WO2003029261A2 - Polymeres de glycosaminoglycane et procedes de preparation et d'utilisation de ces derniers - Google Patents

Polymeres de glycosaminoglycane et procedes de preparation et d'utilisation de ces derniers Download PDF

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WO2003029261A2
WO2003029261A2 PCT/US2002/022386 US0222386W WO03029261A2 WO 2003029261 A2 WO2003029261 A2 WO 2003029261A2 US 0222386 W US0222386 W US 0222386W WO 03029261 A2 WO03029261 A2 WO 03029261A2
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functional acceptor
providing
polymer
sugars
synthase
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PCT/US2002/022386
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English (en)
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WO2003029261A3 (fr
WO2003029261A9 (fr
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Paul L. Deangelis
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Deangelis Paul L
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Priority claimed from US10/142,143 external-priority patent/US7307159B2/en
Application filed by Deangelis Paul L filed Critical Deangelis Paul L
Priority to EP02797026A priority Critical patent/EP1470235A4/fr
Priority to AU2002361549A priority patent/AU2002361549A1/en
Publication of WO2003029261A2 publication Critical patent/WO2003029261A2/fr
Publication of WO2003029261A9 publication Critical patent/WO2003029261A9/fr
Publication of WO2003029261A3 publication Critical patent/WO2003029261A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
    • C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds

Definitions

  • the present invention relates to methodology for the production of polymers, such as oligosaccharides, by a glycosaminoglycan synthase and, more particularly, polymer production utilizing glycosaminoglycan synthases from Pasteurella multocida.
  • oligosaccharides small sugar chains
  • Complex sugars cause biological effects by binding to target proteins including enzymes and receptors.
  • the presently claimed and disclosed invention also relates to a chemoenzymatic synthesis methodology to create both pure, chimeric, and hybrid polymers, such as oligosaccharides, composed of hyaluronan, chondroitin, keratan, dermatan, heparin units, and combinations
  • oligosaccharides are used in parallel using a microarray format or a microtiter
  • Polysaccharides are large carbohydrate molecules comprising from about
  • the polysaccharide's biological function is due to the interaction of the
  • polysaccharide with proteins such as receptors and growth factors.
  • glycosaminoglycan class of polysaccharides and oligosaccharides which includes heparin, chondroitin, dermatan, keratan, and hyaluronic acid, plays major roles in determining cellular behavior (e.g. migration, adhesion) as well
  • oligosaccharides are, therefore, essential for the correct formation and maintenance of the organs of the human body.
  • Group A & C Streptococcus and Type A Pasteurella multocida produce authentic hyaluronic acid capsules, and other
  • Pasteurella multocida Type F and D
  • pathogenic Escherichia coli K4 and
  • the pathogenic microbes form the polysaccharide
  • Enzymes alternatively called synthases, synthetases, or transferases,
  • cDNA complementary DNA
  • Vertebrate tissues and pathogenic bacteria are the sources of more exotic polysaccharides utilized in the medical field - e.g. as
  • glycosaminoglycan polysaccharides glycosaminoglycan polysaccharides, heparin from pig intestinal mucosa and
  • hyaluronic acid from rooster combs are employed in several applications including clot prevention and eye surgery, respectively.
  • bacterial capsules e.g. various Streptococcus pneumoniae strains
  • Streptococcus pneumoniae strains are utilized to vaccinate both children and adults against disease with
  • polymers in vitro utilize: (i) difficult, multistep sugar chemistry, or (ii) reactions
  • polymer contains 25, 100, or even thousands of monomers.
  • polysaccharides are the most abundant biomaterials on earth, yet many of the molecular details of their biosynthesis and function are
  • Hyaluronic acid or "HA” is a linear polysaccharide of the
  • glycosaminoglycan class and is composed of up to thousands of ⁇ (l,4)GlcUA-
  • HA is a major structural element of the
  • HA has a high
  • HA also interacts with proteins
  • CD44 such as CD44, RHAMM, and fibrinogen thereby influencing many natural processes such as angiogenesis, cancer, cell motility, wound healing, and cell
  • HA has been widely used as a viscoelastic replacement for the vitreous humor of the eye in ophthalmic surgery during implantation of intraocular lenses in cataract patients.
  • HA injection directly into joints is also used to alleviate pain
  • adsorbed HA coatings also improve the biocompatibility of medical devices such as catheters and sensors by reducing fouling and tissue abrasion.
  • HA is also made by certain microbes that cause disease in humans and
  • Some bacterial pathogens namely Gram-negative Pasteurella
  • multocida Type A and Gram-positive Streptococcus Group A and C produce an extracellular HA capsule which protects the microbes from host defenses such as phagocytosis.
  • Mutant bacteria that do not produce HA capsules are 10 2 - and
  • PBCV-1 Paramecium bursaria Chlorella virus
  • HAS HA synthases
  • HA hydroxyase
  • Mn Mg
  • Co ion a divalent Mn, Mg, or Co ion
  • the HASs are membrane proteins localized to the lipid bilayer at the cell surface.
  • the HA polymer is transported across the bilayer into the
  • HasA (or spHAS) from Group A Streptococcus pyogenes was the first HA
  • HAS3 and the viral enzyme, A98R are quite similar at the amino acid level to certain regions of the HasA polypeptide chain ( ⁇ 30% identity overall) and were
  • transmembrane segments depending on the species. Very little of these HAS polypeptide chains are expected to be exposed to the outside of the cell.
  • streptococcal enzyme was reported to add sugars to the nonreducing terminus
  • polypeptides from vertebrates and streptococci actually utilize different reaction pathways.
  • chondroitin synthase also is useful, but it adds chondroitin chains to the acceptor's non-reducing
  • the present invention provides a method for the production of polysaccharides
  • the present invention also encompasses the use of modified synthases
  • the present invention also encompasses the methodology of polysaccharide or oligosaccharide polymer grafting, i.e. HA or chondroitin,
  • a hyaluronan synthase (pmHAS) or a chondroitin synthase (pmCS) or a heparin synthase (pmHS, PglA) from various types of P. multocida.
  • pmHAS hyaluronan synthase
  • pmCS chondroitin synthase
  • pmHS, PglA heparin synthase
  • Such grafting methodologies have uses, but are not limited thereto, for the production of sugar libraries (both natural and chimeric or hybrid), protein-testing or cell-testing in microarray or microtiter plate formats.
  • pmHAS HA synthase from the fowl cholera pathogen, Type A P. multocida
  • the pmHAS is an authentic HA synthase.
