Bacterial mediated tnf alpha gene silencing

Abstract

Methods are described for the delivery of one or more small interfering RNAs (siRNAs) to a eukaryotic cell using a bacterium. Methods are also described for using this bacterium to regulate gene expression in eukaryotic cells using RNA interference, and methods for treating an inflammatory disease or disorder. The bacterium 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 . 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 TNFα. 
     
     
         2 . 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 TNFα. 
     
     
         3 . A method of treating or preventing an inflammatory disease or disorder in a mammal, the method comprising regulating the expression of at least one gene in a cell known to increase inflammation 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 TNFα. 
     
     
         4 . Art 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 TNFα. 
     
     
         5 . 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 TNFα. 
     
     
         6 . The method of  claim 1 , wherein said invasive bacterium is a non-pathogenic or non-virulent bacterium. 
     
     
         7 . The method of  claim 1 , wherein said invasive bacterium is a therapeutic bacterium. 
     
     
         8 . The method of  claim 1 , wherein said mammalian cell is in vivo or in vitro. 
     
     
         9 . The method of  claim 1 , wherein said mammalian cell is selected from the group consisting of human, bovine, ovine, porcine, feline, buffalo, canine, goat, equine, donkey, deer, avian, bird, chicken and primate cells. 
     
     
         10 . The method of  claim 1 , wherein the mammalian cell is selected from the group consisting of a gastrointestinal epithelial cell and a macrophage. 
     
     
         11 . The method of  claim 3 , 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. 
     
     
         12 . The method of  claim 3 , wherein the inflammatory disease or disorder is selected from the group consisting of inflammatory bowel disease, Crohn's disease, ulcerative colitis, rheumatoid arthritis and an allergy. 
     
     
         13 . The method of  claim 1 , wherein said one or more DNA molecules encoding said one or more siRNAs are transcribed within the animal cell. 
     
     
         14 . The method of  claim 13 , wherein said one or more siRNAs are transcribed within the animal cell as shRNAs. 
     
     
         15 . The method of  claim 1 , wherein said one or more DNA molecules encoding said one or more siRNAs are transcribed within the bacterium. 
     
     
         16 . The method of  claim 1 , wherein said one or more DNA molecules encoding one or more siRNAs comprise a prokaryotic promoter. 
     
     
         17 . The method of  claim 16 , wherein said prokaryotic promoter is a T7 promoter. 
     
     
         18 . The method of  claim 1 , wherein said animal cells are infected with about 10 3  to 10 11  viable invasive bacteria. 
     
     
         19 . The method of  claim 18 , wherein said animal cells are infected with about 10 5  to 10 9  viable invasive bacteria. 
     
     
         20 . The method of  claim 1 , wherein said animal cells are infected at a multiplicity of infection ranging from about 0.1 to 10 6 . 
     
     
         21 . The method of  claim 20 , wherein said animal cells are infected at a multiplicity of infection ranging from about 10 2  to 10 4 . 
     
     
         22 . The method of  claim 1 , wherein the expressed siRNAs direct the multienzyme complex RNA-induced silencing complex of the cell to interact with the mRNA of TNFα. 
     
     
         23 . The method of  claim 22 , wherein said complex degrades said mRNA. 
     
     
         24 . The method of  claim 1 , wherein expression of TNFα is decreased or inhibited. 
     
     
         25 . The invasive bacterium of  claim 4 , wherein said invasive bacterium is a non-pathogenic or non-virulent bacterium. 
     
     
         26 . The invasive bacterium of  claim 4 , wherein said invasive bacterium is a therapeutic bacterium. 
     
     
         27 . A composition comprising the bacterium of  claim 4  and a pharmaceutically acceptable carrier. 
     
     
         28 . A eukaryotic host cell comprising the bacterium of  claim 4 , and a pharmaceutically acceptable carrier.