Compositions for Bacterial Mediated Gene Silencing and Methods of Using the 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 off 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 of delivering one or more siRNAs to animal cells, the method comprising infecting the animal cells with at least one invasive bacterium containing one or more siRNAs or one or more DNA molecules encoding one or more siRNAs. 
     
     
         2 . A method of regulating gene expression in animal cells, the method comprising infecting the animal cells with 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 the gene to be regulated, thereby regulating expression of said gene. 
     
     
         3 . A method of treating or preventing cancer or a cell proliferation disorder in a mammal, the method comprising regulating the expression of at least one gene in a cell known to increase cell proliferation by infecting the cells of the mammal with at least one invasive bacterium containing one or more siRNAs or one or more DNA molecules encoding one or more siRNAs. 
     
     
         4 . An invasive bacterium comprising one or more siRNAs or one or more DNA molecules encoding one or more siRNAs. 
     
     
         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. 
     
     
         6 . The method of  claims 1 - 3 , wherein said invasive bacterium is a non-pathogenic or non-virulent bacterium 
     
     
         7 . The method of  claim 6 , wherein said invasive bacterium is a therapeutic bacterium. 
     
     
         8 . The method of  claim 6 , wherein said invasive bacterium is an attenuated strain selected from a member of the group consisting of  Listeria, Shigella, Salmonella, E. coli , and  Bifidobacteriae.    
     
     
         9 . The method of  claim 8 , wherein said  Salmonella  strain is an attenuated strain of the  Salmonella typhimurium  species. 
     
     
         10 . The method of  claim 9 , wherein said attenuated strain of the  Salmonella typhimurium  species is SL 7207 or VNP20009. 
     
     
         11 . The method of  claim 8 , wherein said attenuated  E. coli  strain is BM 2710. 
     
     
         12 . The method of  claims 1 - 3 , wherein said 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 . The method of  claim 1 , wherein said animal cell is in vivo or in vitro. 
     
     
         14 . The method of  claims 1 - 2 , wherein said animal cell is a mammalian cell. 
     
     
         15 . The method of  claim 14 , wherein said mammalian cell is a member of the group consisting of human, bovine, ovine, porcine, feline, buffalo, canine, goat, equine, donkey, deer, and primate cells. 
     
     
         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  claims 1 - 3 , 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  claims 1 - 3 , 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  claims 1 - 3 , 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 . The method of  claims 1 - 3 , wherein said animal cells are infected with about 10 3  to 10 11  viable invasive bacteria. 
     
     
         31 . The method of  claim 30 , wherein said animal cells are infected with about 10 5  to 10 9  viable invasive bacteria. 
     
     
         32 . The method of  claims 1 - 3 , wherein said animal cells are infected at a multiplicity of infection ranging from about 0.1 to 10 6 . 
     
     
         33 . The method of  claim 32 , wherein said animal cells are infected at a multiplicity of infection ranging from about 10 2  to 10 4 . 
     
     
         34 . The method of  claim 3 , wherein expressed siRNAs interfere with the mRNA of the gene to be regulated. 
     
     
         35 . The method of  claims 2  or  34 , 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 . The method of  claim 35 , wherein said complex degrades said mRNA 
     
     
         37 . The method of  claim 35 , wherein expression of the gene is decreased or inhibited. 
     
     
         38 . The method of  claims 2 - 3 , 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 cell or a pancreatic cell. 
     
     
         41 . The method of  claim 40 , wherein the colon cell is an SW 480 cell. 
     
     
         42 . The method of  claim 40 , wherein the pancreatic cell is a CAPAN-1 cell. 
     
     
         43 . The invasive bacterium of  claim 4 , wherein said invasive bacterium is a non-pathogenic or non-virulent bacterium. 
     
     
         44 . The invasive bacterium of  claim 43 , wherein said invasive bacterium is a therapeutic bacterium. 
     
     
         45 . A composition comprising the bacterium of  claim 4  and a pharmaceutically acceptable carrier. 
     
     
         46 . A eukaryotic host cell comprising the bacterium of  claim 4 , and a pharmaceutically acceptable carrier. 
     
     
         47 . The bacterium of  claim 4 , wherein said invasive bacterium is an attenuated strain selected from a member of the group consisting of  Listeria, Shigella, Salmonella, E. coli , and  Bifidobacteriae.    
     
     
         48 . The bacterium of  claim 47 , wherein said  Salmonella  strain is an attenuated strain of the  Salmonella typhimurium  species. 
     
     
         49 . The bacterium of  claim 48 , wherein said attenuated strain of the  Salmonella typhimurium  species is SL 7207 or VNP20009. 
     
     
         50 . The bacterium of  claim 47 , wherein said attenuated  E. coli  strain is BM 2710. 
     
     
         51 . The prokaryotic vector of  claim 5 , wherein the RNA-polymerase III promoter is a U6 promoter or an H1 promoter. 
     
     
         52 . The prokaryotic vector of  claim 5 , wherein the prokaryotic promoter is a T7 promoter.