Therapeutic Gene-Switch Constructs and Bioreactors for the Expression of Biotherapeutic Molecules, and Uses Thereof

Abstract

The present invention relates to methods and compositions for treating, ameliorating or preventing a disease or disorder in a subject by introducing into cells of the subject a therapeutic gene switch construct that controls expression of one or more therapeutic products.

1 . (canceled) 
     
     
         2 . A method for expressing a therapeutic polypeptide or therapeutic polynucleotide in a subject, comprising:
 (a) introducing into a subject (1) a first polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor complex through operable association with a therapeutic switch promoter, and (2) a second polynucleotide encoding said therapeutic polypeptide or therapeutic polynucleotide operably associated with a factor-regulated promoter which is activated by said ligand-dependent transcription factor complex, wherein said first and second polynucleotides are introduced so as to permit expression of said ligand-dependent transcription factor complex; and   (b) administering ligand to said subject to induce expression of said therapeutic polypeptide or therapeutic polynucleotide.   
     
     
         3 . The method of  claim 2 , wherein said therapeutic switch promoter is constitutive. 
     
     
         4 . The method of  claim 2 , wherein said therapeutic switch promoter is activated under conditions associated with a disease, disorder, or condition so as to permit expression of said ligand-dependent transcription factor complex under conditions associated with said disease, disorder, or condition. 
     
     
         5 . The method of  claim 4 , wherein said disease, disorder, or condition is responsive to said therapeutic polypeptide or therapeutic polynucleotide. 
     
     
         6 . The method of  claim 2 , wherein said therapeutic polypeptide or therapeutic polynucleotide is expressed and disseminated at a level sufficient to treat, ameliorate, or prevent said disease, disorder, or condition. 
     
     
         7 . The method of  claim 2 , wherein said gene switch is an ecdysone receptor (EcR)-based gene switch. 
     
     
         8 . The method of  claim 7 , wherein said ligand binds to the EcR ligand binding domain. 
     
     
         9 . The method of  claim 8 , wherein said ligand is a diacylhydrazine. 
     
     
         10 . The method of  claim 9 , wherein said ligand is RG-115819, RG-115932, or RG-115830. 
     
     
         11 - 16 . (canceled) 
     
     
         17 . The method of  claim 2 , wherein said first and second polynucleotides are introduced into said subject in one or more modified cells. 
     
     
         18 . The method of  claim 17 , wherein said modified cells are prepared by introducing said first and second polynucleotides into cells that have been isolated from said subject to produce modified autologous cells, which are then re-introduced into said subject. 
     
     
         19 . The method of  claim 17 , wherein said modified cells are modified non-autologous (MNA) cells into which said first and second polynucleotides have been incorporated. 
     
     
         20 - 22 . (canceled) 
     
     
         23 . The method of  claim 19 , wherein said MNA cells are C2C12 mouse myoblast cells, HEK293 human embryonic kidney cells, ARPE-19 cells, hMSC cells, pancreatic islet cells, MDCK cell, a CHO cell, an astrocyte derived cell, an oligodendrocyte derived cell, or a myoblast derived cell. 
     
     
         24 - 25 . (canceled) 
     
     
         26 . The method of  claim 17 , wherein said modified cells have been treated such that said cells, upon introduction into said subject, are protected from the subject's immune system. 
     
     
         27 . The method of  claim 26 , wherein said modified cells are contained within a barrier system which allows dissemination of said therapeutic protein or therapeutic polynucleotide, but which prevents direct contact of said modified cells with cells of the subject's immune system. 
     
     
         28 . The method of  claim 27 , wherein said barrier system comprises modified cells with a conformal coating. 
     
     
         29 . The method of  claim 28 , wherein said conformal coating comprises a polymer. 
     
     
         30 . The method of  claim 29 , wherein said polymer is selected from the group consisting of polyethylene glycol, and hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA). 
     
     
         31 . The method of  claim 27 , wherein said barrier system comprises encapsulated modified cells. 
     
     
         32 . The method of  claim 31 , wherein said modified cells are contained in a macroencapsulation device. 
     
     
         33 . The method of  claim 32 , wherein said macroencapsulation device comprises one or more synthetic membranes. 
     
     
         34 . The method of  claim 33 , wherein said macroencapsulation device comprises two or more synthetic membranes, said synthetic membranes comprising different pore sizes so as to regulate mass transit through said macroencapsulation device. 
     
     
         35 . The method of  claim 32 , wherein said macroencapsulation device comprises a semi-permeable polymer outer membrane and an internal scaffold which supports cells. 
     
     
         36 - 61 . (canceled) 
     
     
         62 . A method for expressing a therapeutic polypeptide or therapeutic polynucleotide in one or more modified cells, comprising:
 (a) introducing into a cell (1) a first polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor complex through operable association with a therapeutic switch promoter, and (2) a second polynucleotide encoding a therapeutic polypeptide or therapeutic polynucleotide operably associated with a factor-regulated promoter which is activated by said ligand-dependent transcription factor complex, thereby producing a modified cell; and   (b) administering ligand to said modified cell to induce expression of said therapeutic polypeptide or therapeutic polynucleotide.   
     
     
         63 - 64 . (canceled) 
     
     
         65 . The method of  claim 62 , wherein said method is carried out in vitro. 
     
     
         66 . The method of  claim 62 , wherein said method is carried out ex vivo in a cell that has been isolated from a subject. 
     
     
         67 . The method of  claim 62 , wherein said method is carried out in vivo. 
     
     
         68 . A nucleic acid composition comprising a first polynucleotide encoding a gene switch, said gene switch comprising at least one transcription factor sequence, wherein said at least one transcription factor sequence encodes a ligand-dependent transcription factor complex through operable association to a therapeutic switch promoter. 
     
     
         69 . (canceled) 
     
     
         70 . The nucleic acid composition of  claim 68 , wherein said therapeutic switch promoter is activated under conditions associated with a disease, disorder, or condition so as to permit expression of said ligand-dependent transcription factor complex under conditions associated with said disease, disorder, or condition. 
     
     
         71 . The nucleic acid composition of  claim 68 , further comprising a second polynucleotide encoding a polypeptide or polynucleotide associated with a disease, disorder, or condition through operable association with a promoter which is activated by said ligand-dependent transcription factor complex. 
     
     
         72 - 90 . (canceled) 
     
     
         91 . A modified cell comprising the nucleic acid composition of  claim 71 . 
     
     
         92 - 99 . (canceled) 
     
     
         100 . A bioreactor device comprising one or more modified cells as recited in  claim 91 . 
     
     
         101 - 142 . (canceled)