Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
  • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P13/00—Drugs for disorders of the urinary system
  • A61P13/12—Drugs for disorders of the urinary system of the kidneys
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
  • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P27/00—Drugs for disorders of the senses
  • A61P27/02—Ophthalmic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4725—Proteoglycans, e.g. aggreccan

Definitions

• the present invention relates to veterinary decorin compositions and methods of their production and use.
• Proteoglycans carrying one or more glycosaminoglycan (GAG) chains form a large gene family that may be classifed into five groups according to the structural properties of the core protein.
• One of the groups is the small Leucine -rich proteoglycan family comprised of decorin (DCN), biglycan, fibromodulin and lumican. These are characterized by 40 kDa core proteins that contain Leucine-rich repeats of approximately 12 amino acids.
• DCN is a prototype of the group and is also referred to as PG-S2, PG40, proteodermatan sulphate and DS-PGII. It contains one dermatan chondroitin sulphate GAG chain covalently linked to a Serine of the mature core protein and is considered to be a multifunctional proteoglycan.
• DCN Decorin protein is present in most all animal tissues. The reduction or absence of decorin leads to problems within the body.
• the proposed functions of DCN include, but are not limited to, regulation of collagen fibrillogenesis, maintenance of tissue integrity via binding with fibronectin and thrombospondin, and a reservoir of transforming growth factor ⁇ (TGF- ⁇ ).
• TGF- ⁇ transforming growth factor ⁇
• the latter function of DCN is achieved through its core protein sequestering the growth factor in the extracellular milieu from receptors expressed on the cell surface.
• Various conditions including all types of surgery, cuts, burns, eye injuries, spinal cord injury, head trauma, lung disease, kidney disease, liver disease and cancer can disrupt the balance of decorin in the effected tissue.
• decorin in humans has been proposed. Examples of such uses include: suppression of cell proliferation by decorin (US 6,046,162); methods for inhibiting TGF-beta activity (US 6,277,812); methods of a pathology or a fibrotic condition by administering decorin (US 6,436,900); methods of preventing or reducing scarring with decorin or biglycan (US 6,509,314), treatment of glomerulonephritis with decorin (US 5,726,149); suppressing tumor cell growth by administering decorin (US 6,524,573); and inhibiting proliferative diseases by inhibiting TGF-beta mediated angiogenesis (US
• the present invention relates to veterinary decorin compositions and methods of their production and use.
• the present invention provides a veterinary decorin core protein molecule that is at least 95% identical to one of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, and 27, with the proviso that the veterinary decorin core protein comprises a mutation at amino acid 4.
• the mutation prevents gagylation of the molecule.
• the mutation is a serine to alanine mutation.
• the protein molecule is at least 98%> identical to one of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, and 27, with the proviso that the veterinary decorin core protein comprises a mutation at amino acid 4.
• the protein molecule is at least 99% identical to one of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, and 27, with the proviso that the veterinary decorin core protein comprises a mutation at amino acid 4. In some embodiments, the protein molecule is 100% identical to one of SEQ ID NOs: 4, 7, 10, 13, 16, 19, 22, and 27, with the proviso that the veterinary decorin core protein comprises a mutation at amino acid 4. In some embodiments, the core molecule is operably linked to an exogenous signal peptide. In some embodiments, the exogenous signal peptide is a bovine lactalbumin signal peptide.
• the present invention provides a composition comprising a veterinary decorin core protein molecule according as described above in combination with a pharmaceutically acceptable carrier.
• the present invention provides a nucleic acid sequence encoding a veterinary decorin core molecule as described above.
• the nucleic acid sequence encoding a veterinary decorin core molecule is operably associated with an exogenous signal peptide.
• the exogenous signal peptide is a bovine lactalbumin signal peptide.
• the present invention provides a host cell comprising the expression vector as described above.
• the present invention provides a method of producing a veterinary decorin protein comprising expressing the expression vector as described above in a host cell to produce the veterinary decorin protein and purifying the veterinary decorin protein.
• the present invention provides a pharmaceutical formulation comprising a veterinary decorin core protein molecule as described above, wherein the formulation is a liquid, powder, spray, gel, ointment, lotion, or eye drop.