  • pmCS chondroitin synthase
  • pmCS is an authentic chondroitin synthase.
  • PglA and pmHS Two unique heparin synthases, PglA and pmHS, from Type A, D, and F P.
  • multocida and Type D P. multocida respectively, have been identified and cloned and are disclosed and claimed in co-pending U.S. Serial No.10/142, 143, filed May 8, 2002, and entitled "Heparin/Heparosan Synthase from P. multocida
  • PglA and the pmHS are authentic heparin synthases.
  • pmCS synthases add sugars to the nonreducing end of a growing polymer chain.
  • the correct rrfonosaccharides are added sequentially in a stepwise
  • pmHAS is more homologous to the amino termini of other bacterial glycosyltransferases that produce different capsular polysaccharides or lipopolysaccharides. Furthermore, pmHAS is about twice as long as any other HAS enzyme.
  • pmHS or PglA are supplied with functional acceptor oligosaccharides, total HA,
  • chondroitin and heparin biosynthesis is increased up to 50-fold over reactions without the exogenous oligosaccharide.
  • the native versions of the pmHAS, pmCS, pmHS, and PglA enzymes isolated from P. multocida do not perform such elongation reactions with exogenous acceptor (or perform with very low
  • a HA- or chondroitin- or heparin-binding site may exist that holds onto the HA or chondroitin or heparin chain during polymerization.
  • HA or chondroitin or heparin oligosaccharides supplied by the hand of man are also capable of occupying this site of the recombinant enzyme and thereafter be
  • HASs insolubility in aqueous solution
  • native HASs CSs, HSs, and PglAs
  • the HAS enzyme from Group A and C Streptococcus bacteria has been detergent-solubilized and purified in an active state in small quantities.
  • pmHAS 1"703 comprises residues 1-703 of the 972 residues of the native pmHAS enzyme.
  • pmHAS 1"703 can be mass-produced in E. coli and purified by chromatography.
  • the pmHAS 1"703 enzyme retains the ability of the
  • parent enzyme to add onto either a long HA polymer, a short HA primer, a long
  • chondroitin polymer a short chondroitin primer, a short chondroitin polymer, as
  • the chondroitin chain may also be sulfated.
  • the purified pmHAS 1"703 enzyme is stable in an optimized buffer for days on ice and for hours at normal reaction temperatures.
  • One formulation of the optimal buffer consists of IM ethylene glycol, 0.1 - 0.2 M ammonium
  • pmCS, pmHAS, pnHS, and PglA possess two separate glycosyltransferase
  • the WGGED sequence motif appears to be involved in GlcNAc-transferase activity because E396 mutants and D370 mutants possessed only GlcUA-transferase activity.
  • the highly homologous (90% identical) pmCS can also be mutated in the same fashion. For example, mutating the homologous DXD motif in the GlcUA site of
  • pmCS results in an enzyme with only GalNAc-transferase activity.
  • Type F P. multocida synthesizes an unsulfated chondroitin ( ⁇ 3N-
  • acetylgalactosamine [GalNAc]- ⁇ 4GlcUA) capsule acetylgalactosamine [GalNAc]- ⁇ 4GlcUA capsule. Domain swapping between pmHAS and the homologous chondroitin synthase, pmCS, has been performed.
  • a chimeric or hybrid enzyme consisting of residues 1-427 of pmHAS and
  • the hexosamine-transferase site resides in the N-terminal domain while the GlcUA-transferase site' resides in the COOH-terminal domain of these GAG
  • the present invention encompasses methods of producing a variety of
  • polysaccharides especially those of the glycosaminoglycan class, serve numerous roles in the body as structural elements and signaling molecules.
  • grafting or making hybrid molecules composed of more than one polymer backbone it is possible to meld distinct physical and biological properties into a
  • the present invention also incorporates the propensity of certain recombinant enzymes, when prepared in a virgin state, to utilize various acceptor molecules as the seed for further polymer growth : naturally occurring forms of the
  • the present invention results in (a) the production of hybrid oligosaccharides or polysaccharides and (b) the formation of polysaccharide
  • Such hybrid polymers can serve as "molecular glue” — i.e. when two
  • Such polysaccharide coatings are useful for integrating a foreign object within a surrounding tissue matrix.
  • a prosthetic device is more
  • the device's artificial components could be any suitable polysaccharide. Additionally, the device's artificial components could be any suitable polysaccharide. Additionally, the device's artificial components could be any suitable polysaccharide. Additionally, the device's artificial components could be any suitable polysaccharide. Additionally, the device's artificial components could be any suitable polysaccharide. Additionally, the device's artificial components could be any suitable polysaccharide. Additionally, the device's artificial components could be any other materials.
  • Another aspect of the present invention is the coating or grafting of GAGs onto various drug delivery matrices or bioadhesives or suitable
  • medicaments to improve and/or alter delivery, half-life, persistence, targeting
  • pmHAS and pmCS and pmHS and PglA to elongate defined oligosaccharide derivatives.
  • the non-reducing end sugar addition allows the reducing end to be modified for other purposes; the addition of GAG chains to small molecules,
  • pmHAS and pmCS and pmHS and PglA appear distinct from most other known HA and chondroitin and heparin synthases based on differences in sequence, topology in the
  • pmHAS the 972-residue membrane-associated hyaluronan synthase, catalyzes the transfer of both GlcNAc and GlcUA to form an HA polymer.
  • pmHAS and pmCS mutants have been analyzed.
  • pmHAS 1"703 is a
  • pmHAS 1"650 is inactive as a HA
  • Pasteurella multocida Type F the minor fowl cholera pathogen, produces an extracellular polysaccharide capsule that is a putative virulence factor.
  • Serator No. 09/842,484 filed April 25, 2002, and entitled “Chondroitin Synthase Gene and Methods of Making and Using Same", the contents of which are hereby expressly incorporated herein in their entirety, the
  • pmCS residue enzyme
  • glycosyltransferase that forms a polysaccharide composed of chondroitin
  • multocida also are a single polypeptide specie that possess both transferase activities to catalyze heparin/heparosan.
  • claimed invention provides for the synthesis of oligosaccharide sugars that have
  • NanoHA N-(nanoHA).
  • nanoHA signifies the very small
  • Metastasis the escape of cancer cells throughout the body, is one of the biggest fears of both the ailing patient and the physician, and this area is a well
  • nanoHA will serve as a supplemental treatment to inhibit cancer growth and metatasis in conjunction
  • HA oligosaccharides are also very promising in an in vivo
  • HA oligosaccharides susceptible to the therapeutic action of HA oligosaccharides.
