Source: https://patents.google.com/patent/US9885072B2/en
Timestamp: 2019-09-20 12:46:40
Document Index: 222552903

Matched Legal Cases: ['§ 119', 'Application No. 13767739', 'Application No. 13767739', 'Application No. 13767739', 'Application No. 13767739', 'Application No. 13767739', 'Application No. 13767739']

US9885072B2 - High molecular weight heparosan polymers and methods of production of use thereof - Google Patents
High molecular weight heparosan polymers and methods of production of use thereof Download PDF
US9885072B2
US9885072B2 US15/467,683 US201715467683A US9885072B2 US 9885072 B2 US9885072 B2 US 9885072B2 US 201715467683 A US201715467683 A US 201715467683A US 9885072 B2 US9885072 B2 US 9885072B2
US15/467,683
US20170198325A1 (en
2015-02-17 Priority to US14/624,354 priority patent/US20150150900A1/en
2017-03-23 Application filed by University of Oklahoma filed Critical University of Oklahoma
2017-03-23 Priority to US15/467,683 priority patent/US9885072B2/en
2017-07-13 Publication of US20170198325A1 publication Critical patent/US20170198325A1/en
2018-02-06 Publication of US9885072B2 publication Critical patent/US9885072B2/en
This application is a continuation of U.S. Ser. No. 14/624,354, filed Feb. 17, 2015, now abandoned; which is a divisional of U.S. Ser. No. 13/854,435, filed Apr. 1, 2013, now U.S. Pat. No. 8,980,608, issued Mar. 17, 2015; which claims benefit under 35 USC § 119(e) of U.S. provisional application Ser. No. 61/617,952, filed Mar. 30, 2012. The entire contents of the above-referenced patents and patent applications are hereby expressly incorporated herein by reference.
BACKGROUND 1. Field of the Inventive Concept(s)
The presently disclosed and/or claimed inventive concept(s) relates to methodology for the production and uses of glycosaminoglycan compositions, and more particularly, to compositions comprising an isolated heparosan polymer of high molecular weight, as well as methods of production and uses thereof.
The presently disclosed and/or claimed inventive concept(s) overcomes the disadvantages and defects of the prior art. The presently disclosed and/or claimed inventive concept(s) is based on a biomaterial comprising heparosan, the natural biosynthetic precursor of heparin and heparan sulfate. This composition has numerous characteristics that provide improvements and advantages over existing products. While heparosan is very similar to HA and heparin, the molecule has greater stability within the body since it is not the natural final form of this sugar and therefore the body has no degradation enzymes or binding proteins that lead to loss of functionality. This property also reduces biofouling, infiltration, scarring and/or clotting. Heparosan is also more hydrophilic than synthetic coatings such as plastics or carbon. Finally, aside from bacterial HA, most other current filler biomaterials are typically animal-derived, which causes concern for side effects such as allergic reactions or stimulating granulation, and such side effects will not be a concern with heparosan. Also, most naturally occuring heparosan polymers are known to have certain size ranges of molecular weight, depending on origin of the heparosan biopolymer such as the biosynthesis pathways utilized, including types of catalysts, hosts, and supporting appratus. As is known in the art, the size distribution of the heparosan biopolymer affects its physical properties, such as viscosity, chain entanglement, and solubility. In the presently disclosed and/or claimed inventive concept(s), we have developed a means to produce extremely high molecular weight (MW) heparosan polymers that have higher viscosity and can be used at lower concentrations (either with or without chemical crosslinking) than the naturally occurring heparosan preparations.
FIG. 1A contains an alignment of two E. coli gene-optimized sequences (SEQ ID NOS: 9 and 10) with the native Pasteurella multocida heparosan synthase gene (SEQ ID NO:1).
FIG. 1B contains an alignment of the two E. coli gene-optimized sequences (SEQ ID NOS: 9 and 10).
Unless otherwise defined herein, scientific and technical terms used in connection with the presently disclosed and/or claimed inventive concept(s) shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989) and Coligan et al. Current Protocols in Immunology (Current Protocols, Wiley Interscience (1994)), which are incorporated herein by reference. The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
All patents, published patent applications, and non-patent publications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this presently disclosed and/or claimed inventive concept(s) pertains. All patents, published patent applications, and non-patent publications referenced in any portion of this application are herein expressly incorporated by reference in their entirety to the same extent as if each individual patent or publication was specifically and individually indicated to be incorporated by reference.
All of the compositions and/or methods disclosed and/or claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the inventive concept(s) as defined by the appended claims.
The term “naturally-occurring” as used herein as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory or otherwise is naturally-occurring. Similarly, a sugar polymer or polysaccharide with instrinsic structural features (such as but not limited to, composition, molecular weight (MW) distribution, etc.) found in native organisms (i.e., unmodified by the hand of man) is termed “naturally occurring”.
The terms “administration” and “administering”, as used herein will be understood to include all routes of administration known in the art, including but not limited to, oral, topical, transdermal, parenteral, subcutaneous, intranasal, mucosal, intramuscular, intraperitoneal, intravitreal and intravenous routes, including both local and systemic applications. In addition, the compositions of the presently disclosed and/or claimed inventive concept(s) (and/or the methods of administration of same) may be designed to provide delayed, controlled or sustained release using formulation techniques which are well-known in the art.
The term “dermal augmentation” in the context of the presently disclosed and/or claimed inventive concept(s) refers to any change of the natural state of a mammal's skin and related areas due to external acts. The areas that may be changed by dermal augmentation include, but not limited to, epidermis, dermis, subcutaneous layer, fat, arrector pill muscle, hair shaft, sweat pore, and sebaceous gland.
The term “biomaterial” as used herein will be understood to refer to any nondrug material that can be used to treat, enhance, protect, or replace any tissue, organ, or function in an organism. The term “biomaterial” also refers to biologically derived material that is used for its structural rather than its biological properties, for example but not by way of limitation, to the use of collagen, the protein found in bone and connective tissues, as a cosmetic ingredient, or to the use of carbohydrates modified with biotechnological processes as lubricants for biomedical applications or as bulking agents in food manufacture. A “biomaterial” is any material, natural or man-made, that comprises whole or part of a living structure or biomedical device that performs, auguments, protects, or replaces a natural function and that is compatible with the body.
As used herein, when the term “isolated” is used in reference to a molecule, the term means that the molecule has been removed from its native environment. For example, a polynucleotide or a polypeptide naturally present in a living organism is not “isolated,” but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is “isolated.” Further, recombinant DNA molecules contained in a vector are considered isolated for the purposes of the presently disclosed and/or claimed inventive concept(s). Isolated RNA molecules include in vivo or in vitro RNA replication products of DNA and RNA molecules. Isolated nucleic acid molecules further include synthetically produced molecules. Additionally, vector molecules contained in recombinant host cells are also isolated. Overall, this also applies to carbohydrates in general. Thus, not all “isolated” molecules need be “purified.”
As used herein, the term “substrate” will be understood to refer to any surface of which a coating may be disposed. Examples of substrates that may be utilized in accordance with the presently disclosed and/or claimed inventive concept(s) include, but are not limited to, silica, silicon, glass, polymers, nanotubes, nanoparticles, organic compounds, inorganic compounds, metals and combinations thereof. When the substrate is a metal, the metal may include, but is not limited to, gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys and combinations thereof.
The terms “gel” and “semi-solid” are used interchangeably herein and will be understood to include a colloidal system, with the semblance of a solid, in which a solid is dispersed in a liquid; the compound may have a finite yield stress. The term “gel” also refers to a jelly like material formed by the coagulation of a colloidal liquid. Many gels have a fibrous matrix and fluid filled interstices: gels are viscoelastic rather than simply viscous and can resist some mechanical stress without deformation. When pressue is applied to gels or semi-solids, they conform to the shape at which the pressure is applied.
A non-limiting example of a type of gene optimization utilized in accordance with the presently disclosed and/or claimed inventive concept(s) is codon optimization. The terms “codon-optimized” and “codon optimization” refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is efficiently expressed in the organism of interest. Although the genetic code is degenerate in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome. In some embodiments, the polynucleotides encoding enzymes may be codon-optimized for optimal production from the host organism selected for expression.
Turning now to the presently disclosed and/or claimed inventive concept(s), compositions that include an isolated high molecular weight (HMW) heparosan polymer are included and described in detail herein, along with methods of producing and using same. In certain embodiments, the composition is a biomaterial composition. In particular embodiments, the isolated heparosan polymer is biocompatible with a mammalian patient and biologically inert in extracellular compartments of the mammalian patient. The heparosan polymer is substantially not susceptible to vertebrate (such as but not limited to, mammalian) hyaluronidases or vertebrate (such as but not limited to, mammalian) heparanses and thereby is not substantially degraded in vivo in extracellular compartments of the mammalian patient. In addition, the heparosan polymer may be recombinantly produced as described in detail herein utilizing a combination of host cell and synthase biosynthesis, where features of both of these factors influence the MW made by the live cell.
