Methods and compositions comprising DNA damaging agents and p53

Abstract

The present invention relates to the use of tumor suppressor genes in combination with a DNA damaging agent or factor for use in killing cells, and in particular cancerous cells. A tumor suppressor gene, p53, was delivered via a recombinant adenovirus-mediated gene transfer both in vitro and in vivo, in combination with a chemotherapeutic agent. Treated cells underwent apoptosis with specific DNA fragmentation. Direct injection of the p53-adenovirus construct into tumors subcutaneously, followed by intraperitoneal administration of a DNA damaging agent, cisplatin, induced massive apoptotic destruction of the tumors. The invention also provides for the clinical application of a regimen combining gene replacement using replication-deficient wild-type p53 adenovirus and DNA-damaging drugs for treatment of human cancer.

1 . A method of killing a cell, comprising contacting a cell with a p53 protein or gene and a DNA damaging agent in a combined amount effective to kill said cell.  
     
     
         2 . The method of  claim 1 , wherein said cell is contacted with a p53 protein or gene in combination with X-ray radiation, UV-irradiation, γ-irradiation, microwaves, adriamycin, 5-fluorouracil, etoposide, camptothecin, actimomycin-D, mitomycin C, or cisplatin.  
     
     
         3 . The method of  claim 2 , wherein said cell is contacted with a p53 protein or gene in combination with cisplatin.  
     
     
         4 . The method of  claim 1 , wherein said cell is contacted with a recombinant vector that expresses a p53 protein in said cell in combination with a DNA damaging agent.  
     
     
         5 . The method of  claim 4 , wherein said p53-expressing recombinant vector is a naked DNA plasmid, a plasmid within a liposome, a retroviral vector, an AAV vector, or a recombinant adenoviral vector.  
     
     
         6 . The method of  claim 5 , wherein said p53-expressing recombinant vector is a recombinant adenoviral vector.  
     
     
         7 . The method of  claim 6 , wherein said p53-expressing recombinant vector is a recombinant adenoviral vector comprising a p53 expression region positioned under the control of the cytomegalovirus IE promoter.  
     
     
         8 . The method of  claim 6 , wherein said recombinant adenoviral vector comprises a p53 expression region, the cytomegalovirus IE promoter and the SV40 early polyadenylation signal.  
     
     
         9 . The method of  claim 6 , wherein at least one gene essential for adenovirus replication is deleted from said adenovirus vector construct and the p53 expression region is introduced in its place.  
     
     
         10 . The method of  claim 9 , wherein the E1A and E1B regions of the adenovirus vector are deleted and the p53 expression region is introduced in their place.  
     
     
         11 . The method of  claim 6 , wherein said recombinant adenoviral vector is present within a recombinant adenovirus.  
     
     
         12 . The method of  claim 1 , wherein said cell is first contacted with a p53 protein or gene and is subsequently contacted with a DNA damaging agent.  
     
     
         13 . The method of  claim 1 , wherein said cell is first contacted with a DNA damaging agent and is subsequently contacted with a p53 protein or gene.  
     
     
         14 . The method of  claim 1 , wherein said cell is simultaneously contacted with a p53 protein or gene and a DNA damaging agent.  
     
     
         15 . The method of  claim 1 , wherein said cell is contacted with a first composition comprising a p53 protein or gene and a second composition comprising a DNA damaging agent.  
     
     
         16 . The method of  claim 15 , wherein said first or second composition is dispersed in a pharmacologically acceptable formulation.  
     
     
         17 . The method of  claim 1 , wherein said cell is contacted with a single composition comprising a p53 protein or gene in combination with a DNA damaging agent.  
     
     
         18 . The method of  claim 17 , wherein said composition is dispersed in a pharmacologically acceptable formulation.  
     
     
         19 . The method of  claim 17 , wherein said cell is contacted with a single composition comprising a recombinant vector that expresses p53 in said cell in combination with a DNA damaging agent.  
     
