Vectors Conditionally Expressing Therapeutic Proteins, Host Cells Comprising the Vectors, and Uses Thereof

Abstract

This invention relates to the field of therapeutics. Most specifically, the invention provides methods of generating conditionally expressing vectors for one or more immunomodulators under the control of a gene expression modulation system in the presence of activating ligand and uses for therapeutic purposes in animals. These vector may be provided to treat a variety of disorders, e.g., neoplastic disorders, through direct injection or through in vitro engineered cells, such as dendritic cells.

1 . A vector for conditionally expressing protein(s) having the function(s) of one or more therapeutic protein comprising a polynucleotide encoding a gene switch, wherein said polynucleotide comprises (1) at least one transcription factor sequence which is operably linked to a promoter, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor, and (2) a polynucleotide encoding one or more proteins having the function of an therapeutic protein operably linked to a promoter which is activated by said ligand-dependent transcription factor, wherein said vector is not contained within a cell upon in vivo administration. 
     
     
         2 . (canceled) 
     
     
         3 . (canceled) 
     
     
         4 . The vector of  claim 1 , wherein said vector is selected from the group consisting of plasmid, adenovirus, retrovirus, adeno-associated virus, pox virus, baculovirus, vaccinia virus, herpes simplex virus, Epstein-Barr virus, adenovirus, geminivirus, caulimovirus, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers. 
     
     
         5 . The vector of  claim 4 , wherein said vector is an adenoviral vector. 
     
     
         6 . The vector of  claim 1 , further comprising a polynucleotide encoding a protein having the function of IL-12. 
     
     
         7 . The vector of  claim 6 , wherein said polynucleotide encoding said one or more proteins having the functions of the immunomodulator and said polynucleotide encoding said protein(s) having the function of IL-12 are under control of a regulated promoter of said gene switch. 
     
     
         8 . The vector of  claim 1 , wherein said gene switch is an ecdysone receptor (EcR)-based gene switch. 
     
     
         9 . The vector of  claim 1 , wherein said polynucleotide encoding a gene switch comprises a first transcription factor sequence under the control of a first promoter and a second transcription factor sequence under the control of a second promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. 
     
     
         10 . The vector of  claim 1 , wherein said polynucleotide encoding a gene switch comprises a first transcription factor sequence and a second transcription factor sequence under the control of a promoter, wherein the proteins encoded by said first transcription factor sequence and said second transcription factor sequence interact to form a protein complex which functions as a ligand-dependent transcription factor. 
     
     
         11 . (canceled) 
     
     
         12 . (canceled) 
     
     
         13 . The vector of  claim 6 , wherein said polynucleotide encoding the protein having the function of IL-12 encodes human IL-12. 
     
     
         14 .- 38 . (canceled) 
     
     
         39 . A pharmaceutical composition comprising the vector of  claim 1 . 
     
     
         40 .- 49 . (canceled) 
     
     
         50 . A method of treating a disease or disorder, comprising administering (1) the vector of  claim 1 , and (2) a therapeutically effective amount of one or more activating ligands to a mammal in need thereof. 
     
     
         51 . The method of  claim 50 , wherein said disease or disorder is a tumor in said mammal. 
     
     
         52 . The method of  claim 50  or  claim 51 , wherein said treating is reducing a size of a tumor or preventing a tumor in said mammal. 
     
     
         53 .- 59 . (canceled) 
     
     
         60 . The method of  claim 51 , wherein said tumor is a benign tumor. 
     
     
         61 . The method of  claim 50 , wherein said tumor is a malignant tumor. 
     
     
         62 . The method of  claim 50 , wherein said tumor is a melanoma. 
     
     
         63 . The method of  claim 50 , wherein said tumor is a malignant melanoma skin cancer. 
     
     
         64 . The method of  claim 50 , wherein said ligand is a diacylhydrazine. 
     
     
         65 . The method of  claim 50 , wherein said ligand is selected from RG-115819, RG-115932, and RG-115830. 
     
     
         66 . The method of  claim 50 , wherein said ligand is an amidoketone or oxadiazoline. 
     
     
         67 .- 70 . (canceled) 
     
     
         71 . A kit comprising the vector of any one of  claims 1 ,  4 - 10  and  13 , and (b) a ligand that activates the gene switch. 
     