The disclosed and/or claimed 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 about 2,000. Polymers of this size are hitherto unreported in the scientific literature and prior art. Each single n unit is approximately 400 Da, and therefore the isolated heparosan polymer has a molecular weight (MW) of greater than or equal to about 800 kDa. In particular embodiments, n is a positive integer in a range of from about 2,000 to about 17,000, and therefore the isolated heparosan polymer has a MW in a range of from about 0.8 MDa to about 6.8 MDa. In addition, n may be a positive integer such as but not limited to, 2,250; 2,500; 2,750; 3,000; 3,250; 3,500; 3,750; 4,000; 4,250; 4,500; 4,750; 5,000; 5,250; 5,500; 5,750; 6,000; 6,250; 6,500; 6,750; 7,000; 7,250; 7,500; 7,750; 8,000; 8,250; 8,500; 8,750; 9,000; 9,250; 9,500; 9,750; 10,000; 10,250; 10,500; 10,750; 11,000; 11,250; 11,500; 11,750; 12,000; 12,250; 12,500; 12,750; 13,000; 13,250; 13,500; 13,750; 14,000; 14,250; 14,500; 14,750; 15,000; 15,250; 15,500; 15,750; 16,000; 16,250; 16,500; 16,750; and 17,000; as well as within a range of any of the above.
The heparosan polymer may be linear or cross-linked. The compositions of the presently disclosed and/or claimed inventive concept(s) may be administered to a patient by any means known in the art; for example, but not by way of limitation, the compositions may be injectable and/or implantable. In addition, the compositions may be in a gel or semi-solid state, a suspension of particles, or the compositions may be in a liquid form.
Alternatively, the heparosan polymer may be attached to a substrate. When attached to a substrate, the isolated heparosan polymer may be covalently (via a chemical bond) or non-covalently (via weak bonds) attached to the substrate. Any substrate known in the art or otherwise contemplated herein may be utilized, so long as the substrate is capable of being attached to the heparosan polymer and functioning in accordance with the presently disclosed and/or claimed inventive concept(s). Examples of substrates that may be utilized in accordance with the presently disclosed and/or claimed inventive concept(s) include, but are not limited to, silica, silicon, semiconductors, glass, polymers, nanotubes, nanoparticles, organic compounds, inorganic compounds, metals, and combinations thereof. Non-limiting examples of metals that may be utilized include gold, copper, stainless steel, nickel, aluminum, titanium, thermosensitive alloys, and combinations thereof.
The presently disclosed and/or claimed inventive concept(s) also comprises biomaterial compositions comprising a cross-linked gel that includes an isolated heparosan polymer and at least one cross-linking agent. The cross-linking agent may be any cross-linking agent known or otherwise contemplated in the art; specific non-limiting examples of cross-linking agents that may be utilized in accordance with the presently disclosed and/or claimed inventive concept(s) include aldehydes, epoxides, polyaziridyl compounds, glycidyl ethers, divinyl sulfones, and combinations and derivatives thereof. An advantage of the currently described inventive concept(s) is that lower concentrations of this high MW (greater than 1 MDa or 1,000 kDa) polymer may be used to produce useful gels than if a lower MW polymer was employed.
Any of the biomaterial compositions of the presently disclosed and/or claimed inventive concept(s) may be a moisturizing biomaterial that protects from dehydration; alternatively, any of the biomaterial compositions of the presently disclosed and/or claimed inventive concept(s) may be a lubricating biomaterial.
Another aspect of the presently disclosed and/or claimed inventive concept(s) is related to kits for in vivo administration of any of the compositions described herein above or otherwise contemplated herein to a mammalian patient. The kit may also include instructions for administering the composition to the mammalian patient. The kit may optionally also contain one or more other compositions for use in accordance with the methods described herein.
The presently disclosed and/or claimed inventive concept(s) is further directed to a method of recombinantly producing a high MW heparosan polymer. In the method, a recombinant host cell containing a nucleotide sequence encoding a heparosan synthase, the enzyme that polymerizes the monosaccharides from UDP-sugar precursors into heparosan polysaccharide or sugar polymer, is cultured under conditions appropriate for the expression of the heparosan synthase. The heparosan synthase produces the high MW heparosan polymer, which is then isolated.
Any heparosan synthase known in the art or otherwise contemplated herein may be utilized in accordance with the presently disclosed and/or claimed inventive concept(s), so long as the heparosan synthase is capable of producing a high MW heparosan polymer in an appropriate host under the appropriate culture conditions. Non-limiting examples of heparosan synthases that may be utilized in accordance with the presently disclosed and/or claimed inventive concept(s) are described in greater detail herein below.
Any host cell known in the art or otherwise contemplated herein may be utilized in accordance with the presently disclosed and/or claimed inventive concept(s), so long as the host cell is capable of being made recombinant with a heparosan synthase gene and producing a high MW heparosan polymer upon expression of the heparosan synthase gene under the appropriate culture conditions. Non-limiting examples of host cells that may be utilized in accordance with the presently disclosed and/or claimed inventive concept(s) are described in greater detail below.
In one embodiment, the presently disclosed and/or claimed inventive concept(s) shows that Pasteurella heparosan synthases (or catalyst with sequence similarity or key motifs) will perform the ultra-high MW heparosan biosynthesis operation in an E. coli host cell with the proper UDP-sugar and transport infrastructure. Most available E. coli strains employed in laboratories as well as most wild-type isolates are therefore not useful without further manipulation. The presently disclosed and/or claimed inventive concept(s) demonstrates that an E. coli K5 host (or strains that contain similar infrastructure) is amenable to high MW heparosan polymer production.
In theory, at least simple two models for controlling the size of a polymer are possible: (A) host cell-controlled biosynthesis or (B) synthase-controlled biosynthesis. In the former model, the nature of the supporting apparatus (e.g., UDP-sugar precursors, transporters) defines the final size distribution made by the live cell. In the latter model, the intrinsic properties of the polymerizing catalyst (e.g., elongation rate, processivity) control the polymer size distribution made by the live cell. A third model (C), combinatorial host cell/synthase biosynthesis, is possible where features of both factors influence the MW made by a live cell; this model is also the most complex, unpredictable, and non-obvious to decipher. For the presently disclosed and/or claimed inventive concept(s), models A & B are inconsistent with the observed data; neither the Escherichia coli K5 host cell's product size (˜50-80 kDa) nor the Pasteurella heparosan synthase product size (˜100-300 kDa) is similar to the heparosan made in the inventive concept(s) (>800 kDa) and should be considered a non-predictable outcome that has not been reported in the patent or scientific literature to date.
Certain embodiments of the presently disclosed and/or claimed inventive concept(s) also include the use of alternative hosts with the potential for glycosaminoglycan production, including bacteria from both Gram-negative (e.g., Pseudomonas, etc.) and Gram-positive classes (e.g., Bacilli, Lactoctococci, etc.), as well as other microbes (fungi, archae, etc). The basic requirements of a recombinant host for use in heparosan production in accordance with the presently disclosed and/or claimed inventive concept(s) include: (a) the glycosyltransferase(s) that produce heparosan, and (b) the UDP-sugar precursors UDP-GlcNAc and UDP-GlcUA. It should be noted that the latter requirement can be met by either native genes or introduced recombinant genes. The required genes can be either episomally and/or chromosomally located.
In certain embodiments, the host cell further comprises at least one gene encoding an enzyme for synthesis of a heparosan sugar precursor (i.e., UDP-GlcNAc or UDP-GlcUA). Non-limiting examples of genes encoding an enzyme for synthesis of a heparosan sugar precursor that may be utilized in accordance with the presently disclosed and/or claimed inventive concept(s) include pyrophosphorylases, transferases, mutases, dehydrogenases, and epimerases.
The ultra-high MW (= or >1 MDa) heparosan polymer is not known in nature and not been shown or reported by others. As well-known in the polymer field, the size distribution affects its physical properties (e.g., viscosity, chain entanglement, solubility). The >1 MDa heparosan described and claimed in the inventive concept(s) is preferred over the naturally occurring heparosan with respect to performance in production of certain biomaterials, such as but not limited to, viscoelastics and hydrogels.
The presently disclosed and/or claimed inventive concept(s) is also related to methods of augmenting tissue in a mammalian patient. In such methods, an effective amount of any of the biomaterial compositions described herein above or otherwise contemplated herein is administered to the mammalian patient. The biomaterial composition may be administered to the patient by any method known in the art, including, but not limited to, injection and/or implantation. When injected, the biomaterial composition may be in a liquid state or a suspension of particles, whereas when implanted, the biomaterial composition may be in a gel or semi-solid state, or may be attached to a substrate.
The presently disclosed and/or claimed inventive concept(s) also relates to methods of repairing voids in tissues of mammals. In the method, any of the biomaterial compositions described herein above or otherwise contemplated herein is administered into the voids. In certain embodiments, the biomaterial composition may be injected and/or implanted into the voids.
The presently disclosed and/or claimed inventive concept(s) also relates to methods of creating voids or viscus in tissues of mammals. In the method, any of the biomaterial compositions described herein above or otherwise contemplated herein are disposed into a tissue or a tissue engineering construct to create the voids or viscus. In certain embodiments, the biomaterial composition may be injected and/or implanted into the tissue/tissue engineering construct to create the voids or viscus.
