Methods to enhance and confine expression of genes

Abstract

The present invention provides a novel approach to gene therapy of restricted areas such as tumors. The methods introduced here comprise: (a) placing a gene of interest in a plasmid vector driven by a heat or light inducible promoter; (b) modifying this vector by including a tetracycline responsive fusion protein which acts as a transcriptional activator, thus permitting regulation of gene expression by varying the levels of drug and; (c) modifying this vector by including DNA sequences that reduce or eliminate expression of genes in normal bystander cells. Also provided are a set of vectors for both sustained and regulable expression. There is also presented novel vectors for the gene therapy treatment of local and metastatic breast, ovarian and prostate cancer.

What is claimed is:  
     
         1 . A method of achieving localized, temporal expression of a gene under control of a heat inducible promoter, comprising the steps of: 
 inserting said gene into a cloning site of a pDATH-X (Dominant negative, Antisense, TET-ON controllable Heat shock promoter plasmid) vector, said vector comprising:    a) cassette 1 comprising TET-ON expressed under the control of a heat shock promoter and a tet operator, wherein said TET-ON consists of a fusion of the coding sequences for amino acids 1-207 of tetracycline repressor and the C-terminus last 130 amino acid transcription activation domain of VP16 protein of the herpes simplex virus, wherein said heat shock promoter consists of heat shock response elements (−260 to 30) of the human heat shock 70 gene promoter linked to a minimal cytomegalovirus promoter, pCMV; wherein said tet operator consists of 19 base pair inverted repeats of operator O2 of TN10 to which said tet repressor and TET-ON bind;    b) cassette 2 comprising a cloning site for a therapeutic gene downstream of a tetp-CMV promoter consisting of a tet operator linked to a minimal cytomegalovirus promoter, pCMV, wherein said tet operator consists of 19 base pair inverted repeats of operator O2 of TN10 to which said tet repressor and TET-ON bind;    c) cassette 3 comprising antisense TET-ON under the control of pCMV promoter, wherein said antisense TET-ON consists of an antisense sequence complementary to the first 80 nucleotides of the TET-ON sequence including the ATG start codon; and    d) cassette 4 comprising a dominant negative TET-ON under the control of pCMV promoter, wherein said dominant negative TET-ON consists of a tet repressor without a VP16 transactivation domain;    introducing the vector containing said gene into the host organism; and    applying heat energy to a location on said host organism where expression of said gene is desired.    
     
     
         2 . The method of  claim 1 , where said host organism is a human.  
     
     
         3 . A recombinant vector, pDATE-X (Dominant negative, Antisense, TET-ON controllable EGR promoter expression plasmid), said vector comprising the cassettes: 
 (a) cassette 1 comprising the TET-ON sequence under the control of the EGRp, the tetracycline operator binding site and pCMV;    (b) cassette 2 comprising a therapeutic gene X under the control of the tetp-pCMV promoter;    (c) cassette 3 comprising antisense TET-ON under the control of the pCMV promoter; and    (d) cassette 4 comprising dominant negative TET-ON under the control of the pCMV promoter.    
     
     
         4 . A recombinant vector, pRIBs-X, (Radiation-Inducible, Breast-specific Promoter) expression vector, said vector comprising the cassettes: 
 (a) cassette 1 comprising “Gal-DBD-mx” which is a fusion open reading frame encoding the N-terminus (amino acids 1-147) DNA-binding domain of the yeast GAL4 protein (Gal-DBD) fused to the basic helix-loop-helix-leucine zipper domain of Max (amino acids 8-112) followed by SV40 poly A, wherein the resulting fusion gene GAL-DBD-mx is controlled by the radiation inducible Egr-1 promoter;    (b) cassette 2 comprising the minimal CMV promoter, “antisense Gal-DBD-mx”, which is an antisense construct complementary to the Gal-DBD-mx sequence, an internal ribosomal entry site (IRES) and “Gal-DBD” which competes with the Gal-DBD-mx for the pGAL binding site;    (c) cassette 3 comprising “VP16-TA-mc” which is a fusion ORF encoding at the N-terminus the first 11 amino acids of Gal4 (amino acids 1-147), followed by the nuclear localization signal of the SV40 large T antigen, the 130 amino acid C-terminus transactivation domain of the herpes simplex viral protein VP16, the basic helix-loop-helix-leucine zipper domain of c-Myc (amino acids 350-439), followed by SV40 polyA, wherein the resulting fusion gene, VP16-TA-mc, is under the control of the c-erbB2 promoter “perB2” up to the first ATG;    (d) cassette 4 comprising “Galp”, five copies of a 17-mer DNA-binding site for Gal4, wherein a TET-ON sequence is placed under the control of the GAPp-ptet promoter and a therapeutic gene X is linked to the TET-IN via an IRES;    (e) cassette comprising an antisense TET-ON which is a sequence consisting of the complementary sequence to the first 80 bases of the TET-ON sequence including the ATG under the control of the pCMV promoter; and    (f) cassette 6 comprising a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207.    
     
