Genetic inhibition by double-stranded RNA

• US 6,506,559 B1
• Filed: 12/18/1998
• Issued: 01/14/2003
• Est. Priority Date: 12/23/1997
• Status: Expired due to Term

• Current Assignee: Carnegie Institution of Washington, University Of Massachusetts
• Original Assignee: Carnegie Institution of Washington
• Inventors: Mello, Craig C., Fire, Andrew, Montgomery, Mary, Timmons, Lisa, Kostas, Stephen, Xu, SiQun, Driver, Samuel E., Tabara, Hiroaki
• Primary Examiner(s): Wang, Andrew
• Assistant Examiner(s): Lacourciere, Karen A
• Application Number:
  US09/215,257
• Time in Patent Office: 1,488 Days
• Field of Search: 514/44, 435/6, 435/91.1, 435/325
• US Class Current: 435/6.16
• CPC Class Codes: A01K 2217/05 Animals comprising random inserted nucleic acids (transgenic)
• CPC Class Codes: A01K 2227/703 Worms, e.g. Caenorhabdities elegans
• CPC Class Codes: A61K 31/7105 Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
• CPC Class Codes: A61K 31/713 Double-stranded nucleic acids or oligonucleotides
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 35/02 specific for leukemia
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/8218 Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
• CPC Class Codes: C12N 15/8285 for nematode resistance
• CPC Class Codes: C12N 15/85 for animal cells
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/14 interfering N.A.
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/50 Physical structure
• CPC Class Codes: C12N 2310/53 partially self-complementary or closed

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.

Claims

1. A method to inhibit expression of a target gene in a cell in vitro comprising introduction of a ribonucleic acid (RNA) into the cell in an amount sufficient to inhibit expression of the target gene, wherein the RNA is a double-stranded molecule with a first strand consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second strand consisting essentially of a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene, wherein the first and the second ribonucleotide strands are separate complementary strands that hybridize to each other to form said double-stranded molecule, and the double-stranded molecule inhibits expression of the target gene.

2. The method of claim 1 in which the target gene is a cellular gene.

3. The method of claim 1 in which the target gene is an endogenous gene.

4. The method of claim 1 in which the target gene is a transgene.

5. The method of claim 1 in which the target gene is a viral gene.

6. The method of claim 1 in which the cell is from an animal.

7. The method of claim 1 in which the cell is from a plant.

8. The method of claim 6 in which the cell is from an invertebrate animal.

9. The method of claim 8 in which the cell is from a nematode.

10. The method of claim 1 in which the first ribonucleotide sequence comprises at least 25 bases which correspond to the target gene and the second ribonucleotide sequence comprises at least 25 bases which are complementary to the nucleotide sequence of the target gene.

11. The method of claim 1 in which the target gene expression is inhibited by at least 10%.

12. A method to inhibit expression of a target gene in an invertebrate organism comprising:

(a) providing an invertebrate organism containing a target cell, wherein the target cell contains the target gene and the target cell is susceptible to RNA interference, and the target gene is expressed in the target cell;

(b) contacting said invertebrate organism with a ribonucleic acid (RNA), wherein the RNA is a double-stranded molecule with a first strand consisting essentially of a ribonucleotide sequence which corresponds to a nucleotide sequence of the target gene and a second strand consisting essentially of a ribonucleotide sequence which is complementary to the nucleotide sequence of the target gene, wherein the first and the second ribonucleotide sequences are separate complementary strands that hybridize to each other to form the double-stranded molecule; and

(c) introducing the RNA into the target cell, thereby inhibiting expression of the target gene.

13. The method of claim 12 in which the organism is a nematode.

14. The method of claim 13 in which a formulation comprised of the RNA is applied on or adjacent to a plant, and disease associated with nematode infection of the plant is thereby reduced.

15. The method of claim 12 in which said double-stranded ribonucleic acid structure is at least 25 bases in length and each of the ribonucleic acid strands is able to specifically hybridize to a deoxyribonucleic acid strand of the target gene over the at least 25 bases.

16. The method of claim 12 in which the expression of the target gene is inhibited by at least 10%.

17. The method of claim 12 in which the RNA is introduced within a body cavity of the organism and outside the target cell.

18. The method of claim 12 in which the RNA is introduced by extracellular injection into the organism.

19. The method of claim 12 in which the organism is contacted with the RNA by feeding the organism food containing the RNA.

20. The method of claim 19 in which the food comprises a genetically-engineered host transcribing the RNA.

21. The method of claim 12 in which at least one strand of the RNA is produced by transcription of an expression construct.

22. The method of claim 21 in which the organism is a nematode and the expression construct is contained in a plant, and disease associated with nematode infection of the plant is thereby reduced.