     
         72 . The kit of  claim 71 , wherein the ligand is RG-115819, RG-115830 or RG-115932. 
     
     
         73 . (canceled) 
     
     
         74 . (canceled) 
     
     
         75 . The method of  claim 50 , wherein the disease is selected from the group consisting of chronic renal disease, osteoarthritis, oncology, viral upper respiratory infection, feline plasma cell stomatitis, feline eosinophillic granulomas, feline leukemia virus infection, canine distemper infection, systemic fungal infections, cardiomyopathy, mucopolysaccharidosis VII, and infectious disease. 
     
     
         76 . The method of  claim 50 , wherein the infectious disease is selected from the group consisting of Bovine respiratory disease, Porcine respiratory disease, Avian influenza, Avian infectious bronchitis, Bovine spongiform encephalopathy, Canine leishmaniasis, Chronic wasting disease, Classical swine fever, Echinococcus, Enzootic pneumonia, FIP, Foot-and-mouth disease, Jaagsiekte, Maedi-Visna, Mastitis in animals, Microsporum canis, Orf (animal disease), Peste des petits ruminants, Pox diseases, Psittacine beak and feather disease, Rabies, Mediterranean fever (Brucellosis) or Bang's disease or undulant fever, Malta fever, contagious abortion, epizootic abortion,  Salmonella  food poisoning, enteric paratyphosis, Bacillary dysentery, Pseudotuberculosis, plague, pestilential fever, Tuberculosis, Vibrios, Circling disease, Weil's disease (Leptospirosis) or canicola fever, Hemorrhagic jaundice (Leptospira icterohaemorrhagiae), dairy worker fever (L. hardjo), Relapsing fever, tick-borne relapsing fever, spirochetal fever, vagabond fever, famine fever, Lyme arthritis, Bannworth's syndrome (lime disease), tick-borne meningopolyneuritis, erythema chronicum migrans, Vibriosis, Colibacteriosis, colitoxemia, white scours, gut edema of swine, enteric paratyphosis, Staphylococcal alimentary toxicosis, staphylococcal gastroenteritis, Canine Corona Virus (CCV) or canine parvovirus enteritis, feline infectious peritonitis virus, transmissible gastroenteritis (TGE) virus, Hagerman Redmouth Disease (ERMD), Infectious Hematopoietic necrosis (IHN), porcine  Actinobacillus  ( Haemophilus ) pleuropneumonia, Hansen's disease, Streptotrichosis, Mycotic Dermatitis of Sheep, Pseudoglanders, Whitmore's disease, Francis' disease, deer-fly fever, rabbit fever, O'Hara disease, Streptobacillary fever, Haverhill fever, epidemic arthritic erythema, sodoku, Shipping or transport fever, hemorrhagic septicemia, Ornithosis, Parrot Fever, Chlamydiosis, North American blastomycosis, Chicago disease, Gilchrist's disease, Cat Scratch Fever, Benign Lymphoreticulosis, Benign nonbacterial Lymphadenitis, Bacillary Angiomatosis, Bacillary Peliosis Hepatis, Query fever, Balkan influenza, Balkan grippe, abattoir fever, Tick-borne fever, pneumorickettsiosis, American Tick Typhus, Tick-borne Typhus Fever, Vesicular Rickettsiosis, Kew Gardens Spotted Fever, Flea-borne Typhus Fever, Endemic Typhus Fever, Urban Typhus, Ringworm, Dermatophytosis, Tinea, Trichophytosis, Microsporosis, Jock Itch, Athlete's Foot,  Sporothrix schenckii , dimorphic fungus, Cryptococcosis and histoplasmosis, Benign Epidermal Monkeypox, BEMP, Herpesvirus simiae, Simian B Disease, Type C lethargic encephalitis, Yellow fever, Black Vomit, hantavirus pulmonary syndrome, Korean Hemorrhagic Fever, Nephropathia Epidemica, Epidemic Hemorrhagic Fever, Hemorrhagic Nephrosonephritis, lymphocytic choriomeningitis, Venezuelan equine encephalitis, California encephalitis/La crosse encephalitis, African Hemorrhagic Fever, Green or Vervet Monkey Disease, Hydrophobia, Lyssa, Infectious hepatitis, Epidemic hepatitis, Epidemic jaundice, Rubeola, Morbilli, Swine and Equine Influenza, Fowl Plague, Newcastle disease, Piroplasmosis, toxoplasmosis, African Sleeping Sickness, Gambian Trypanosomiasis, Rhodesian Trypanosomiasis, Chagas's Disease, Chagas-Mazza Disease, South American Trypanosomiasis, Entamoeba histolytica, Balantidial dysentery, cryptosporidiosis, giardiasis, Cutaneous leishmaniasis: Chiclero ulcer, espundia, pianbols, uta, and buba (in the Americas); oriental sore, Aleppo boil (in the Old World); Bagdad boil, Delhi boil, Baum ulcer, Visceral leishmaniasis: kala-azar, Microsporidiosis, Anisakiasis, Trichinosis, Angiostrongylosis, eosinophilic meningitis or meningoencephalitis (A. cantonensis), abdominal angiostrongylosis (A. costaricensis), Uncinariasis, Necatoriasis, Hookworm Disease, Capillariasis, Brugiasis, Toxocariasis, Oesophagostomiasis, Strongyloidiasis, Trichostrongylosis, Ascaridiasis, Diphyllobothriasis, Sparganosis, Hydatidosis, Hydatid Disease, Echinococcus granulosis, Cystic hydatid disease, Tapeworm Infection, Schistosoma, Burkitt lymphoma caused by EBV, Rous sarcoma caused by Rous retrovirus, Kaposi' sarcoma caused by herpes virus type 8, adult T-cell leukemia caused by HTLV-I retrovirus, and hairy cell leukemia caused by HTLV-II. 
     