The presently disclosed and/or claimed inventive concept(s) also relates to methods of reparative surgery or plastic surgery. In the method, any of the biomaterial compositions described herein above or otherwise contemplated herein is administered to a patient and serves as a filling material at the site to which it is administered. In certain embodiments, the biomaterial composition may be injected and/or implanted into the patient.
The presently disclosed and/or claimed inventive concept(s) further relates to methods of dermal augmentation and/or treatment of skin deficiency in a patient. In the method, any of the biomaterial compositions described herein above or otherwise contemplated herein is administered to the patient. In certain embodiments, the biomaterial composition may be injected and/or implanted into the patient. The biomaterial composition is biocompatible, swellable, hydrophilic, and substantially non-toxic, and the biomaterial composition swells upon contact with physiological fluids at the administration/injection/implantation site.
The dermal augmentation method of the presently disclosed and/or claimed inventive concept(s) is especially suitable for the treatment of skin contour deficiencies, which are often caused by various conditions/exposures, including but not limited to, aging, environmental exposure, weight loss, child bearing, injury, surgery, in addition to diseases such as acne and cancer. Non-limiting examples of contour deficiencies that may be treated in accordance with the presently disclosed and/or claimed inventive concept(s) include frown lines, worry lines, wrinkles, crow's feet, marionette lines, stretch marks, and internal and external scars resulted from injury, wound, bite, surgery, or accident.
In addition, the presently disclosed and/or claimed inventive concept(s) also relates to methods of medical or prophylactic treatment of a mammalian patient. In the method, any of the compositions described herein above or otherwise contemplated herein is administered to the mammalian patient in need of such a treatment. In certain embodiments, the composition may be injected and/or implanted into the mammalian patient.
Further, the presently disclosed and/or claimed inventive concept(s) also relates to methods of treatment or prophylaxis of tissue augmentation in a mammalian patient. In the method, a medical or prophylactic composition comprising a polysaccharide gel composition that includes any of the biomaterial compositions described herein above or otherwise contemplated herein is administered to the mammalian patient.
The presently disclosed and/or claimed inventive concept(s) is further related to a delivery system for a substance having biological or pharmacological activity. The system comprising a molecular cage formed of a cross-linked gel of heparosan or a mixed cross-linked gel of heparosan and at least one other hydrophilic polymer co-polymerizable therewith. The system further includes a substance having biological or pharmacological activity dispersed therein, wherein the substance is capable of being diffused therefrom in a controlled manner.
The biomaterials of the presently disclosed and/or claimed inventive concept(s) may be utilized in any methods of utilizing biomaterials known or otherwise contemplated in the art. For example but not by way of limitation, the biomaterial compositions of the presently disclosed and/or claimed inventive concept(s) may be utilized in any of the methods of utilizing other known biomaterials that are described in U.S. Pat. No. 4,582,865, issued to Balazs et al. on Apr. 15, 1986; U.S. Pat. No. 4,636,524, issued to Balazs et al. on Jan. 13, 1987; U.S. Pat. No. 4,713,448, issued to Balazs et al. on Dec. 15, 1987; U.S. Pat. No. 5,137,875, issued to Tsununaga et al. on Aug. 11, 1992; U.S. Pat. No. 5,827,937, issued to Ang on Oct. 27, 1998; U.S. Pat. No. 6,436,424, issued to Vogel et al. on Aug. 20, 2002; U.S. Pat. No. 6,685,963, issued to Taupin et al. on Feb. 3, 2004; and U.S. Pat. No. 7,060,287, issued to Hubbard et al. on Jun. 13, 2006. The entire contents of such patents are hereby expressly incorporated herein by reference, and therefore any of the methods described therein, when utilized with the novel biomaterial compositions of the presently disclosed and/or claimed inventive concept(s), also fall within the scope of the presently disclosed and/or claimed inventive concept(s).
Other specific examples of uses for the biomaterial compositions of the presently disclosed and/or claimed inventive concept(s) include, but are not limited to: (a) a persistent lubricating coating on a surface, such as, but not limited to, surgical devices; (b) a long lasting moisturizer; (c) a viscoelastic supplement for joint maladies; and (d) a non-thrombotic, non-occluding blood conduit (such as, but not limited to, a stent or artificial vessel, etc.). In addition, any of the biomaterial compositions of the presently disclosed and/or claimed inventive concept(s) may be utilized in tissue engineering to form a viscus or vessel duct or lumen by using the biomaterial compositions of the presently disclosed and/or claimed inventive concept(s) as a three-dimensional space maker; in this instance, the surrounding cells will not bind to the biomaterial compositions of the presently disclosed and/or claimed inventive concept(s), thereby making such biomaterial compositions well suited for this technology.
In addition, the presently disclosed and/or claimed inventive concept(s) further includes methods of doing business by producing any of the compositions described or otherwise contemplated herein by the methods described herein above and selling and delivering the compositions to a customer or providing such compositions to a patient.
In one embodiment of the presently disclosed and/or claimed inventive concept(s), the compositions of the presently disclosed and/or claimed inventive concept(s) may be produced using recombinant heparosan synthases as described or otherwise known in the art, including but not limited to, the heparosan synthases disclosed in the inventor's prior patents U.S. Pat. No. 7,307,159, issued Dec. 11, 2007; U.S. Pat. No. 7,771,981, issued May 8, 2002; and U.S. Pat. No. 8,088,604, issued Jan. 3, 2012; as well as the heparosan synthases disclosed in the inventor's published patent applications US 2008/0226690, published Sep. 18, 2008; US 2010/0036001, published Feb. 11, 2010; and US 2012/0108802, published May 3, 2012. The entire contents of the above-referenced patents and patent applications, and especially the sequence listings thereof, are expressly incorporated herein by reference as if explicitly disclosed herein.
Non-limiting examples of heparosan synthases that may be utilized in accordance with the presently disclosed and/or claimed inventive concept(s) include: a recombinant heparosan synthase having an amino acid sequence as set forth in at least one of SEQ ID NOS: 2, 4, and 6-8; a recombinant heparosan synthase encoded by the nucleotide sequence of at least one of SEQ ID NOS: 1, 3, 5, and 9-11; a recombinant heparosan synthase encoded by a nucleotide sequence capable of hybridizing to a complement of the nucleotide sequence of at least one of SEQ ID NOS: 1, 3, 5, and 9-11 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.; a recombinant heparosan synthase encoded by a nucleotide sequence capable of hybridizing to a complement of a nucleotide sequence encoding an amino acid sequence as set forth in at least one of SEQ ID NOS: 2, 4, and 6-8 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.; a recombinant heparosan synthase encoded by a nucleotide sequence capable of hybridizing to a complement of the nucleotide sequence of at least one of SEQ ID NOS: 1, 3, 5, and 9-11 under hybridization conditions comprising hybridization at a temperature of 30° C. in 5×SSC, 5×Denhardts reagent, 30% formamide for about 20 hours followed by washing twice in 2×SSC, 0.1% SDS at about 30° C. for about 15 min followed by 0.5×SSC, 0.1% SDS at about 30° C. for about 30 minutes; and a recombinant heparosan synthase encoded by a nucleotide sequence capable of hybridizing to a complement of a nucleotide sequence encoding an amino acid sequence as set forth in of at least one of SEQ ID NOS: 2, 4, and 6-8 under hybridization conditions comprising hybridization at a temperature of 30° C. in 5×SSC, 5×Denhardts reagent, 30% formamide for about 20 hours followed by washing twice in 2×SSC, 0.1% SDS at about 30° C. for about 15 min followed by 0.5×SSC, 0.1% SDS at about 30° C. for about 30 minutes.
Additional non-limiting examples of heparosan synthases that may be utilized in accordance with the presently disclosed and/or claimed inventive concept(s) include: a recombinant heparosan synthase that is at least 60% identical to at least one of SEQ ID NOS: 2, 4, and 6-8; a recombinant heparosan synthase that is at least 70% identical to at least one of SEQ ID NOS: 2, 4, and 6-8; a recombinant heparosan synthase that is at least 80% identical to at least one of SEQ ID NOS: 2, 4, and 6-8; a recombinant heparosan synthase that is at least 85% identical to at least one of SEQ ID NOS: 2, 4, and 6-8; a recombinant heparosan synthase that is at least 90% identical to at least one of SEQ ID NOS: 2, 4, and 6-8; a recombinant heparosan synthase that is at least 95% identical to at least one of SEQ ID NOS: 2, 4, and 6-8; a recombinant heparosan synthase encoded by a nucleotide sequence that is at least 60% identical to at least one of SEQ ID NOS: 1, 3, 5, and 9-11; a recombinant heparosan synthase encoded by a nucleotide sequence that is at least 70% identical to at least one of SEQ ID NOS: 1, 3, 5, and 9-11; a recombinant heparosan synthase encoded by a nucleotide sequence that is at least 80% identical to at least one of SEQ ID NOS: 1, 3, 5, and 9-11; a recombinant heparosan synthase encoded by a nucleotide sequence that is at least 85% identical to at least one of SEQ ID NOS: 1, 3, 5, and 9-11; a recombinant heparosan synthase encoded by a nucleotide sequence that is at least 90% identical to at least one of SEQ ID NOS: 1, 3, 5, and 9-11; and a recombinant heparosan synthase encoded by a nucleotide sequence that is at least 95% identical to at least one of SEQ ID NOS: 1, 3, 5, and 9-11.