• the present invention provides methods of treating a veterinary subject in need thereof comprising administering to the subject a veterinary decorin core protein according to claim 1 in an effective amount.
• the veterinary decorin core protein is administered by a method selected from the group consisting of enteral, parenteral and topical administration.
• the veterinary decorin core protein is administered by a method selected from the group consisting of oral administration, intravenous administration, intradermal administration, subcutaneous administration, transdermal administration, nasal administration, intramuscular
• administration intrathecal administration, intraocular administration, intravitreal
• administration intravaginal administration, and transmucosal administration.
• the veterinary subject is suffering from a wound or other injury to the skin and the veterinary decorin core protein is administered to inhibit scar formation.
• the wound is the result of cosmetic or general surgery, injury to the skin, or injury causing proud flesh.
• the scar is a keloid scar.
• the veterinary subject is suffering from an injury or disease to the eye.
• the injury to the eye is the result of corneal surgery, an eye burn, an eye infection, and an abrasive injury.
• the veterinary subject is suffering from a lung disease.
• the lung disease is selected from the group consisting of interstitial lung disease and pulmonary fibrosis.
• the veterinary subject is suffering from kidney disease.
• the kidney disease is selected from the group consisting of diabetic nephropathy and renal fibrosis.
• the veterinary subject is suffering from liver disease.
• the liver disease is selected from the group consisting of cirrhosis and hepatic fibrosis.
• cancer is an EGF Receptor or IGF-I receptor positive cancer.
• the veterinary subject is suffering from heart disease. In some embodiments, the veterinary subject is suffereing from a neurological trauma. In some embodiments, the neurological trauma is selected from a brain injury and a spinal cord injury. DESCRIPTION OF THE FIGURES
• Figure 1 Canine decorin coding sequence and the flanking DNA cloning junctions in the final retrovector expression construct are shown. Kozak translation initiation sequence is highlighted. The cloning sites Hindlll and Xhol are also shown.
• Figure 2 Map of Canine Decorin in GPEx® Vector.
• FIG. 3 Feline Decorin coding sequence and the flanking DNA cloning junctions in the final retrovector expression construct are shown. The Kozak translation initiation sequence is highlighted. The cloning sites Hindlll and Xhol are also shown.
• FIG. 5 SDS-PAGE gel of media from the pooled CHO cell line expressing Canine decorin mature protein. Lane 1. Molecular weight standards. Lane 2. Non-reduced media sample. Lane 3. Reduced media sample. The decorin protein shows as a doublet in both conditions similar to the purified human decorin shown in Figure 7.
• FIG. 6 SDS-PAGE gel of media from the pooled CHO cell line expressing Feline decorin mature protein. Lane 1. Molecular weight standards. Lane 2. Non-reduced media sample. Lane 3. Reduced media sample. The decorin protein shows as a doublet in both conditions similar to the purified human decorin shown in Figure 7.
• FIG. 1 SDS-PAGE gel of purified human decorin mature protein. Lane 1.
• veterinary decorin refers to a decorin molecule from a companion or stock animal, e.g., poultry such as a chicken, cows, goats, sheep, pigs, horses, dogs and cats.
• veterinary decorin core protein refers to a veterinary decorin protein molecule that has a mutation at amino acid 4 of mature decorin and that substantially lacks modification with a glycosaminoglycan (GAG; i.e., is non-gagylated) at amino acid 4.
• GAG glycosaminoglycan
• veterinary subject encompasses stock and companion animals, including, but not limited to, cows, sheep, horses, pigs, goats, chickens, turkeys, dogs and cats.
• the term "host cell” refers to any eukaryotic cell (e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells), whether located in vitro or in vivo.
• eukaryotic cell e.g., mammalian cells, avian cells, amphibian cells, plant cells, fish cells, and insect cells
• cell culture refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro, including oocytes and embryos.
• vector refers to any genetic element, such as a plasmid, phage, transposon, cosmid, chromosome, virus, virion, etc., which is capable of replication when associated with the proper control elements and which can transfer gene sequences between cells.
