Cellular glycosaminoglycan compositions and methods of making and using

Abstract

Disclosed herein are substantially pure glycosaminoglycan compositions derived from genetically modified cells.

1 - 58 . (canceled) 
     
     
         59 . A method of preparation of a substantially pure glycosaminoglycan selected from the group consisting of heparan sulfate, chondroitin sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid comprising: (a) growing a cell deficient or transgenic in one or more genes recited in Tables 1, 2, 4 or 5, using an appropriate growth media, (b) isolating the growth media from the cells by centrifugation 
     
     
         60 . (canceled) 
     
     
         60 - 69 . The method of  claim 59 , wherein the method comprises fractionating the mixture by ion exchange column. 
     
     
         71 . The method of  claim 59 , wherein the method comprises removing contaminating nucleic acids by nuclease digestion. 
     
     
         72 . The method of  claim 59 , wherein the method comprises removing contaminating proteins by protease digestion. 
     
     
         73 . The method of  claim 59 , wherein the method comprises fractionating the resulting product by ion exchange. 
     
     
         74 . The method of  claim 59 , wherein the method comprises desalting. 
     
     
         75 . The method of  claim 59 , wherein the method does not require the use of an enzyme. 
     
     
         76 . The method of  claim 59 , wherein the method does not require the use of a chondroitinase. 
     
     
         77 . The method of  claim 59 , wherein the glycosaminoglycan is at least 95% free from protein and nucleic acid contamination. 
     
     
         78 - 90 . (canceled) 
     
     
         91 . A method of treating a thrombosis, an inflammation, a cancer, a microbial infection, a neurodegenerative disorder, or a wound in an individual in need thereof comprising administering an effective amount of the composition of any one of claims  1  to  37  or the pharmaceutical composition of claim  38  a glycosaminoglycan prepared according to the method of  claim 59 . 
     
     
         92 . The method of  claim 91 , wherein the thrombosis comprises, venous thrombosis, deep vein thrombosis, portal vein thrombosis, renal vein thrombosis, jugular vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease, Cerebral venous sinus thrombosis, Cavernous sinus thrombosis, arterial thrombosis, stroke, myocardial infarction, or Hepatic artery thrombosis. 
     
     
         93 . The method of  claim 91 , wherein the inflammation comprises, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, multiple sclerosis (MS), encephalomyelitis, myasthenia gravis, systemic lupus erythematosus (SLE), asthma, allergic asthma, autoimmune thyroiditis, atopic dermatitis, eczematous dermatitis, psoriasis, Sjogren's Syndrome, Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis (UC), inflammatory bowel disease (IBD), cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves' disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, interstitial lung fibrosis, Hashimoto's thyroiditis, autoimmune polyglandular syndrome, insulin-dependent diabetes mellitus (IDDM, type I diabetes), insulin-resistant diabetes mellitus (type 2 diabetes), immune-mediated infertility, autoimmune Addison's disease, pemphigus vulgaris, pemphigus foliaceus, dermatitis herpetiformis, autoimmune alopecia, vitiligo, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura, pernicious anemia, Guillain-Barre syndrome, stiff-man syndrome, acute rheumatic fever, sympathetic ophthalmia, Goodpasture's syndrome, systemic necrotizing vasculitis, antiphospholipid syndrome or an allergy, Behcet's disease, X-linked lymphoproliferative syndrome (SH2D1A/SAP deficiency), hyper IgE syndrome, or Graft vs. Host Disease (GVHD). 
     
