High molecular weight heparosan polymers and methods of production and use thereof

Abstract

High molecular weight heparosan polymers are described, as are methods of producing and using the high molecular weight heparosan polymers.

What is claimed is: 
     
         1 . A method for recombinantly producing a high molecular weight heparosan polymer, the method comprising the steps of:
 culturing a recombinant host cell containing a nucleotide sequence encoding a polypeptide having heparosan synthase activity under conditions appropriate for the expression of the heparosan synthase, wherein at least one of:
 (a) the polypeptide having heparosan synthase activity is at least 90% identical to at least one of SEQ ID NOS:2, 4, and 6-8, and the nucleotide sequence encoding the polypeptide has been gene-optimized for expression in the recombinant host cell; 
 (b) the polypeptide having heparosan synthase activity has 1-20 amino acid additions, deletions, and/or substitutions when compared to at least one of SEQ ID NOS:2, 4, and 6-8, and the nucleotide sequence encoding the polypeptide has been gene-optimized for expression in the recombinant host cell; 
 (c) the polypeptide is encoded by the nucleotide sequence of at least one of SEQ ID NOS:9-11; 
 (d) the polypeptide is encoded by a nucleotide sequence that is at least 90% identical to at least one of SEQ ID NOS:9-11; and 
   isolating heparosan polymer produced by the heparosan synthase, wherein the isolated heparosan polymer is biocompatible with a mammalian patient and biologically inert within extracellular compartments of a mammalian patient, and wherein the isolated heparosan polymer is represented by the structure (-GlcUA-beta1,4-GlcNAc-alpha-1,4-) n , wherein n is a positive integer greater than or equal to 2,000.   
     
     
         2 . The method of  claim 1 , wherein the isolated heparosan polymer is further defined as having a value for n in a range of from about 2,000 to about 17,000. 
     
     
         3 . The method of  claim 1 , wherein the recombinant host cell further comprises at least one gene encoding an enzyme for synthesis of a heparosan sugar precursor, wherein the at least one gene encoding an enzyme for synthesis of a heparosan sugar precursor is selected from the group consisting of a pyrophosphorylase, a transferase, a mutase, a dehydrogenase, and an epimerase, capable of producing UDP-GlcNAc or UDP-GlcUA. 
     
     
         4 . The method of  claim 1 , further comprising the step of crosslinking the isolated heparosan polymer. 
     
     
         5 . The method of  claim 1 , further comprising the step of covalently and/or non-covalently attaching the isolated heparosan polymer to at least a portion of a surface of a substrate. 
     
     
         6 . The method of  claim 5 , wherein the substrate is selected from the group consisting of silica, silicon, semiconductors, glass, polymers, nanotubes, nanoparticles, organic compounds, inorganic compounds, metals and combinations thereof. 
     
     
         7 . The method of  claim 6 , wherein at least a portion of the substrate is a metal selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof. 
     
     
         8 . The method of  claim 1 , wherein the isolated heparosan polymer is substantially not susceptible to mammalian hyaluronidases or heparanases and thereby is not substantially degraded in vivo in extracellular compartments of a mammalian patient. 
     
     
         9 . The method of  claim 1 , wherein the recombinant host cell is an  E. coli  recombinant host cell. 
     
     
         10 . An isolated nucleotide sequence encoding a polypeptide having heparosan synthase activity, wherein at least one of:
 (a) the polypeptide having heparosan synthase activity is at least 90% identical to at least one of SEQ ID NOS:2, 4, and 6-8, and the nucleotide sequence encoding the polypeptide has been gene-optimized for expression in the recombinant host cell; and   (b) the polypeptide having heparosan synthase activity has 1-20 amino acid additions, deletions, and/or substitutions when compared to at least one of SEQ ID NOS:2, 4, and 6-8, and the nucleotide sequence encoding the polypeptide has been gene-optimized for expression in the recombinant host cell;   (c) the polypeptide is encoded by the nucleotide sequence of at least one of SEQ ID NOS:9-11; and   (d) the polypeptide is encoded by a nucleotide sequence that is at least 90% identical to at least one of SEQ ID NOS:9-11.   
     
     
         11 . The isolated nucleotide sequence of  claim 10 , wherein the host cell for which the nucleotide sequence has been gene-optimized is  E. coli.    
     
     
         12 . A recombinant host cell, comprising the isolated nucleotide sequence of  claim 10 . 
     
     
         13 . The recombinant host cell of  claim 12 , further defined as an  E. coli  recombinant host cell. 
     
     
         14 . A method of augmenting tissue in a mammalian patient, comprising the steps of:
 administering an effective amount of a biomaterial composition to a mammalian patient, the biomaterial composition comprising an isolated heparosan polymer that is biocompatible with the mammalian patient and biologically inert in extracellular compartments of the mammalian patient, wherein the isolated heparosan polymer is represented by the structure (-GlcUA-beta1,4-GlcNAc-alpha-1,4-) n , wherein n is a positive integer greater than or equal to 2,000, and wherein the isolated heparosan polymer is produced according to the method of  claim 1 .   
     
     
         15 . The method of  claim 14 , wherein the biomaterial composition is not substantially susceptible to hyaluronidases or heparanases and thereby is not substantially degraded in extracellular compartments of the mammalian patient. 
     
     
         16 . The method of  claim 14 , wherein the biomaterial composition is in a gel, semi-solid, liquid, and/or particulate state. 
     
     
         17 . The method of  claim 14 , wherein the biomaterial composition further comprises a substrate, and wherein the isolated heparosan polymer is covalently and/or non-covalently attached to the substrate. 
     
     
         18 . The method of  claim 17 , wherein the substrate is selected from the group consisting of silica, silicon, semiconductors, glass, polymers, nanotubes, nanoparticles, organic compounds, inorganic compounds, metals, and combinations thereof. 
     
     
         19 . The method of  claim 18 , wherein at least a portion of the substrate is a metal selected from the group consisting of gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys, and combinations thereof. 
     
     
         20 . The method of  claim 14 , wherein the step of administering an effective amount of the biomaterial composition to the mammalian patient is further defined as implanting and/or injecting an effective amount of the biomaterial composition into the mammalian patient.