Self-enhancing, pharmacologically controllable expression systems

Abstract

Self-enhancing, pharmacologically controllable expression systems The invention relates to a nucleic acid construct which constitutes a self-enhancing expression system and which comprises the following components: at least one first structural gene that encodes an active compound; at least one second structural gene that encodes a transcription factor protein; and at least one activation sequence comprised of at least one sequence that binds the transcription factor protein and at least one promoter sequence; wherein each activation sequence activates the expression of a structural gene and the expression of the transcription factor protein; and to the use of the nucleic acid construct for preparing a drug for treating diseases.

1 . A nucleic acid construct that comprises: 
 at least one first structural gene that encodes an active compound;    at least one second structural gene that encodes a transcription factor protein; and    at least one activation sequence comprised of at least one sequence that binds the transcription factor protein and at least one promoter sequence;    wherein each activation sequence activates the expression of a structural gene and the expression of the transcription factor protein.    
     
     
         2 . A nucleic acid construct according to  claim 1 , wherein an activation sequence is attached to the 5′ end of the first structural gene and an activation sequence is attached to the 5′ end of the transcription factor protein gene.  
     
     
         3 . A nucleic acid construct according to  claim 1 , that comprises two identical transcription factor protein binding sequences and two non-identical promoters.  
     
     
         4 . A nucleic acid construct according to  claim 1 , further comprising an internal ribosomal entry site wherein said internal ribosomal entry site participates in activating expression of the transcription factor protein by the activation sequence.  
     
     
         5 . A nucleic acid construct according to  claim 1 , further comprising: 
 a nuclear export signal sequence appended to the first structural gene;    a third promoter; and    a nuclear export factor gene sequence.    
     
     
         6 . A nucleic acid construct according to  claim 1 , wherein at least two of the structural genes are mutually linked by an IRES sequence or by an activation sequence.  
     
     
         7 . A nucleic acid construct according to  claim 1 , wherein at least two transcription factor protein genes are mutually linked by an IRES sequence or by an activation sequence.  
     
     
         8 . A nucleic acid construct according to  claim 7 , wherein said transcription factor protein genes are non-identical and transcription factor proteins produced from said genes binds said transcription factor protein binding sequences in the nucleic acid construct.  
     
     
         9 . A nucleic acid construct according to  claim 1 , further comprising a pharmacological control module that comprises, in serial order: 
 at least one promoter;    at least one fusion protein gene that comprises an activation domain of a transcription factor protein, and    a sequence that binds a coupling protein;    at least one promoter;    at least one fusion protein gene wherein the fusion protein comprises    a DNA-binding protein and a protein that binds a coupling substance; and    at least one activation sequence that comprises a site for the DNA-binding protein.    
     
     
         10 . A nucleic acid construct that comprises: 
 at least one first structural gene that encodes an active compound;    at least one second structural gene that encodes a transcription factor protein; and    at least one activation sequence comprised of at least one sequence that binds said transcription factor protein and at least one pharmacological control module that comprises, in serial order, at least one promoter, at least one fusion protein gene coding for an activation domain of a transcription factor protein and coding for a coupling substance protein, at least one promoter, at least one fusion protein gene coding for a DNA-binding protein and coding for a second coupling substance protein, and at least one activation sequence that comprises a site for the DNA-binding protein    wherein each activation sequence activates the expression of a structural gene and the expression of the transcription factor protein.    
     
     
         11 . A nucleic acid construct that comprises: 
 at least one first structural gene that encodes an active compound;    at least one second structural gene that encodes at least one first fusion protein that comprises an activation domain of a transcription factor protein, and a sequence that binds a coupling substance;    at least one third structural gene that encodes at least one second fusion protein that comprises a protein that binds a coupling substance and a DNA-binding protein;    at least one activation sequence comprised of at least one sequence that binds said second fusion protein coupled to said first fusion protein by a coupling substance and at least one promoter sequence;    wherein each activation sequence activates the expression of at least one of said structural genes.    
     
     
         12 . A nucleic acid construct according to  claim 1 , wherein said activation sequence comprises 
 a sequence for binding a transcription factor protein, the sequence selected from the group consisting of the Gal4 protein gene, the LexA protein gene, the Lac I repressor protein gene, the tetracyclin repressor protein gene, and the ZFHD-1 protein gene;    a promoter sequence selected from the group consisting of the basal c-fos promoter in combination with the HSV-1 VPI6 transactivation domain, the U2 sn RNA promoter in combination with a sequence of the Oct-2 activation domain, and the HSV TK promoter;    and a transcription factor protein gene selected from the group consisting of    the DNA-binding domain of the Gal4 protein, the DNA-binding domain of the LexA protein, the Lac I repressor protein gene, the tetracycline repressor protein gene and the ZFHD1 protein gene.    
     
     
         13 . A nucleic acid construct according to  claim 12 , wherein said transcription factor protein comprises the SV40 nuclear localization signal and the HSV-1 VP16 acid transactivation domain.  
     
