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. 
     
     
         2 . The vector of  claim 1 , wherein the therapeutic protein is selected from the group consisting of erythropoetin, ghrelin, osteoprotegerin, RANKL, RANKL decoy, TNF-α antagonist, an IL-1 antagonist, G-CSF, GM-CSF, IFN-α, IFN-γ, angiostatin, endostatin, TNF-α, PP1DCY-LSRLOC, β-glucuronidase, IL-12, 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, iuduronate sulfatase, and ceruloplasmin. 
     
     
         3 . The vector of  claim 1 , wherein the therapeutic protein is one or more immunomodulators selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-7, IL-8, IL-9, IL-10R DN or a subunit thereof, IL-15, IL-18, IL-21, IL-23, IL-24, IL-27, GM-CSF, IFN-alpha, IFN-gamma, IFN-alpha 1, IFN alpha 2, IL-15-R-alpha, CCL3 (MIP-1a), CCL5 (RANTES), CCL7 (MCP3), XCL1 (lymphotactin), CXCL1 (MGSA-alpha), CCR7, CCL19 (MIP-3b), CXCL9 (MIG), CXCL10 (IP-10), CXCL12 (SDF-1), CCL21 (6Ckine), OX40L, 4-1BBL, CD40, CD70, GITRL, LIGHT, b-Defensin, HMGB1, Flt3L, IFN-beta, TNF-alpha, dnFADD, BCG, TGF-alpha, PD-L1 RNAi, a PD-L1 antisense oligonucleotide, TGFbRII DN, ICOS-L, S100, CD40L, p53, survivin, p53-survivin fusion, MAGE3, PSA and PSMA. 
     
     
         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 . (canceled) 
     
     
         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 . The vector of  claim 10 , wherein said first transcription factor sequence and said second transcription factor sequence are connected by an EMCV internal ribosomal entry site (IRES). 
     
     
         12 . The vector of  claim 1 , wherein said polynucleotide encoding the one or more proteins having the functions of the therapeutic protein encodes the human protein(s). 
     
     
         13 . The vector of  claim 5 , wherein said polynucleotide encoding the protein having the function of IL-12 encodes human IL-12. 
     
     
         14 . The vector of  claim 3 , wherein said immunomodulator is TNF-alpha. 
     
     
         15 . The vector of  claim 14 , wherein said immunomodulator is human TNF-alpha. 
     
     
         16 . The vector of  claim 3 , wherein said immunomodulator comprises an amino acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 37 (human TNF-alpha). 
     
     
         17 - 21 . (canceled) 
     
     
         22 . The vector of  claim 1 , further comprising 5′ untranslated region (UTR). 
     
     
         23 - 24 . (canceled) 
     
     
         25 . The vector of  claim 1 , which further comprises 3′ regulatory region. 
     
     
         26 - 31 . (canceled) 
     
     
         32 . A method of producing a population of immune cells or therapy support cells (TSC) expressing protein(s) having the function of one or more therapeutic proteins, comprising modifying the immune cells with the vector of  claim 1 . 
     
     
         33 . The method of  claim 32 , wherein said cells are human dendritic cells. 
     
     
         34 . The method of  claim 33 , wherein said dendritic cells are bone marrow dendritic cells. 
     
     
         35 . A population of immune cells or TSC expressing protein(s) having the function of one or more therapeutic proteins, comprising the vector of  claim 1 . 
     
     
         36 . The population of immune cells or TSC of  claim 35 , wherein said cells are human dendritic cells. 
     
     
         37 . An in vitro engineered immune cell or a TSC comprising the vector of  claim 1 . 
     
     
         38 . The in vitro engineered immune cell or TSC of  claim 37 , wherein said immune cell or TSC is a human dendritic cell. 
     
     
         39 . A pharmaceutical composition comprising the vector of  claim 1 . 
     
     
         40 . (canceled) 
     
     
         41 . The composition of  claim 39 , further comprising a pharmaceutically acceptable carrier. 
     
     
         42 - 102 . (canceled)