Nucleic acid constructs containing hybrid promoters for use in gene therapy

Abstract

Nucleic acid constructs containing hybrid promoters for use in gene therapy and genetic manipulation. The invention relates to a nucleic acid construct for the precise, regulated expression of genes in host cells, which construct exhibits at least one mutation which inhibits the proper expression of the expressed gene and exhibits at least one additional second mutation which relieves the inhibition due to the first mutation, to an isolated cell which harbors the nucleic acid construct, and to the use of the nucleic acid construct for preparing pharmaceuticals and for treating diseases with excessive cell proliferation.

We claim:  
     
         1 . A nucleic acid construct for the regulated expression of a transgene in a host cell comprising: 
 at least one nucleic acid sequence containing a first mutation which inhibits the proper expression of said transgene, and    at least one nucleic acid sequence containing a second mutation which abolishes the inhibition due to the first mutation.    
     
     
         2 . The nucleic acid construct of  claim 1  for the regulated expression of a transgene in a host cell, wherein: 
 (i) when said nucleic acid sequence containing said first mutation is a transgene (b) containing a mutation which inhibits the transcription and/or the translation of said transgene or inhibits the function of the pharmacologically active compound, then said nucleic acid construct further comprises a first promoter or enhancer sequence (a), which is located upstream from the 5′ end of said transgene, or  
 (i′) when said nucleic acid sequence containing said first mutation is a first promoter or enhancer sequence (a′), which contains a mutation which inhibits the function of the first promoter, then said nucleic acid construct further comprises a transgene (b′) encoding a pharmacologically active compound.  
 
     
     
         3 . A nucleic acid construct for the regulated expression of a transgene in a host cell comprising in a 5′ to 3′ end direction reading frame, the following components: 
 (i) a first promoter or enhancer sequence (a), which activates the transcription of said transgene or a first promoter or enhancer sequence (a′) which contains a mutation which inhibits the function of the first promoter (a′);  
 (ii) a transgene (b′) encoding a pharmacologically active compound or a transgene (b) containing a mutation which inhibits the transcription and/or the translation of said transgene or inhibits the function of the pharmacologically active compound encoded by said transgene;  
 (iii) a second promoter or enhancer sequence (c), which activates the basal transcription of the component (d) or which contains a mutation which inhibits the function of the second promoter; and  
 (iv) a gene (d) encoding a tRNA or a regulatory protein for abolishing the mutation in at least one of the promoters (a) and (c) or in the transgene (b).  
 
     
     
         4 . The nucleic acid construct of  claim 3 , wherein the promoters (a) and (c) are nonspecific, cell-specific or virus-specific, and wherein at least one of the promoters (a) and (c) can be activated by at least one of several activation mechanisms including nonspecifically, virus-specifically, metabolically, by tetracycline, by hypoxia, cell-specifically and cell cycle-specifically.  
     
     
         5 . The nucleic acid construct of  claim 3 , wherein the transgene (b) contains a nuclear retention signal (NRS) which is linked, at its 5′ end, directly or indirectly, to the 3′ end of the transgene, and wherein the transcription product of the NRS provides a structure for binding a nuclear export factor (NEF).  
     
     
         6 . The nucleic acid construct of  claim 5 , further comprising the following components: 
 (v) a third promoter or enhancer sequence (i) which activates the basal transcription of the NEF; and    (vi) a nucleic acid sequence encoding the NEF (k) which binds to the transcription product of the NRS, thereby mediating transport of the transcription product of the transgene out of the cell nucleus into the cytoplasm.    
     
     
         7 . The nucleic acid construct of  claim 4 , wherein the promoter or enhancer sequences (a) and (c) are identical.  
     
     
         8 . The nucleic acid construct of  claim 7 , wherein at least one of the promoter or enhancer sequences (a) and (c) is a chimeric promoter containing the promoter module CDE-CHR or E2FBS-CHR, in which said promoter module exerts an effect on the expression of a downstream gene and interacts with a neighboring, upstream, activator sequence.  
     
     
         9 . The nucleic acid construct of  claim 8 , wherein the expression of the downstream gene is cell cycle specifically inhibited.  
     
