BACTERIA-MEDIATED GENE MODULATION VIA microRNA MACHINERY

Abstract

The present invention provides a method of synthesizing, processing, and/or delivering miRNA or its precursors to eukaryotic cells using bacteria, preferably non-pathogenic or therapeutic strains of bacteria, to effect gene modulation in eukaryotic cells.

1 . A DNA vector that encodes at least a microRNA (miRNA) or a miRNA precursor, wherein said miRNA is capable of modulating the expression of at least one eukaryotic, prokaryotic, or viral target gene. 
     
     
         2 . The vector of  claim 1  wherein said miRNA precursor is a pri-miRNA. 
     
     
         3 . The vector of  claim 1  wherein said miRNA precursor is a pre-miRNA. 
     
     
         4 . The vector of  claim 1  wherein said vector encodes duplex miRNAs, wherein said two miRNA strands comprise a substantially complementary region. 
     
     
         5 . The vector of  claim 1  wherein said miRNA is a mature miRNA. 
     
     
         6 . The vector of  claim 1  further comprising a prokaryotic promoter. 
     
     
         7 . The vector of  claim 1  further comprising a eukaryotic promoter. 
     
     
         8 . The vector of  claim 1  wherein said at least one target gene is a cancer-related gene. 
     
     
         9 . The vector of  claim 1  further encodes an Hly gene. 
     
     
         10 . A bacterium comprising a microRNA (miRNA), a miRNA precursor, or a DNA molecule encoding at least said miRNA or said precursor, wherein said miRNA is capable of modulating the expression of at least one eukaryotic, prokaryotic, or viral target gene. 
     
     
         11 . The bacterium of  claim 10  wherein the precursor is pre-miRNA. 
     
     
         12 . The bacterium of  claim 10  wherein the precursor is pri-miRNA. 
     
     
         13 . The bacterium of  claim 10  wherein the bacterium is a live invasive bacterium. 
     
     
         14 . The bacterium of  claim 10  wherein the bacterium is a derivate of a live invasive bacterium. 
     
     
         15 . The bacterium of  claim 10  wherein the bacterium is non-pathogenic and non-virulent. 
     
     
         16 . The bacterium of  claim 10 , wherein the bacterium is an attenuated strain selected from the group consisting of  Listeria, Shigella, Salmonella, E. coli , and Bifidobacteriae. 
     
     
         17 . The bacterium of  claim 10 , wherein the bacterium is selected from 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.,  Pseiidomonas  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., and enteroinvasive  E. coli  spp. 
     
     
         18 . The bacterium of  claim 10  further comprising an enzyme or ribozyme that is capable of processing the precursor closer to a mature miRNA. 
     
     
         19 . The bacterium of  claim 18  wherein the enzyme is an endonuclease. 
     
     
         20 . The bacterium of  claim 10  further comprising at least one of a bacterial RNase III, a Dicer, a Dicer-like enzyme, Drosha, and Pasha. 
     
     
         21 . The bacterium of  claim 10  further comprising an enzyme that assists in transporting genetic materials, upon their release from the bacterium, into the cytoplasm of a target eukaryotic cell. 
     
     
         22 . The bacterium of  claim 21  wherein said enzyme is an Hly protein. 
     
     
         23 . The bacterium of  claim 10  further comprising a prokaryotic promoter controlling the expression of said DNA molecule. 
     
     
         24 . The bacterium of  claim 23 , wherein the promoter is T7 promoter. 
     
     
         25 . The bacterium of  claim 10 , further comprising a eukaryotic promoter controlling the expression of said DNA molecule. 
     
     
         26 . The bacterium of  claim 10  wherein the DNA molecule further encodes an Hly gene. 
     
     
         27 . The bacterium of  claim 10  wherein the eukaryotic gene is an animal gene. 
     
     
         28 . The bacterium of  claim 10  wherein the eukaryotic gene is mammalian or avian gene. 
     
     
         29 . The bacterium of  claim 10  wherein the at least one target gene is a cancer-related gene. 
     
     
         30 . A method of delivering a microRNA (miRNA) or a miRNA precursor to an animal cell, said method comprising infecting said animal cell with a bacterium comprising a microRNA (miRNA), a miRNA precursor, or a DNA molecule encoding at least said miRNA or said precursor, wherein said miRNA is capable of modulating the expression of at least one eukaryotic, prokaryotic, or viral target gene. 
     
     
         31 . The method of  claim 30 , wherein said animal cell is a human cell. 
     
     
         32 . The method of  claim 30 , further comprising lysing said bacterium after infecting. 
     
     
         33 - 47 . (canceled)