Methods of producing hyaluronic acid and chimeric and hybrid glycosaminoglycan polymers

• US 7,741,091 B2
• Filed: 01/25/2007
• Issued: 06/22/2010
• 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: Jing, Wei, DeAngelis, Paul L.
• Primary Examiner(s): Fronda; Christian L
• Application Number:
  US11/698,311
• Publication Number:
  US 20070117188A1
• Time in Patent Office: 1,244 Days
• Field of Search: None
• US Class Current: 435/193
• 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 29/085 Macromolecular materials
• CPC Class Codes: C08B 37/0063 Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
• 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 19/26 Preparation of nitrogen-containing carbohydrates
• CPC Class Codes: C12P 19/28 N-glycosides

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 multocida, in order to create a variety of glycosaminoglycan oligosaccharides having a natural or chimeric or hybrid sugar structure.

Claims

1. A method for enzymatically producing a hyaluronic acid polymer in vitro, wherein the hyaluronic acid polymer is composed of less than about 150 sugars, comprising the steps of:

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

providing a hyaluronic acid synthase capable of elongating the functional acceptor, wherein the hyaluronic acid synthase is selected from the group consisting of;

(a) a hyaluronic acid synthase having the amino acid sequence as set forth in SEQ ID NO;

2;

(b) a hyaluronic acid synthase encoded by the nucleotide sequence as set forth in SEQ ID NO;

1;

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

20, 27-30 and 71;

(d) a truncated form of (a) having the amino acid sequence as set forth in SEQ ID NO;

9;

(e) a mutated form of (a) encoded by the nucleotide sequence as set forth in SEQ ID NO;

42; and

(f) a hyaluronic acid synthase encoded by a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of SEQ ID NO;

1 under hybridization conditions selected from the group consisting of;

(i) 5×

SSC/5×

Denhardt'"'"'s solution/1.0% SDS at 68°

C., followed with washing in 0.2×

SSC/0.1% SDS at room temperature;

(ii) 1.8×

HPB at about 30°

C. to about 45°

C. followed by washing in 0.2-0.5×

HPB at about 45°

C.; and

(iii) 5×

SSC/5×

Denhardt'"'"'s solution/1% SDS at 68°

C., followed with washing in 3×

SSC at 42°

C.; and

providing UDP-GlcUA and UDP-GlcNAc sugars such that the hyaluronic acid synthase elongates the functional acceptor to provide a hyaluronic acid polymer wherein the hyaluronic acid polymer is composed of less than about 150 sugars.

2. The method of claim 1 wherein, in the step of providing a functional acceptor, uronic acid is further defined as a uronic acid selected from the group consisting of GlcUA, IdoUA, and GalUA, and hexosamine is further defined as a hexosamine selected from the group consisting of GlcNAc, GalNAc, GlcN and GalN.

3. The method of claim 1, wherein the hyaluronic acid polymer is composed of from about 50 to about 150 sugars.

4. The method of claim 1, wherein the hyaluronic acid polymer is composed of from about 15 to 50 sugars.

5. The method of claim 1, wherein the hyaluronic acid polymer is composed of from about 10 to 14 sugars.

6. The method of claim 1 wherein, in the step of providing the functional acceptor, the functional acceptor has at least three sugar units.

7. The method of claim 1 wherein the hyaluronic acid synthase is immobilized and the at least one of UDP-GlcUA and UDP-GlcNAc are in liquid phase.

8. The method of claim 1 wherein the functional acceptor is immobilized and the at least one of UDP-GlcUA and UDP-GlcNAc and the hyaluronic acid synthase are in liquid phase.

9. The method of claim 1, further comprising the step of providing a divalent metal ion selected from the group consisting of manganese, magnesium, cobalt, nickel and combinations thereof, and wherein the method occurs in a buffer having a pH of from about 6 to about 8.

10. A method for producing a chimeric or hybrid glycosaminoglycan having a non-natural structure, wherein the chimeric or hybrid glycosaminoglycan comprises at least one sugar not found in a single naturally occurring glycosaminoglycan, the method comprising the steps of:

providing a functional acceptor, wherein the functional acceptor has at least two sugar units selected from the group consisting of uronic acid and hexosamine, and wherein at least one of the at least two sugar units is a sugar unit other than GlcUA and GlcNAc;

providing at least one hyaluronic acid synthase selected from the group consisting of;

(a) a hyaluronic acid synthase having the amino acid sequence as set forth in SEQ ID NO;

2;

(b) a hyaluronic acid synthase encoded by the nucleotide sequence as set forth in SEQ ID NO;

1;

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

20, 27-30 and 71;

(d) a truncated form of (a) having the amino acid sequence as set forth in SEQ ID NO;

9;

(e) a mutated form of (a) encoded by the nucleotide sequence as set forth in SEQ ID NO;

42;

(f) a hyaluronic acid synthase encoded by a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of SEQ ID NQ;

1 under hybridization conditions selected from the group consisting of;

(i) 5×

SSC/5×

Denhardt'"'"'s solution/1.0% SDS at 68°

C., followed with washing in 0.2×

SSC/0.1% SDS at room temperature;

(ii) 1.8×

HPB at about 30°

C. to about 45°

C. followed by washing in 0.2-0.5×

HPB at about 45°

C.; and

(iii) 5×

SSC/5×

Denhardt'"'"'s solution/1% SDS at 68°

C., followed with washing in 3×

SSC at 42°

C.; and

(g) at least one modified hyaluronic acid synthase, wherein the at least one modified hyaluronic acid synthase is a single action glycosyltransferase capable of adding only one of GlcUA or GlcNAc to a functional acceptor, and wherein the at least one modified hyaluronic acid synthase is selected from the group consisting of;

(i) a modified hyaluronic acid synthase having the nucleotide sequence as set forth in any of SEQ ID NQS;

10-12, 16-19, 31-41, and 43-50; and

(ii) a modified hyaluronic acid synthase encoded by a nucleotide sequence capable of hybridizing to the complement of the nucleotide sequence of SEQ ID NO;

1 under the hybridization conditions of (f);

providing at least one of UDP-GlcUA and UDP-GlcNAc such that the at least one hyaluronic acid synthase elongates the functional acceptor in a manner so as to provide a chimeric or hybrid glycosaminoglycan having a non-natural structure whereby the chimeric or hybrid glycosaminoglycan comprises at least one sugar not found in a naturally occurring hyaluronic acid polymer.