     
         77 . The method of  claim 75 , wherein the infectious disease is selected from the group consisting of Bovine respiratory disease, Porcine respiratory disease, and Avian influenza. 
     
     
         78 . The method of  claim 75 , wherein the oncology is selected from the group consisting of osteosarcoma, leukemia, and lymphoma. 
     
     
         79 . The method of  claim 50 , wherein the one or more proteins is selected from the group consisting of erythropoetin, ghrelin, osteoprotegerin, RANKL, RANKL decoy, TNF-a antagonist, an IL-1 antagonist, G-CSF, GM-CSF, IFN-α, IFN-γ, angiostatin, endostatin, TNF-α, PP1DCY-LSRLOC, β-glucuronidase, and IL-12. 
     
     
         80 . (canceled) 
     
     
         81 . The method of  claim 50 , wherein said disease or disorder is a lysosomal storage disorder. 
     
     
         82 . The method of  claim 81 , wherein the lysosomal storage disorder is selected from the group consisting of Pompe disease/Glycogen storage disease type II, Gaucher Disease (Type I, Type II, Type III), Fabry disease, Mucopolysaccharidosis II (Hunter syndrome), Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome), Mucopolysaccharidosis I, Metachromatic Leukodystrophy, Neuronal Ceroid Lipofuscinoses or CLN6 disease (Atypical Late Infantile, Late Onset variant, Early Juvenile, Finnish Variant Late Infantile CLN5, Jansky-Bielschowsky disease/Late infantile CLN2/TPP1 Disease, Kufs/Adult-onset NCL/CLN4 disease, Northern Epilepsy/variant late infantile CLN8, Santavuori-Haltia/Infantile CLN1/PPT disease, Beta-mannosidosis), Batten-Spielmeyer-Vogt/Juvenile NCL/CLN3 disease, Sanfilippo syndrome Type A, Sanfilippo syndrome Type B, Sanfilippo syndrome Type C, Sanfilippo syndrome Type D, MPSI Hurler Syndrome, Niemann-Pick Disease (Type A, Type B, Type C, Type D), Activator Deficiency/GM2 Gangliosidosis, Alpha-mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, Cystinosis, Danon disease, Farber disease, Fucosidosis, Galactosialidosis (Goldberg Syndrome), GM1 gangliosidosis (Infantile, Late infantile/Juvenile, Adult/Chronic), I-Cell disease/Mucolipidosis II, Infantile Free Sialic Acid Storage Disease/ISSD, Juvenile Hexosaminidase A Deficiency, Krabbe disease (Infantile Onset, Late Onset), Mucopolysaccharidoses disorders (Pseudo-Hurler polydystrophy/Mucolipidosis IIIA, Scheie Syndrome, MPS I Hurler-Scheie Syndrome, Morquio Type A/MPS IVA, Morquio Type B/MPS IVB, MPS IX Hyaluronidase Deficiency, Sly Syndrome (MPS VII), Mucolipidosis I/Sialidosis, Mucolipidosis IIIC, Mucolipidosis type IV), Multiple sulfatase deficiency, Pycnodysostosis, Sandhoff disease/Adult Onset/GM2 gangliosidosis, Sandhoff disease/GM2 gangliosidosis—Infantile, Sandhoff disease/GM2 gangliosidosis—Juvenile, Schindler disease, Salla disease, Infantile Sialic Acid Storage Disease, Tay-Sachs/GM2 gangliosidosis, Wolman disease, Asparylglucosaminuria, and prosaposin. 
     