     
         10 . The nucleic acid construct of  claim 6 , wherein at least one of the promoter or enhancer sequences (a), (c) and (i) is an activator-responsive promoter unit which comprises the following components: 
 (vii) at least one promoter or enhancer sequence (e) which can be activated by at least one of several activation methods including nonspecifically, virus-specifically, metabolically, by tetracycline, cell-specifically and cell cycle-specifically;    (viii) at least one activator subunit (f) which is situated downstream from the promoter or enhancer sequence (e), and wherein the basal transcription of the activator subunit is activated by the promoter or enhancer sequence (e); and    (ix) an activator-responsive promoter (g) which is activated by the expression products of the activator subunit (f) or of several identical (f) subunits or by different (f′) activator subunits.    
     
     
         11 . The nucleic acid construct of  claim 10 , in which at least one of the promoter or enhancer sequences (a), (c) or (i), and the activator-responsive promoter (g) is a chimeric promoter, and the activator subunit (f) is a gene encoding at least one transcription factor which activates the chimeric promoter.  
     
     
         12 . The nucleic acid construct of  claim 10 , wherein the activator-responsive promoter (g) is monomers or multimers of the LexA operator in combination with the SV40 promoter and is activated by two activator-subunits (f) and (f′); wherein 
 the activator subunit (f) comprises the cDNA encoding the LexA DNA-binding protein whose 3′ end is linked to the 5′ end of the cDNA encoding the Gal80 protein; and  
 the activator subunit (f) comprises in a 5′ to 3′ end reading frame, the cDNA encoding the Gal80-binding domain of the Gal4 protein, the cDNA encoding the SV40 large T antigen, and the cDNA encoding the transactivating domain of HSV-1 VP16.  
 
     
     
         13 . The nucleic acid construct of  claim 12 , wherein the cDNA encoding the LexA DNA-binding protein encodes amino acids 1-81 or 1-202 of the LexA DNA-binding protein, the cDNA encoding the Gal80 protein encodes amino acids 1435 of the Gal80 protein, the cDNA encoding the Gal80-binding domain of the Gal4 protein encodes amino acids 851-881 of the Gal4 protein, the cDNA encoding the SV40 large T antigen encodes amino acids 126-132 of the SV40 large T antigen, and the cDNA encoding the transactivating domain encodes amino acids 406-488 of HSV-1 VP16.  
     
     
         14 . The nucleic acid construct of  claim 12 , wherein the monomers or multimers of the LexA operator are replaced with monomers or multimers of the Gal4 binding region and the cDNA encoding the LexA DNA-binding protein is replaced with the cDNA encoding the DNA-binding domain of the Gal4 protein.  
     
     
         15 . The nucleic acid construct of  claim 10 , wherein the activator-responsive promoter (g) is monomers or multimers encoding the binding sequence for the Gal4 binding protein and the activator subunit (f) comprises the nuclear localization signal (NLS) from SV40 large T antigen, and the acid transactivating domain (TAD) from HSV-1 VP16, or the activator subunit (f′) contains the nuclear localization signal (NLS) from SV40 large T; the cDNA encoding the DNA-binding domain of the Gal4 protein, and the cDNA encoding the CD4-binding sequence of the p56 Ick protein.  
     
     
         16 . The nucleic acid construct of  claim 15 , wherein NLS encodes SEQ ID NO.: 1, and the TAD encodes amino acids 406-488 from HSV-1 VP16, the cDNA for the DNA-binding domain of the Gal4 protein encodes amino acids 1-147 of the Gal4 protein, and the cDNA for the CD4-binding sequence encodes amino acids 1-71 of the p56 Ick protein.  
     
     
         17 . The nucleic acid construct of  claim 6 , wherein the nucleic acid sequence encoding the NRS is selected from the group consisting of the Rev-responsive element (RRE) from HIV-1 or V-2, the RRE-equivalent retention signal from retroviruses other than HIV-1 and HIV-2, and the RRE-equivalent retention signal from HBV.  
     
     
         18 . The nucleic acid construct of  claim 6 , wherein the NEF (k) is selected from the group consisting of the rev gene from retrovirus, the gene encoding the hnRNP-A1 protein or the gene encoding the transcription factor TFIII-A.  
     
     
         19 . The nucleic acid construct of  claim 18 , wherein the retrovirus is selected from the group consisting of HIV-1, HIV-2, visna-maedi-virus, caprine arthritis encephalitis virus, equine infectious anemia virus, feline immunodeficiency virus and HTLV.  
     
     
         20 . The nucleic acid construct of  claim 10 , wherein at least one TATA sequence in at least one of the promoters (a), (c), (g), and (i) is mutated and component (d) is a gene encoding a TATA-binding protein (TBP) which is mutated and binds to the mutated TATA box, enabling transcription.  
     