• vector includes cloning and expression vehicles, as well as viral vectors.
• the terms “complementary” or “complementarity” are used in reference to polynucleotides (i.e., a sequence of nucleotides) related by the base-pairing rules.
• sequence “5'-A-G-T-3', M is complementary to the sequence "3 -T-C-A-5'.”
• Complementarity may be “partial,” in which only some of the nucleic acids' bases are matched according to the base pairing rules. Or, there may be “complete” or “total” complementarity between the nucleic acids.
• the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of hybridization between nucleic acid strands. This is of particular importance in amplification reactions, as well as detection methods that depend upon binding between nucleic acids.
• a partially complementary sequence is one that at least partially inhibits a completely complementary sequence from hybridizing to a target nucleic acid sequence and is referred to using the functional term "substantially homologous.”
• the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency.
• a substantially homologous sequence or probe i.e., an oligonucleotide which is capable of hybridizing to another oligonucleotide of interest
• conditions of low stringency are such that non-specific binding is permitted; low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction.
• the absence of non-specific binding may be tested by the use of a second target which lacks even a partial degree of
• operable combination refers to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced.
• the term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
• the term "signal sequence” refers to any DNA sequence which, when operably linked to a recombinant DNA sequence, encodes a signal peptide which is capable of causing the secretion of the recombinant polypeptide.
• the signal peptides comprise a series of about 15 to 30 hydrophobic amino acid residues (See, e.g., Zwizinski et al, J. Biol. Chem. 255(16): 7973-77 [1980], Gray et al, Gene 39(2): 247-54 [1985], and Martial et al, Science 205: 602-607 [1979]).
• secretion signal sequences are preferably derived from genes encoding polypeptides secreted from the cell type targeted for tissue-specific expression (e.g., secreted milk proteins for expression in and secretion from mammary secretory cells).
• Secretory DNA sequences are not limited to such sequences.
• Secretory DNA sequences from proteins secreted from many cell types and organisms may also be used (e.g., the secretion signals for t-PA, serum albumin, lactoferrin, and growth hormone, and secretion signals from microbial genes encoding secreted polypeptides such as from yeast, filamentous fungi, and bacteria).
• purified refers to molecules, either nucleic or amino acid sequences, that are removed from their normal environment, isolated or separated.
• An "isolated nucleic acid sequence” is therefore a purified nucleic acid sequence.
• substantially purified molecules are at least 60% free, preferably at least 75% free, and more preferably at least 90% free from other components with which they are normally associated.
• the present invention relates to veterinary decorin compositions and methods of their production and use.
• Native decorin is a glycoprotein with an attached glycosaminoglycan and an average molecular weight of 90-140 kD.
• the present invention contemplates the production and use recombinant veterinary decorin.
• the veterinary decorin is a veterinary decorin core protein, i.e., a substantially non-gagylated veterinary decorin.
• the veterinary decorin core protein comprises a mutation at amino acid 4 (i.e., the 4 th amino acid from the N-terminus) of the mature veterinary decorin core protein molecule.
• the mutation is a serine to alanine mutation.
• the veterinary decorin core protein molecule is a protein molecule having a sequence at least 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of SEQ ID NOS:4 (chicken), 7 (bovine), 10 (canine), 13 (caprine), 16 (equine), 19 (porcine), 22 (ovine), and 27 (feline) provided that that the decorin core protein comprises a mutation at amino acid 4 (i.e., the 4 th amino acid from the N-terminus) of the mature decorin core protein molecule.
• Decorin is commonly expressed as a pre-pro-protein.
• the present invention provides veterinary decorin fusion molecules comprising a heterologous signal sequence in operable association with the veterinary decorin pro-peptide and mature peptide sequences.
• the heterologous signal polypeptide is an alpha-lactalbumin signal polypeptide.
• the alpha-lactalbumin signal polypeptide is a bovine alpha- lactalbumin signal polypeptide.
• the heterologous signal polypeptide is at least 80%, 90%, or 100% identical to MMSFVSLLLVGILFHATQA (SEQ ID NO:23).
