Viral agents

Abstract

A viral DNA construct, and virus encoded thereby, is provided having one or more tumour specific transcription factor binding sites in place of one or more wild type transcription factor binding sites operatively positioned in the promoter region which controls expression of early genes responsible for viral nucleic acid replication. Preferred constructs place the tumour specific transcription factor binding sites in operative relation to DNA polymerase, DNA terminal protein and/or DNA binding protein. Compositions and constructs contained therein are provided, particularly for use in therapy. Methods of treating patients for neoplasms are also provided.

1 . A viral DNA construct encoding for a virus capable of replication in a human or animal tumour cell and causing death of such tumour cells characterised in that it comprises one or more selected transcription factor binding sites operatively positioned together with one or more early viral protein gene open reading frames such as to promote expression of these open reading frames in the presence of said selected transcription factor, the protein products of those open reading frames being mechanistically directly involved in viral construct nucleic acid replication and wherein the selected transcription factor binding sites are for a transcription factor the level or activity of which is increased in a human or animal tumour cell relative to that of a normal human or animal cell of the same type.  
     
     
         2 . A viral DNA construct as claimed in  claim 1  having a nucleic acid sequence corresponding to that of a wild type virus sequence characterised in that it has one or more wild type transcription factor binding sites replaced by one or more selected transcription factor binding sites, these selected sites being operatively positioned in one or more promoter regions which control expression of early genes such as to promote expression in the presence of said selected transcription factor, the protein products of those genes being mechanistically directly involved in viral nucleic acid replication and wherein the selected transcription factor binding sites are for a transcription factor the level or activity of which is increased in a human or animal tumour cell relative to that of a normal human or animal cell of the same type.  
     
     
         3 . A viral DNA construct as claimed in  claim 1  characterised in that the selected transcription factor binding sites are for a transcription factor whose activity or level is specifically increased by causal oncogenic mutations.  
     
     
         4 . A construct as claimed in  claim 2  characterised in that its nucleic acid sequence corresponds to that of the genome of an adenovirus, lentivirus, polyomavirus, vaccinia virus, herpes virus or parvovirus with the selected transcription factor binding sites operatively positioned to control expression of the early viral genes.  
     
     
         5 . A construct as claimed in  claim 1  characterised in that its nucleic acid sequence, other than the selected sites, corresponds to that of the genome of adenovirus Ad5, Ad40 or Ad41, or incorporates DNA encoding for fibre protein from Ad5, Ad40 or Ad41, optionally with 15 to 25 lysines added to the end thereof.  
     
     
         6 . A construct as claimed in  claim 1  characterised in that the genes controlled by the tumour specific transcription factor binding site are DNA polymerase, DNA terminal protein and/or DNA binding protein.  
     
     
         7 . A construct as claimed in  claim 1  characterised in that its nucleic acid sequence corresponds to that of an adenovirus having a wild type E2 early promoter transcription factor binding site replaced by the tumour cell specific transcription factor binding site.  
     
     
         8 . A construct as claimed in  claim 1  characterised in that it encodes a functional viral RNA export capacity.  
     
     
         9 . A construct as claimed in  claim 1  having an E1 region wherein the E1B 55K gene is functional and/or intact.  
     
     
         10 . A construct as claimed in  claim 1  characterised in that the tumour specific transcription factor binding site used in place of wild type site is selected from Tcf-4, RBPJκ, Gli-1, HIF1alpha and telomerase promoter binding sites.  
     
     
         11 . A construct as claimed in  claim 1  characterised in that the replaced transcription factor binding site is selectively activated in tumour cells containing oncogenic APC and β-catenin mutations.  
     
     
         12 . A construct as claimed in  claim 1  characterised in that the replacement sites are single or multiples of a Tcf-4 binding site sequence.  
     
     
         13 . A construct as claimed in  claim 12  characterised in that it comprises from 2 to 20 Tcf-4 binding site sequences at each replaced promoter site.  
     
     
         14 . A construct as claimed in  claim 1  characterised in that its sequence corresponds to that of an adenovirus genome having mutations in one or more residues in the NF1, NFκB, AP1 and ATF regions of the E3 promoter.  
     