The use of truncated heparosan synthase genes to produce any of the compositions described or otherwise contemplated herein also falls within the scope of the presently disclosed and/or claimed inventive concept(s). For instance, the removal of the last 50 residues or the first 77 residues of PmHS1 (SEQ ID NOS: 7 and 8, respectively) does not inactivate its catalytic function (Kane et al., 2006). Those of ordinary skill in the art would appreciate that simple amino acid removal from either end of the heparosan synthase sequence can be accomplished. The truncated versions of the sequence simply have to be checked for activity in order to determine if such a truncated sequence is still capable of producing heparosan.
Similarly, the use of fusion proteins that add other polypeptide segments (to either termini or internally) to the heparosan synthase sequence also falls within the scope of the presently disclosed and/or claimed inventive concept(s). The fusion protein partner (such as but not limited to, maltose-binding protein, thioredoxin, etc.) can increase stability, increase expression levels in the cell, and/or facilitate the purification process, but the catalytic activity for making the heparosan polymer intrinsic to the inventive concept(s) remains the same.
The recombinant heparosan synthase utilized in accordance with the presently disclosed and/or claimed inventive concept(s) also encompass sequences essentially as set forth in SEQ ID NOS: 1-8. The term “a sequence essentially as set forth in SEQ ID NO:X” means that the sequence substantially corresponds to a portion of SEQ ID NO:X and has relatively few amino acids or codons encoding amino acids which are not identical to, or a biologically functional equivalent of, the amino acids or codons encoding amino acids of SEQ ID NO:X. The term “biologically functional equivalent” is well understood in the art and is further defined in detail herein, as a gene having a sequence essentially as set forth in SEQ ID NO:X, and that is associated with the ability of prokaryotes to produce HA or a heparosan polymer in vitro or in vivo. In the above examples, X refers to either SEQ ID NO:1-11 or any additional sequences set forth herein, such as the truncated or mutated versions of pmHS1 that are contained generally in SEQ ID NOS: 7-8.
These references and countless others indicate that one of ordinary skill in the art, given a nucleic acid sequence or an amino acid sequence, could make substitutions and changes to the nucleic acid/amino acid sequence without changing its functionality (specific examples of such changes are given hereinafter and are generally set forth in SEQ ID NOS: 7-8). Also, a substituted nucleic acid segment may be highly identical and retain its enzymatic activity with regard to its unadulterated parent, and yet still fail to hybridize thereto. As such, variations of the sequences that fall within the above-defined functional limitations have been disclosed in the applications incorporated by reference. As such, the presently disclosed and/or claimed inventive concept(s) should not be regarded as being solely limited to the use of the specific sequences disclosed and/or incorporated by reference herein. Even further, if smaller regions or sequence motifs contain the active site residues or important functional units, this similarity is also indicative of function. The presently disclosed and/or claimed inventive concept(s) may utilize nucleic acid segments encoding an enzymatically active HS from P. multocida—pmHS1 and/or PmHS2. One of ordinary skill in the art would appreciate that substitutions can be made to the pmHS1 or PmHS2 nucleic acid segments listed in SEQ ID NO:1, 3 and 5, respectively, without deviating outside the scope and claims of the presently disclosed and/or claimed inventive concept(s). Standardized and accepted functionally equivalent amino acid substitutions are presented in Table 1. In addition, other analogous or homologous enzymes that are functionally equivalent to the disclosed synthase sequences would also be appreciated by those skilled in the art to be similarly useful in the methods of the presently disclosed and/or claimed inventive concept(s), that is, a new method to control precisely the size distribution of the heparosan polymer.
Amino Acid Group Semi-Conservative Substitutions
Therefore, the presently disclosed and/or claimed inventive concept(s) also includes the use of heparosan synthases that have amino acid sequences that differ from at least one of SEQ ID NOS: 2, 4, and 6-8 by at least one of the following: the presence of 1-60 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8; the presence of 1-55 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8; the presence of 1-50 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8; the presence of 1-45 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8; the presence of 1-40 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8; the presence of 1-35 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8; the presence of 1-30 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8; the presence of 1-25 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8; the presence of 1-20 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8; the presence of 1-15 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8; the presence of 1-10 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8; and the presence of 1-5 amino acid additions, deletions, or substitutions when compared to at least one of SEQ ID NOS: 2, 4, and 6-8.
The hybridizing portion of the hybridizing nucleic acids is typically at least about 14 nucleotides in length, and preferably between about 14 and about 100 nucleotides in length. The hybridizing portion of the hybridizing nucleic acid is at least about 60%, e.g., at least about 80% or at least about 90%, identical to a portion or all of a nucleic acid sequence encoding a heparin/heparosan synthase or its complement, such as SEQ ID NO:1, 3, 5, 9, 10, or 11, or the complement thereof. Hybridization of the oligonucleotide probe to a nucleic acid sample typically is performed under standard or stringent hybridization conditions. Nucleic acid duplex or hybrid stability is expressed as the melting temperature or Tm, which is the temperature at which a probe nucleic acid sequence dissociates from a target DNA. This melting temperature is used to define the required stringency conditions. If sequences are to be identified that are related and substantially identical to the probe, rather than identical, then it is useful to first establish the lowest temperature at which only homologous hybridization occurs with a particular concentration of salt (e.g., Standard Saline Citrate (SSC), Saline Sodium Phosphate EDTA (SSPE), or High Phosphate Buffer (HPB) solutions). Then, assuming that 1% mismatching results in a 1° C. decrease in the Tm, the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if sequences having >95% identity with the probe are sought, the final wash temperature is decreased by about 5° C.). In practice, the change in Tm can be between about 0.5° C. and about 1.5° C. per 1% mismatch. Examples of standard stringent hybridization conditions include hybridizing at about 68° C. in 5×SSC/5×Denhardt's solution/1.0% SDS, followed with washing in 0.2×SSC/0.1% SDS at room temperature or hybridizing in 1.8×HPB at about 30° C. to about 45° C. followed by washing a 0.2-0.5×HPB at about 45° C. Moderately stringent conditions include hybridizing as described above in 5×SSC\5×Denhardt's solution 1% SDS washing in 3×SSC at 42° C. The parameters of salt concentration and temperature can be varied to achieve the optimal level of identity between the probe and the target nucleic acid. Additional guidance regarding such conditions is readily available in the art, for example, by Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, (Cold Spring Harbor Press, N.Y.); and Ausubel et al. (eds.), 1995, Current Protocols in Molecular Biology, (John Wiley & Sons, N.Y.). Several examples of low stringency protocols include: (A) hybridizing in 5×SSC, 5×Denhardts reagent, 30% formamide at about 30° C. for about 20 hours followed by washing twice in 2×SSC, 0.1% SDS at about 30° C. for about 15 min followed by 0.5×SSC, 0.1% SDS at about 30° C. for about 30 min (FEMS Microbiology Letters, 2000, vol. 193, p. 99-103); (B) hybridizing in 5×SSC at about 45° C. overnight followed by washing with 2×SSC, then by 0.7×SSC at about 55° C. (J. Viological Methods, 1990, vol. 30, p. 141-150); or (C) hybridizing in 1.8×HPB at about 30° C. to about 45° C.; followed by washing in 1×HPB at 23° C.
The DNA segments that may be utilized to produce the compositions of the presently disclosed and/or claimed inventive concept(s) encompass DNA segments encoding biologically functional equivalent HS proteins and peptides. Such sequences may arise as a consequence of codon redundancy and functional equivalency which are known to occur naturally within nucleic acid sequences and the proteins thus encoded. Alternatively, functionally equivalent proteins or peptides may be created via the application of recombinant DNA technology, in which changes in the protein structure may be engineered, based on considerations of the properties of the amino acids being exchanged. Changes designed by man may be introduced through the application of site-directed mutagenesis techniques, e.g., to introduce improvements to the enzyme activity or to antigenicity of the HS protein or to test HS mutants in order to examine HS activity at the molecular level or to produce HS mutants having changed or novel enzymatic activity and/or sugar substrate specificity.
The presently disclosed and/or claimed inventive concept(s) also include the use of nucleotide sequences encoding any of the heparosan synthases described or otherwise contemplated herein, wherein the nucleotide sequences are synthetic sequences that have been gene-optimized for expression in a particular host cell. Specific, non-limiting examples of gene-optimized heparosan synthase encoding nucleotide sequences are provided in SEQ ID NOS: 9-11. SEQ ID NOS: 9-10 include nucleotide sequences encoding the heparosan synthase of SEQ ID NO:2 and which have been gene-optimized for expression in E. coli. SEQ ID NO:11 includes a nucleotide sequence encoding the heparosan synthase of SEQ ID NO:2 and which have been gene-optimized for expression in Bacillus.