• the propeptide sequence is at least 80%>, 90%>, or 100% identical to the propeptide sequences identified in SEQ ID NOs:3, 6, 9, 12, 15, 18, 21 and 26.
• the decorin core protein portion of the fusion polypeptide is at least 90%>, 95% , 99% or 100% identical to SEQ ID NO: 1 (mature decorin core protein), provided that that the decorin core protein comprises a mutation at amino acid 4 (i.e., the 4 th amino acid from the N-terminus) of the mature decorin core protein molecule.
• the fusion protein is 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one of SEQ ID NOS:3 (chicken), 6 (bovine), 9 (canine), 12 (caprine), 15 (equine), 18 (porcine), 21 (ovine), and 26 (feline)(signal-propeptide veterinary decorin core protein), provided that that the decorin core protein comprises a mutation at amino acid 4 (i.e., the 4 th amino acid from the N-terminus) of the mature decorin core protein molecule.
• the present invention further provides nucleic acid sequences encoding the fusion proteins, as well as vectors comprising the nucleic acid sequences.
• the heterologous signal polypeptide is at least 80%, 90%, or 100% identical to ATGATGTCCTTTGTCTCTCTGCTCCTGGTAGGCATCCTATTCCATGCCACCCAGGC
• the propeptide nucleic acid sequence is at least 80%, 90%, or 100% identical to the propeptide sequences identified in SEQ ID NOs: l, 5, 8, 11, 14, 17, 20 and 25.
• the veterinary decorin core protein portion of the fusion polypeptide is at least 90%>, 95% , 99% or 100% identical to the veterinary decorin core protein portion identified in SEQ ID NOs: l, 5, 8, 11, 14, 17, 20, and 25 provided that that the decorin core protein nucleic acid sequence comprises a mutation at amino acid 4 (i.e., the 4 th amino acid from the N-terminus) of the mature decorin core protein molecule.
• the fusion protein is at least 90%, 95%, 99% or 100% identical to one of SEQ ID NOs: 1, 5, 8, 11, 14, 17, 20 and 25 (signal-propeptide veterinary decorin core protein nucleic acid sequences), provided that that the decorin core protein nucleic acid sequence comprises a mutation at amino acid 4 (i.e., the 4 th amino acid from the N-terminus) of the mature decorin core protein molecule.
• the veterinary decorin polynucleotides of the present invention may be employed for producing veterinary decorin polypeptides by recombinant techniques.
• the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
• vectors include, but are not limited to, retroviral vectors, chromosomal, nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40, bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies). It is contemplated that any vector may be used as long as it is replicable and viable in the host. In some preferred, retroviral vectors, chromosomal, nonchromosomal and synthetic DNA sequences (e.g., derivatives of SV40, bacterial plasmids, phage DNA; baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, and viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies). It is
• the vectors are retroviral vectors as described in US Pat. Nos. 6,852,510 and 7,332,333 and U.S. Pat. Publ. Nos. 200402335173 and 20030224415, all of which are incorporated herein by references in their entirety. In some especially preferred
• the vectors are pseudotyped retroviral vectors.
• some embodiments of the present invention provide recombinant constructs comprising one or more of the sequences as broadly described above (e.g., one of SEQ ID NOs: 1, 5, 8, 11, 14, 17, 20 and 25).
• the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
• the heterologous structural sequence (e.g., one of SEQ ID NOs: 1, 5, 8, 11, 14, 17, 20 and 25) is assembled in appropriate phase with translation initiation and termination sequences.
• the appropriate DNA sequence is inserted into the vector using any of a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art.
• vectors include, but are not limited to, the following vectors: 1) Bacterial— pQE70, pQE60, pQE-9 (Qiagen), pBS, pDIO, phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia); 2) Eukaryotic-pWLNEO, pSV2CAT, pOG44, PXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia); and 3) Baculovirus- pPbac and pMbac (Stratagene).
• Bacterial— pQE70, pQE60, pQE-9 Qiagen
• mammalian expression vectors comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking non-transcribed sequences.
• DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
• the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis.
