Improved gene silencing methods

Abstract

Provided are methods and means to obtain improved gene silencing of target nucleic acids whereby at least two inhibitory RNA molecules are provided which are targeted to the same nucleic acid, but which are processed into short interfering RNA molecules through different processing pathways. Also provided are methods and means to obtain improved gene silencing of target nucleic acids whereby at least two inhibitory RNA molecules are provided which are targeted to different nucleic acids, but which are processed into short interfering RNA molecules through different processing pathways.

1 . A method to reduce the expression of a target nucleic acid in a eukaryotic cell or organism comprising the step of introducing a combination of at least two inhibitory RNA molecules each capable of reducing the expression of a first target nucleic acid in said eukaryotic cell or organism, wherein said inhibitory RNA molecules are processed into oligonucleotides of 21 to 24 nucleotides in length predominantly via different RNAi molecule processing pathways in the cell or organism. 
     
     
         2 . A method to reduce the expression of first and second target nucleic acids in a eukaryotic cell or organism comprising the step of introducing a combination of at least two inhibitory RNA molecules, wherein one of said inhibitory RNA molecules is capable of reducing the expression of the first target nucleic acid in said eukaryotic cell or organism and the other of said inhibitory RNA molecules is capable of reducing the expression of the second target nucleic acid in said eukaryotic cell or organism, and wherein said inhibitory RNA molecules are processed into oligonucleotides of 21 to 24 nucleotides in length predominantly via different RNAi molecule processing pathways in the cell or organism. 
     
     
         3 . The method according to  claim 1 , wherein the two inhibitory RNA molecules target the same region, overlapping regions, or non-overlapping regions of the first target nucleic acid. 
     
     
         4 . The method according to  claim 1 , wherein the two inhibitory RNA molecules are not covalently joined. 
     
     
         5 . The method according to  claim 1 , wherein the two inhibitory RNA molecules are expressed from different promoters, such as PolII- and PolIII-dependent promoters. 
     
     
         6 . The method according to  claim 1 , wherein said inhibitory RNA molecules are cleaved into oligonucleotides of 21 to 24 nucleotides in length predominantly via different dicer or Dicerlike proteins. 
     
     
         7 . The method according to  claim 1 , wherein said eukaryotic cell is a plant cell or said eukaryotic organism is a plant. 
     
     
         8 . The method according to  claim 1  wherein said eukaryotic cell is an animal cell or said eukaryotic organism is an animal. 
     
     
         9 . The method according to  claim 1 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL1 or an RNAse with similar function and specificity and the other of said inhibitory RNA molecules is predominantly cleaved via DCL4 or an RNAse with similar function and specificity. 
     
     
         10 . The method according to  claim 1 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL3 or an RNAse with similar function and specificity and the other of said inhibitory RNA molecules is predominantly cleaved via DCL4 or an RNAse with similar function and specificity. 
     
     
         11 . The method according to  claim 1 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL1 or an RNAse with similar function and specificity and the other of said inhibitory RNA molecules is predominantly cleaved via DCL2 or an RNAse with similar function and specificity. 
     
     
         12 . The method according to  claim 1 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL1 or an RNAse with similar function and specificity and the other of said inhibitory RNA molecules is predominantly cleaved via DCL3 or an RNAse with similar function and specificity. 
     
     
         13 . The method according to  claim 1 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL2 or an RNAse with similar function and specificity and the other of said inhibitory RNA molecules is predominantly cleaved via DCL3 or an RNAse with similar function and specificity. 
     
     
         14 . The method according to  claim 1 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL2 or an RNAse with similar function and specificity and the other of said inhibitory RNA molecules is predominantly cleaved via DCL4 or an RNAse with similar function and specificity. 
     
     
         15 . The method according  claim 1 , wherein one of said inhibitory RNA molecules is a miRNA molecule, a pre-microRNA molecule or a pri-miRNA molecule capable of reducing the expression of the first target nucleic acid, and wherein said other inhibitory RNA molecule is a double stranded RNA molecule comprising a complementary or essentially complementary first and second RNA region, said first RNA region comprising at least 19 consecutive nucleotides corresponding to a nucleotide sequence of the first or second target nucleic acid and said second RNA region comprising at least 19 consecutive nucleotides corresponding to a nucleotide sequence complementary to the nucleotide sequence of the first or second target nucleic acid, wherein said first and second RNA regions hybridize to each other with at least 18 or 19 basepairs. 
     
     
         16 . The method according to  claim 1 , wherein one of said inhibitory RNA molecules comprises at least 19 consecutive nucleotides from a promoter region of said target nucleic acid molecule, and said other inhibitory RNA molecule comprises at least 19 consecutive nucleotides from the region of said target nucleic acid molecule which is transcribed into a RNA molecule. 
     