     
         94 . The method of  claim 91 , wherein the cancer comprises Acanthoma, Acinic cell carcinoma, Acoustic neuroma, Acral lentiginous melanoma, Acrospiroma, Acute eosinophilic leukemia, Acute lymphoblastic leukemia, Acute megakaryoblastic leukemia, Acute monocytic leukemia, Acute myeloblastic leukemia with maturation, Acute myeloid dendritic cell leukemia, Acute myeloid leukemia, Acute promyelocytic leukemia, Adamantinoma, Adenocarcinoma, Adenoid cystic carcinoma, Adenoma, Adenomatoid odontogenic tumor, Adrenocortical carcinoma, Adult T-cell leukemia, Aggressive NK-cell leukemia, AIDS-Related Cancers, AIDS-related lymphoma, Alveolar soft part sarcoma, Ameloblastic fibroma, Anal cancer, Anaplastic large cell lymphoma, Anaplastic thyroid cancer, Angioimmunoblastic T -cell lymphoma, Angiomyolipoma, Angiosarcoma, Appendix cancer, Astrocytoma, Atypical teratoid rhabdoid tumor, Basal cell carcinoma, Basal-like carcinoma, B-cell leukemia, B-cell lymphoma, Bellini duct carcinoma, Biliary tract cancer, Bladder cancer, Blastoma, Bone Cancer, Bone tumor, Brain Stem Glioma, Brain Tumor, Breast Cancer, Brenner tumor, Bronchial Tumor, Bronchioloalveolar carcinoma, Brown tumor, Burkitt's lymphoma, Cancer of Unknown Primary Site, Carcinoid Tumor, Carcinoma, Carcinoma in situ, Carcinoma of the penis, Carcinoma of Unknown Primary Site, Carcinosarcoma, Castleman's Disease, Central Nervous System Embryonal Tumor, Cerebellar Astrocytoma, Cerebral Astrocytoma, Cervical Cancer, Cholangiocarcinoma, Chondroma, Chondrosarcoma, Chordoma, Choriocarcinoma, Choroid plexus papilloma, Chronic Lymphocytic Leukemia, Chronic monocytic leukemia, Chronic myelogenous leukemia, Chronic Myeloproliferative Disorder, Chronic neutrophilic leukemia, Clear-cell tumor, Colon Cancer, Colorectal cancer, Craniopharyngioma, Cutaneous T -cell lymphoma, Degos disease, Dermatofibrosarcoma protuberans, Dermoid cyst, Desmoplastic small round cell tumor, Diffuse large B cell lymphoma, Dysembryoplastic neuroepithelial tumor, Embryonal carcinoma, Endodermal sinus tumor, Endometrial cancer, Endometrial Uterine Cancer, Endometrioid tumor, Enteropathy -associated T-cell lymphoma, Ependymoblastoma, Ependymoma, Epithelioid sarcoma, Erythroleukemia, Esophageal cancer, Esthesioneuroblastoma, Ewing Family of Tumor, Ewing Family Sarcoma, Ewing's sarcoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Extrahepatic Bile Duct Cancer, Extramammary Paget's disease, Fallopian tube cancer, Fetus in fetu, Fibroma, Fibrosarcoma, Follicular lymphoma, Follicular thyroid cancer, Gallbladder Cancer, Gallbladder cancer, Ganglioglioma, Ganglioneuroma, Gastric Cancer, Gastric lymphoma, Gastrointestinal cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumor, Gastrointestinal stromal tumor, Germ cell tumor, Germinoma, Gestational choriocarcinoma, Gestational Trophoblastic Tumor, Giant cell tumor of bone, Glioblastoma multiforme, Glioma, Gliomatosis cerebri, Glomus tumor, Glucagonoma, Gonadoblastoma, Granulosa cell tumor, Hairy Cell Leukemia, Hairy cell leukemia, Head and Neck Cancer, Head and neck cancer, Heart cancer, Hemangioblastoma, Hemangiopericytoma, Hemangiosarcoma, Hematological malignancy, Hepatocellular carcinoma, Hepatosplenic T-cell lymphoma, Hereditary breast-ovarian cancer syndrome, Hodgkin Lymphoma, Hodgkin's lymphoma, Hypopharyngeal Cancer, Hypothalamic Glioma, Inflammatory breast cancer, Intraocular Melanoma, Islet cell carcinoma, Islet Cell Tumor, Juvenile myelomonocytic leukemia, Kaposi Sarcoma, Kaposi's sarcoma, Kidney Cancer, Klatskin tumor, Krukenberg tumor, Laryngeal Cancer, Laryngeal cancer, Lentigo maligna melanoma, Leukemia, Leukemia, Lip and Oral Cavity Cancer, Liposarcoma, Lung