• Promoters useful in the present invention include, but are not limited to, the LTR or SV40 promoter, the E. coli lac or trp, the phage lambda PL and PR, T3 and T7 promoters, and the cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, and mouse metallothionein-I promoters and other promoters known to control expression of gene in prokaryotic or eukaryotic cells or their viruses.
• recombinant expression vectors include origins of replication and selectable markers permitting transformation of the host cell (e.g.,
• dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli are examples of dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or tetracycline or ampicillin resistance in E. coli.
• transcription of the DNA encoding the polypeptides of the present invention by higher eukaryotes is increased by inserting an enhancer sequence into the vector.
• Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increase its transcription.
• Enhancers useful in the present invention include, but are not limited to, the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
• the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
• the vector may also include appropriate sequences for amplifying expression.
• the present invention provides host cells containing the above-described constructs.
• the host cell is a higher eukaryotic cell (e.g., a mammalian or insect cell).
• the host cell is a lower eukaryotic cell (e.g., a yeast cell).
• the host cell can be a prokaryotic cell (e.g., a bacterial cell).
• a prokaryotic cell e.g., a bacterial cell.
• host cells include, but are not limited to, Escherichia coli, Salmonella typhimurium, Bacillus subtilis, and various species within the genera
• the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
• introduction of the construct into the host cell can be accomplished by retroviral transduction, calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (See e.g., Davis et al.
• polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
• Proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.
• protein is secreted and cells are cultured for an additional period.
• cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
• microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
• the present invention also provides methods for recovering and purifying decorin from recombinant cell cultures including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
• the present invention provides improved methods for the purification of decorin, especially decorin core protein.
• the processes comprise two column
• cation exchange chromatography is used to capture decorin from medium containing decorin, preferably a clarified medium.
• the cation exchange chromatography medium is SP-Sepharose FF.
• the cation exchange medium is equilibrated at about 5 to 15 mM sodium phosphate, preferably 10 mM sodium phosphate, and 20 to 70 mM NaCL, preferably about 50 mM NaCL at a neutral pH.
• the cation exchange medium is washed.
• the wash buffer comprises about 5 to 15 mM sodium phosphate, preferably 10 mM sodium phosphate, and 20 to 70 mM NaCL, preferably about 50 mM NaCL.
• decorin is then eluted from the cation exchange medium.
• the elution buffer comprises about 5 to 15 mM sodium phosphate, preferably 10 mM sodium phosphate, and 150 to 250 mM NaCL, preferably about 200 mM NaCL.
• the eluate containing decorin from the cation exchange chromatography step is applied to a hydroxyapatite medium.
• the hydroxyapatite medium is CHT Type 1.
• the hydroxyapatite medium is equilibrated at about 5 to 15 mM sodium phosphate, preferably 10 mM sodium phosphate, and 150 to 250 mM NaCL, preferably about 200 mM NaCL.
• the decorin containing medium is applied to the hydroxyapatite medium.
• the wash buffer comprises about 5 to 15 mM sodium phosphate, preferably 10 mM sodium phosphate, and 150 to 250 mM NaCL, preferably about 200 mM NaCL.
• decorin is then eluted from the hydroxyapatite medium.
• the elution buffer comprises about 0.2 to 0.4 M sodium phosphate, preferably 0.3 M sodium phosphate, and 150 to 250 mM NaCL, preferably about 200 mM NaCL.
• the eluate containing decorin from the hydroxyapatite chromatography step is buffer exchanged and applied to an ion exchange membrane.
• the ion exchange membrane is a Q ion exchange membrane, for examples a Mustang Q ion exchange medium.
• the membrane is equilibrated with from about 30 mM to 70 mM Tris-HCl, preferably about 50 mM Tris-HCl.
• the membrane is washed and the decorin passes through the membrane.
• the wash buffer comprises from about 30 mM to 70 mM Tris-HCl, preferably about 50 mM Tris-HCl.
• the decorin is preferably concentrated to a desired concentration, for example by flow filtration.
• solutions of containing the decorin are treated to inactivate or remove viruses.
• solutions comprising decorin are treated with a surfactant to inactivate viruses.