The use of gene-optimized sequences is known and used in the art to increase expression of the gene sequence within the heterologous host. However, a novel product was unexpectedly produced from the heparosan synthase expressed in E. coli when the Pasteurella gene sequence was gene-optimized and expressed in E. coli. The inventive concept(s) disclose the production of mega-Dalton molecular weight heparosan polymers, and this novel species has never before been reported in any known microbes. One of ordinary skill in the art would assume that optimization of a gene sequence encoding an enzyme would result in increased expression of that enzyme in the heterologous host, thereby resulting in increased production of the same enzyme-derived product (i.e., higher amounts of the heparosan polymer of the typical size found in the native microbes) produced in the native host. Unexpectedly, the expression of gene-optimized Pasteurella multocida heparosan synthase in E. coli resulted in a new species of product—an ultra-high molecular weight heparosan polymer. Production of heparosan polymers of this size have not been reported for any other microbe. In addition, the heparosan polymers produced in accordance with the presently disclosed and/or claimed inventive concept(s) exhibit superior and advantageous properties compared to the lower molecular weight products currently known in the art. These properties provide enhanced utility for the heparosan polymer in the biomaterials field. For example but not by way of limitation, the ultra-high molecular weight (MW) heparosan polymers produced in accordance with the presently disclosed and/or claimed inventive concept(s) exhibit enhanced solution viscosity and can be used at lower concentrations (either with or without chemical crosslinking) than the naturally occurring heparosan preparations.
The presently disclosed and/or claimed inventive concept(s) further includes isolated nucleotide sequences, along with recombinant host cells containing same, that contain any of the gene-optimized heparosan synthase sequences disclosed or otherwise contemplated herein.
Heparosan, a sugar polymer that is the natural biosynthetic precursor of heparin and heparan sulfate, has numerous characteristics that indicate that this material exhibits enhanced performance in a variety of medical applications or medical devices. In comparison to HA and heparin, two very structurally similar polymers used in many current applications in several large markets, heparosan is more stable in the body, as no naturally occurring enzymes degrade heparosan, and therefore the biomaterial compositions of the presently disclosed and/or claimed inventive concept(s) should have longer lifetimes compared to presently used biomaterials. In addition, heparosan interacts with fewer proteins (thus less fouling) and cells (thus less infiltration, scarring, or clotting) when compared to existing biomaterials.
In comparison to synthetic plastics or carbon, the natural hydrophilicity (aka water-loving) characteristics of heparosan also enhance tissue compatibility. Animal-derived proteins (e.g., collagen, bovine serum albumin) and calcium hydroxyapatite often have side effects, including but not limited to, eliciting an allergic response and/or stimulating granulation (5). On the other hand, even certain pathogenic bacteria use heparosan to hide in the body since this polymer is non-immunogenic (8-10). The biomaterial compositions of the presently disclosed and/or claimed inventive concept(s) produced from a non-animal source also promise to be free of adventitious agents (e.g., vertebrate viruses, prions) that could potentially contaminate animal- or human-derived sources.
Certain carbohydrates play roles in forming and maintaining the structures of multicellular organisms in addition to more familiar roles as nutrients for energy. Glycosaminoglycans [GAGS], long linear polysaccharides consisting of disaccharide repeats that contain an amino sugar, are well-known to be essential in vertebrates (9, 11-15). The GAG structures possess many negative groups and are replete with hydroxyl groups; therefore these sugars have a high capacity to adsorb water and ions. Heparin/heparan (backbone [β4GlcUA-α4GlcNAc]n), chondroitin (backbone [β4GlcUA-β3GalNAc]n), and hyaluronan (HA; backbone [β4GlcUA-β3GlcNAc]n) are the three most prevalent GAGs in humans. Depending on the tissue and cell type, the GAGs are structural, adhesion, and/or signaling elements. A few clever microbes also produce extracellular polysaccharide coatings, called capsules, composed of GAG chains that serve as virulence factors (9, 10). The capsule is thought to assist in the evasion of host defenses such as phagocytosis and complement. As the microbial polysaccharide is identical or very similar to the host GAG, the antibody response is either very limited or non-existent.
Artificial plastics (poly[lactide] in SCULPTRA® (Sanofi-Aventis) or poly[methylmethacrylate] in ARTECOLL® (Artes Medical, Inc., San Diego, Calif.), ceramics (calcium hydroxyapatite in RADIESSE® (Bioform Medical, Inc., San Mateo, Calif.)) or pure carbon have utility for many therapeutic applications (1,5,7,18), but in many respects, their chemical and physical properties are not as optimal as polysaccharides for the targeted goals of dermal fillers or surface coatings. The most critical issues are lack of good wettability (due to poor interaction with water) and/or hardness (leading to an unnatural feel or brittleness). The presently disclosed and/or claimed inventive concept(s) is related to the use of heparosan to replace and supplant useful sugar polymers that are hydrophilic (water loving) and may be prepared in a soft form.
HA and heparin have been employed as biomaterial coatings for vascular prosthesis and stents (artificial blood vessels and supports), as well as coatings on intraocular lenses and soft-tissue prostheses (7, 22). The rationale is to prevent blood clotting, enhance fouling resistance, and prevent post-surgery adhesion (when organs stick together in an undesirable fashion). The biomaterial compositions of the presently disclosed and/or claimed inventive concept(s) should also be suitable as a coating, as described in greater detail herein after.
A key advantage with heparosan is that it has increased biostability in the extracellular matrix when compared to other GAGs. As with most compounds synthesized in the body, new molecules are made, and after serving their purpose, are broken down into smaller constituents for recycling. Heparin and heparan sulfate are eventually degraded and turned over by a single enzyme known as heparanase (23, 24). Experimental challenge of heparosan and N-sulfo-heparosan with heparanase, however, shows that these polymers lacking 0-sulfation are not sensitive to enzyme action in vitro (25, 26). These findings demonstrate that heparosan is not fragmented enzymatically in the body. Overall, this indicates that heparosan is a very stable biomaterial.
Gene-optimized pmHS1 sequences for expression in E. coli and Bacillus. Three gene-optimized sequences encoding the Pasteurella multocida heparosan synthase of SEQ ID NO:2 were obtained. Two of the sequences (SEQ ID NOS: 9 and 10) were gene-optimized for expression in E. coli, while the third sequence (SEQ ID NO:11) was gene-optimized for expression in Bacillus.
FIG. 1A contains an alignment of the two E. coli gene-optimized sequences, SEQ ID NOS: 9 and 10, with the native Pasteurella multocida heparosan synthase gene (SEQ ID NO:1). FIG. 1B contains an alignment of only the two E. coli gene-optimized sequences, SEQ ID NOS: 9 and 10. FIG. 1C contains an alignment of the Bacillus gene-optimized sequence (SEQ ID NO:11) with the native Pasteurella multocida heparosan synthase gene (SEQ ID NO:1).
Table 2 illustrates the percent identity between the two gene-optimized sequences of SEQ ID NOS: 9-10 and the native Pasteurella multocida gene sequence (SEQ ID NO:1). Note that all three sequences encode amino acid sequences that are 100% identical to the amino acid sequence of SEQ ID NO:2. As can be seen, the two gene-optimized sequences are approximately 74% identical to the native Pasteurella gene sequence. It is also noted that the two gene-optimized sequences are only 95% identical to each other, so there is some variation obtained from the algorithm that is being used to generate the optimized sequence.
Percent Identities of Gene-optimized and
Native Heparosan Synthase Gene Sequences
pmHS1-opt2
pmHS1 pmHS1-opt1 (SEQ ID NO:
(SEQ ID NO: 1) (SEQ ID NO: 9) 10)
pmHS1 74.3% 73.7%
Production of High MW Heparosan Polysaccharide.
There are two types of naturally occurring micobes, (a) certain Pasteurella multocida bacteria (Type D) and their related brethren such as certain Avibacteria, and (b) Escherichia coli K5 and their related brethren that make an extracellular coating composed of unsulfated heparosan polymer that is readily harvested from the culture media. An unexpected and advantageous characteristic has been discovered for the recombinant (gene-optimized Pasteurella gene in an E. coli host) heparosan over both natural bacterial heparosan and mammalian heparin; the heparosan produced in accordance with the presently disclosed and/or claimed inventive concept(s) has a higher molecular weight of approximately 1 to 6.8 MDa (1,000 to 6,800 kDa); therefore, gels or liquid viscoelastics formed of this recombinant heparosan should be easier to produce.
Various advantages of the presently disclosed and/or claimed inventive concept(s) are outlined in Tables 3 and 4.