     
         17 . The method according to  claim 1 , wherein said inhibitory RNA molecules are transcribed from inhibitory RNA encoding genes introduced in said eukaryotic cell. 
     
     
         18 . The method according to  claim 17 , wherein one of said inhibitory RNA molecules is transcribed from an inhibitory RNA-encoding gene under control of a promoter recognized by RNA polymerase II and said other inhibitory RNA is transcribed from an inhibitory RNA-encoding gene under control of a promoter recognized by RNA polymerase III. 
     
     
         19 . The method according to  claim 1 , wherein said target nucleic acid is a viral nucleic acid or a gene or transcribed region thereof. 
     
     
         20 . The method according to  claim 1 , wherein said target nucleic acid is a transgene or a region thereof. 
     
     
         21 . The method according to  claim 1 , wherein said target nucleic acid is an endogenous gene in the cell, a promoter thereof, a transcript thereof or a region thereof. 
     
     
         22 . The method according to  claim 1 , wherein the combination of two inhibitory RNA molecules reduces the expression of the first target nucleic acid to a greater extent than either inhibitory RNA molecule alone in said eukaryotic cell or organism. 
     
     
         23 . The method according to  claim 1 , wherein the combination of two inhibitory RNA molecules reduces the expression of the first target nucleic acid more stably than either inhibitory RNA molecule alone in said eukaryotic cell or organism. 
     
     
         24 . The method according to  claim 1 , wherein the combination of two inhibitory RNA molecules reduces the expression of the first target nucleic acid such that the reduced expression is less sensitive to external factors than the reduced expression of the target nucleic acid in the presence of either inhibitory RNA molecules alone in said eukaryotic cell or organism. 
     
     
         25 . A eukaryotic cell comprising at least two inhibitory RNA molecules capable of reducing the expression of a first target nucleic acid in said eukaryotic cell, wherein said inhibitory RNA molecules are processed into oligonucleotides of 21 to 24 nucleotides in length via different RNAi molecule processing pathways in the cell. 
     
     
         26 . A eukaryotic cell comprising at least two inhibitory RNA molecules capable of reducing the expression of first and second target nucleic acids in said eukaryotic cell, wherein one of said inhibitory RNA molecules is capable of reducing the expression of the first target nucleic acid in said eukaryotic cell or organism and the other of said inhibitory RNA molecules is capable of reducing the expression of the second target nucleic acid in said eukaryotic cell or organism, and wherein said inhibitory RNA molecules are processed into oligonucleotides of 21 to 24 nucleotides in length via different RNAi molecule processing pathways in the cell. 
     
     
         27 . The cell according to  claim 25 , wherein the two inhibitory RNA molecules target the same region, overlapping regions, or non-overlapping regions of the first target nucleic acid. 
     
     
         28 . The cell according to  claim 25 , wherein the two inhibitory RNA molecules are not covalently joined. 
     
     
         29 . The cell according to  claim 25 , wherein the two inhibitory RNA molecules are expressed from different promoters, such as PolII- and PolIII-dependent promoters. 
     
     
         30 . The eukaryotic cell according to  claim 25 , wherein said inhibitory RNA molecules are cleaved into oligonucleotides of 21 to 24 nucleotides in length predominantly via different dicer or Dicerlike proteins. 
     
     
         31 . The eukaryotic cell according to  claim 25  which is in a plant. 
     
     
         32 . The eukaryotic cell according to  claim 25  which is in an animal. 
     
     
         33 . The eukaryotic cell according to  claim 25 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL1 or a similar RNAse and the other of said inhibitory RNA molecules is predominantly cleaved via DCL4 or a similar RNAse. 
     
     
         34 . The eukaryotic cell according to  claim 25 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL3 or a similar RNAse and the other of said inhibitory RNA molecules is predominantly cleaved via DCL4 or a similar RNAse. 
     
     
         35 . The eukaryotic cell according to  claim 25 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL1 or a similar RNAse and the other of said inhibitory RNA molecules is predominantly cleaved via DCL2 or a similar RNAse. 
     
     
         36 . The eukaryotic cell according to  claim 25 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL1 or a similar RNAse and the other of said inhibitory RNA molecules is predominantly cleaved via DCL3 or a similar RNAse. 
     
     
         37 . The eukaryotic cell according to  claim 25 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL2 or a similar RNAse and the other of said inhibitory RNA molecules is predominantly cleaved via DCL3 or a similar RNAse. 
     
