Apobec3 mediated dna editing

Abstract

The present invention relates to methods and compositions for preventing the occurance or progression of a cancer or pre-cancerous condition associated with expression, or over-expression of human cytidine deaminases of the APOBEC3 family. The invention also relates to drug screening assays designed to identify compounds that regulate the activity, or level of expression, of hA3A, hA3C and hA3H. The invention further relates to transgenic mice, as well as cells derived from said mice, that have been genetically engineered to express, or over-express hA3A, hA3C and/or hA3H. Such mice may be utilized to screen for, or identify, compounds that modulate the activity, or expression, of the human cytidine deaminases. The present invention also provides topical compositions such as cosmetic lotion, crème, or sunscreen for use on the skin, which comprise one or more inhibitors of human cytidine deaminase activity. The present invention relates to a double stranded DNA obtained following opening up of its duplex structure, said DNA being edited with cellular protein normally or abnormally expressed in the nucleus of an eukaryotic cell. The mono stranded DNA derived from the said double stranded DNA is a part of the present invention.

What is claimed: 
     
         1 . A method for identifying a compound that inhibits hA3A, hA3C or hA3H enzyme activity comprising (i) contacting a cell expressing hA3A, hA3C or hA3H with a test compound and substrate and measuring the level of cytidine deaminase activity, (ii) in a separate experiment, contacting a cell expressing hA3A, hA3C or hA3H and substrate and measuring the level of cytidine deaminase activity, where the conditions are essentially the same as in part (i) and then (iii) comparing the level of cytidine deaminase activity measured in part (i) with the level of cytidine deaminase activity in part (ii), wherein a decrease level of cytidine deaminase activity in the presence of the test compound indicates that the test compound is a cytidine deaminase activity inhibitor. 
     
     
         2 . A method for identifying a compound that increases hA3A, hA3C or hA3H enzyme activity comprising (i) contacting a cell expressing hA3A, hA3C or hA3H with a test compound and substrate and measuring the level of cytidine deaminase activity, (ii) in a separate experiment, contacting a cell expressing hA3A, hA3C or hA3H and substrate and measuring the level of cytidine deaminase activity, where the conditions are essentially the same as in part (i) and then (iii) comparing the level of cytidine deaminase activity measured in part (i) with the level of cytidine deaminase activity in part (ii), wherein an increase level of cytidine deaminase activity in the presence of the test compound indicates that the test compound is a cytidine deaminase activity activator. 
     
     
         3 . A method of inhibiting DNA editing in a cell comprising contacting said cell with a compound selected according to  claim 1 . 
     
     
         4 . A transgenic animal expressing a nucleic acid encoding human hA3A, hA3C or hA3H, wherein expression of said hA3A, hA3C or hA3H results in an increase in DNA editing. 
     
     
         5 . A method for identifying a compound that induces expression of hA3A, hA3C or hA3H in the transgenic mouse of  claim 4 , comprising contacting said mouse with a test compound and determining whether said test compound increases the level of hA3A, hA3C or hA3H expression. 
     
     
         6 . A method of identifying a compound that induces expression of hA3A, hA3C or hA3H comprising contacting a cell derived from the transgenic mouse of  claim 4  with a test compound and determining whether said test compound increases the level of hA3A, hA3C or hA3H expression. 
     
     
         7 . A method for identifying a carcinogenic compound comprising (i) contacting a cell expressing hA3A, hA3C or hA3H with a compound suspected of being carcinogenic and substrate and measuring the level of cytidine deaminase activity, (ii) in a separate experiment, contacting a cell expressing hA3A, hA3C or hA3H and substrate and measuring the level of cytidine deaminase activity, where the conditions are essentially the same as in part (i) and then (iii) comparing the level of cytidine deaminase activity measured in part (i) with the level of cytidine deaminase activity in part (ii), wherein an increase level of cytidine deaminase activity in the presence of the test compound indicates that the test compound is carcinogenic compound. 
     
     
         8 . A double stranded DNA following opening up of its duplex structure, said DNA being edited with a cellular protein normally or abnormally expressed in the nucleus of an eukaryotic cell. 
     
     
         9 . A double stranded DNA according to  claim 8  is a double stranded cellular DNA following opening up of its duplex structure, said DNA being edited with a cellular protein normally or abnormally expressed in the nucleus of an eukaryotic cell. 
     
     
         10 . An edited DNA according to  claim 8  which is an origin of replication of a gene or a gene or a part of a gene or an exon or a siRNA gene. 
     
     
         11 . A single stranded DNA obtained or derived from the double stranded DNA according to  claims 8  and  10 , said double stranded DNA being obtained after the opening up of its duplex structure and editing by a cellular protein normally or abnormally expressed in the nucleus of an eukaryotic cell. 
     
     
         12 . A mutated or hypermutated double or single stranded viral DNA according to  claims 8  and  10 , edited following opening up of its duplex structure with a cellular protein normally or abnormally expressed in the nucleus of an eukaryotic cell, the percentage of mutation or hypermutation being compared with the normal nucleic acids of reference. 
     
     
         13 . A double stranded or single stranded DNA according to  claims 8  to  12 , are derived or mutated from the normal viral nucleic acids sequences which are comprised among the sequences of the viral genomes or the cDNA from at least one of the viruses: cytomegalovirus, herpes simplex 1 and 2, Epstein-Barr virus, human herpes virus 1, 2, 3, 4, 5, 6, 7 and 8, adenoviruses, human and animal papillomaviruses, BK virus and JC virus. 
     
     
         14 . An edited double stranded or single stranded DNA according to  claim 13 , is related to a part of the genome of a human papillomavirus. 
     
     
         15 . A method for detecting cytidine deamination in HPV DNA comprising:
 (i) transfecting a human cell with HPV plasmid DNA, and an APOBEC3 gene selected from APOBEC3A, APOBEC3C and APOBEC3H;   (ii) contacting or not the transfected cell with a compound to test;   (iii) recovering total DNA after at least 72 hours of incubation;   (iv) performing 3D-PCR on total DNA;   (v) cloning and sequencing 3D-PCR products; and   (vi) counting cytidine deamination of both strands of HPV DNA,   
       Wherein a decrease of cytindine deamination of both strands of HPV DNA in the presence of the compound to test, compared to to in the absence thereof, indicates that the compound inhibits cytidine deamination in HPV DNA.