Tissue-specific and pathogens-specific toxic agents, ribozymes, dnazymes and antisense oligonucleotides, and methods of use thereof

Abstract

The present invention relates to the discovery, identification and characterization of toxic agents which are lethal to pathogens and methods for targeting such toxic agents to a pathogen or pathogen infected cells in order to treat and/or eradicate the infection. In particular, the present invention relates to toxic agents which target bacteria at different stages of the bacterial life cycle, which are delivered alone or in combination to bacteria or bacteria-infected cells. The invention relates to toxic agents which are lethal to diseased cells and methods for targeting such toxic agent to a diseased cell in order to treat and/or eradicate the disease. The present invention relates to promoter elements which are pathogen-specific or tissue-specific and the use of such promoter elements to achieve pathogen-specific or tissue-specific expression of the toxic agent(s) and/or ribozyme(s) of the present invention. Specifically, the invention relates to the delivery of one or more toxic gene products, antisense RNAs, or ribozymes, or combination thereof. The invention provides a novel system by which multiple pathogenic targets may be simultaneously targeted to cause the death of a pathogen, or cell infected with a pathogen. Further, the invention has important implications in the eradication of drug-resistant bacterium and bacterial pathogens. The invention provides a novel system by which multiple targets may be simultaneously targeted to cause the death of a diseased cell. The invention has important implications in the eradication of drug-resistant pathogens (such as antibiotic resistant bacteria) and drug-resistant diseased cells (such as drug-resistant cancer cells).

1 . A purified preparation of at least one nucleic acid molecule that specifically hybridizes under physiological conditions to mRNA encoding at least one viral protein associated with transformation or plasmid copy number control or which hybridizes to a viral polyadenylation signal or a core, pre-core or polymerase-encoding sequence, wherein at least one sequence is selected from the group consisting of SEQ ID NOs:37-66 or a homologous sequence thereof.  
     
     
         2 . A purified preparation of at least one nucleic acid molecule of  claim 1 , wherein said mRNA is E6/E7 mRNA.  
     
     
         3 . A purified preparation of at least one nucleic acid molecule of  claim 1 , wherein said mRNA is papilloma viral RNA or hepatitis B viral RNA.  
     
     
         4 . A purified preparation of at least one nucleic acid molecule that specifically hybridizes under physiological conditions to a sequence selected from the group consisting of SEQ ID NOs:37-66 or a homologous sequences in a related HPV or HBV strain.  
     
     
         5 . A purified preparation of at least one nucleic acid molecule of  claim 1 , wherein said nucleic acid molecules are DNAzymes, antisense oligonucleotides or ribozymes.  
     
     
         6 . A purified preparation of at least one nucleic acid molecule of  claim 1 , wherein said nucleic acid molecules are DNAzymes.  
     
     
         7 . A purified preparation of at least one nucleic acid molecule of  claim 1 , wherein said nucleic acid molecules are antisense oligonucleotides.  
     
     
         8 . A purified preparation of at least one nucleic acid molecule of  claim 1 , wherein said nucleic acid molecules are ribozymes.  
     
     
         9 . A purified preparation of at least one nucleic acid molecule of  claim 1 , wherein said nucleic acid molecules are resistant to nuclease degradation.  
     
     
         10 . A purified preparation of at least one nucleic acid molecule of  claim 9 , wherein said nucleic acid molecules are modified at their 3′ ends, are resistant to nuclease degradation and have a 3′-3′ inverted T at their 3′ ends.  
     
     
         11 . A pharmaceutical composition comprising any of the nucleic acid molecules of  claim 1  and a pharmaceutically acceptable carrier.  
     
     
         12 . A method of treating papilloma virus-induced conditions or hepatitis virus-induced conditions comprising administering to a subject a pharmaceutical compositions of  claim 11 .  
     
     
         13 . A method of  claim 12 , wherein said administration comprises topical application to the cervix.  
     
     
         14 . A method of  claim 12 , wherein said administration comprises topical application to the epidermis.  
     
     
         15 . A pharmaceutical composition of  claim 11 , wherein said nucleic acid molecules are formulated as a cosmetic formulation.  
     
     
         16 . A pharmaceutical composition of  claim 11 , wherein said nucleic acid molecules are formulated in an amount sufficient to produce a cytotoxic or cytostatic effect in cells infected with papilloma virus or hepatitis B virus.  
     
     
         17 . A pharmaceutical composition of  claim 16 , wherein said cells are transformed by a papilloma virus.  
     
     
         18 . A pharmaceutical composition of  claim 16 , wherein said cells are transformed by a hepatitis B virus.  
     
     
         19 . A pharmaceutical composition of  claim 16 , wherein said nucleic acid molecules are formulated for topical administration.  
     
     
         20 . A pharmaceutical composition of  claim 19 , wherein said nucleic acids are formulated into an ointment, salve, gel, cream or lotion.  
     
     
         21 . A pharmaceutical composition of  claim 11 , wherein said nucleic acid molecules are formulated into a liposome preparation or a lipid preparation.  
     
     
         22 . A method of  claim 12 , wherein said papilloma virus-induced condition is selected from the group consisting of warts, cervical dysplasia, cervical carcinoma, carcinoma in situ and laryngeal papilloma.  
     
     
         23 . A method of  claim 22 , wherein said administration is to epithelial cells.  
     
     
         24 . A method of  claim 23 , wherein said epithelial cells are selected from the group consisting of squamous epithelia, cutaneous epithelia and mucosal epithelia.  
     
     
         25 . The pharmaceutical composition of  claim 11  wherein said one or more nucleic acids are formulated for delivery in a liposome.  
     
     
         26 . The pharmaceutical composition of  claim 25  wherein said liposome is capable of tissue-specific uptake in the liver.  
     
     
         27 . The pharmaceutical composition of  claim 26  wherein said liposome is modified using asialofetuin or one or more sugars.