Bacteria mediated gene silencing

Abstract

Methods are described for the delivery of one or more small interfering RNAs (siRNAs) to a eukaryotic cell using a bacterium or BTP. Methods are also described for using this bacterium to regulate gene expression in eukaryotic cells using RNA interference, and methods for treating viral diseases and disorders. The bacterium or BTP includes one or more siRNAs or one or more DNA molecules encoding one or more siRNAs. Vectors are also described for use with the bacteria of the invention for causing RNA interference in eukaryotic cells.

1 . An invasive bacterium comprising one or more siRNAs or one or more DNA molecules encoding one or more siRNAs, wherein the expressed siRNAs interfere with the mRNA of one or more HPV oncogenes. 
     
     
         2 . A prokaryotic vector comprising at least one DNA molecule encoding one or more siRNAs and at least one RNA-polymerase III compatible promoter or at least one prokaryotic promoter, wherein the expressed siRNAs interfere with the mRNA of one or more HPV oncogenes. 
     
     
         3 . A method of delivering one or more siRNAs to mammalian cells, the method comprising introducing to said mammalian cells at least one invasive bacterium containing one or more siRNAs or one or more DNA molecules encoding one or more siRNAs, wherein the expressed siRNAs interfere with the mRNA of one or more HPV oncogenes. 
     
     
         4 . A method of regulating gene expression in mammalian cells, the method comprising introducing to said mammalian cells at least one invasive bacterium containing one or more siRNAs or one or more DNA molecules encoding one or more siRNAs, wherein the expressed siRNAs interfere with the mRNA of one or more HPV oncogenes. 
     
     
         5 . A method of treating or preventing a viral disease or disorder in a mammal, the method comprising regulating the expression of at least one gene in a cell known to increase proliferation, growth or dysplasia, by introducing to the cells of the mammal at least one invasive bacterium containing one or more siRNAs or one or more DNA molecules encoding one or more siRNAs, wherein the expressed siRNAs interfere with the mRNA of one or more HPV oncogenes. 
     
     
         6 . The invasive bacterium of  claim 1 , wherein said invasive bacterium is a non-pathogenic or non-virulent bacterium. 
     
     
         7 . The invasive bacterium of  claim 1 , wherein said invasive bacterium is a therapeutic bacterium. 
     
     
         8 . A composition comprising the invasive bacterium of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         9 . A eukaryotic host cell comprising the invasive bacterium of  claim 1 , and a pharmaceutically acceptable carrier. 
     
     
         10 . The method of  claim 4 , wherein said mammalian cells are in vivo or in vitro. 
     
     
         11 . The method of  claim 4 , wherein said mammalian cells are selected from the group consisting of human, bovine, ovine, porcine, feline, buffalo, canine, goat, equine, donkey, deer, avian, bird, chicken, and primate cells. 
     
     
         12 . The method of  claim 4 , wherein said invasive bacterium is a non-pathogenic or non-virulent bacterium. 
     
     
         13 . The method of  claim 4 , wherein said invasive bacterium is a therapeutic bacterium. 
     
     
         14 . The method of  claim 4 , wherein said one or more DNA molecules encoding said one or more siRNAs are transcribed within the mammalian cell. 
     
     
         15 . The method of  claim 14 , wherein said one or more siRNAs are transcribed within the mammalian cell as shRNAs. 
     
     
         16 . The method of  claim 4 , wherein said one or more DNA molecules encoding said one or more siRNAs are transcribed within the bacterium. 
     
     
         17 . The method of  claim 4 , wherein said one or more DNA molecules encoding one or more siRNAs comprise a prokaryotic promoter. 
     
     
         18 . The method of  claim 17 , wherein said prokaryotic promoter is selected from the group consisting of a T7 promoter, a P gapA  promoter, a P araBAD  promoter, a P tac  promoter, a P lacUV5  promoter and a recA promoter. 
     
     
         19 . The method of  claim 4 , wherein said one or more DNA molecules encoding one or more siRNAs further comprise at least one of enhancer sequence, terminator sequence, invasion factor sequence or lysis regulation sequence. 
     
     
         20 . The method of  claim 4 , wherein said mammalian cells are infected with about 10 3  to 10 11  viable invasive bacteria. 
     
     
         21 . The method of  claim 20 , wherein said mammalian cells are infected with about 10 5  to 10 9  viable invasive bacteria. 
     
     
         22 . The method of  claim 4 , wherein said mammalian cells are infected at a multiplicity of infection ranging from about 0.1 to 10 6 . 
     
     
         23 . The method of  claim 22 , wherein said mammalian cells are infected at a multiplicity of infection ranging from about 10 2  to 10 4 . 
     
     
         24 . The method of  claim 4 , wherein the expressed siRNAs direct the multienzyme complex RNA-induced silencing complex of the cell to interact with the mRNA of one or more HPV oncogenes. 
     
     
         25 . The method of  claim 24 , wherein said complex degrades said mRNA. 
     
     
         26 . The method of  claim 4 , wherein expression of one or more HPV oncogenes is decreased or inhibited. 
     
     
         27 . The method of  claim 5 , wherein the viral disease or disorder is selected from the group consisting of infection, epithelial dysplasia and cancer caused by HPV infection. 
     
     
         28 . The method of  claim 5 , wherein said mammal is selected from the group consisting of human, bovine, ovine, porcine, feline, buffalo, canine, goat, equine, donkey, deer, avian, bird, chicken, and primate. 
     
     
         29 . The method of  claim 5 , wherein the mammalian cells are selected from the group consisting of a gastrointestinal epithelial cell, a macrophage, a cervical epithelial cell, a rectal epithelial cell and a pharyngeal epithelial cell. 
     
     
         30 . The invasive bacterium of  claim 1 , wherein said invasive bacterium contains the pNJSZc plasmid.