Associated Barrier
of Current Innovative Approaches
Key Variable Project Target Current Practice Procedure Disclosed Herein
Coating Long lasting HA, heparin, Degraded by body's Use heparosan, a
Stability (weeks- Bovine serum natural enzymes polymer that is not
months) albumin (BSA) enzymatically digested
Carbon (C) — in human body
Lipids (L) Shed from surface
Wettability Freely interacts BSA, HA, heparin, L — Use water-loving
with water C Hydrophobic heparosan polymer
Clotting not bind clotting factors bind biologically inert
proteins or BSA, C, L — heparosan polymer
Transmission animal virus or HA [bacterial], PP, — bacterially derived
prions CHP heparosan
of Current Approaches
Formation long-lasting Collagen Gel (CG) polymer that is not
(>12-24 Plastic Particles (PP) Grainy appearance enzymatically
months), but & too long lifetime digested in human
not permanent Ca Hydroxyapatite Grainy appearance body, and is not a
gel Particles (CHP) too long lifetime, & coarse, hard
cannot inject easily material
Immunogenicity, No antibody HA [bacterial], PP, — Use heparosan
Allergenicity generation CHP polymer that looks
HA [chicken], Immune or allergic ‘human’ and does
CG [bovine >human] response not trigger immune
adhesion bind polymer that lacks
and/or signaling PP, CHP — known adhesion
CG Cells bind domains or
Transmission human or HA [bacterial], PP, — bacterially derived
animal virus CHP heparosan
and/or prions
Compatible marked areas PP, CHP Obscures images transparent
Resource not overly supply or cell via bacterial
expensive to culture derived fermentation
produce (costly)
HA, CHP, PP, CHP —
Production of Mega-Dalton Molecular Weight Heparosan.
Production of Mega-Dalton Molecular Weight Heparosan in E. coli BL21(DE3).
Production of Mega-Dalton Molecular Weight Heparosan in E. coli BL21 Express Iq.
Effect of Deletion of Heparosan Production in E. coli K5 on Production of Mega-Dalton Molecular Weight Heparosan.
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
(e) the nucleotide sequence encodes a Pasteurella heparosan synthase; 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 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.
3. The method of claim 1, further comprising at least one of the steps of:
(a) crosslinking the isolated heparosan polymer; and/or
(b) covalently and/or non-covalently attaching the isolated heparosan polymer to at least a portion of a surface of a substrate; optionally where the substrate is selected from the group consisting of silica, silicon, semiconductors, glass, polymers, nanotubes, nanoparticles, organic compounds, inorganic compounds, metals, and combinations thereof; optionally 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.
4. The method of claim 1, wherein the recombinant host cell is an E. coli recombinant host cell.
5. 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.
6. 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.
US15/467,683 2012-03-30 2017-03-23 High molecular weight heparosan polymers and methods of production of use thereof Active US9885072B2 (en)
US15/889,096 Continuation US20180251803A1 (en) 2012-03-30 2018-02-05 High molecular weight heparosan polymers and methods of production and use thereof
US20170198325A1 US20170198325A1 (en) 2017-07-13
US9885072B2 true US9885072B2 (en) 2018-02-06
US8580290B2 (en) * 2001-05-08 2013-11-12 The Board Of Regents Of The University Of Oklahoma Heparosan-based biomaterials and coatings and methods of production and use thereof
AR099900A1 (en) * 2014-04-01 2016-08-24 Merz Pharma Gmbh & Co Kgaa Soft tissue fillers polysaccharide with improved persistence, kit, method, use
EP0544592A2 (en) 1991-11-28 1993-06-02 Sanofi High molecular weight N,O-sulfated heparosans, process for producing the same and pharmaceutical compositions containing them
US5407911A (en) 1991-09-17 1995-04-18 Asahi Kasei Kogyo Kabushiki Kaisha Parathyroid hormone-containing emulsion for nasal administration
AU2002256501A1 (en) 2001-05-08 2003-05-01 The Board Of Regents Of The University Of Oklahoma Heparin/heparosan synthase and methods of making and using same
US20140107066A1 (en) 2012-03-30 2014-04-17 The Board Of Regents Of The University Of Oklahoma High molecular weight heparosan polymers and methods of production and use thereof
Ahn, J., et al.; "Cloning of the Putative Tumour Suppressor Gene for Hereditary Multiple Exostoses (EXT1)," Nature Genetics (1995), vol. 11, No. 2, pp. 137-143.
Ambrosio, et al.; "Rheological Study on Hyaluronic Acid and Its Derivative Solutions," Journal of Macromolecular Science-Part A, Pure and Applied Chemistry (1999), vol. 7&8, pp. 991-1000.
Ambrosio, et al.; "Rheological Study on Hyaluronic Acid and Its Derivative Solutions," Journal of Macromolecular Science—Part A, Pure and Applied Chemistry (1999), vol. 7&8, pp. 991-1000.
Ausubel et al.; "Hybridization Analysis of DNA Blots," Current Protocols in Molecular Biology (1993) Supplement 21 2.10-2.10.16.
Bio Tie Therapies; BioHeparin "The Only Semi-synthetically Produced Low Molecular Weight Heparin," printed from www.biotie.com on Feb. 19, 2004. 7 pages.
Boyce, J.D., et al.; "Pasteurella Multocida Capsule: Composition, Function and Genetics," Journal of Biotechnology (2000), vol. 83, pp. 153-160.
Bronner, D., et al.; "Synthesis of the K5 (group II) Capsular Polysaccharide in Transport-deficient Recombinant Escherichia coli," FEMS Microbiology Letters 113 (1993), pp. 273-284.
Casu, B., et al.; "Heparin-Like Compounds Prepared by Chemical Modification of Capsular Polysaccharide from E. coli," Elsevier Science Carbohydrate Research 263 (1994), pp. 271-284.
Chavaroche et al.; "In Vitro Synthesis of Heparosan Using Recombinant Pasteurella Multocida Heparsan Synthase PmHS2," Applied Microbioligy Biotechnology (2010) vol. 85, pp. 1881-1891.
Chavaroche et al.; "Production Methods for Heparosan, a Precursor of Heparin and Heparan Sulfate," Carbohydrate Polymers ( 2013) vol. 93, pp. 38-48.
Cheung, P.K., et al.; "Etiological Point Mutations in the Hereditary Multiple Exostoses Gene EXT1: A Functional Analysis of Heparan Sulfate Polymerase Activity," Am. J. Hum. Genet. (2001), vol. 69, pp. 55-66.
Chica, R.A., et al.; "Semi-Rational Approaches to Engineering Enzyme Activity: Combining the Benefits of Directed Evolution and Rational Design," Elsevier Current Opinion in Biotechnology (2005) vol. 16, pp. 378-384.
Clines, G.A., et al.; "The Structure of the Human Multiple Exostoses 2 Gene and Characterization of Homologs in Mouse and Caenorhabditis Elegans," Genome Research by Cold Spring Harbor Laboratory Press (1997) vol. 7, pp. 359-367, ISSN: 1054-9803/97.
Crawford, B.E., et al.; "Cloning, Golgi Localization, and Enzyme Activity of the Full-length Heparin/Heparan Sulfate-Glucuronic Acid C5-epimerase," Journal of Biol. Chem. (2001) vol. 276, No. 24, pp. 21538-21543.
DeAngelis, P.L. et al.; "Identification of a Distinct, Cryptic Heparosan Synthase from Pasteurella Multocida Types A, D, and F," Journal of Bacteriology (2004) vol. 186, No. 24, pp. 8529-8532.
DeAngelis, P.L., et al.; "Identification and Molecular Cloning of a Heparosan Synthase from Pasteurella Multocida Type D," The Journal of Biological Chemistry (2002) vol. 277, No. 9, ISSN: Mar. 1, pp. 7209-7213.
DeAngelis, P.L., et al.; "Identification of the Capsular Polysaccharides of Type D and F Pasteurella Multocida as Unmodified Heparin and Chondroitin, Respectively," Carbohydrate Research (2002), vol. 337, pp. 1547-1552.
DeAngelis, P.L.; "Microbial Glycosaminoglycan Glycosyltransferases," Glysobiology (2002) vol. 12, No. 1, pp. 9R-16R.
Duncan, G., et al.; "The Link Between Heparan Sulfate and Hereditary Bone Disease: Finding a Function for the EXT Family of Putative Tumor Suppressor Proteins," The Journal of Clinical Investigation (2001) vol. 108, No. 4, pp. 511-516.
EP Application No. 13767739.9, DeAngelis, et al. filed Oct. 30, 2014; EPO Communication pursuant to Article 94(3) EPC, dated Oct. 25, 2016.
EP Application No. 13767739.9, DeAngelis, et al. filed Oct. 30, 2014; EPO Communication Pursuant to Rules 70(2) and 70a(2) EPC dated Mar. 31, 2016.
EP Application No. 13767739.9, DeAngelis, et al. filed Oct. 30, 2014; EPO Communication Under Rule (3) EPC dated Feb. 20, 2017.
EP Application No. 13767739.9, DeAngelis, et al.; filed Oct. 30, 2014; Extended European Search Report dated Mar. 11, 2016.
EP Application No. 13767739.9, DeAngelis, et al.; filed Oct. 30, 2014; Response to Communication Under Rules 70(2) and 70a(2) EPC filed Oct. 10, 2016.
EP Application No. 13767739.9, DeAngelis, et al.; filed Oct. 30, 2014; Response to Examination Report filed Dec. 2, 2016.