     
         5 . The recombinant vector of  claim 4 , wherein the perbB2 promoter of cassette 3 is replaced with the whey acidic protein promoter.  
     
     
         6 . The recombinant vector of  claim 4 , wherein the perbB2 promoter of cassette 3 is replaced with the stromelysin 3 promoter.  
     
     
         7 . The recombinant vector of  claim 4 , wherein said gene X is a gene encoding tumor necrosis factor alpha.  
     
     
         8 . A method of treating local and metastatic breast and ovarian cancer comprising the step of: 
 administering the expression vector of  claim 4  to an individual in need of such treatment.    
     
     
         9 . A method of treating local and metastatic breast and ovarian cancer comprising the step of: 
 administering the expression vector of  claim 4  to an individual in need of such treatment.    
     
     
         10 . A method of treating local and metastatic breast and ovarian cancer comprising the step of: 
 administering the expression vector of  claim 6  to an individual in need of such treatment.    
     
     
         11 . A recombinant pRIPs-X (Radiation-Inducible, Prostate-specific Promoter) expression vector, said vector comprising the cassettes: 
 (a) cassette 1 comprising “Gal-DBD-mx” which is a fusion open reading frame encoding the N-terminus (amino acids 1-147) DNA-binding domain of the yeast GAL4 protein fused to the basic helix-loop-helix leucine zipper domain of Max (amino acids 8-112) followed by SV40 polyA, wherein the resulting fusion gene GAL-DBD-mx is controlled by the radiation inducible Egr-1 promoter;    (b) cassette 2 comprising the minimal CMV promoter, antisense Gal-DBD-mx, which is an antisense construct complementary to the Gal-DBD-mx sequence, IRES, which is an internal ribosomal entry site and Gal-DBD which competes with the Gal-DBD-mx for the pGAL binding site;    (c) cassette 3 comprising “VP16-TA-mc”, a fusion open reading frame encoding at the N-terminus the first 11 amino acids of Gal4, followed by the nuclear localization signal of the SV40 large T antigen, the 130 amino acid C-terminus transactivation domain of the herpes simplex viral protein VP16, the basic helix-loop-helix leucine zipper domain of c-Myc (amino acids 350-439), followed by SV40 polyA, wherein the resulting fusion gene, VP16-TA-mc, is under the control of the probasin gene promoter “pProbasin” up to the first ATG;    (d) cassette 4 comprising GALp, five copies of the 17-mer DNA-binding site for Gal4, wherein the TET-ON sequence is under the control of the GALp-ptet promoter and a therapeutic gene X is linked to the TET-ON via an internal ribosomal entry site;    (e) cassette 5 comprising an antisense TET-ON which is a sequence consisting of the complementary sequence to the first 80 bases of the TET-ON sequence including the ATG, under the control of the pCMV promoter; and    (f) cassette 6 comprising a dominant negative TET-ON consisting of the coding sequence for amino acids 1-207.    
     
     
         12 . The recombinant vector of  claim 11 , wherein said probasin promoter of cassette 3 is replaced with the prostate specific antigen promoter.  
     
     
         13 . The recombinant vector of  claim 11 , wherein said gene X is tumor necrosis factor alpha.  
     
     
         14 . A method of treating local and metastatic prostate cancer comprising the step of: 
 administering the expression vector of  claim 11  to an individual in need of such treatment.    
     
     
         15 . A method of treating local and metastatic prostate cancer comprising the step of: 
 administering the expression vector of  claim 12  to an individual in need of such treatment.    
     