     
         21 . The nucleic acid construct of  claim 20 , wherein the TATA box is mutated to TGTAAA and the gene encoding the TBP is mutated to T in N862, to A in N889, to C in N890 and to G in N895.  
     
     
         22 . The nucleic acid construct of  claim 21 , comprising a hybrid promoter containing in a 5′ to 3′end direction reading frame, the following elements: 
 Element I comprising: 
 the promoter of the VEGF receptor I gene  
 
  containing nucleotides −1195 to +100, wherein the nucleotides of the TATA box of TATAAA in position −31 to −26 are mutated to TGTAAA; 
 the sequence GCCACC;  
 nucleotide sequence 63 to 107 of the cDNA encoding the immunoglobulin signal peptide; and  
 nucleotide sequence 93 to 1982 of the cDNA encoding β-glucuronidase; and  
 
 Element II comprising: 
 nucleotides −487 to +121 of the promoter of the cdc25C gene;  
 nucleotide sequence +1 to +1001 of the TATA box-binding protein, which is mutated in nucleotides 862 (A replaced with T), 889 and 890 (GT replaced with AC) and 895 (C replaced with G).  
 
 
     
     
         23 . The nucleic acid construct of  claim 21 , comprising a hybrid promoter containing in a 5′ to 3′ direction reading frame, the following elements: 
 Element III 
 the promoter of the VEGF receptor I gene containing nucleotides −1195 to 100, and wherein the nucleotides of the TATA box of TATAAA in position −31 to −26 are mutated to TGTAAA;  
 the sequence GCCACC;  
 nucleotide sequence 63 to 107 of the cDNA encoding the immunoglobulin signal peptide; and  
 nucleotide sequence 93 to 1982 the cDNA encoding β-glucuronidase; and  
 nucleotide sequence 7357 to 7602 of the cDNA encoding HIV-1 virus RER as the nuclear retention signal (NRS); and  
 
 Element IIa 
 nucleotides −290 to +121 of the promoter of the cdc25C gene; and  
 nucleotide sequence +1 to +1001 of the TATA box-binding protein, which is mutated in nucleotides 862 (A replaced with T), 889 and 890 (GT replaced with AC) and 895 (C replaced with G); and  
 
 Element IV 
 nucleotides −487 to +247 of the promoter of the von Willebrand factor (vWF) gene; and  
 the cDNA for HIV-1 virus REV encoding amino acid sequences 1-117 as the nuclear export factor (NEF).  
 
 
     
     
         24 . The nucleic acid construct of  claim 21 , comprising a hybrid promoter and an activator-responsive promoter unit containing in a 5′ to 3′ end direction reading frame, which contains the following elements: 
 Element V comprising: 
 1) Activator subunit A comprising: 
 nucleotides −290 to +121 of the promoter of the cdc25C gene;  
 the cDNA for the DNA-binding domain of amino acids 1 to 147 of Gal4 protein; and  
 the cDNA encoding amino acids 1 to 435 Gal80;  
 
 2) Activator subunit B comprising: 
 the promoter of the VEGF receptor I gene containing nucleotides −1195 to +100 with TGTAAA in −31 to −26 nucleotides;  
 the cDNA for the Gal80-binding domain of amino acids 851 to 881 of Gal4;  
 the nuclear localization signal (NLS) encodes SEQ ID NO.: 1; and  
 the acid transactivating domain (TAD) encoding amino acids 406 to 488 of HSV-1 VP16; and  
 
 3) Activator-responsive promoter comprising: 
 the binding sequence for Gal4 having the nucleotide sequence SEQ ID NO.: 2 operably linked to nucleotides 48 to 5191 of the SV40 basal promoter;  
 the sequence GCCACC;  
 nucleotide sequence 63 to 107 of the cDNA encoding the immunoglobulin signal peptide; and  
 nucleotide sequence 93 to 1982 of the cDNA for β-glucuronidase; and  
 
 
 Element VI comprising: 
 nucleotides −487 to +247 of the promoter of the von Willebrand factor gene; and  
 the gene for the TATA box-binding protein (nucleotide sequence 1-1001) which is mutated in nucleotides 862 (A replaced with T); 889 and 890 (GT replaced with AC) and 895 (C replaced with G).  
 
 
     
     
         25 . The nucleic acid construct of  claim 20 , wherein at least one gene in at least one of the components including the transgene (b), the nuclear export factor (k), TBP (d) and the binding proteins (f) and/or (f′) of the activator-responsive promoter (g) is mutated such that the expressed protein is unable to function, and wherein component d) is a gene for a tRNA which possesses an anticodon which is complementary to the mutation or carries an end group which takes up the correct amino acid for abolishing the mutation in said components.  
     