cancer, Luteoma, Lymphangioma, Lymphangiosarcoma, Lymphoepithelioma, Lymphoid leukemia, Lymphoma, Macroglobulinemia, Malignant Fibrous Histiocytoma, Malignant fibrous histiocytoma, Malignant Fibrous Histiocytoma of Bone, Malignant Glioma, Malignant Mesothelioma, Malignant peripheral nerve sheath tumor, Malignant rhabdoid tumor, Malignant triton tumor, MALT lymphoma, Mantle cell lymphoma, Mast cell leukemia, Mediastinal germ cell tumor, Mediastinal tumor, Medullary thyroid cancer, Medulloblastoma, Medulloblastoma, Medulloepithelioma, Melanoma, Melanoma, Meningioma, Merkel Cell Carcinoma, Mesothelioma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Metastatic urothelial carcinoma, Mixed Mullerian tumor, Monocytic leukemia, Mouth Cancer, Mucinous tumor, Multiple Endocrine Neoplasia Syndrome, Multiple Myeloma, Multiple myeloma, Mycosis Fungoides, Mycosis fungoides, Myelodysplastic Disease, Myelodysplastic Syndromes, Myeloid leukemia, Myeloid sarcoma, Myeloproliferative Disease, Myxoma, Nasal Cavity Cancer, Nasopharyngeal Cancer, Nasopharyngeal carcinoma, Neoplasm, Neurinoma, Neuroblastoma, Neuroblastoma, Neurofibroma, Neuroma, Nodular melanoma, Non-Hodgkin Lymphoma, Non-Hodgkin lymphoma, Nonmelanoma Skin Cancer, Non-Small Cell Lung Cancer, Ocular oncology, Oligoastrocytoma, Oligodendroglioma, Oncocytoma, Optic nerve sheath meningioma, Oral Cancer, Oral cancer, Oropharyngeal Cancer, Osteosarcoma, Osteosarcoma, Ovarian Cancer, Ovarian cancer, Ovarian Epithelial Cancer, Ovarian Germ Cell Tumor, Ovarian Low Malignant Potential Tumor, Paget's disease of the breast, Pancoast tumor, Pancreatic Cancer, Pancreatic cancer, Papillary thyroid cancer, Papillomatosis, Paraganglioma, Paranasal Sinus Cancer, Parathyroid Cancer, Penile Cancer, Perivascular epithelioid cell tumor, Pharyngeal Cancer, Pheochromocytoma, Pineal Parenchymal Tumor of Intermediate Differentiation, Pineoblastoma, Pituicytoma, Pituitary adenoma, Pituitary tumor, Plasma Cell Neoplasm, Pleuropulmonary blastoma, Poly embryoma, Precursor T-lymphoblastic lymphoma, Primary central nervous system lymphoma, Primary effusion lymphoma, Primary Hepatocellular Cancer, Primary Liver Cancer, Primary peritoneal cancer, Primitive neuroectodermal tumor, Prostate cancer, Pseudomyxoma peritonei, Rectal Cancer, Renal cell carcinoma, Respiratory Tract Carcinoma Involving the NUT Gene on Chromosome 15, Retinoblastoma, Rhabdomyoma, Rhabdomyosarcoma, Richter's transformation, Sacrococcygeal teratoma, Salivary Gland Cancer, Sarcoma, Schwannomatosis, Sebaceous gland carcinoma, Secondary neoplasm, Seminoma, Serous tumor, Sertoli-Leydig cell tumor, Sex cord-stromal tumor, Sezary Syndrome, Signet ring cell carcinoma, Skin Cancer, Small blue round cell tumor, Small cell carcinoma, Small Cell Lung Cancer, Small cell lymphoma, Small intestine cancer, Soft tissue sarcoma, Somatostatinoma, Soot wart, Spinal Cord Tumor, Spinal tumor, Splenic marginal zone lymphoma, Squamous cell carcinoma, Stomach cancer, Superficial spreading melanoma, Supratentorial Primitive Neuroectodermal Tumor, Surface epithelial-stromal tumor, Synovial sarcoma, T-cell acute lymphoblastic leukemia, T-cell large granular lymphocyte leukemia, T-cell leukemia, T-cell lymphoma, T-cell prolymphocytic leukemia, Teratoma, Terminal lymphatic cancer, Testicular cancer, Thecoma, Throat Cancer, Thymic Carcinoma, Thymoma, Thyroid cancer, Transitional Cell Cancer of Renal Pelvis and Ureter, Transitional cell carcinoma, Urachal cancer, Urethral cancer, Urogenital neoplasm, Uterine sarcoma, Uveal melanoma, Vaginal Cancer, Verner Morrison syndrome, Verrucous carcinoma, Visual Pathway Glioma, Vulvar Cancer, Waldenstrom's macroglobulinemia, Warthin's tumor, or Wilms' tumor. 
     