     
         16 . A recombinant expression vector, pHIBs-X (Heat Inducible, Breast-specific promoter), said vector comprising the cassettes: 
 (a) cassette 1 comprising Gal-DBD-mx which is a fusion open reading frame encoding the N-terminus (amino acids 1-147) DNA-binding domain of the yeast GAL4 protein fused to the basic helix-loop-helix leucine zipper domain of Max (amino acids 8-112) followed by SV40 polyA, wherein the resulting fusion gene GAL-DBD-mx is controlled by the heat inducible heat shock protein promoter;    (b) cassette 2 comprising the minimal CMV promoter, antisense Gal-DBD-mx, a construct complementary to the Gal-DBD-mx sequence, an internal ribosomal entry site and Gal-DBD, which competes with the Gal-DBD-mx for the pGAL binding site;    (c) cassette 3 comprising “VP16-TA-mc” which is a fusion open reading frame encoding at the N-terminus the first 11 amino acids (amino acids 1-147), followed by the nuclear localization signal of the SV40 large T antigen, the 130 amino acid C-terminus transactivation domain of the herpes simplex viral protein VP16, the basic helix-loop-helix leucine zipper domain of c-Myc (amino acids 350-439), followed by SV40 polyA, wherein the resulting fusion gene VP16-TA-mc is under the control of the c-erbB2 gene promoter “perbB2” up to the first ATG;    (d) cassette 4 contains GALp, five copies of a 17-mer DNA-binding site for Gal4, wherein the TET-ON sequence is under the control of the GALp-ptet promoter and a therapeutic gene, X, is linked to the TET-ON via an internal ribosomal entry site;    (e) cassette 5 comprising an antisense TET-ON which is a sequence consisting of the complementary sequence to the first 80 bases of the TET-ON sequence including the ATG, under the control of the pCMV promoter; and    (f) cassette 6 comprising a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207.    
     
     
         17 . The recombinant vector of  claim 16 , wherein the perbB2 promoter of cassette 3 is replaced with the whey acidic protein promoter.  
     
     
         18 . The recombinant vector of  claim 16 , wherein the perbB2 promoter of cassette 3 is replaced with the stromelysin 3 promoter.  
     
     
         19 . The method of  claim 16 , wherein said therapeutic gene is tumor necrosis factor alpha.  
     
     
         20 . A method of treating local and metastatic breast and ovarian cancer comprising the step of: 
 administering the expression vector of  claim 16  to an individual in need of such treatment.    
     
     
         21 . A method of treating local and metastatic breast and ovarian cancer comprising the step of: 
 administering the expression vector of  claim 17  to an individual in need of such treatment.    
     
     
         22 . A method of treating local and metastatic breast and ovarian cancer comprising the step of: 
 administering the expression vector of  claim 18  to an individual in need of such treatment.    
     
     
         23 . A recombinant vector, pHIPs-X (Heat-Inducible, Prostate-specific Promoter), said vector comprising the cassettes: 
 (a) cassette 1 comprising Gal-DBD-mx which is a fusion open reading frame encoding the N-terminus (amino acids 1-147) DNA-binding domain of the yeast GAL4 protein fused to the basic helix-loop-helix leucine zipper domain of Max (amino acids 8-112) followed by SV40 polyA, wherein the resulting fusion gene GAL-DBD-mx is controlled by the heat inducible heat shock protein promoter;    (b) cassette 2 comprising the minimal CMV promoter (mCMVp), antisense Gal-DBD-mx, a construct complementary to the Gal-DBD-mx sequence, an internal ribosomal entry site and Gal-DBD, which competes with the Gal-DBD-mx for the pGAL binding site;    (c) cassette 3 comprising “VP16-TA-mc”, a fusion open reading frame encoding at the N-terminus the first 11 amino acids of Gal4, followed by the nuclear localization signal of the SV40 large T antigen, the 130 amino acid C-terminus transactivation domain of the herpes simplex viral protein VP16, the basic helix-loop-helix leucine zipper domain of c-Myc (amino acids 350-439), followed by SV40 polyA, wherein the resulting fusion gene, VP16-TA-mc, is under the control of the probasin gene promoter “pProbasin” up to the first ATG;    (d) cassette 4 comprising GALp, five copies of a 17-mer DNA-binding site for Gal4, wherein the TET-ON sequence is under the control of the GALp-ptet promoter and a therapeutic gene, X, is linked to the TET-ON via an internal ribosomal entry site;    (e) cassette 5 comprising an antisense TET-ON which is a sequence consisting of the complementary sequence to the first 80 bases of the TET-ON sequence including the ATG, under the control of the pCMV promoter; and    (f) cassette 6 comprising a dominant negative TET-ON consisting of the coding sequences for amino acids 1-207.    
     
     
         24 . The recombinant vector in  claim 23 , wherein the probasin promoter is replaced with the prostate-specific antigen promoter.  
     
     
         25 . The recombinant vector of  claim 23 , wherein said therapeutic gene is tumor necrosis alpha.  
     
     
         26 . A method of treating local and metastatic prostate cancer comprising the step of: 
 administering the expression vector of  claim 23  to an individual in need of such treatment.    
     
     
         27 . A method of treating local and metastatic prostate cancer comprising the step of: 
 administering the expression vector of  claim 25  to an to individual in need of such treatment.