  • a very desirable attribute of HA-oligosaccharides for therapeutics is that
  • metastasis are HA chains composed of 10 to 14 sugars.
  • oligosaccharides are extremely limited and will not allow the medical potential
  • Small HA molecules are presently made either by: (1) partially depolymerizing costly large polymers with degradative
  • the enzyme wants to degrade the polymer to the 4 sugar end stage product, but this sugar is inactive for cancer treatment.
  • the use of acid hydrolysis also removes a fraction of the acetyl groups from the
  • the partial depolymerization method only yields fragments of the original
  • HA polymer and is essentially useless for creating novel sugars beyond simple derivatizations (e.g. esterifying some fraction of GlcUA residues in an
  • hyaluronic acid or chondroitin or heparin enzyme catalysts and substrates are examples of hyaluronic acid or chondroitin or heparin enzyme catalysts and substrates.
  • FIG. 1 is a graphical representation showing that an HA tetramer stimulates pmHAS polymerization.
  • FIG. 2 is a graphical plot showing that HA polymerization is effected by HA oligosaccharides.
  • FIG. 3 is a graphical plot showing HA tetramer elongation into larger
  • FIG. 4 is a graphical representation of a thin layer chromatography analysis of pmHAS extension of HA tetramer.
  • FIG. 5 is a graphical representation of thin layer chromatography analysis
  • FIG. 6 is an electrophoresis gel showing the purification of pmHAS 1"703 .
  • FIG. 7 is a pictorial representation of the pmHAS truncation mutants.
  • FIG. 8 is a graphical representation of a mutant combination assay.
  • FIG. 9 is a tabular representation showing enzyme activity of the pmHAS-
  • FIG. 10 is a graphical representation of a high-throughput assay for
  • FIG. 11 is a Western Blot analysis showing the expression of pmHAS and its truncated forms. Either whole cell lysates (pmHAS 437"972 , pmHAS 1"567 , and pmHAS 152"756 ) or membrane preparations (pmHAS 437"756 , pmHAS 1"567 , rl-972,
  • FIG. 12 is a pictorial representation of domains Al and A2 of pmHAS.
  • A The approximate relative positions of domain Al and A2 in pmHAS and
  • FIG. 13 is a graphical representation of the complementation of the HAS activity of mutant enzymes in vitro. HAS enzyme assays with HA-derived
  • FIG. 14 is a pictorial representation of a model of the two putative
  • FIG. 15 is a sequence alignment of pmCS and pmHAS.
  • the two Pasteurella GAG synthases are highly homologous. Identical residues are denoted with the hyphen. The numbering scheme corresponds to the slightly
  • domains correspond to regions important for hexosamine transferase or for glucuronic acid transferase activity, respectively (33). Most sequence differences are found in the amino-terminal half of the polypeptides.
  • FIG. 16 is a Western Blot Analysis of Truncated Recombinant Pasteurella
  • V vector control
  • Std Prestained standards
  • FIG. 17 is a graphical plot of gel filtration analysis of radiolabeled polymer
  • pmCS 1"704 extract (1 mg total protein) was incubated with chondroitin acceptor oligosaccharide (5 mg), UDP-[ 14 C]GlcUA and UDP- [ 3 H]GalNAc (580 mM 0.16 mCi each) in a reaction volume of 200 ml for 30
  • dotted line measured by the in-line detector is presented as disintegrations per second (dps).
  • the double-headed arrow corresponds to a response of 20 dps.
  • A untreated polymer, peak 15.9 min
  • B Flavobacterium chondroitinase AC lyase-treated polymer, peak, 19.2 min
  • C HA lyase-treated polymer, peak 15.9
  • FIG. 18 is a pictorial representation of a model of the two putative glycosyltransferase sites of pmHAS and pmCS.
  • PmHAS and pmCS contain two
  • FIG. 19 graphically depicts Sequence Similarity of pmHS with KfiA and KfiC. Elements of the Pasteurella heparosan synthase, HS1 (containing
  • residues 91-240 and HS2 are very similar to portions of two proteins from the E. coli K5 capsular locus (A, residues 75-172 of KfiA; C, residues 262- 410 of KfiC) as shown by this modified Multalin alignment (ref. 21; numbering scheme corresponds to the pmHS sequence).
  • the HSl and HS2 elements may be important for hexosamine transferase or for glucuronic acid transferase activities, respectively, (con, consensus symbols:
  • FIG. 20 depicts pmHS Activity Dependence on Acceptor and Enzyme
  • FIG. 21 Gel Filtration Analysis of Radiolabeled Polymer Synthesized in
  • FIG. 22(A-D) graphically depicts the alignment of the pmHS (two clones:
  • FIG. 23 depicts chimeric constructs of pm-EG, pm-FH, pm-IK, and pm-JL. PCR-overlap-extensio ⁇ was performed. Pm-EG contains residues 1-265 from
  • Pm-FH contains
  • Pm-IK contains residues 1-221 from pmHAS and residues 215-704 from pmCS and is a Glc-UA-Tase.
  • Pm-JL contains residues 1-214 from
  • pmCS and residues 222-703 from pmHAS and is an active HA synthase is an active HA synthase.
  • FIG. 24 depicts a comparison of partial primary sequences of pmHAS and different pmCSs. Primary sequences of presumably chondroitin synthases from
  • FIG. 25 depicts chimeric constructs of pmHAS 1"221 -CS 215"258 -HAS 266*703 and
  • pmHAS 1"221 -CS 215"258 -HAS 266"703 A very interesting result was that pmCS 1"214 - HAS 222"265 -CS 258"704 can transfer both GalNAc and GlcNAc to HA oligomer acceptor; this enzyme displays relaxed sugar specificity.
  • FIG. 26 depicts a summary of enzyme activities of chimeric proteins.
  • the enzymes are drawn as bars. Black bars represent pmCS.
  • White bars represent pmHAS. +, active; -, inactive.
  • PmCHC represents pmCS 1"214 -HAS 222"265 -CS 258"704 . The roles of the two domains are confirmed and we have
  • FIG. 27 depicts a mass spectra of F-HA12 product. This fluorescent HA oligosaccharide was synthesized using the twin bioreactor scheme. A peak with
  • FIG. 28 is a pictorial representation of a hyalose biocatalytic scheme
  • GAGs Glycosaminoglycans
  • Hyaluronan [HA], chondroitin, and heparan sulfate/heparin contain a uronic acid as the other component of the
  • the GAGs are
  • Vertebrates may contain all four types of GAGs, but the polysaccharide chain is often further modified after sugar polymerization.