Esko, J.D. et al.; "Molecular Diversity of Heparin Sulfate," J. Clin. Invest. (2001), vol. 108, pp. 169-173.
Fareed, J.; "Heparin, Its Fractions, Fragments and Derivatives-Some Newer Perspectives," Seminars in Thrombosis and Hemostasis, (1985), vol. 11, No. 1, 1985, pp. 1-9.
Fareed, J.; "Heparin, Its Fractions, Fragments and Derivatives—Some Newer Perspectives," Seminars in Thrombosis and Hemostasis, (1985), vol. 11, No. 1, 1985, pp. 1-9.
Finke, A., et al.; "Biosynthesis of the Escherichia coli K5 Polysaccharide, a Representative of Group II Capsular Polysaccharides: Polymerization in Vitro and Characterization of the Product," Journal of Bacteriology(1991), pp. 4088-4094.
Griffiths, G., et al.; "Characterization of the Glycosyltransferase Enzyme from the Escherichia coli K5 Capsule Gene Cluster and Identification and Charaterization of the Glucuronyl Active Site," The Journal of Biological Chemistry (1998), vol. 273, No. 19, pp. 11752-11757.
Guo, H.H., et al.; "Protein Tolerance to Random Amino Acid Change," PNAS (2004) vol. 101, No. 25, pp. 9205-9210.
Hagner-McWhirter A., et al.; "Biosynthesis of Heparin/Heparan Sulfate: Kinetic Studies of the Glucuronyl C5-Epimerase with N-Sulfated Derivatives of the Escherichia coli K5 Capsular Polysaccharide as Substrates," Glycobiology (2000) vol. 10, No. 2, pp. 159-171.
Hänfling, P., et al.; "Analysis of the Enzymatic Cleavage (β Elimination) of the Capsular K5 Polysaccharide of Escherichia coli by the K5-Specific Coliphage: a Reexamination," Journal of Bacteriology (1996) vol. 178, No. 15, pp. 4747-4750.
Hill, A.L., et al.; "Identification of the Xenopus Laevis cDNA for EXT1: A Phylogenetic Perspective," DNA Sequence (2002), vol. 13, No. 2, pp. 85-92; ISSN: 1042-5179; Taylor & Francis, Ltd.
Hodson, N., et al.; "Identification That KfiA, a Protein Essential for the Biosynthesis of the Escherichia coli K5 Capsular Polysaccharide, Is an α-UDP-GlcNAc Glycosyltransferase," The Journal of Biological Chemistry (2000) vol. 275, No. 35, pp. 27311-27315.
Jing, W., et al.; "Dissection of the Two Transferase Activities of the Pasteurella Multocida Hyaluronan Synthase: Two Active Sites Exist in One Polypeptide," Glycobiology (2000) vol. 10, No. 9, pp. 883-889.
Jing, W., et al.; "Structure Function Analysis of Pasteurella Glycosaminoglycan Synthesis," Annual Conference of the Society for Glycobiology (188), p. 705.
Kane, T.A., et al.; "Functional Characterization of PmHS1, a Pasteurella Multocida Heparosan Synthase," Journal of Biol. Chem. (2006) vol. 281, No. 44, pp. 33192-33197.
Katada, T., et al.; "cDNA Cloning and Distribution of XEXT1, the Xenopus Homologue of EXT1," Dev Genes Evol. (2002), vol. 212, pp. 248-250.
Kim, B.T., et al.; "Human Tumor Suppressor EXT Gene Family Members EXTL1 and EXTL3 Encode α1,4-N-Acetylglucosaminyltransferases That Likely Are Involved in Heparan Sulfate/Heparin Biosynthesis," PNAS (2001), vol. 98, No. 13, pp. 7176-7181.
Kim, B-T, et al.; "Demonstration of a Novel Gene DEXT3 of Drosophila melanogaster as the Essential N-Acetylglucosamine Transferase in the Heparan Sulfate Biosynthesis," The Journal of Biological Chemistry (2002) vol. 277, No. 16, pp. 13659-13665.
Kitagawa, H., et al.; "Rib-2, a Caenorhabditis Elegans Homolog of the Human Tumor Suppressor EXT Genes Encodes a Novel α1,4-N-Acetylglucosaminyltransferase Involved in the Biosynthetic Initiation and Elongation of Heparan Sulfate." The Journal of Biological Chemistry (2001) vol. 276, No. 7; pp. 4834-4838.
Kitagawa, H., et al.; "The Tumor Suppressor EXT-like Gene EXTL2 Encodes an α1, 4-N-Acetylhexosaminyltransferase That Transfers N-Acetylgalactosamine and N-Acetylglucosamine to the Common Glycosaminoglycan-Protein Linkage Region," The Journal of Biological Chemistry (1999)m, vol. 274, No. 20, pp. 13933-139337.
Kröncke, K.D., et al.; "Expression of the Escherichia coli K5 Capsular Antigen: Immunoelectron Microscopic and Biochemical Studies with Recombinant E. coli," Journal of Bacteriology (1990), pp. 1085-1091.
Kusche, M., et al.; "Biosynthesis of Heparin-Use of Escherichia coli K5 Capsular Polysaccharide as a Model Substrate in Enzymic Polymer-Modification Reactions," Biochem J. (1991) vol. 275, pp. 151-158.
Kusche, M., et al.; "Biosynthesis of Heparin—Use of Escherichia coli K5 Capsular Polysaccharide as a Model Substrate in Enzymic Polymer-Modification Reactions," Biochem J. (1991) vol. 275, pp. 151-158.
Leali, D., et al.; "Fibroblast Growth Factor-2 Antagonist Activity and Angiostatic Capacity of Sulfated Escherichia coli K5 Polysaccharide Derivatives," The Journal of Biological Chemistry (2001) vol. 276, No. 41, pp. 37900-37908.
Legeai-Mallet L., et al.; "EXT 1 Gene Mutation Induces Chondrocyte Cytoskeletal Abnormalities and Defective Collagen Expression in the Exostoses," Journal of Bone and Mineral Research (2000) vol. 15, No. 8, pp. 1489-1500.
Lidholt, K., et al.; "Biosynthesis of Heparin. The D-Glucuronosyl- and N-Acetyl-D-Glucosaminyltransferase Reactions and their Relation to Polymer Modification," Biochem J. (1992), vol. 287, pp. 21-29.
Lidholt, K., et al.; "Substrate Specificities of Glycosyltransferases Involved in Formation of Heparin Precursor and E. coli K5 Capsular Polysaccharides," Carbohydrate Research (1994), vol. 255, pp. 87-101.
Lin, X, et al.: "Expression and Functional Analysis of Mouse EXT1, a Homolog of the Human Multiple Exostoses Type 1 Gene," Biochemical and Biophysical Research Communications (1998) vol. 248, No. 3, pp. 738-743.
Lin, X, et al.; "Disruption of Gastrulation and Heparan Sulfate Biosynthesis in EXT1-Deficient Mice," Developmental Biology (2000), vol. 224, pp. 299-311.
Lind, T., et al.; "Biosynthesis of Heparin/Heparan Sulfate," The Journal of Biological Chemistry (1993) vol. 268, No. 28, pp. 20705-20708.
Lind, T., et al.; "The Putative Tumor Suppressors EXT1 and EXT2 Are Glycosyltransferases Required for the Biosynthesis of Heparan Sulfate.," The Journal of Biological Chemistry (1998) vol. 273, No. 41, pp. 26265-26268.
Linhardt, R.J., et al.; "Isolation and Characterization of Human Heparin," Biochemistry (1992), vol. 31, No. 49, pp. 12441-12445, Abstract only.
Linhardt, R.J., et al.; "Production and Chemical Processing of Low Molecular Weight Heparins," Seminars in Thrombosis and Hemostasis (1999), vol. 25, pages Suppl 3, pp. 5-16.
Manzoni, M., et al.; "Production of K5 Polysaccharides of Different Molecular Weight by Escherichia coli," Journal of BioActive and Compatible Polymers (1996) vol. 11, pp. 301-311.
May, B.J. et al.; "Complete Genomic Sequence of Pasteurella Multocida, Pm70," PNAS (2001), vol. 98, No. 6 pp. 3460-3465.
McCormick, C., et al.; "The Putative Tumor Suppressor EXT1 Alters the Expression of Cell-Surface Heparan Sulfate," Nature Genetics (1998), vol. 19, No. 2, pp. 158-161.
McCormick, C., et al.; "The Putative Tumor Suppressors EXT1 and EXT2 Form a Stable Complex That Accumulates in the Golgi Apparatus and Catalyzes the Synthesis of Heparan Sulfate," PNAS ( 2000) vol. 97, No. 2, pp. 668-673.
Nader, H.B., et al.; "New Insights on the Specificity of Heparin and Heparan Sulfate Lyases from Flavobacterium Heparinum Revealed by the Use of Synthetic Derivatives of K5 Polysaccharide from E. coli and 2-0-Desulfated Heparin," Glycoconjugate Journal (1999), vol. 16, pp. 265-270.