• the surfactant is
• the surfactant treating step is performed after the cation exchange chromatography step.
• the solutions comprising decorin are filtered to remove viruses.
• the solutions are filtered through a a viral Filter (e.g., a Virosart viral filter).
• the filtrations step is performed after the hydroxyapatite chromatography step.
• compositions suitable for clinical use in human patients.
• the compositions comprise a purified decorin core protein comprising a mutation at position 4 of the mature decorin core protein so that the decorin protein is substantially non-gagylated.
• the compositions provide purified decorin proteins, and the compositions are characterized in comprising less than about 100, 50, 20, 10, 5 or 2 ng residual host cell protein/mg decorin core protein in the composition and/or less than about 20, 10, 5, or 2 pg residual host cell DNA/mg decorin core protein.
• the decorin is provided in a aqueous solution.
• the aqueous solution is phosphate buffered saline (e.g., lOmM sodium phosphate, 150mM sodium chloride), with a pH of from about 6.5 to 7.5, preferably about 7.0.
• phosphate buffered saline e.g., lOmM sodium phosphate, 150mM sodium chloride
• the present invention further provides pharmaceutical compositions comprising veterinary decorin purified as described above.
• Pharmaceutically acceptable carriers are well known in the art and include aqueous solutions such as physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, vegetable oils (e.g., olive oil) or injectable organic esters.
• Further pharmaceutically acceptable carriers include, for example, hyaluronic acid, and aqueous solutions such as bicarbonate buffers, phosphate buffers, Ringer's solution and physiological saline supplemented with 5% dextrose or human serum albumin, if desired.
• a pharmaceutically acceptable carrier can be used to administer the decorin polypeptide to a cell in vitro or to a subject in vivo.
• a pharmaceutically acceptable carrier can contain a physiologically acceptable compound that acts, for example, to stabilize the polypeptide or to increase or decrease the absorption of the agent.
• a physiologically acceptable compound can include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients.
• Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives, which are particularly useful for preventing the growth or action of microorganisms.
• Various preservatives are well known and include, for example, phenol and ascorbic acid.
• a pharmaceutically acceptable carrier including a physiologically acceptable compound, depends, for example, on the route of administration of the polypeptide and on the particular physio-chemical characteristics of the specific polypeptide.
• a physiologically acceptable compound such as aluminum monosterate or gelatin is particularly useful as a delaying agent, which prolongs the rate of absorption of a pharmaceutical composition administered to a subject.
• a parmaceutical composition comprising an effective amount of veterinary decorin can be administered to a subject by various routes including, for example, topically, orally or parenterally, such as intravenously, intramuscularly, subcutaneously, intraperitoneally or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively.
• Topical administration can be passive, for example, by direct application of an ointment or powder, or active, for example, using a nasal spray or inhalant.
• one component of the composition is an appropriate propellant.
• the pharmaceutical composition also can be incorporated, if desired, into liposomes, microspheres or other polymer matrices
• Liposomes for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.
• An effective amount of the pharmaceutical composition comprising veterinary decorin is generally in the range of about 0.01 to 100 mg/kg body weight. An effective amount can be determined using methods known to those in the art.
• the total effective amount can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which the multiple doses are administered over a more prolonged period of time.
• a fractionated treatment protocol in which the multiple doses are administered over a more prolonged period of time.
• the veterinary decorin-containing compositions of the present invention can be combined with additional active agents.
• agents include, but are not limited to, decorin- synthesis enhencers, collagen-synthesis enhencers, matrix metalloproteinases (MMP) inhibitors, antioxidants, collagen modulators, anti-wrinkle or anti-aging agents, antibiotics, depigmenting agents, analgesics, antimicrobials, anti-inflammatory agents, moisturers, skin lightening agents, corticosteroids, or sun-block agents.
• MMP matrix metalloproteinases
• the veterinary decorin-containing compositions can be combined with a cosmetically or pharmaceutically or dermatologically acceptable carrier.
• the carriers include, but are not limited to, water, mineral oil, ethylene glycol, propylene glycol, lanolin, glyceryl stearate, sorbitan stearate, isopropyl myristate, isopropyl palmitate, acetone, glycerol,
• phosphatidylcholine sodium cholate, or ethanol.