  • One or more of GAGs may contain all four types of GAGs, but the polysaccharide chain is often further modified after sugar polymerization.
  • chondroitin and heparan sulfate/heparin chains in vertebrates are initially synthesized by elongation of a xylose-containing linkage tetrasaccharide attached to a variety of proteins. Keratan is either O-linked or N-linked to certain proteins depending on the
  • HA and all of the known bacterial GAGs are not part of the classification of proteins known as glycoproteins. All GAGs except HA are found covalently linked to a core protein, and such combination is referred to as a
  • proteoglycan Glycoproteins are usually much smaller than proteoglycans and
  • proteoglycan is also usually a glycoprotein, therefore usually contains other oligosaccharide chains besides the GAGs.
  • GAGs and their derivatives are currently used in the medical field as ophthalmic and viscoelastic supplements, adhesion surgical aids to prevent
  • tissue engineering matrices immune and neural cell modulators, and drug targeting agents.
  • GAGs Complex carbohydrates, such as GAGs, are information rich molecules.
  • a major purpose of the sugars that make up GAGs is to allow communication
  • proteins bind to particular sugar chains in a very selective fashion.
  • a protein may simply adhere to the sugar, but quite often the protein's
  • intrinsic activity may be altered and/or the protein transmits a signal to the cell
  • heparin binding to inhibitory proteins helps shuts down the clotting response.
  • HA binds to cells via the CD44 receptor that stimulates the cells to migrate and to proliferate.
  • oligosaccharides can be
  • HA polysaccharide plays structural roles in the eye, skin, and joint
  • HA-oligosaccharides composed of 10 to 14 sugars [HA ⁇ o- ⁇ ] have promise for inhibition of cancer cell growth and metastasis.
  • mice injected with various invasive and virulent tumor cell lines have promise for inhibition of cancer cell growth and metastasis.
  • HA oligosaccharides (melanoma, glioma, carcinomas from lung, breast and ovary) develop a number of large tumors and die within weeks. Treatment with HA oligosaccharides greatly reduced the number and the size of tumors. Metastasis, the escape of cancer cells throughout the body, is one of the biggest fears of both the ailing patient and the physician. HA or HA-like oligosaccharides appear to serve as a
  • the preliminary mode of action of the HA-oligosaccharide sugars is thought to be mediated by binding or interacting with one of several important HA-binding proteins (probably CD44 or RHAM) in the mammalian body.
  • CD44 or RHAM important HA-binding proteins
  • CD44 protein molecules in a cancer cell can bind simultaneously to a long HA polymer. This multivalent HA binding causes CD44 activation (perhaps
  • each CD44 molecule individually binds a different HA molecule in a monovalent manner such that no dimerization/patching event
  • the optimal HA-sugar size is 10 to 14 sugars.
  • the optimal HA size or oligosaccharide composition may be found to
  • HA-oligosaccharides for therapeutics are natural by-products that can occur in small amounts in the
  • Enhancement of wound-healing and resupplying cardiac oxygenation may be additional applications that harness the
  • Dendritic cells possess adjuvant activity in stimulating specific CD4
  • dendritic cells are targets in vaccine development strategies for the prevention and treatment of infections, allograft
  • heparin is so potent it must be used in a hospital setting and require careful monitoring in order to avoid hemorrhage. Newer, processed lower molecular
  • heparin is responsible for the ATIII-anticoagulant effect. But since heparin is a
  • the pentasaccharide can also be prepared in a pure state.
  • VEGF vascular endothelial growth factor
  • HBEGF heparen-binding epidermal growth factor
  • FGF fibroblast growth factor
  • heparin part by heparin; therefore, diseases such as cancer and atherosclerosis are potential targets. Abnormal or unwanted proliferation would be curtailed if the growth factor was prevented from stimulating target disease-state cells by interacting with a heparin-like oligosaccharide analog instead of a surface- bound receptor. Alternatively, in certain cases, the heparin oligosaccharides alone have been shown to have stimulatory effects.
  • Chondroitin is the most abundant GAG in the human body, but all of its
  • chondroitin or similar molecules are of utility in re-wiring synaptic connections after degenerative diseases (e.g. Alzheimer's) or paralytic
  • coagulation proteins such as heparin cofactor II.
  • glycosyltransferases with unparalleled control and superb efficiency is currently being developed by several universities and companies.
  • a major obstacle is the production of useful catalyst because the vast majority of glycosyltransferases
  • glycosyltransferase enzymes that synthesize the alternating sugar repeat backbones in microbes and in vertebrates utilize UDP-sugar precursors and divalent metal cofactors (e.g. magnesium, cobalt and/or manganese ion) near neutral pH according to the overall reaction: nUDP-GlcUA + nUDP-HexNAc - 2nUDP + [GlcUA-HexNAc] n
  • HexNAc GlcNAc or GalNAc.
  • n the degree of polymerization
  • the enzyme is called a synthase or co-polymerase.
  • P. multocida infects fowl, swine, and cattle as well as many
  • the enzymes are: a HA synthase, or (pmHAS); a chondroitin synthase, or (pmCS); and two heparosan synthases, or (pmHS and PglA).
  • recombinant pmHAS can elongate exogeneously-
  • the pmHAS synthase has been shown to add monosaccharides one at a time in a step-wise fashion to the growing chain.
  • heparosan is Beta4GlcUA-alpha4GlcNAc.
  • the pmHS and PglA enzymes were
  • pmHAS possesses two independent catalytic sites in one polypeptide. Mutants were created that transferred only GlcUA, and distinct mutants were also created that transferred only GlcNAc. These mutants cannot polymerize HA chains individually, but if the two types of mutants are mixed together in the
  • chondroitin synthase, pmCS has a similar sequence and similar two-domain structure.
  • the heparosan synthases, pmHS and PglA also contain regions for the two active sites. Single action mutants have also been created for the chondroitin synthase, pmCS, and are described hereinafter in detail.
  • the Pasteurella GAG synthases are very specific glycosyltransferases with respect to the sugar transfer reaction; only the correct monosaccharide from the authentic UDP-sugar is added onto acceptors.
  • the epimers or other closely structurally related precursor molecules e.g. UDP-glucose are not utilized.
  • the GAG synthases do, however, utilize certain heterologous acceptor sugars.