     
         83 . The method of  claim 82 , wherein the lysosomal storage disorder is selected from the group consisting of Pompe disease/Glycogen storage disease type II, Gaucher Disease (Type I, Type II, Type III), Fabry disease, Mucopolysaccharidosis II (Hunter syndrome), Mucopolysaccharidosis VI (Maroteaux-Lamy syndrome), Mucopolysaccharidosis I, and Metachromatic Leukodystrophy. 
     
     
         84 . The method of  claim 81 , wherein the one or more proteins is selected from the group consisting of a-galactosidase A, Arylsulfatase A, a-glucosidase, b-glucosidase, glucocerebrosidase, CLN6 protein, Juvenile associated with CLN3, N-sulfoglucosamine sulfohyrolase (SGSH), a-N-acetylglucosaminidase, acetyl-CoA-glucosaminide acetyltransferase, N-acetylglucosamine-6-sulfatase, a-L-iduronidase, arylsulfatase B, acid sphingomyelinase, and iuduronate sulfatase. 
     
     
         85 . The method  claim 50 , wherein said disease or disorder is a liver disease. 
     
     
         86 . The method of  claim 85 , wherein the liver disease is Hepatitis B. 
     
     
         87 . The method of  claim 85 , wherein the liver disease is Hepatitis C. 
     
     
         88 . The method of  claim 85 , wherein the protein is by IFN-α. 
     
     
         89 . The method of  claim 85 , wherein the protein is ceruloplasmin. 
     
     
         90 . The method  claim 50  wherein said disease or disorder is an ocular disease. 
     
     
         91 . The method of  claim 90 , wherein said ocular disease is selected from the group consisting of: glaucoma, Open Angle Glaucoma, Angle Closure Glaucoma, Aniridic Glaucoma, Congenital Glaucoma, Juvenile Glaucoma, Lens-Induced Glaucoma, Neovascular Glaucoma, Post-Traumatic Glaucoma, Steroid-Induced Glaucoma, Sturge-Weber Syndrome Glaucoma, and Uveitis-Induced Glaucoma, diabetic retinopathy, macular degeneration, macular degeneration, choroidal neovascularization, vascular leak, and/or retinal edema, bacterial conjunctivitis, fungal conjunctivitis, viral conjunctivitis, uveitis, keratic precipitates, macular edema, inflammation response after intra-ocular lens implantation, uveitis syndromes, retinal vasculitis, sarcoidosis, Eales disease, acute retinal necrosis, Vogt Koyanaki Harada syndrome, occular toxoplasmosis, radiation retinopathy, proliferative vitreoretinopathy, endophthalmitis, ocular glaucomas, ischemic optic neuropathy, thyroid associated orbitopathy, orbital pseudotumor, pigment dispersion syndrome (pigmentary glaucoma), scleritis, episcleritis choroidopathies, retinopathies (for example, cystoid macular edema, central serous choroidopathy and presumed ocular histoplasmosis syndrome, retinal vascular disease, retinal artery occlusions, retinal vein occlusions, retinopathy of prematurity, retinitis pigmentosa, familial exudative vitreoretinopathy (FEVR), idiopathic polypoidal choroidal vasculopathy, epiretinal macular membranes and cataracts. 
     
     
         92 .- 102 . (canceled)