Naggi, A., et al.; "Toward a Biotechnological Heparin Through Combined Chemical and Enzymatic Modification of the Escherichia coli K5 Polysaccharide," Seminars in Thrombosis and Hemostasis (2001), vol. 27, No. 5, pp. 437-443.
Otto, N.J., et al.; "Structure/Function Analysis of Pasteurella Multocida Heparosan Synthases," Journal of Biological Chemistry (2012), vol. 287, No. 10, pp. 7203-7212.
Pandit, K.K.; "Capsular Hyaluronic Acid in Pasteurella Multocida Type A and its Counterpart in Type D," Research in Veterinary Science (1993), No. 54, pp. 20-24.
Pedersen, L.C., et al.; "Heparan/Chondroitin Sulfate Biosynthesis.," The Journal of Biological Chemistry (2000), vol. 275, No. 44, pp. 34580-34585.
Peppas, N.A., et al.; "New Challenges in Biomaterials," Science (1994) vol. 263, pp. 1715-1720.
Petit, C., et al.; "Region 2 of the Escherichia coli K5 Capsule Gene Cluster Encoding Proteins for the Biosynthesis of the K5 Polysaccharide," Molecular Microbiology (1995), vol. 17, No. 4, pp. 611-620.
Poggi., et al.; "Inhibition of B16-BL6 Melanoma Lung Colonies by Semisynthetic Sulfaminoheparosan Sulfates from E. coli K5 Polysaccharide," Seminars in Thrombosis and Hemostasis (2002), vol. 28, No. 4, pp. 383-391.
Razz, N., et al.; "Structural and Functional Properties of Heparin Analogues Obtained by Chemical Sulphation of Escherichia coli K5 Capsular Polysaccharide," Biochem J. (1995) vol. 309, pp. 465-472.
Rigg, G.P., et al.; "The Localization of KpsC, S and T, and KfiA, C and D Proteins Involved in the Biosynthesis of the Escherichia coli K5 Capsular Polysaccharide: Evidence for a Membrane-Bound Complex," Microbiology (1998), vol. 144, pp. 2905-2914.
Rimler, R.B., et al.; "Influence of Chondroitinase on Indirect Hemagglutination Titers and Phagocytosis of Pasteurella Multocida Serogroups A, D and F," Veterinary Microbiology (1995) vol. 47 pp. 287-294.
Rimler, R.B.; "Presumptive Identification of Pasteurella Multocida Serogroups A, D and F by Capsule Depolymerisation with Mucopolysaccharidases"; Veterinary Record (1994), vol. 134, pp. 191-192.
Roberts, I., et al.; "Molecular Cloning and Analysis of Genes for Production of K5, K7, K12, and K92 Capsular Polysaccharides in Escherichia coli," Journal of Bacteriology (1986), vol. 168, No. 3, pp. 1228-1233.
Roberts, I.S., et al.; "Common Organization of Gene Clusters for Production of Different Capsular Polysaccharides (K Antigens) in Escherichia coli," Journal of Bacteriology (1988), pp. 1305-1310.
Sasisekharan, R., et al.; "Heparin and Heparan Sulfate: Biosynthesis, structure and function," Current Opinion in Chemical Biology (2000), vol. 4, pp. 626-631.
Seffernick, J.L., et al.; "Melamine Deaminase and Atrazine Chlorohydrolase: 98 Percent Identical But Functionally Different," Journal of Bacteriology (2001) vol. 183, No. 8, pp. 2405-2410.
Senay, C., et al.; "The EXT1/EXT2 Tumor Suppressors: Catalytic Activities and Role in Heparan Sulfate Biosynthesis," EMBO Reports (2000) vol. 1, No. 3, pp. 282-286.
Simmons, A.D., et al.; "A Direct interaction Between EXT Proteins and Glycosyltransferases is Defective in Hereditary Mulitple Exostoses," Hum. Mol. Genet. (1999), vol. 8, No. 12, pp. 2155-2164.
Sismey-Ragatz, et al.; "Chemoenzymatic Synthesis with Distinct Pasteurella Heparosan Synthases, Monodisperse Polymers and Unnatural Structures," The Journal of Biological Chemistry (2007), vol. 282, No. 39, pp. 28321-28327.
Smith, A.N., et al.; "Molecular Analysis of the Escherichia coli K5 kps Locus: Identification and Characterization of an Inner-Membrane Capsular Polysaccharide Transport System," Molecular Microbiology (1990), vol. 4, No. 11, pp. 1863-1869.
Soldani, G., et al.; "Experimental and Clinical Pharmacology of Glycosaminoglycans (GAGs)," Drugs Exptl. Clin. Res. (1991) vol. XVII, No. I, pp. 81-85.
Song, G., et al.; "Identification of Mutations in the Human EXT1 and EXT2 Genes," Pub Med National Library of Medicine (1999), vol. 16. No. 4, pp. 208-210.
Stickens, D., et al.; "The EXT2 Multiple Exostoses Gene Defines a Family of Putative Tumour Suppressor Genes," Nature Genetics (1996), vol. 14 pp. 25-32.
Sugahara, K., et al.; "Heparin and Heparan Sulfate Biosynthesis," Life (2002) vol. 54, pp. 163-175.
The Thrombosis Interest Group of Canada; "Practical Treatment Guidelines-Heparin." Printed from http://www.tigc.org, 7 pages.
The Thrombosis Interest Group of Canada; "Practical Treatment Guidelines—Heparin." Printed from http://www.tigc.org, 7 pages.
Townsend, K.M. et al.; "Genetic Organization of Pasteurella Multocida Cap Loci and Development of a Multiplex Capsular PCR Typing System," Journal of Clinical Microbiology, (2001), vol. 39, No. 3, pp. 924-929.
Toyoda, H., et al.; "Structural Analysis of Glycosaminoglycans in Drosophila and Caenorhabditis Elegans and Demonstrations That tout-velu, a Drosophila Gene Related to EXT Tumor Suppressors, Affects Heparan Sulfate in Vivo," The Journal of Biological Chemistry (2000), vol. 275, No. 4; pp. 2269-2275.
Tsung, et al.; "Biodegradable Polymers in Drug Delivery Systems," Fundamentals and Applications of Controlled Release Drug Delivery, edited by J. Siepmann, R.A. Siegal, and M.J. Rathbone (2012), pp. 107-123.
U.S. Appl. No. 13/855,046; Paul L. DeAngelis; Office Action dated Apr. 6, 2015.
U.S. Appl. No. 14/060,077; Paul L. DeAngelis, Office Action dated Aug. 26, 2015.
Van Aken, H., et al.; "Anticoagulation: The Present and Future," Clin. Appl. Thrombosis/Hemostasis (2001) vol. 7 No. 3, pp. 195-204.
Van Hul, W., et al.; "Identification of a Third EXT-like Gene (EXTL3) Belonging to the EXT Gene Family," Genomics (1998), vol. 47, pp. 230-237.
Vann, W.F., et al.; "The Structure of the Capsular Polysaccharide (K5 Antigen) of Urinary-Tract-Infective Escherichia coli 010:K5:H4," Eur. J. Biochem. (1981), vol. 116, pp. 359-364.
Vicenzi, E., et al.; "Broad Spectrum Inhibition of HIV-1 Infection by Sulfated K5 Escherichia coli Polysaccharide Derivatives," AIDS (2003) vol. 17, pp. 177-181; ISSN: 0269-9370 Lippincott Williams & Wilkins.
Wei, G., et al.; "Location of the Glucuronosyltransferase Domain in the Heparan Sulfate Copolymerase EXT1 by Analysis of Chinese Hamster Ovary Cell Mutants," The Journal of Biological Chemistry (2000), vol. 275, No. 36, pp. 27733-27740.
Wise, C.A., et al.; "Identification and Localization of the Gene for EXTL, a Third Member of the Multiple Exostoses Gene Family," Genome Research by Cold Spring Harbor Laboratory Press (1997), vol. 7, pp. 10-16, ISSN: 1054-9803/97.
Witkowski, A., et al.; "Conversion of a β-Ketoacyl Synthase to a Malonyl Decarboxylase by Replacement of the Active-Site Cysteine with Glutamine," Biochemistry (1999) vol. 38, pp. 11643-11650.
Wyatt Technology Corporation; "Heparin Characterization," Printed from www.tigc.org, on Apr. 5, 1997.
Zak, B.M., et al.; "Hereditary Multiple Exostoses and Heparan Sulfate Polymerization," Biochimica et Biophysica Acta 1573 (2002), pp. 346-355.
US8980608B2 (en) 2015-03-17
Noble 2002 Hyaluronan and its catabolic products in tissue injury and repair
AU2006311794B2 (en) 2012-09-20 Heparan sulfate glycosaminoglycan lyase and uses thereof
US20030073221A1 (en) 2003-04-17 Hyaluronan synthase gene and uses thereof
EP1270599B1 (en) 2004-03-31 Salmon-origin chondroitin sulfate
Boeriu et al. 2013 Production methods for hyaluronan
Kimata et al. 1973 Cytodifferentiation and proteoglycan biosynthesis