Genetic Inhibition by Double-Stranded RNA

Abstract

A process is provided of introducing an RNA into a living cell to inhibit gene expression of a target gene in that cell. The process may be practiced ex vivo or in vivo. The RNA has a region with double-stranded structure. Inhibition is sequence-specific in that the nucleotide sequences of the duplex region of the RNA and of a portion of the target gene are identical. The present invention is distinguished from prior art interference in gene expression by antisense or triple-strand methods.

1 .- 39 . (canceled) 
     
     
         40 . A method to inhibit expression of a target gene in a plant cell comprising synthesis of a first ribonucleic acid (RNA) and second RNA in the plant cell, wherein the first and second RNAs hybridize to each other to form a double-stranded structure in the cell, the first RNA consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and the second RNA consisting essentially of a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene, wherein said first and said second RNA are synthesized in the plant cell in an amount sufficient to inhibit the expression of the target gene. 
     
     
         41 . A method to inhibit expression of a target gene in a plant cell comprising introducing into said cell double-stranded ribonucleotide (RNA) comprising a first RNA consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second RNA which consists essentially of a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene and wherein said first and second RNAs are present in an amount sufficient to inhibit the expression of the target gene. 
     
     
         42 . The method of  claim 40  or  claim 41  wherein the target gene is an endogenous gene or a transgene. 
     
     
         43 . The method of  claim 42  wherein the expression of the target gene is inhibited in the cell in vitro. 
     
     
         44 . The method of  claim 40  or  claim 41  wherein the target gene expression is reduced by at least 10%. 
     
     
         45 . A kit comprising reagents for inhibiting expression of a target gene in a plant cell, wherein said kit comprises a means for introduction of a ribonucleic acid (RNA) into the plant cell in an amount sufficient to inhibit expression of the target gene, and wherein the RNA has a double-stranded structure with an identical nucleotide sequence as compared to a portion of the target gene. 
     
     
         46 . A method of inhibiting expression of a target gene in a plant cell in a plant comprising providing at least one ribonucleic acid (RNA) to the plant cell in an amount sufficient to inhibit the expression of a target gene, wherein said RNA is provided to the plant cell by synthesizing said RNA in said plant cell, wherein the RNA comprises or forms a double-stranded structure containing a first strand consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second ribonucleotide sequence which is complementary to the target gene, wherein the first and the second ribonucleotide sequences are complementary sequences that hybridize to each other to comprise or form said double-stranded structure, and wherein the RNA comprising or forming the double-stranded structure inhibits expression of target gene. 
     
     
         47 . The method of  claim 46 , wherein said RNA is transcribed from an expression construct. 
     
     
         48 . The method of  claim 46 , wherein said double-stranded structure is formed by a single self-complementary RNA strand comprising the first and second ribonucleotide sequences. 
     
     
         49 . The method of  claim 48 , wherein said single self-complementary RNA strand is transcribed from an expression construct. 
     
     
         50 . A method of inhibiting the expression of a target gene in a plant cell, comprising contacting said plant cell with an expression construct, wherein said expression construct comprises an inverted duplication for a segment of the target gene, wherein said segment of the target gene comprises a nucleotide sequence substantially identical to at least one portion of the target gene, wherein a promoter drives expression of said inverted-duplication, and wherein said inverted-duplication forms a double-stranded RNA structure which inhibits expression of the target gene. 
     
     
         51 . The method of  claim 50 , wherein the double-stranded RNA structure is partially double-stranded. 
     
     
         52 . An expression construct comprising an inverted-duplication for a segment of a target gene in a plant cell, wherein said segment of a target gene comprises a nucleotide sequence substantially identical to at least a portion of the target gene, wherein a promoter drives expression of said inverted-duplication, and wherein said inverted-duplication forms a double-stranded RNA structure which is capable of inhibiting expression of the target gene in a plant cell. 
     
     
         53 . An expression construct comprising a nucleotide sequence comprising a regulatory region, wherein said nucleotide sequence is capable of transcribing a single self-complementary RNA strand which forms a double-stranded RNA structure, and wherein said double-stranded RNA structure is capable of inhibiting expression of a target gene in a plant cell.