     
         95 . The method of  claim 91 , wherein the microbial infection comprises a viral infection, a bacterial infection, or a parasitic infection. 
     
     
         96 . The method of  claim 91 , wherein the neurodegenerative disorder comprises Alzheimer's disease, Parkinson's disease, Huntington's disease, Amyotrophic lateral sclerosis, Dementia, Transmissible spongiform encephalopathy, Dentatorubropallidoluysian atrophy, Spinal and bulbar muscular atrophy, Spinocerebellar ataxia Type 1, Spinocerebellar ataxia Type 2, Spinocerebellar ataxia Type 3, Spinocerebellar ataxia Type 6, Spinocereb ellar ataxia Type 7, or Spinocerebellar ataxia Type 17. 
     
     
         97 . The method of  claim 91 , wherein the wound comprises an incision, a laceration, an abrasion, an avulsion, a puncture wound, a penetration wound, a gunshot wound, a hematoma, or a crush injury. 
     
     
         98 . (canceled) 
     
     
         99 . The method of  claim 59 , wherein the cell is deficient in one or more of chondroitin sulfate synthase 1 (ChSy), Chondroitin Sulfate N-Acetylgalactosaminyltransferase 2 (CSGalNAcT2), Chondroitin Polymerizing Factor (ChPF), heparan sulfate 2-O-sulphotransferase (HS2ST), glucuronic acid epimerase (GLCE), heparan sulfate N-deacetylase/sulfotransferase-1 (HSNDST1), heparan sulfate N-deacetylase/sulfotransferase-2 (HSNDST2), Sulfatase 1 (Sulf1), Sulfatase (Su1f2), Beta-glucuronidase (GUSB), Galactosamine-6 sulfatase (GALNS), Alpha-L-iduronidase (IDUA), Sulfamidase (SGSH), N-acetyltransferase (AANAT, ARD1A, GNPNAT1, HGSNAT, MAK 10, NAT1, NAT2, NAT5, NAT6, NAT8, NAT8L, NAT9, NAT 10, NATI 1, NAT12, NAT13, NAT14,NAT14), Uronate-2-sulfatase (IDS), Alpha-N-acetylglucosaminidase (NAGLU), PAPS synthase (PAPSS1, PAPSS2), Xylosyltransferase 1 (XYLT1), Xylosyltransferase 2 (XYLT2), Galactosyltransferase 1 (B4GALT1), Galactosyltransferase 2 (B4GALT2), Glucuronyltransferase 1 (UDPGT), Exostosin-Like Glycosyltransferase 3 (EXTL3), Exostosin Glycosyltransferase 1 (EXT1), Exostosin Glycosyltransferase 2 (EXT2), Heparanase (HPSE), Glypican 1 (GPC1), Glypican 2 (GPC2), Glypican 3 (GPC3), Glypican 4 (GPC4), Glypican 5 (GPCS), Glypican 6 (GPC6), Syndecan 1 (SDC1), Syndecan 2 (SDC2), Syndecan 3 (SDC3), Syndecan 4 (SDC4), Betaglycan (BGCAN/TGFBR3), CD44V3 (CD44V3), Neuropillin 1 (NRP1), Serglycin (SRGN), Perlecan (PLC), Agrin (AGRN), Collagen 18 (COL18A1), GalNAc transferase 2 (CSGalNAcT2), Chondroitin sulfate synthase 1 (Chsy 1), Chondroitin sulfate synthase 3 (Chsy3), Chondroitin sulfate polymerizing factor (Chpf), Chondroitin sulfate polymerizing factor (Chpf2), Chondroitn 4-O-sulfotransferase 1 (Chstl 1), Chondroitin 4-O-sulfotransferase 2 (Chst12), Chondroitin 4-O-sulfotransferase 3 (Chst13), Chondroitin 4-sulfate 6-O-sulfotransferase (Chst15;), Chondroitin 6 sulfotransferase-1 (Chst3), Chondroitin 6-O-sulfotransferase 2 (Chst7), Dermatan sulfate glucuronyl C5 epimerase 1 (Dse), Dermatan sulfate glucuronyl C5 epimerase-like (Dse1), Dermatan sulfate 4-O-sulfotransferase (Chst14), Aggrecan (CSPG1) (Agc1), Versican/PG-M (CSPG2) (Vcan), Neurocan (CSPG3) (Ncan), Brevican (BCAN) (Bean), Epiphycan (Dspg3) (Epyc), Procollagen, type IX, alpha 2 (Col9a2), DSD-1-proteoglycan, Phosphacan (Ptprz1), Thrombomodulin (Thbd), Endocan (Esm1), Leprecan (Prolyl 3-hydroxylase 1) (Lepre1), Decorin (Dcn), Biglycan (Bgn), Testican 1 (Spockl;
 osteonectin1) (Spock1), osteonectin2 (Spock2), Testican 2 (Spock3; osteonectn3) (Spock3), Proteoglycan-4 (Lubricin) (Prg4), NG2 (CSPG4) (Cspg4), or Invariant chain (Cd74).   
     
