a range of from about 1.0 to about 1.005.

32. The method of claim 1, wherein the defined glycosaminoglycan polymers so produced are capable of acting as a bioadhesive sealant, a tissue engineering aid, a cell matrix mimetic, a cell behavior or growth modulator, a drug delivery agent, or combinations thereof.

33. A method for enzymatically producing defined glycosaminoglycan polymers comprising the steps of:

providing at least one functional acceptor, wherein the functional acceptor is selected from the group consisting of an HA polymer, a chondroitin polymer, a chondroitin sulfate polymer, a heparin, heparan or heparosan polymer, mixed GAG chains, analog containing chains and combinations thereof;

providing at least one recombinant glycosaminoglycan transferase having an empty acceptor site and being capable of elongating the at least one functional acceptor in a controlled fashion to form extended glycosaminoglycan molecules, the at least one recombinant glycosaminoglycan transferase selected from the group consisting of;

(a) a recombinant glycosaminoglycan transferase having an amino acid sequence as set forth in SEQ ID NO;

2;

(b) a recombinant glycosaminoglycan transferase encoded by a nucleotide sequence as set forth in SEQ ID NO;

1;

(c) a truncated form of (a) encoded by a nucleotide sequence as set forth in any of SEQ ID NOS;

10, 20, 27-32 and 71;

(d) a mutated form of (a) encoded by a nucleotide sequence as set forth in any of SEQ ID NOS;

11, 12, 16-19, 33-50;

(e) a recombinant glycosaminoglycan transferase encoded by a nucleotide sequence capable of hybridizing to the nucleotide sequence of SEO ID NO;

1 under hybridization conditions comprising hybridization at a temperature of 68°

C. in 5×

SSC/5×

Denhardt'"'"'s solution/1.0% SDS, followed with washing in 3×

SSC at 42°

C.; and

providing at least one UDP-sugar selected from the group consisting of UDP-GlcUA, UDP-GlcNAc, and UDP-GlcN in a stoichiometric ratio to the at least one functional acceptor such that the at least one recombinant glycosaminoglycan transferase elongates the at least one functional acceptor to provide glycosaminoglycan polymers wherein the glycosaminoglycan polymers have a desired size distribution greater than 1 MDa, and wherein the desired size distribution is obtained by controlling the stoichiometric ratio of UDP-sugar to functional acceptor.

34. The method of claim 33 wherein, in the step of providing at least one functional acceptor, the functional acceptor is an HA polymer having a mass in a range of from about 3.5 kDa to about 2 MDa.

35. The method of claim 33 wherein, in the step of providing at least one recombinant glycosaminoglycan transferase, the at least one recombinant glycosaminoglycan transferase comprises a recombinant single action glycosyltransferase capable of adding only one sugar unit selected from the group consisting of GlcUA, GlcNAc and GlcN.

36. The method of claim 33, wherein the at least one recombinant glycosaminoglycan transferase is immobilized and the at least one functional acceptor and the at least one UDP-sugar are in a liquid phase.

37. The method of claim 33, wherein the at least one functional acceptor is immobilized and the at least one UDP-sugar are in a liquid phase.

38. The method of claim 33, further comprising the step of providing a divalent metal ion.

39. The method of claim 38, wherein the divalent metal ion is selected from the group consisting of manganese, magnesium, cobalt, nickel and combinations thereof.

40. The method of claim 33, wherein the method occurs in a buffer having a pH from about 6 to about 8.

41. The method of claim 33 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor comprises a moiety selected from the group consisting of a fluorescent tag, a radioactive tag, an affinity tag, a detection probe, a medicant, and combinations thereof.

42. The method of claim 33 wherein, in the step of providing at least one UDP-sugar, at least one UDP-sugar is radioactively labeled.

43. The method of claim 33 wherein the glycosaminoglycan polymers are chimeric or hybrid glycosaminoglycans comprising more than one type of polymer backbone.

44. The method of claim 33 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor is a plurality of functional acceptors immobilized on a substrate.

45. The method of claim 33 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor is a plurality of functional acceptors in a liquid phase.

46. The method of claim 33 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor is immobilized on a microtiter plate.

47. The method of claim 33 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor is immobilized on a microarray slide.

48. The method of claim 33 wherein, in the step of providing at least one functional acceptor, the at least one functional acceptor is sulfated or is a modified oligosaccharide.

49. The method of claim 33 wherein the ratio of UDP-sugar to functional acceptor is low to produce products having a molecular weight less than about 0.5 MDa.

50. The method of claim 33 wherein the ratio of UDP-sugar to functional acceptor is high to produce products having a molecular weight greater than about 0.5 MDa.

51. The method of claim 33 wherein the substantially monodisperse glycosaminoglycan polymers have a polydispersity value in a range of from about 1.0 to about 1.005.

52. A method for enzymatically producing defined glycosaminoglycan polymers comprising the steps of:

providing at least one functional acceptor, wherein the functional acceptor has at least two sugar units selected from the group consisting of uronic acid and hexosamine;

providing at least one recombinant glycosaminoglycan transferase having an empty acceptor site and being capable of elongating the at least one functional acceptor in a controlled fashion to form extended glycosaminoglycan molecules, the at least one recombinant glycosaminoglycan transferase selected from the group consisting of;

(a) a recombinant glycosaminoglycan transferase having an amino acid sequence as set forth in SEQ ID NO;

2;

(b) a recombinant glycosaminoglycan transferase encoded by a nucleotide sequence as set forth in SEQ ID NO;

1 ;

(c) a truncated form of (a) encoded by a nucleotide sequence as set forth in any of SEQ ID NOS;

10, 20, 27-32 and 71;

(d) a mutated form of (a) encoded by a nucleotide sequence as set forth in any of SEQ ID NOS;

11, 12, 16-19, 33-50;

e) a recombinant glycosaminoglycan transferase encoded by a nucleotide sequence capable of hybridizing to the nucleotide sequence of SEQ ID NO;

1 under hybridization conditions comprising hybridization at a temperature of 68°

C. in 5×

SSC/5×

Denhardt'"'"'s solution/1.0% SDS, followed with washing in 3×

SSC at 42°

C.; and

;

providing at least one UDP-sugar selected from the group consisting of UDP-GlcUA, UDP-GlcNAc, and UDP-GlcN in a stoichiometric ratio to the at least one functional acceptor such that the at least one recombinant glycosaminoglycan transferase elongates the at least one functional acceptor to provide glycosaminoglycan polymers wherein the glycosaminoglycan polymers have a desired size distribution such that the glycosaminoglycan polymers are substantially monodisperse in size such that the glycosaminoglycan polymers have a polydispersity value in a range of from 1.0 to 1.1, and wherein the desired size distribution is obtained by controlling the stoichiometric ratio of UDP-sugar to functional acceptor.