Genetic inhibition by double-stranded RNA
View Patent ↗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. A method of delaying, inhibiting or otherwise reducing the expression of a target gene in an animal cell, the method comprising introducing into said animal cell an expression construct, wherein said expression construct comprises an inverted-duplication of a segment of said target gene wherein said inverted-duplication of the target gene segment comprises a nucleotide sequence substantially identical to at least a portion of the target gene and the complement of said portion of the target gene, and wherein a single promoter is used to drive expression of said inverted-duplication of the target gene segment, and wherein said inverted-duplication contains at least one copy of said portion of the target gene segment in the sense orientation.
2. The method according to claim 1 , wherein said inverted-duplication contains at least one copy of said portion of the target gene segment in the antisense orientation.
3. A method of delaying, inhibiting or otherwise reducing the expression of a target gene in an animal cell, comprising expressing in said animal cell an expression construct, wherein said expression construct comprises a first and a second copy of a segment of the target gene, wherein each copy of said target gene segment is identical and separately placed under the control of a first and a second promoter, respectively, which are operable in said cell, and wherein said target gene segment comprises a nucleotide sequence which is substantially identical to at least a portion of the target gene, and wherein the first copy of said target gene segment is placed operably in the sense orientation under the control of the first promoter and wherein the second copy of said target gene segment is placed operably in the antisense orientation under the control of the second promoter.
4. The method according to any one of claims 1 , 2 or 3 , wherein said target gene segment is at least 25 nucleotides long.
5. The method according to claim 1 , wherein said target gene segment comprises a nucleotide sequence that is identical to said portion of the target gene.
6. The method of claim 1 or 3 , wherein the animal cell is not a mammalian cell.
7. A method of delaying, inhibiting or otherwise reducing the expression of a target gene in an animal cell, the method comprising introducing into said animal cell an expression construct, wherein said expression construct comprises an inverted-duplication of a segment of the target gene wherein said inverted-duplication of the target gene segment comprises a nucleotide sequence substantially identical to a nucleotide sequence of 25-50 consecutive nucleic acids in length of the target gene segment and the complement of said 25-50 consecutive nucleic acids of the target gene segment, and wherein a single promoter is used to drive expression of said inverted-duplication of the target gene segment, and wherein said inverted-duplication contains at least one copy of said 25-50 consecutive nucleic acids of the target gene segment in the sense orientation.
8. The method according to claim 7 , wherein said inverted-duplication contains at least one copy of said target gene segment in the antisense orientation.
9. A method of delaying, inhibiting or otherwise reducing the expression of a target gene in an animal cell, comprising expressing in said animal cell an expression construct, wherein said expression construct comprises a first and a second identical copy of a segment of the target gene that is substantially identical to 25-50 consecutive nucleic acids of the target gene segment, wherein each copy of said segment of the target gene is separately placed under the control of a first and a second promoter, respectively, which are operable in said cell, and wherein the first copy of said target gene segment is placed operably in the sense orientation under the control of the first promoter and the second copy of said target gene segment is placed operably in the antisense orientation under the control of the second promoter.
10. The method according to claim 7 or claim 9 , wherein said target gene segment comprises a nucleotide sequence that is identical to said portion of the target gene.
11. The method of claim 1 , wherein a spacer nucleotide sequence is located between the nucleotide sequence substantially identical to at least a portion of the target gene and the complement of said portion of the target gene.
12. The method of claim 7 , wherein a spacer nucleotide sequence is located between the nucleotide sequence substantially identical to the nucleotide sequence of 25-50 consecutive nucleic acids in length of the target gene segment and the complement of said 25-50 consecutive nucleic acids of the target gene segment.
13. The method of claim 1 , wherein the target gene is a cellular gene, an endogenous gene, a viral gene or a transgene.
14. The method of claim 1 or claim 7 , wherein the animal cell is a mammalian cell.