  • pmHAS will elongate short chondroitin acceptors with long HA chains.
  • pmHS will also add long heparosan chains onto HA acceptor
  • nucleic acid segment and “DNA segment” are used interchangeably and refer to a DNA molecule which has been isolated free
  • a "purified" DNA or nucleic acid segment as used herein refers to a DNA segment which contains a
  • HAS Hyaluronate Synthase
  • CS Chondroitin Synthase
  • DNA segment includes DNA segments and smaller fragments of such segments, and also recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like.
  • a DNA segment comprising an isolated or purified pmHAS or
  • pmCS or pmHS or PglA gene refers to a DNA segment including HAS or CS or HS coding sequences isolated substantially away from other naturally occurring genes or protein encoding sequences.
  • the term "gene" is used
  • substantially away from other coding sequences means that the gene of interest, in this case pmHAS or pmCS or pmHS or PglA forms the significant part of the coding region of the DNA segment, and that the DNA segment does
  • prokaryotic enzyme gene is sought to be employed.
  • advantages include (a) the ease of isolation of a prokaryotic gene because of the relatively small size of the genome and, therefore, the reduced amount of
  • DNA sequences in accordance with the present invention will further include genetic control regions which allow the expression of the sequence in a selected recombinant host.
  • the genetic control region may be native to the cell from which the gene was isolated, or may be native to the recombinant host cell, or may be an exaggerous segment that is compatible with and recognized by the transcriptional machinery of the selected
  • control region employed will generally vary depending on the particular use (e.g., cloning host) envisioned.
  • the invention concerns isolated DNA segments
  • HAS or CS or HS pepetides or peptide fragment thereof an amino acid sequence encoding HAS or CS or HS pepetides or peptide fragment thereof, and in particular to a HAS or CS or HS peptide or peptide
  • DNA segment or vector encodes a full length HAS or CS or
  • HS protein or is intended for use in expressing the HAS or CS or HS protein, preferred sequences are those which are essentially as set forth in SEQ ID NO: 1
  • Truncated pmHAS gene (such as, but not limited to, pmHAS 1"703 , SEQ ID NO: 1
  • Nucleic acid segments having HAS or CS or HS activity may be isolated by
  • SEQ ID NO:X means that the sequence substantially corresponds to a portion
  • SEQ ID NO:X 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.
  • SEQ ID NO:X SEQ ID NO:X, and that is associated with the ability of prokaryotes to produce HA or a hyaluronic acid or chondroitin or heparin polymer in vitro or in vivo.
  • X refers to either SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 69, 70 or 71 or any additional sequences set forth herein, such as the
  • pmHAS 1"703 truncated or mutated versions of pmHAS 1"703 that are contained generally in SEQ ID NOS: 10-60.
  • nucleic acid segment i.e. encoding conserved or semi-conserved
  • 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. Additionally, the
  • present application discloses 4 enzymes and numerous mutants of these enzymes that still retain at least 50% of the enzymatic activity of the
  • unmutated parent enzyme - i.e. Vi of the dual action transferase activity of the unadulterated parent.
  • the invention discloses nucleic acid segments encoding an enzymatically
  • nucleic acid segment that encodes a protein in accordance with SEQ ID NO:i or 3 or 5 or 7 or 71, respectively, further defined as a recombinant vector.
  • the term "recombinant vector” refers to a vector that has been modified
  • the recombinant vector may be further defined as an expression vector comprising a promoter operatively linked to said HAS- or CS- or HS- encoding nucleic acid segment.
  • a further preferred embodiment of the present invention is a host cell, made recombinant with a recombinant vector comprising an HAS or CS or HS
  • the preferred recombinant host cell may be a prokaryotic cell.
  • the recombinant host cell is an eukaryotic cell.
  • engineered or recombinant cell is intended to refer to a cell
  • cells are distinguishable from naturally occurring cells which do not contain a recombinantly introduced gene.
  • Engineered cells are thus cells having a gene
  • genes positioned adjacent to a promoter associated or not naturally associated with the particular introduced gene include genes positioned adjacent to a promoter associated or not naturally associated with the particular introduced gene.
  • the HAS- or CS- or HS- encoding DNA In preferred embodiments, the HAS- or CS- or HS- encoding DNA
  • segments further include DNA sequences, known in the art functionally as origins of replication or "replicons", which allow replication of contiguous sequences by the particular host.
  • origins of replication or "replicons”
  • Such origins allow the preparation of extrachromosomally localized and replicating chimeric or hybrid segments or
  • the employed origin is one capable of replication in bacterial hosts suitable for biotechnology applications.
  • the invention may thus be defined in terms of a recombinant transformation vector which includes the
  • HAS- or CS- or HS- coding gene sequence together with an appropriate replication origin and under the control of selected control regions.
  • HAS or CS or HS gene or cDNA may be used to obtain the HAS or CS or HS gene or cDNA, in light of the
  • polymerase chain reaction or RT-PCR produced DNA fragments may be obtained which contain full complements of genes or cDNAs from a number of sources, including other strains of Pasteurella
  • acids should encode a biologically functional equivalent HAS or CS or HS.
  • Typical useful vectors include plasmids and
  • eukaryotic organisms examples include pKK223-3, pSA3, recombinant
  • Gram-negative bacteria e.g. Bacillus, Lactococcus, or E. coli
  • Vectors such as these exemplified by the pSA3 vector of Dao and Ferretti or the pAT19 vector of Trieu-Cuot, et al., allow one to perform clonal colony
  • the recombinant vector is employed to make the functional GAG synthase for in vitro use. These are benign and well studied organisms used in the production
  • GRAS generally recognized as safe
  • HAS or CS or HS gene copy number HAS or CS or HS gene copy number.
  • Another technique would include integrating at least one copy of the HAS or CS or HS gene into chromosomal DNA. This extra amplification would be especially
  • the chromosomal DNA-ligated vector is employed to transfect the host that is selected for clonal screening purposes such as E. coli, through the use of a vector that is capable of expressing the inserted DNA in the chosen host.
