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 - 47 . (canceled) 
     
     
         48 . A method of regulating the expression of at least one target gene in an animal cell, said method comprising:
 infecting an animal cell with a bacterium comprising a microRNA (miRNA), a miRNA precursor, or a DNA molecule encoding at least said miRNA or said miRNA precursor, said bacterium further comprising: (i) a ribozyme or a first enzyme selected from the group consisting of an endonuclease, a bacterial RNase III, a Dicer, a Dicer-like enzyme, Drosha, and Pasha for processing any miRNA precursor to a mature miRNA, and (ii) a second enzyme or genetic material producing said second enzyme which, in turn, effects transporting of miRNA and miRNA precursor, upon their release from the bacterium, into the cytoplasm of said target animal cell; and   lysing said bacterium to release its content, thereby allowing said miRNA from said content or produced from said content to modulate the expression of at least one eukaryotic, prokaryotic, or viral target gene in said animal cell.   
     
     
         49 . The method of  claim 48 , wherein said miRNA precursor is pre-miRNA. 
     
     
         50 . The method of  claim 48 , wherein said miRNA precursor is pri-miRNA. 
     
     
         51 . The method of  claim 48 , wherein said DNA molecule encodes duplex miRNAs, and wherein said two miRNA strands comprise a substantially complementary region. 
     
     
         52 . The method of  claim 48 , wherein said bacterium is a live invasive bacterium or a derivate of a live invasive bacterium. 
     
     
         53 . The method of  claim 48 , wherein said bacterium is non-pathogenic and non-virulent. 
     
     
         54 . The method of  claim 48 , wherein said bacterium is an attenuated strain selected from the group consisting of  Listeria, Shigella, Salmonella, E. coli , and Bifidobacteriae. 
     
     
         55 . The method of  claim 48 , wherein said bacterium is selected from the group consisting of  Yersinia  spp.,  Escherichia  spp.,  Klebsiella  spp.,  Bordetella  spp.,  Neisseria  spp.,  Aeromonas  spp.,  Franciesella  spp.,  Corynebacteriiim  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. 
     
     
         56 . The method of  claim 48 , wherein said second enzyme is an Hly protein. 
     
     
         57 . The method of  claim 48 , wherein said bacterium further comprises a prokaryotic promoter controlling the expression of said DNA molecule. 
     
     
         58 . The method of  claim 57 , wherein said promoter is a T7 promoter. 
     
     
         59 . The method of  claim 48 , wherein said bacterium further comprises a eukaryotic promoter controlling the expression of said DNA molecule. 
     
     
         60 . The method of  claim 48 , wherein said DNA molecule further encodes an Hly gene. 
     
     
         61 . The method of  claim 48 , wherein said eukaryotic gene is an animal gene. 
     
     
         62 . The method of  claim 48 , wherein said eukaryotic gene is a mammalian or avian gene. 
     
     
         63 . The method of  claim 48 , wherein said at least one target gene is a cancer-related gene. 
     
     
         64 . The method of  claim 48 , wherein said animal cell is a human cell. 
     
     
         65 . The method of  claim 48 , wherein, upon lysing said bacterium to release its content, said miRNA regulates the expression of at least one target gene through mRNA translation repression, mRNA degradation or both. 
     
     
         66 . The method of  claim 48 , wherein said bacterium expresses and processes the miRNA precursor into an miRNA within said animal cell. 
     
     
         67 . The method of  claim 48  for treating or preventing a disorder in said animal, wherein said at least one target gene is known to be involved in the disorder. 
     
     
         68 . The method of  claim 67 , wherein said disorder is caused by at least one defective miRNA in said animal, and wherein said bacteria further comprises a functional version of said defective miRNA, an RNA precursor to said functional version, or a DNA molecule encoding at least said functional version or said RNA precursor. 
     
     
         69 . The method of  claim 67 , wherein said disorder is caused by at least one upregulated miRNA in said animal, and wherein said bacteria further comprises an antisense version of said upregulated miRNA, an RNA precursor to said antisense version, or a DNA molecule encoding at least said antisense version or said RNA precursor. 
     
     
         70 . The method of  claim 48  for treating or preventing cancer in an animal, wherein the at least one target gene is known to be involved in the cancer. 
     
     
         71 . A method of regulating the expression of at least one target gene in an animal cell, said method comprising:
 infecting an animal cell with a live, non-pathogenic and non-virulent bacterium comprising a DNA molecule encoding at least a microRNA (miRNA) or a miRNA precursor miRNA, said bacterium further comprising: (i) a ribozyme or a first enzyme selected from the group consisting of an endonuclease, a bacterial RNase III, a Dicer, a Dicer-like enzyme, Drosha, and Pasha for processing any miRNA precursor to a mature miRNA, and (ii) an Hly protein or genetic material producing said Hly protein for transporting miRNA and miRNA precursor, upon their release from the bacterium, into the cytoplasm of said target animal cell; and   lysing said bacterium to release its content, thereby allowing said miRNA from said content or produced from said content to modulate the expression of at least one animal gene in said animal cell.