• the veterinary decorin-containing compositions can be combined with a skin penentration enhancer.
• the enhancers helping to transport the active components through the normal intact skin, include, but are not limited to, liposomes, mixed lipid micelles, ethosomes, transfersomes, niosomes, ethanol, amides, ethers, glycols, hydrocarbon oils, sodium lauryl sulfate, oleic acid, hydroalcoholic solution, and soya phosphatidylcholine or their combinations.
• Other skin penetration enhancement includes different pH values, co- solvents, surfactants, cyclodextrins, and iontophoresis.
• a suitable carrier or vehicle or enhancer will include the formulation of gels, creams, lotions, solutions, colloidal dispersions, emulsions (oil-in-water or water-in-oil), foams, sprays, suspensions, sunscreens, liquid and various skin care preparations for topical application to the skin.
• the decorin-containing compositions can be prepared in any formula for topical application to the skin.
• the formulation mentioned above can also be combined with other ingredients, depending on the intended use of the formulation.
• these ingredients include, but are not limited to, preservatives, vitamins, polymers, fragrances, water- or oil-soluble film formers, or flavoring agents.
• the veterinary decorin compositions of the present invention find a variety of uses. While the methods utilizing veterinary decorin are generally applicable, specific examples of pathologies which can be treated include cancer, a fibrotic disease, and glomerulonephritis. In fibrotic cancer, for example, decorin can be used to bind TGF- ⁇ , destroying TGF-P's growth stimulating activity on the cancer cell.
• the verterinary decorin compositions further find use in treating tumors which are positive for the EGF rececptor and/or IGF-1 receptor.
• compositions are used to treat or inhibit scarring in the proud flesh of horses.
• the compositions are used to treat injuries to the eye, for example, injury to the eye resulting from corneal surgery, eye burns (chemical or thermal), eye infections, and abrasive injuries to the eye.
• the decorin compositions are used to treat heart disease.
• the decorin compositions are used to treat neurological traumas, such as brain or spinal cord injuries.
• Expression constructs have been designed for a mutant form of veterinary decorin from a number of different species of companion animals and stock animals. A serine to alanine modification was made at amino acid 4 of mature decorin for each of the different species. The mutation prevents a GAG from being attached to the decorin molecule.
• the expression constructs use the bovine a-lactalbumin signal peptide instead of the endogenous signal peptide for protein production and secretion.
• the constructs are outlined below.
• Chicken Decorin Expression Gene Sequence SEQ ID NO:l
• bovine a -lactalbumin signal peptide coding region is shown in boldface type
• the chicken decorin pro-peptide coding region is underlined
• the mutated mature decorin coding region is shown in standard type
• bovine a -lactalbumin signal peptide is shown in boldface type
• the chicken decorin pro-peptide is underlined
• the mutated mature decorin coding region is shown in standard type (Amino acid #4 of mature decorin was mutated from a serine to an alanine to prevent addition of the GAG moiety to the mature protein. This amino acid is shown in boldface type as well as underlined).
