ENABLING THE USE OF LONG dsRNA FOR GENE TARGETING IN MAMMALIAN AND OTHER SELECTED ANIMAL CELLS

Abstract

The present invention provides a method of enabling the use of long dsRNA for gene silencing in mammalian cells through bacteria, preferably non-pathogenic or therapeutic strains of bacteria. DNA that encodes long double-strand RNAs are transformed into bacteria and processed in the bacterial cells into a mixture of smaller RNA duplexes and then released into the cytoplasm of the target cells, resulting in modulation of gene expression in the target cells. The methods overcome the incompatibility between long strong dsRNA and mammalian cells by eliminating, or mitigating, the non-specific innate immune response. The eukaryotic cells can be mammalian cells or avian cells. The gene of interest can be a mammalian, avian, bacterial, eukaryotic, or viral gene.

1 . A bacterium comprising a long double-strand RNA (dsRNA), or a DNA molecule encoding the RNA,
 wherein the dsRNA comprises a sequence substantially complementary to a messenger RNA (mRNA) sequence of a target eukaryotic gene or viral gene.   
     
     
         2 . The bacterium of  claim 1  wherein said bacterium is capable of processing the dsRNA into a mixture of shorter RNA duplexes. 
     
     
         3 . The bacterium of  claim 2  wherein the mixture of short RNA duplexes is capable of modulating the expression of the eukaryotic gene or viral gene. 
     
     
         4 . The bacterium of  claim 1  further comprising an enzyme or ribozyme capable of processing the dsRNA into a mixture of shorter RNA duplexes. 
     
     
         5 . The bacterium of  claim 4  wherein the enzyme is an endonuclease. 
     
     
         6 . The bacterium of  claim 1  wherein said bacterium further comprises a bacterial RNase III, a Dicer, or a Dicer-like enzyme. 
     
     
         7 . The bacterium of  claim 1  wherein said bacterium is capable of modulating the expression of the eukaryotic gene or viral gene in a eukaryotic cell after being introduced into a eukaryotic cell. 
     
     
         8 . The bacterium of  claim 1  wherein the dsRNA is at least 40 bp in length. 
     
     
         9 . The bacterium of  claim 1  wherein the dsRNA is at least of a length selected from the group consisting of 70, 100, 200, 400 and 1000 bp. 
     
     
         10 - 13 . (canceled) 
     
     
         14 . The bacterium of  claim 1  wherein the DNA molecule comprises one or more prokaryotic promoters controlling the expression of the dsRNA. 
     
     
         15 . The bacterium of  claim 14  wherein at least one of the one or more prokaryotic promoters is a T7 promoter. 
     
     
         16 . The bacterium of  claim 14  wherein at least one of the one or more prokaryotic promoters is endogenous to said bacterium. 
     
     
         17 . The bacterium of  claim 1  wherein the DNA molecule comprises two prokaryotic promoters controlling the expression of the two strands of the DNA molecule. 
     
     
         18 . The bacterium of  claim 1  wherein said eukaryotic gene is an animal gene. 
     
     
         19 . The bacterium of  claim 1  wherein said eukaryotic gene is a mammalian gene or an avian gene. 
     
     
         20 . The bacterium of  claim 1 , wherein said eukaryotic gene is a mammalian disease gene. 
     
     
         21 . The bacterium of  claim 1 , wherein said eukaryotic gene is a cancer gene. 
     
     
         22 . The bacterium of  claim 1 , wherein said eukaryotic gene is β-catenin. 
     
     
         23 . The bacterium of  claim 1 , wherein said eukaryotic gene is k-Ras. 
     
     
         24 . The bacterium of  claim 1 , wherein said viral gene is an HIV gene. 
     
     
         25 . The bacterium of  claim 1  wherein said bacterium is invasive. 
     
     
         26 . The bacterium of  claim 1  wherein said bacterium is live. 
     
     
         27 . The bacterium of  claim 1  wherein said bacterium is a half-killed bacterium. 
     
     
         28 . The bacterium of  claim 1  wherein said bacterium is non-pathogenic. 
     
     
         29 . The bacterium of  claim 1  wherein said bacterium is a therapeutic bacterium. 
     
     
         30 . The bacterium of  claim 1 , wherein said bacterium is an attenuated strain selected from the group consisting of  Listeria, Shigella, Salmonella, E. coli , and Bifidobacteriae. 
     
     
         31 . The bacterium of  claim 1  wherein the DNA molecule comprises a circular, double-strand plasmid. 
     
     
         32 . The bacterium of  claim 1  wherein the DNA molecule is integrated into a bacterial chromosome. 
     
     
         33 . The bacterium of  claim 1  wherein the dsRNA comprises a sequence that is perfectly complementary to the mRNA sequence of a eukaryotic gene or viral gene. 
     
     
         34 . A bacterium comprising a mixture of short RNA duplexes capable of modulating the expression of a eukaryotic gene or viral gene. 
     
     
         35 . The bacterium of  claim 34  wherein the mixture of short RNA duplexes is produced from a long double-strand RNA (dsRNA). 
     
     
         36 - 69 . (canceled) 
     
     
         70 . The bacterium of  claim 1  further comprising an enzyme that assists in transporting genetic materials, upon their release from the bacterium, into the cytoplasm of a target eukaryotic cell. 
     
     
         71 . The bacterium of  claim 70  wherein said enzyme is an Hly protein. 
     
     
         72 . (canceled)