     
         14 . A nucleic acid construct according to  claim 9 , wherein said pharmacological control module comprises: 
 a protein coding sequence selected from the group consisting of the herpes virus VP16 transactivation domain, the p65 subunit of the NF-KB transcription factor, and the Oct-2 N-terminal glutamine-rich domain which is directly or indirectly linked to the Oct-2 C-terminal proline-rich Oct-2 domain;    a protein coding sequence selected from the group consisting of the DNA-binding domain of the Gal4 protein, the DNA-binding domain of the LexA protein, the Lac I repressor protein, the tetracycline repressor protein, and the ZFHD1 protein; and    at least one nucleic acid sequence that binds a protein selected from the group consisting of the Gal4 protein, the LexA protein, the Lac I repressor protein, the tetracycline repressor protein, and the ZFHD1 protein;    wherein the nucleic acid sequence that binds a protein is linked to a promoter selected from the group consisting of the basal SV40 promoter, the c-fos promoter in combination with the HSV-1 VP16 transactivation domain, the U2 sn RNA promoter in combination with the HSV-1 VP16 transactivation domain or with at least a sequence of the Oct-2 activation domain, and the HSV TK promoter.    
     
     
         15 . A nucleic acid construct according to  claim 14 , further comprising an SV40 nuclear localization signal and an HSV-1 VP16 acid transactivation domain wherein the SV40 nuclear localization signal and HSV-1 VP16 acid transactivation domain are present in a gene that encodes a fusion protein.  
     
     
         16 . A nucleic acid construct according to  claim 9  wherein at least one fusion protein comprises an antibody or antibody fragment.  
     
     
         17 . A nucleic acid construct according to  claim 16 , wherein said antibody fragment comprises a single-chain Fv fragment having a variable chain and a light chain where the variable and light chains are linked covalently by a short peptide sequence.  
     
     
         18 . A nucleic acid construct according to  claim 9 , wherein at least one fusion protein comprises a binding domain of a naturally occurring protein.  
     
     
         19 . A nucleic acid construct according to  claim 1 , wherein at least one promoter is selected from the group consisting of RNA polymerase III, RNA polymerase II, CMV promoter and enhancer, SV40 promoter, an HBV promoter, an HCV promoter, an HSV promoter, an HPV promoter, an EBV promoter, an HTLV promoter, an HIV promoter, an cdc25C promoter, a cyclin A promoter, a cdc2 promoter, a bmyb promoter, a DHFR promoter and an E2F-1 promoter.  
     
     
         20 . A nucleic acid construct according to  claim 5 , wherein the nuclear export signal and the corresponding nuclear export factor are selected from a rev-responsive element/rev protein of a retrovirus selected from the group consisting of HIV-1, HIV-2, HTLV-1 and HBV.  
     
     
         21 . A nucleic acid construct according to  claim 1 , wherein the structural gene encodes a compound selected from the group consisting of inhibitors of cell proliferation, cytostatic or cytotoxic proteins, enzymes for cleaving prodrugs, antibodies, fusion proteins between antibody fragments and other proteins, cytokines, growth factors, hormones, receptors for cytokines and growth factors, cytokine antagonists, inflammation inducers, coagulation-inducing factors, coagulation inhibitors, fibrinolysis-inducing proteins, angiogenesis inhibitors, angiogenesis factors, hypotensive peptides, blood plasma proteins, insulin receptor, LDL receptor, enzymes whose absence leads to metabolic diseases or immunosuppression, viral antigens, bacterial antigens, parasitic antigens or tumour antigens, antiidiotype antibodies for these antigens, and a fusion protein derived from any combination of these.  
     
     
         22 . A nucleic acid construct according to  claim 9 , wherein at least two—structural genes are mutually linked by an IRES sequence or by an activation sequence.  
     
     
         23 . A vector comprising the nucleic acid construct of  claim 1 .  
     
     
         24 . A pharmaceutical composition comprising a nucleic acid construct of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         25 . A pharmaceutical composition according to  claim 24 , further comprising a coupling substance that binds to a fusion protein.  
     
     
         26 . A pharmaceutical composition according to  claim 25 , wherein said coupling substance penetrates cell membranes and enters cells.  
     
     
         27 . A pharmaceutical composition according to  claim 25 , wherein said coupling substance is selected from the group consisting of rapamycin, FK506, cyclosporin A, methotrexate, folic acid, retinoic acid, penicillin, 4-hydroxy tamoxifen, tamoxifen, tetracycline, and a tetracycline/isopropyl-β-D-thiogalactoside conjugate.  
     
     
         28 . A cell which comprises a nucleic acid construct as described in  claim 1 .  
     
     
         29 . A process for preparing a nucleic acid construct as described in  claim 1 , comprising: 
 linking a sequence that binds a transcription factor protein to a promoter sequence to form an activation sequence; and    linking the activation sequence to at least one structural gene and to at least one gene that encodes a transcription factor protein.    
     
     
         30 . A method of treating a disease in a patient comprising administering the nucleic acid construct of  claim 1  to a patient in need thereof, wherein the nucleic acid construct is administered in an amount sufficient to ameliorate the disease.  
     
     
         31 . A method for vaccination of a patient comprising administering the nucleic acid construct of  claim 1  to a patient in need thereof, wherein the nucleic acid construct is administered in an amount sufficient to prevent the disease.  
     
     
         32 . A method for preparing a pharmaceutical composition comprising: 
 transforming a cell with a DNA construct as described in  claim 1;     culturing the transformed cell to obtain multiple copies of the DNA construct; and    purifying DNA from the cultured cells.