  • the invention concerns isolated DNA segments and recombinant vectors that include within their sequence a nucleic acid
  • nucleic acid sequence substantially corresponds to a portion of SEQ ID NO: 1,3,5,7,69, or 71 and has relatively few codons which are not identical, or functionally equivalent, to the
  • codon is used herein to refer to codons that encode the same amino acid, such
  • additional residues such as additional N- or C-terminal amino acids or
  • terminal sequences particularly applies to nucleic acid sequences which may, for example, include various non-coding sequences flanking either of the 5' or 3' portions of the coding region or may include various internal sequences, which are known to occur within genes. Furthermore, residues may be removed from
  • nucleotides of SEQ ID NO: 1,3,5,7,69, or 71 will be sequences which are "essentially as set forth" in SEQ ID NO: 1,3,5,7,69, or 71. Sequences which are essentially the
  • hybridization conditions will be well known to those of skill in the art and are clearly set forth hereinbelow. In a preferred embodiment, standard stringent hybridization conditions or less stringent hybridization conditions are utilized. The terms "standard stringent hybridization conditions," "moderately
  • hybridization such as pH; temperature; salt concentration; the presence of
  • 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
  • a nucleic acid sequence encoding a HAS or chondroitin or heparin synthase or its complement such as SEQ ID NO: 1,3,5,7,69, or 71 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 T m , which is the temperature at which a probe nucleic acid sequence dissociates from a target DNA. This melting temperature is used to
  • hybridization occurs with a particular concentration of salt (e.g., SSC, SSPE, or HPB). Then, assuming that 1% mismatching results in a 1°C decrease in the T m ,
  • the temperature of the final wash in the hybridization reaction is reduced accordingly (for example, if sequences having >95% identity with the probe are
  • the final wash temperature is decreased by about 5°C). In practice, the final wash temperature is decreased by about 5°C). In practice, the final wash temperature is decreased by about 5°C). In practice, the
  • T m can be between about 0.5°C and about 1.5°C per 1% mismatch.
  • Examples of standard stringent hybridization conditions include hybridizing at
  • Moderately stringent conditions include hybridizing as described above in
  • 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
  • the present invention also encompasses DNA segments which are complementary, or essentially complementary, to the sequences set forth in SEQ ID NO: l or 3 or 5 or 7 or 69 or 71. Nucleic acid sequences which are
  • ATAGCG-3' is complementary to the sequence 5'-CGCTAT-3" because when the two sequences are aligned, each "T” is able to base-pair with an "A", which each "G” is able to base pair with a "C".
  • complementary sequences means nucleic acid sequences which are
  • nucleic acid segment of SEQ ID NO: 1,3,5,7, or 69, or 71 under standard stringent, moderately stringent, or less stringent hybridizing conditions.
  • nucleic acid segments of the present invention regardless of the
  • length of the coding sequence itself may be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction
  • nucleic acid fragment of almost any length may be employed, with the total length preferably being
  • coding regions bearing selected alterations or modifications in the basic coding region may encode larger polypeptides which nevertheless include HAS or CS or HS coding regions or may encode biologically functional equivalent
  • proteins or peptides which have variant amino acid sequences are proteins or peptides which have variant amino acid sequences.
  • DNA segments of the present invention encompass DNA segments
  • polysaccharide must comprise at least 20-100 sugar units.
  • exogenous polymers have been previously available, but typically add only one sugar unit.
  • the unique enzymes described in the present invention e.g.
  • pmHAS, pmCS, pmHS, and PglA form polymers of at least 100-400 sugar units in length.
  • one embodiment of the presently claimed and disclosed invention results in long, defined linear polymers composed of only natural glycosidic linkages.
  • Pasteurella multocida bacteria normally make polymers similar to or identical to vertebrate polymers. These bacteria employ the polysaccharide, either HA (Type A bacteria), chondroitin (Type F bacteria), or heparosan (unsulfated,
  • the recombinant enzyme may be any suitable enzyme that catalyzes the oxidation of the recombinant enzyme.
  • the recombinant enzyme may be any suitable enzyme that catalyzes the oxidation of the recombinant enzyme.
  • the recombinant enzyme may be any suitable enzyme that catalyzes the oxidation of the recombinant enzyme.
  • pmHAS (SEQ ID NO: 2), a 972 amino acid residue protein from Pasteurella multocida, is made in a functional state in recombinant Escherichia coli.
  • the pmHAS gene is given in SEQ ID NO: l.
  • Other functional derivatives of pmHAS for example an enzyme called pmHAS 1"703 (SEQ ID NO:9) and the pmHAS 1'703
  • the preferred E. coli strains do not have an UDP- Glc dehydrogenase and therefore the recombinant enzyme does not make HA
  • the enzyme is in a "virgin" state since the
  • empty acceptor site can be occupied with foreign polymers.
  • the empty acceptor site can be occupied with foreign polymers.
  • recombinant enzyme may be incubated in a mixture comprising from about 10
  • Tris pH 7.2 from about 20 mM MnCI 2 , from about 0.1 to about 15 mM UDP-GlcUA, from about 0.1 to about 15 mM UDP-GlcNAc, and a suitable
  • acceptors can be any functional acceptor, such as a glycosaminoglycan acceptor or sugar acceptor, for example, but not by limitation, short HA chains (two or more sugar units sucK as HA 4 ) or short chondroitin sulfate chains (5 sugar
  • the length of the HA chain added onto the acceptor is controlled by altering the concentration of UDP-sugars and/or the reaction time.
  • Immobilized acceptors, such as beads or other solid objects with bound acceptor are controlled by altering the concentration of UDP-sugars and/or the reaction time.
  • oligosaccharides can also be extended by the pmHAS enzyme using UDP- sugars.
  • the pmHAS enzyme (or its derivatives) can be used to
  • Type A P. multocida produces HA capsule [GlcUA-GlcNAc repeats]
  • Type F P. multocida produces a chondroitin or chondroitin-like polymer capsule [GlcUA-GalNAc
  • PmCS the P. multocida chondroitin synthase
  • chimeric or hybrid polysaccharide materials can serve as a biocompatible molecular glue for cell/cell interactions in artificial tissues or organs and the HA/chondroitin/heparin hybrid mimics natural proteoglycans that normally
  • a recombinant HA/chondroitin/heparin chimeric or hybrid polysaccharide, devoid of such an intermediary protein, is desirous since molecules from animal sources are potentially immunogenic — the chimeric or
  • the recombinant polymers can be
  • glycosyltransferase remains in association with the nascent chain.
  • the nascent chain is covalently attached to the enzyme during its synthesis.
  • the nascent chain is covalently attached to the enzyme during its synthesis.
  • the nascent polymer chain is still covalently attached.
  • a nascent HA chain means for a nascent HA chain to be held at or near the active site.
  • pmHAS it appears ' that a HA-binding site exists near or at the sugar
  • oligosaccharides that vary in size and composition are used to discern the nature of the interaction between pmHAS and the sugar chain.