     
         100 . The method of  claim 59 , wherein the cell is transgenic for one or more of chondroitin sulfate synthase 1 (ChSy), Chondroitin Sulfate N-Acetylgalactosaminyltransferase 2 (CSGalNAcT2), Chondroitin Polymerizing Factor (ChPF), heparan sulfate 2-O-sulphotransferase (HS2ST), glucuronic acid epimerase (GLCE), heparan sulfate N-deacetylase/sulfotransferase-1 (HSNDST1), heparan sulfate N-deacetylase/sulfotransferase-2 (HSNDST2), Sulfatase 1 (Sulf1), Sulfatase (Su1f2), Beta-glucuronidase (GUSB), Galactosamine-6 sulfatase (GALNS), Alpha-L-iduronidase (IDUA), Sulfamidase (SGSH), N-acetyltransferase (AANAT, ARD1A, GNPNAT1, HGSNAT, MAK10, NAT1, NAT2, NATS, NAT6, NAT8, NAT8L, NAT9, NAT 10, NATI 1, NAT12, NAT13, NAT14, NAT14), Uronate-2-sulfatase (IDS), Alpha-N-acetylglucosaminidase (NAGLU), PAPS synthase (PAPSS1, PAPSS2), Xylosyltransferase 1 (XYLT1), Xylosyltransferase 2 (XYLT2), Galactosyltransferase 1 (B4GALT1), Galactosyltransferase 2 (B4GALT2), Glucuronyltransferase 1 (UDPGT), Exostosin-Like Glycosyltransferase 3 (EXTL3), Exostosin Glycosyltransferase 1 (EXT1), Exostosin Glycosyltransferase 2 (EXT2), Heparanase (HPSE), Glypican 1 (GPC1), Glypican 2 (GPC2), Glypican 3 (GPC3), Glypican 4 (GPC4), Glypican 5 (GPCS), Glypican 6 (GPC6), Syndecan 1 (SDC1), Syndecan 2 (SDC2), Syndecan 3 (SDC3), Syndecan 4 (SDC4), Betaglycan (BGCAN/TGFBR3), CD44V3 (CD44V3), Neuropillin 1 (NRP1), Serglycin (SRGN), Perlecan (PLC), Agrin (AGRN), Collagen 18 (COL18A1), GalNAc transferase 2 (CSGalNAcT2), Chondroitin sulfate synthase 1 (Chsy1), Chondroitin sulfate synthase 3 (Chsy3), Chondroitin sulfate polymerizing factor (Chpf), Chondroitin sulfate polymerizing factor (Chpf2), Chondroitn 4-O-sulfotransferase 1 (Chst11), Chondroitin 4-O-sulfotransferase 2 (Chst12), Chondroitin 4-O-sulfotransferase 3 (Chst13), Chondroitin 4-sulfate 6-O-sulfotransferase (Chst15;), Chondroitin 6 sulfotransferase-1 (Chst3), Chondroitin 6-O-sulfotransferase 2 (Chst7), Dermatan sulfate glucuronyl C5 epimerase 1 (Dse), Dermatan sulfate glucuronyl C5 epimerase-like (Dse1), Dermatan sulfate 4-O-sulfotransferase (Chst14), Aggrecan (CSPG1) (Agc1), Versican/PG-M (CSPG2) (Vcan), Neurocan (CSPG3) (Ncan), Brevican (BCAN) (Bean), Epiphycan (Dspg3) (Epyc), Procollagen, type IX, alpha 2 (Col9a2), DSD-1-proteoglycan, Phosphacan (Ptprz1), Thrombomodulin (Thbd), Endocan (Esm1), Leprecan (Prolyl 3-hydroxylase 1) (Lepre1), Decorin (Dcn), Biglycan (Bgn), Testican 1 (Spockl; osteonectin1) (Spock1), osteonectin2 (Spock2), Testican 2 (Spock3; osteonectn3) (Spock3), Proteoglycan-4 (Lubricin) (Prg4), NG2 (CSPG4) (Cspg4), or Invariant chain (Cd74).