Compositions for bacterial mediated gene silencing and methods of using same

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 cancer of cell proliferative disorders. 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 for delivering one or more siRNAs to animal cells, the method comprising infecting the animal cells with live invasive bacteria containing one or more siRNAs or one or more DNA molecules encoding one or more siRNAs. 
     
     
         2 . A method for regulating gene expression in animal cells, the method comprising infecting the animal cells with live invasive bacteria 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 the gene to be regulated, thereby regulating expression of said gene. 
     
     
         3 . A method for treating or preventing cancer or a cell proliferation disorder in a mammal, the method comprising regulating the expression of a gene in a cell known to increase cell proliferation by infecting the cells of the mammal with live invasive bacteria containing one or more siRNAs or one or more DNA molecules encoding one or more siRNAs, wherein the one or more siRNAs are targeted to reduce expression of the gene. 
     
     
         4 . (canceled) 
     
     
         5 . (canceled) 
     
     
         6 . The method of  claim 1 , wherein said live invasive bacteria are non-pathogenic or non-virulent bacteria. 
     
     
         7 . (canceled) 
     
     
         8 . The method of  claim 6 , wherein said live invasive bacteria are an attenuated strain selected from a member of the group consisting of  Listeria, Shigella, Salmonella, E. coli , and Bifidobacteriae. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 1 , wherein said live invasive bacterium is a member of the group consisting of  Yersinia  spp.,  Escherichia  spp.,  Klebsiella  spp.,  Bordetella  spp.,  Neisseria  spp.,  Aeromonas  spp.,  Franciesella  spp.,  Corynebacterium  spp.,  Citrobacter  spp.,  Chlamydia  spp.,  Hemophilus  spp.,  Brucella  spp.,  Mycobacterium  spp.,  Legionella  spp.,  Rhodococcus  spp.,  Pseudomonas  Spp.,  Helicobacter  spp.,  Salmonella  spp.,  Vibrio  spp.,  Bacillus  spp.,  Leishmania  spp. and  Erysipelothrix  spp. which have been genetically engineered to mimic the invasion properties of  Shigella  spp.,  Listeria  spp.,  Rickettsia  spp., or enteroinvasive  E. coli  spp. 
     
     
         13 . (canceled) 
     
     
         14 . The method of  claim 1 , wherein said animal cell is a mammalian cell. 
     
     
         15 . (canceled) 
     
     
         16 . The method of  claim 14 , wherein said mammalian cell is a human cell. 
     
     
         17 . The method of  claim 3 , wherein said mammal is a human. 
     
     
         18 . The method of  claim 1 , wherein said one or more DNA molecules encoding said one or more siRNAs are transcribed within the animal cell. 
     
     
         19 . The method of  claim 18 , wherein said one or more siRNAs are transcribed within the animal cell as shRNAs. 
     
     
         20 . The method of  claim 18 , wherein said one or more DNA molecules encoding said one or more siRNAs comprise an RNA-polymerase III promoter. 
     
     
         21 . The method of  claim 20 , wherein said RNA-polymerase III promoter is a U6 promoter or an H1 promoter. 
     
     
         22 . The method of  claim 1 , wherein said one or more DNA molecules encoding said one or more siRNAs are transcribed within the bacterium. 
     
     
         23 . The method of  claim 22 , wherein said one or more DNA molecules encoding one or more siRNAs comprise a prokaryotic promoter. 
     
     
         24 . The method of  claim 23 , wherein said prokaryotic promoter is a T7 promoter. 
     
     
         25 . The method of  claim 1 , wherein said one or more DNA molecules are introduced to the cell through type III export or bacterial lysis. 
     
     
         26 . The method of  claim 25 , wherein said bacterial lysis is triggered by the addition of an intracellular active antibiotic. 
     
     
         27 . The method of  claim 26 , wherein said antibiotic is tetracycline. 
     
     
         28 . The method of  claim 25 , wherein said bacterial lysis is triggered through bacterial metabolic attenuation. 
     
     
         29 . The method of  claim 28 , wherein said metabolic attenuation is auxotrophy. 
     
     
         30 . (canceled) 
     
     
         31 . (canceled) 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . The method of  claim 3 , wherein expressed siRNAs interfere with the mRNA of the gene to be regulated. 
     
     
         35 . The method of  claim 2 , wherein the expressed siRNAs direct the multienzyme complex RNA-induced silencing complex of the cell to interact with the mRNA of the gene to be regulated. 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 35 , wherein expression of the gene is decreased or inhibited. 
     
     
         38 . The method of  claim 2 , wherein said gene is ras or β-catenin. 
     
     
         39 . The method of  claim 38 , wherein said ras is k-Ras. 
     
     
         40 . The method of  claim 3 , wherein said cell is a colon cancer cell or a pancreatic cancer cell. 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 - 52 . (canceled)