     
         26 . The nucleotide construct of  claim 25 , in which at least one of the following codons UAU, UUG, UAC, UCG, CAG, AAA, AAG or UGG is mutated to UAG, UAA, UAG, UGA or UGG, and the suppressor tRNA (component d)) is the gene sup F(su+3); sup C (su+4), sup D (su+1); sup E (su+2); sup G (su+5) or sup U (su+7).  
     
     
         27 . The nucleic acid construct of  claim 1 , wherein the nucleic acid is DNA.  
     
     
         28 . The nucleic acid construct of  claim 27 , wherein the nucleic acid construct is a vector.  
     
     
         29 . The nucleic acid construct of  claim 28 , wherein the vector is a plasmid vector.  
     
     
         30 . The nucleic acid construct of  claim 28 , wherein the vector is a viral vector.  
     
     
         31 . The nucleic acid construct of  claim 3 , wherein the transgene (b) is a structural gene which encodes a pharmacologically active compound which is selected from the group consisting of cytokines, interferons, growth factors, antibodies, antibody fragments, receptors for cytokines or growth factors, proteins having an antiproliferative, apoptotic, cytostatic or cytolytic effect, angiogenesis inhibitors, thrombosis-inducing proteins, coagulation inhibitors, proteins having a fibrinolytic effect, blood plasma proteins, complement-activating proteins, bacterial proteins, virus coat proteins, bacterial and parasitic antigens, tumor antigens, proteins having an effect on the blood circulation, peptide hormones, enzymes, fusion proteins composed of a ligand and an active compound, antisense RNA and ribozymes.  
     
     
         32 . The nucleic acid construct of  claim 31 , wherein the transgene (b) is a structural gene which encodes an enzyme which cleaves a precursor of a drug to form a drug.  
     
     
         33 . The nucleic acid construct of  claim 31 , wherein the transgene (b) is a structural gene which encodes a ligand/enzyme fusion protein.  
     
     
         34 . The nucleic acid construct of  claim 33 , wherein the ligand binds to proliferating endothelial cells and is selected from the group consisting of antibodies and their fragments, proteins which contain mannose terminally, cytokines, growth factors, and adhesion molecules.  
     
     
         35 . The nucleic acid construct of  claim 34 , wherein the cytokines are IL-1 or TNF, the growth factors are PDGF, bFGF, VEGF or TGFβ, and adhesion molecules are sLex, LFA-1, MAC-1, LECAM-1, VLA-1 or VLA-4.  
     
     
         36 . The nucleic acid construct of  claim 31 , wherein the ligand binds to tumor cells.  
     
     
         37 . An isolated cell containing the nucleic acid construct of  claim 1 .  
     
     
         38 . The isolated cell of  claim 37 , wherein the cell is a macrophage, a lymphocyte, an endothelial cell or a tumor cell.  
     
     
         39 . A method of inhibiting cell proliferation by contacting cells with a cell proliferation inhibiting amount of the nucleic acid construct of  claim 1 .  
     
     
         40 . A method of treating a subject having a disease involving excessive cell proliferation, said method comprising administering to said subject a cell proliferation inhibiting amount of the nucleic acid construct of  claim 1 .  
     
     
         41 . The method of  claim 40 , wherein said disease is tumors or cardiovascular diseases involving proliferation of cells in blood vessels.  
     
     
         42 . A method for treating a subject having a medical condition selected from the group consisting of autoimmune disease, allergies, inflammation, organ rejection, arthritis, infectious disease and neuron disease, said method comprising administering to said subject a medical condition treating amount of the nucleic acid construct of  claim 1 .  
     
     
         43 . The method of  claim 40 , wherein said nucleic acid construct is contained in a non-viral or viral vector.  
     
     
         44 . The method of  claim 42 , wherein said nucleic acid construct is contained in a non-viral or viral vector.  
     
     
         45 . The method of  claim 40 , wherein said nucleic acid construct is administered to the subject locally, intravenously, intraarterially, into a body cavity, into an organ or subcutaneously.  
     
     
         46 . The method of  claim 42 , wherein said nucleic acid construct is administered to the subject locally, intravenously, intraarterially, into a body cavity, into an organ or subcutaneously.  
     
     
         47 . A pharmaceutical composition containing a cell proliferation inhibiting amount of the nucleic acid construct of  claim 1  in a pharmaceutically acceptable carrier.