• bovine a -lactalbumin signal peptide coding region is shown in boldface type
• bovine decorin pro-peptide coding region is underlined
• the mutated mature decorin coding region is shown in standard type
• bovine a -lactalbumin signal peptide is shown in boldface type
• the bovine decorin pro-peptide is underlined
• the mutated mature decorin coding region is shown in standard type (Amino acid #4 of mature decorin was mutated from a serine to an alanine to prevent addition of the GAG moiety to the mature protein. This amino acid is shown in boldface type as well as underlined)
• Mature cow decorin protein sequence (SEQ ID NO:7):
• bovine a -lactalbumin signal peptide coding region is shown in boldface type
• the mutated mature decorin coding region is shown in standard type
• bovine a -lactalbumin signal peptide is shown in boldface type
• the dog decorin pro-peptide is underlined
• the mutated mature decorin coding region is shown in standard type (Amino acid #4 of mature decorin was mutated from a serine to an alanine to prevent addition of the GAG moiety to the mature protein. This amino acid is shown in boldface type as well as underlined)
• Mature dog decorin sequence (SEQ ID NO: 10):
• bovine a -lactalbumin signal peptide coding region is shown in boldface type
• the goat decorin pro-peptide coding region is underlined
• the mutated mature decorin coding region is shown in standard type
• bovine a -lactalbumin signal peptide is shown in boldface type
• the mutated mature decorin coding region is shown in standard type (Amino acid #4 of mature decorin was mutated from a serine to an alanine to prevent addition of the GAG moiety to the mature protein. This amino acid is shown in boldface type as well as underlined)
• bovine a -lactalbumin signal peptide coding region is shown in boldface type
• the mutated mature decorin coding region is shown in standard type
• bovine a -lactalbumin signal peptide is shown in boldface type
• the horse decorin pro-peptide is underlined
• the mutated mature decorin coding region is shown in standard type (Amino acid #4 of mature decorin was mutated from a serine to an alanine to prevent addition of the GAG moiety to the mature protein. This amino acid is shown in boldface type as well as underlined)
• bovine a -lactalbumin signal peptide coding region is shown in boldface type
• the pig decorin pro-peptide coding region is underlined
• the mutated mature decorin coding region is shown in standard type
• bovine a -lactalbumin signal peptide is shown in boldface type
• the pig decorin pro-peptide is underlined
• the mutated mature decorin coding region is shown in standard type (Amino acid #4 of mature decorin was mutated from a serine to an alanine to prevent addition of the GAG moiety to the mature protein. This amino acid is shown in boldface type as well as underlined)
• the sheep decorin pro-peptide coding region is underlined
• the mutated mature decorin coding region is shown in standard type
• the mutated mature decorin coding region is shown in standard type (Amino acid #4 of mature decorin was mutated from a serine to an alanine to prevent addition of the GAG moiety to the mature protein. This amino acid is shown in boldface type as well as underlined)
• Mature sheep decorin protein sequence (SEQ ID NO:22): DEAAGIGPEERFHEVPELEPMGPVCPFRCQCHLRVVQCSDLGLEKVPKDLPPDTALL DLQ NKITEIKDGDFK LK LHTLILI NKISKISPGAFAPLVKLERLYLSK QLKELP EKMPKTLQELRVHENEITKVRKSVFNGLNQMIVVELGTNPLKSSGIENGAFQGMKKL SYIRIADTNITTIPQGLPPSLTELHLDGNKITKVDAASLKGL NLAKLGLSFNSISAVDN GSLANTPHLRELHL NNKLVKVPGGLADHKYIQVVYLHNNNISAIGSNDFCPPGYNT KKASYSGVSLFSNPVQYWEIQPSTFRCVYVRAAVQLGNYK
• the mutated mature decorin coding region is shown in standard type
• bovine a -lactalbumin signal peptide is shown in boldface type
• the mutated mature decorin coding region is shown in standard type (Amino acid #4 of mature decorin was mutated from a serine to an alanine to prevent addition of the GAG moiety to the mature protein. This amino acid is shown in boldface type as well as underlined)
• Canine and Feline Decorin DNA construction and cloning The Canine and Feline gene sequences of decorin were synthesized with the bovine alpha-lactalbumin signal peptide attached. Both DNA sequences were cloned into Catalent's GPEx® expression vectors
• Retrovector Production The expression constructs outlined above were introduced into a HEK 293 cell line that constitutively produces the MLV gag, pro, and pol proteins. An envelope containing expression plasmid was also co-tranfected with the decorin gene construct. The co-transfection resulted in the production of replication incompetent high titer retrovector that was concentrated by ultracentrifugation and used for cell transductions (1 ,2).
• GCHO Cells with Retrovector The Canine and Feline Decorin pooled cell lines were made by performing multiple rounds of transduction of the GPEx® Chinese Hamster Ovary (GCHO) parental cell line with retrovector made from the gene constructs developed to express the two decorin molecules. Three independent transductions were performed to generate a pooled cell line for each of the two products.
• GCHO Chinese Hamster Ovary

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