  • heparosan pentamer which only differs in the glycosidic linkages from HA-derived oligosaccharides, does not serve as an acceptor.
  • chondroitin [GlcUA-GalNAc repeat] does serve as an acceptor for pmHAS.
  • the cells were hyaluronidase-treated to give copious amounts of HA polysaccharide.
  • the cells were hyaluronidase-treated to
  • Both heparin and chondroitin are synthesized by the addition of sugar units to the nonreducing end of the polymer chain.
  • glycosyltransferases initiate chain elongation on at least primer
  • monosachharides More preferably tetrasaccharides such as xylose-galactose-
  • sugar of the disaccharide unit is usually not added and processive elongation to
  • heparosan indicates that a pair of proteins can transfer both sugars to the
  • pmHAS adds single monosaccharides in a sequential fashion to the nonreducing termini of the nascent HA chain. Elongation of HA polymers containing hundreds of sugars has been demonstrated in vitro. The simultaneous formation of the disaccharide repeat unit is not necessary for
  • glycosyltransferase may be stopped and started at
  • pmHAS 1"650 (SEQ. ID NO: 10) can only add
  • pmHAS as well as pmHS or PglA either alone or in combination with pmCS and
  • membrane preparations from recombinant E. coli containing a pmHAS protein had HA synthase activity as judged by incorporation of radiolabel from UDP-[ 14 C]GlcUA into polymer when co-
  • HA oligosaccharides serve as acceptors for GlcUA and GlcNAc transfer.
  • Addition of unlabeled even-numbered HA tetramer (from testicular hyaluronidase digests) to reaction mixtures with recombinant pmHAS 1"703 stimulates incorporation of radiolabel from UDP-[ 14 C]GlcUA into HA polymer by ⁇ 20- to 60-fold in comparison to reactions without oligosaccharides as shown in FIG. 1.
  • pmHAS 1"703 has been shown to add sugars onto a
  • chondroitin pentamer acceptor The pmHAS 1"703 and reagents were prepared in the same manner as shown in Fig. l, except that a chondroitin pentamer was used as the acceptor molecule. The results of this experiment are shown in TABLE III.
  • the pmHAS 1"703 can utilize molecules other than the
  • the HA polymerizing activity of recombinant pmHAS 1"703 is dependent on the simultaneous incubation with both UDP-sugar precursors and a Mn 2+ ion.
  • the level of incorporation is dependent on protein concentration, on HA oligosaccharide concentration, and on incubation time as shown in FIG. 2.
  • FIG. 2 two parallel reactions containing pmHAS 1"703 with even-numbered HA
  • oligosaccharides (105 ⁇ g membrane protein/point with a mixture of HA
  • pmHAS 1"703 without oligosaccharide acceptor (630 ⁇ g protein/point; open circles) were compared.
  • the enzyme preparations were added to prewarmed
  • HA synthesized in the presence or the absence of HA oligosaccharides is sensitive to HA lyase (>95% destroyed) and has a molecular weight of ⁇ 1-
  • the native pmHAS 1"703 enzyme has an attached or bound nascent
  • recombinant enzyme on the other hand, lacks such a nascent HA chain since the E. coli host does not produce the UDP-GlcUA precursor needed to make HA
  • the exogenous HA-derived oligosaccharide has
  • the various recombinant enzymes were tested for their ability to convert HA tetramer into molecular weight products.
  • the reactions contained radiolabeled HA tetramer (5-8 x 10 5 dpm), 750 ⁇ M UDP-
  • X cation and pH values used for each enzyme were: pmHAS 1"703 , Mn/7.2; Xenopous DG42, Mg/7.6; Group A streptococcal HasA, Mg/7.0), and enzyme (units/reaction listed).
  • pmHAS 1"703 Mn/7.2
  • Xenopous DG42 Mg/7.6
  • Group A streptococcal HasA Mg/7.0
  • FIG. 4 demonstrates that pmHAS 1"703
  • control membranes prepared from host cells with a vector plasmid did not alter the mobility of the radiolabeled HA tetramer under any
  • pmHAS 1"703 did not utilize labeled chitopentaose as an acceptor.
  • pmHAS extended an HA tetramer.
  • radiolabeled HA tetramer (HA4 8xl0 3 dpm 3 H) with a GlcUA at the nonreducing
  • HA-derived oligosaccharides with either GlcUA or GlcNAc at the nonreducing terminus served as acceptors for pmHAS 1"703 (FIG. 5).
  • radiolabeled HA pentamer HA5, 5xl0 3 dpm 3 H
  • HA tetramer HA4, 25xl0 3 dpm 3 H
  • autoradiogram (1 mo. exposure) shows the single addition of a sugar onto an acceptor when only the appropriate precursor is supplied (HA4, N lane and HA5, A lane). If both UDP-sugars are supplied (+AN lanes), then a ladder of
  • the HA products are so large that they do not migrate from the application zone.
  • the pmHAS 1"703 enzyme transfers individual monosaccharides sequentially during a
  • Membrane preparations containing recombinant pmHAS (GenBank AF036004) (SEQ. ID NOS: 1 and 2) were isolated from E. coli SURE(pPmHAS).
  • Group A streptococcal HasA or Xenopus DG42 produced in the yeast Saccharomyces cerevisiae were selected from the group consisting of Group A streptococcal HasA or Xenopus DG42 produced in the yeast Saccharomyces cerevisiae.
  • Uronic acid was quantitated by the carbazole method. Even-numbered HA oligosaccharides [(GlcNAc-GlcUA) n ] were generated by degradation of HA (from Group A Streptococcus) with either bovine testicular hyaluronidase Type
  • capsular polysaccharide is ⁇ 4GlcUA- ⁇ 4GlcNAc; this carbohydrate has the same
  • composition as HA but the glycosidic linkages between the monosaccharides are
  • the chitin-derived oligosaccharides, chitotetraose and chitopentaose are ⁇ 4GlcNAc polymers made of 4 or 5 monosaccharides, respectively.
  • An anti-pmHAS monospecific antibody reagent has also been identified that routinely monitors the protein by Western blots or immunoassays; this
  • the reagent can be used to normalize protein expression levels.
  • the DNA inserts encoding the enzyme sequence from interesting mutants picked up in screens can be subcloned and completely sequenced to verify and to identify the mutation site.
  • membrane protein were created and are tabulated (with functionality) in Table V that produce proteins with altered physical properties (i.e. proteins that are more conducive to high-level expression and purification) and altered function
  • the truncation series was generated and tested for activity. All proteins were made at the expected molecular weight, but not all proteins were active.