     
         15 . A construct as claimed in  claim 14  characterised in that the mutations reduce E2 gene transcription caused by E3 promoter activity.  
     
     
         16 . A construct as claimed in  claim 14  characterised in that the mutations are silent mutations, being such as not to alter the predicted protein sequence of any viral protein, but which alter the activity of one or more viral promoters.  
     
     
         17 . A construct as claimed in  claim 1  characterised in that its sequence corresponds to that of an adenovirus genome wherein a region in the E2 early promoter, which is not overlapped by coding sequence, is replaced with multiple Tcf-4 binding site sequence.  
     
     
         18 . A construct as claimed in  claim 1  characterised in that its sequence corresponds to that of an adenovirus genome wherein the transcription factor binding sites in the E3-promoter are modified or deleted.  
     
     
         19 . A construct as claimed in  claim 1  characterised in that its sequence corresponds to that of an adenovirus genome wherein the E2 late promoter has been inactivated with silent mutations.  
     
     
         20 . A virus comprising or encoded by a DNA construct as claimed in  claim 1 .  
     
     
         21 . A virus encoding only wild type proteins other than one or more optional non-wild type non-cytotoxic marker proteins, for use in therapy.  
     
     
         22 . A viral DNA construct, or a virus, as claimed in  claim 1  for use in therapy.  
     
     
         23 . A viral DNA construct, or a virus, as claimed in  claim 21  characterised in that the therapy is of patients having neoplasms.  
     
     
         24 . Use of a viral DNA construct, or a virus, encoding only wild type proteins, other than one or more optional non-wild type non-cytotoxic marker proteins, in the manufacture of a medicament for the treatment of neoplasms.  
     
     
         25 . Use of a viral construct, or a virus, as claimed in  claim 1  in the manufacture of a medicament for the treatment of neoplasms.  
     
     
         26 . A pharmaceutical composition comprising a viral DNA construct, or a virus encoded thereby, encoding only wild type proteins other than one or more optional non-wild type non-cytotoxic marker proteins, together with a physiologically acceptable carrier, wherein the composition is sterile and pyrogen free with the exception of the presence of the DNA or virus encoded thereby.  
     
     
         27 . A composition comprising a viral construct, or a virus, as claimed in  claim 1  together with a physiologically acceptable carrier.  
     
     
         28 . A composition as claimed in  claim 27  characterised in that it is sterile and pyrogen free with the exception of the presence of the viral construct or virus encoded thereby.  
     
     
         29 . A composition as claimed in  claim 27  characterised in that the carrier is a physiologically acceptable saline.  
     
     
         30 . A method of manufacture of a viral DNA construct or a virus encoded thereby as claimed in  claim 1  characterised in that it comprises transforming a viral genome having one or more wild type transcription factor binding sites controlling transcription of genes having protein products directly mechanistically involved in viral nucleic acid replication such as to replace one or more of these by tumour specific transcription factor binding sites,  
     
     
         31 . A method as claimed in  claim 30  characterised in that the viral genome is cloned by gap repair in a circular YAC/BAC in yeast.  
     
     
         32 . A method as claimed in  claim 30  characterised in that the genome is modified by two step gene replacement.  
     
     
         33 . A method as claimed in  claim 30 , characterised in that the modified genome is transferred to a prokaryote for production of viral construct DNA.  
     
     
         34 . A method of manufacture of a virus characterised in that viral construct DNA produced by a method as claimed in  claim 30 , is transferred to a mammalian cell for production of virus.  
     
     
         35 . A method for treating a patient suffering from neoplasms wherein a virus encoding only wild type proteins other than one or more optional non-wild type non toxic-marker proteins is caused to infect tissues of the patient, including or restricted to those of the neoplasm, and allowed to replicate such that neoplasm cells are caused to be killed.  
     
     
         36 . A method for treating a patient suffering from neoplasms wherein a viral DNA construct or virus as claimed in  claim 1  is caused to infect tissues of the patient, including or restricted to those of the neoplasm, and allowed to replicate such that neoplasm cells are caused to be killed.