     
         38 . The eukaryotic cell according to  claim 25 , wherein one of said inhibitory RNA molecules is predominantly cleaved via DCL2 or a similar RNAse and the other of said inhibitory RNA molecules is predominantly cleaved via DCL4 or a similar RNAse. 
     
     
         39 . The eukaryotic cell according to  claim 25 , wherein one of said inhibitory RNA molecules is a miRNA molecule, a pre-microRNA molecule or a pri-miRNA molecule capable of reducing the expression of said target nucleic acid, and wherein said other inhibitory RNA molecule is a double stranded RNA molecule comprising a complementary or essentially complementary first and second RNA region, said first RNA region comprising at least 19 consecutive nucleotides corresponding to a nucleotide sequence of the first or second target nucleic acid and said second RNA region comprising at least 19 consecutive nucleotides corresponding to a nucleotide sequence complementary to the nucleotide sequence of the first or second target nucleic acid, wherein said first and second RNA regions hybridize to each other with at least 18 or 19 basepairs. 
     
     
         40 . The eukaryotic cell according to  claim 25 , wherein one of said inhibitory RNA molecules comprises at least 19 consecutive nucleotides from a promoter region of said target nucleic acid molecule, and said other inhibitory RNA molecule comprises at least 19 consecutive nucleotides from the region of said target nucleic acid molecule which is transcribed into a RNA molecule. 
     
     
         41 . The eukaryotic cell according to  claim 25 , wherein said inhibitory RNA molecules are transcribed from inhibitory RNA encoding genes introduced in said eukaryotic cell. 
     
     
         42 . The eukaryotic cell according to  claim 25 , wherein one of said inhibitory RNA molecules is transcribed from an inhibitory RNA encoding gene under control of a promoter recognized by RNA polymerase II and said other inhibitory RNA is transcribed from an inhibitory RNA encoding gene under control of a promoter recognized by RNA polymerase III. 
     
     
         43 . The eukaryotic cell according to  claim 25 , wherein said target nucleic acid is a viral nucleic acid or a gene or transcribed region thereof. 
     
     
         44 . The eukaryotic cell according to  claim 25 , wherein said target nucleic acid is a transgene or a region thereof. 
     
     
         45 . The eukaryotic cell according to  claim 25  wherein said target nucleic acid is an endogenous gene in the cell, a promoter thereof, a transcript thereof or a region thereof. 
     
     
         46 . A non-human eukaryotic organism consisting essentially or completely of the cells according to  claim 25 . 
     
     
         47 . The non-human eukaryotic organism of  claim 46 , wherein the combination of two inhibitory RNA molecules reduces the expression of the first target nucleic acid to a greater extent than either inhibitory RNA molecule alone in said eukaryotic cell or organism in said eukaryotic organism. 
     
     
         48 . The non-human eukaryotic organism of  claim 46 , wherein the combination of two inhibitory RNA molecules reduces the expression of the first target nucleic acid more stably than either inhibitory RNA molecule alone in said eukaryotic organism. 
     
     
         49 . The non-human eukaryotic organism of  claim 46 , wherein the combination of two inhibitory RNA molecules reduces the expression of the first target nucleic acid such that the reduced expression is less sensitive to external factors than the reduced expression of said target nucleic acid in the presence of either inhibitory RNA molecules alone in said eukaryotic organism. 
     
     
         50 . A composition of matter comprising at least two inhibitory RNA molecules as described in  claim 1 , capable of reducing the expression of a first target nucleic acid in a eukaryotic cell or first and second target nucleic acids in a eukaryotic cell, wherein said inhibitory RNA molecules are processed into oligonucleotides of 21 to 24 nucleotides in length predominantly via different RNAi molecule processing pathways in the cell. 
     
     
         51 . The composition of  claim 50 , wherein the target gene is selected from the group consisting of a pathogenic animal virus genes, a cancer-related gene, an oncogene, an immunomodulatory gene, a gene encoding a cytokine, growth factor, enzyme or transcription factor. 
     
     
         52 . The composition of  claim 50 , further comprising a pharmaceutically acceptable carrier, a veterinarily acceptable carrier, or an agriculturally acceptable carrier. 
     
     
         53 . A kit comprising at least two inhibitory RNA molecules as described in  claim 1  as a combined preparation for simultaneous, separate or sequential use in reduction of the expression of a first target nucleic acid in a eukaryotic organism or first and second target nucleic acids in a eukaryotic cell. 
     
     
         54 . A composition of matter according to  claim 50  for use as a medicament. 
     
     
         55 . (canceled) 
     
     
         56 . A method of treating or preventing a disease in an animal, the method comprising administering a composition according to  claim 50  to an animal in need thereof.