  • pmHAS 1 80-kDa protein
  • pmHAS 1"703 was purified by sequential chromatography steps shown in FIG. 6.
  • FIG. 6 a soluble, active form of the
  • HA synthase was constructed with molecular biological techniques.
  • the recombinant enzyme from E. coli was purified by conventional chromatography with yields of up to 20 mg/liter of cell culture.
  • FIG. 6 is a stained
  • the pmHAS 1"703 is highly active and at least 95% pure as assessed by
  • Synthetic DNA oligonucleotides and multiple rounds of extension with Pfu DNA polymerase were used to add mutations to the coding region using the Quick-Change system from Stratagene.
  • the mutant enzymes are useful for adding on a single GlcNAc or a single GlcUA onto the appropriate acceptor oligosaccharide. It appears that pmHAS 1"703 has two domains or two modules for transferring each sugar.
  • FIG. 8 is a graphical representation of an experiment where combining two single action mutants rescued dual enzymatic activity.
  • Extracts of the mutants were used for all three kinds of assays: for HA polymer production, for GlcUA-Tase activity and for GlcNAc-Tase activity.
  • HA chondroitin
  • heparin or chimeric or hybrid molecules
  • additional substrates may be metal or metalized - i.e. having a metal coating on the surface of a second material (or a laminate material) such as plastic or silica.
  • the metal substrate may be, but is not
  • thermosensitive metal alloys and combinations thereof to name but a few.
  • any metal could be used as the substrate as long as it had a surface layer capable of having an

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Abstract

La présente invention concerne une méthodologie de greffe polymère par une synthase de polysaccharide et, plus particulièrement, la greffe polymère effectuée à l'aide de synthases de hyaluronate ou de chondroïtine ou d'héparine/héparosane issues de Pasteurella multocida, qu'on effectue afin de créer une variété d'oligosaccharides de glycosaminoglycane ayant une structure de sucre naturelle ou chimérique ou bien hybride.
PCT/US2002/022386 2001-07-13 2002-07-12 Polymeres de glycosaminoglycane et procedes de preparation et d'utilisation de ces derniers WO2003029261A2 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1392843A2 (fr) * 2001-05-08 2004-03-03 Board Of Regents Of The University Of Oklahoma Heparine/heparosan synthases et procedes de fabrication correspondant
US8088604B2 (en) 1998-04-02 2012-01-03 The Board Of Regents Of The University Of Oklahoma Production of defined monodisperse heparosan polymers and unnatural polymers with polysaccharide synthases
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
US8697398B2 (en) 2010-03-01 2014-04-15 Dsm Ip Assets B.V. Compositions and methods for bacterial production of chondroitin
US8980608B2 (en) 2012-03-30 2015-03-17 The Board Of Regents Of The University Of Oklahoma High molecular weight heparosan polymers and methods of production and use thereof
US9603945B2 (en) 2008-03-19 2017-03-28 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
US9925209B2 (en) 2008-03-19 2018-03-27 The Board Of Regents Of The University Of Oklahoma Heparosan-polypeptide and heparosan-polynucleotide drug conjugates and methods of making and using same

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000027437A2 (fr) * 1998-11-11 2000-05-18 The Board Of Regents Of The University Of Oklahoma Greffage de polymères par polysaccharide synthases
AU2001253805B2 (en) * 2000-04-25 2006-08-03 De Angelis, Paul L Chondroitin synthase gene and methods of making and using same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DEANGELIS ET AL: 'Identification and Molecular Cloning of a Chondroitin Synthase from Pasteurella Multocida Type F' JOURNAL OF BIOLOGICAL CHEMISTRY vol. 275, no. 31, 04 August 2000, pages 24124 - 24129, XP002163608 *
See also references of EP1470235A2 *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9200024B2 (en) 1998-04-02 2015-12-01 The Board Of Regents Of The University Of Oklahoma Production of defined monodisperse heparosan polymers and unnatural polymers with polysaccharide synthases
US8088604B2 (en) 1998-04-02 2012-01-03 The Board Of Regents Of The University Of Oklahoma Production of defined monodisperse heparosan polymers and unnatural polymers with polysaccharide synthases
EP1392843A4 (fr) * 2001-05-08 2005-09-21 Regents For The University Of Heparine/heparosan synthases et procedes de fabrication correspondant
US7307159B2 (en) 2001-05-08 2007-12-11 The Board Of Regents Of The University Of Oklahoma Heparin/heparosan synthase from P. multocida and methods of making and using same
US7771981B2 (en) 2001-05-08 2010-08-10 The Board Of Regents Of The University Of Oklahoma Heparin/heparosan synthase from P. multocida and methods of making and using same
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
EP1392843A2 (fr) * 2001-05-08 2004-03-03 Board Of Regents Of The University Of Oklahoma Heparine/heparosan synthases et procedes de fabrication correspondant
US9629914B2 (en) 2001-05-08 2017-04-25 The Board Of Regents Of The University Of Oklahoma Heparosan-based biomaterials and coatings and methods of production and use thereof
US9687559B2 (en) 2008-03-19 2017-06-27 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
US9603945B2 (en) 2008-03-19 2017-03-28 The Board Of Regents Of The University Of Oklahoma Heparosan polymers and methods of making and using same for the enhancement of therapeutics
US9925209B2 (en) 2008-03-19 2018-03-27 The Board Of Regents Of The University Of Oklahoma Heparosan-polypeptide and heparosan-polynucleotide drug conjugates and methods of making and using same
US9175293B2 (en) 2010-03-01 2015-11-03 Seikagaku Corporation Compositions and methods for bacterial and genetically modified microorganism production of chondroitin
US8697398B2 (en) 2010-03-01 2014-04-15 Dsm Ip Assets B.V. Compositions and methods for bacterial production of chondroitin
US8980608B2 (en) 2012-03-30 2015-03-17 The Board Of Regents Of The University Of Oklahoma High molecular weight heparosan polymers and methods of production and use thereof
US9885072B2 (en) 2012-03-30 2018-02-06 The Board Of Regents Of The University Of Oklahoma High molecular weight heparosan polymers and methods of production of use thereof
US10392642B2 (en) 2012-03-30 2019-08-27 The Board Of Regents Of The University Of Oklahoma High molecular weight heparosan polymers and methods of production and use thereof

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WO2003029261A9 (fr) 2004-06-10

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