     
         20 . The method of  claim 19 , wherein said cell is contacted with a single composition comprising a recombinant adenovirus containing a recombinant vector that expresses p53 in said cell in combination with a DNA damaging agent.  
     
     
         21 . The method of  claim 1 , wherein said cell is a human cell.  
     
     
         22 . The method of  claim 1 , wherein said cell is a malignant cell.  
     
     
         23 . The method of  claim 22 , wherein said cell is a lung cancer cell.  
     
     
         24 . The method of  claim 22 , wherein said cell is a breast cancer cell.  
     
     
         25 . The method of  claim 22 , wherein said cell has a mutation in a p53 gene.  
     
     
         26 . The method of  claim 1 , wherein said cell is located within an animal and said p53 protein or gene and DNA damaging agent are administered to the animal in a pharmacologically acceptable form.  
     
     
         27 . A method of treating cancer, comprising administering to an animal with cancer a therapeutically effective combination of a p53 protein or gene and a DNA damaging agent.  
     
     
         28 . The method of  claim 27 , comprising injecting into a tumor site a therapeutically effective amount of a pharmaceutical composition comprising a recombinant adenovirus containing a recombinant vector that expresses p53 in the tumor cell, and contacting the tumor with a DNA damaging agent.  
     
     
         29 . The method of  claim 28 , wherein the tumor is contacted with a DNA damaging agent by irradiating the tumor site with X-ray radiation, UV-irradiation, T-irradiation or microwaves.  
     
     
         30 . The method of  claim 28 , wherein the tumor is contacted with a DNA damaging agent by administering to the animal a therapeutically effective amount of a pharmaceutical composition comprising a DNA damaging compound.  
     
     
         31 . The method of  claim 28 , wherein the DNA damaging compound is cisplatin.  
     
     
         32 . A composition comprising a p53 protein or gene in combination with a DNA damaging agent.  
     
     
         33 . The composition of  claim 32 , comprising a p53 protein or gene in combination with adriamycin, 5-fluorouracil, etoposide, camptothecin, actimomycin-D, mitomycin C, or cisplatin.  
     
     
         34 . The composition of  claim 33 , comprising a p53 protein or gene in combination with cisplatin.  
     
     
         35 . The composition of  claim 32 , comprising a recombinant vector that expresses a p53 protein in an animal cell in combination with a DNA damaging agent.  
     
     
         36 . The composition of  claim 35 , wherein said recombinant vector is a naked DNA plasmid, a plasmid within a liposome, a retroviral vector, an AAV vector, or a recombinant adenoviral vector.  
     
     
         37 . The composition of  claim 36 , wherein said recombinant vector is a recombinant adenoviral vector.  
     
     
         38 . The composition of  claim 37 , wherein said recombinant vector is a recombinant adenoviral vector is present within a recombinant adenovirus particle.  
     
     
         39 . The composition of  claim 32 , comprising a recombinant adenoviral vector present within a recombinant adenovirus particle in combination with cisplatin.  
     
     
         40 . The composition of  claim 32 , dispersed in a pharmacologically acceptable formulation.  
     
     
         41 . The composition of  claim 40 , formulated for intralesional administration.  
     
     
         42 . A therapeutic kit comprising, in suitable container means, a pharmaceutical formulation of a recombinant vector that expresses a p53 protein in an animal cell and a pharmaceutical formulation of a DNA damaging agent.  
     
     
         43 . The kit of  claim 42 , wherein said recombinant vector and said DNA damaging agent are present within a single container means.  
     
     
         44 . The kit of  claim 42 , wherein said recombinant vector and said DNA damaging agent are present within distinct container means.  
     
     
         45 . The kit of  claim 42 , comprising a pharmaceutical formulation of a recombinant adenovirus including a recombinant vector that expresses a p53 protein in an animal cell and a pharmaceutical formulation of cisplatin.