Methods and compositions for the specific inhibition of gene expression by double-stranded RNA
View Patent ↗The invention provides compositions and methods for selectively reducing the expression of a gene product from a desired target gene, as well as treating diseases caused by expression of the gene. The method involves introducing into the environment of a cell an amount of a double-stranded RNA (dsRNA) such that a sufficient portion of the dsRNA can enter the cytoplasm of the cell to cause a reduction in the expression of the target gene. The dsRNA has a first oligonucleotide sequence that is between 26 and about 30 nucleotides in length and a second oligonucleotide sequence that anneals to the first sequence under biological conditions. In addition, a region of one of the sequences of the dsRNA having a sequence length of from about 19 to about 23 nucleotides is complementary to a nucleotide sequence of the RNA produced from the target gene.
1. A method for reducing expression of a target gene in a mammalian cell comprising: contacting a cell in vitro with an isolated double stranded nucleic acid in an amount sufficient to reduce expression of a target gene in said cell, wherein said double stranded nucleic acid comprises a first oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus and wherein said double stranded nucleic acid comprises blunt ends, wherein each of said first and said second strands consists of the same number of nucleotide residues and is 25-30 nucleotides, wherein the ultimate and penultimate residues of said 3′ terminus of said first strand and the ultimate and penultimate residues of said 5′ terminus of said second strand form one or two mismatched base pairs, and wherein said second oligonucleotide strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell, thereby reducing expression of the target gene in said mammalian cell.
2. The method of claim 1 , wherein each of said first and said second strands has a length which is at least 26 and at most 30 nucleotides.
3. The method of claim 1 , wherein said isolated double stranded nucleic acid is a double stranded RNA and wherein said double stranded RNA is cleaved endogenously in said cell by Dicer.
4. The method of claim 3 , wherein the cleavage facilitates incorporation of the second oligonucleotide strand into RISC.
5. The method of claim 1 , wherein the amount of said isolated double stranded nucleic acid sufficient to reduce expression of the target gene is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less, and 50 picomolar or less in the environment of said cell.
6. The method of claim 1 , wherein target gene expression is reduced by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.
7. The method of claim 1 , wherein each of said first and said second strands has a length of 27 nucleotides.
8. The method of claim 5 , wherein each of said first and said second strands has a length which is at least 26 and at most 30 nucleotides.
9. The method of claim 5 , wherein each of said first and said second strands has a length of 27 nucleotides.
10. A method for reducing expression of a target gene in a mammal comprising: administering an isolated double stranded nucleic acid to a mammal in an amount sufficient to reduce expression of a target gene in the mammal, wherein said double stranded nucleic acid comprises a first oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus and wherein said double stranded nucleic acid comprises blunt ends, wherein each of said first and said second strands consists of the same number of nucleotide residues and is 25-30 nucleotides, wherein the ultimate and penultimate residues of said 3′ terminus of said first strand and the ultimate and penultimate residues of said 5′ terminus of said second strand form one or two mismatched base pairs, and wherein said second oligonucleotide strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell, thereby reducing expression of the target gene in the mammal.
11. The method of claim 10 , wherein said isolated double stranded nucleic acid is administered at a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said mammal per day, 100 micrograms to 0.5 milligrams per kilogram of said mammal per day, 0.001 to 0.25 milligrams per kilogram of said mammal per day, 0.01 to 20 micrograms per kilogram of said mammal per day, 0.01 to 10 micrograms per kilogram of said mammal per day, 0.10 to 5 micrograms per kilogram of said mammal per day, and 0.1 to 2.5 micrograms per kilogram of said mammal per day.
12. The method of claim 10 , wherein said administering step comprises a mode selected from the group consisting of intravenous injection, intramuscular injection, intraperitoneal injection, infusion, subcutaneous injection, transdermal, aerosol, rectal, vaginal, topical, oral and inhaled delivery.
13. The method of claim 10 , wherein each of said first and said second strands has a length which is at least 26 and at most 30 nucleotides.
14. The method of claim 10 , wherein each of said first and said second strands has a length of 27 nucleotides.
15. The method of claim 11 , wherein each of said first and said second strands has a length which is at least 26 and at most 30 nucleotides.
16. The method of claim 11 , wherein each of said first and said second strands has a length of 27 nucleotides.
17. The method of claim 10 , wherein said isolated double stranded nucleic acid is a double stranded RNA and wherein said double stranded RNA is cleaved endogenously in said cell by Dicer.
18. The method of claim 17 , wherein the cleavage facilitates incorporation of the second oligonucleotide strand into RISC.
19. A formulation comprising an isolated double stranded nucleic acid, wherein said isolated double stranded nucleic acid comprises a first oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus and wherein said double stranded nucleic acid comprises blunt ends, wherein each of said first and said second strands consists of the same number of nucleotide residues and is 25-30 nucleotides, wherein the ultimate and penultimate residues of said 3′ terminus of said first strand and the ultimate and penultimate residues of said 5′ terminus of said second strand form one or two mismatched base pairs, and wherein said second oligonucleotide strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell, wherein said double stranded nucleic acid is present in an amount effective to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell in vitro by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.
20. The formulation of claim 19 , wherein said effective amount is selected from the group consisting of 1 nanomolar or less, 200 picomolar or less, and 50 picomolar or less in the environment of said cell.
21. A formulation comprising an isolated double stranded nucleic acid, wherein said isolated double stranded nucleic acid comprises a first oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus and wherein said double stranded nucleic acid comprises blunt ends, wherein each of said first and said second strands consists of the same number of nucleotide residues and is 25-30 nucleotides, wherein the ultimate and penultimate residues of said 3′ terminus of said first strand and the ultimate and penultimate residues of said 5′ terminus of said second strand form one or two mismatched base pairs, and wherein said second oligonucleotide strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell, wherein said double stranded nucleic acid is present in an amount effective to reduce target gene expression when said double stranded nucleic acid is introduced into a cell of a mammalian subject by an amount (expressed by %) selected from the group consisting of at least 10%, at least 50% and at least 80-90%.
22. The formulation of claim 21 , wherein said effective amount is a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said subject per day, 100 micrograms to 0.5 milligrams per kilogram of said subject per day, 0.001 to 0.25 milligrams per kilogram of said subject per day, 0.01 to 20 micrograms per kilogram of said subject per day, 0.01 to 10 micrograms per kilogram of said subject per day, 0.10 to 5 micrograms per kilogram of said subject per day, and 0.10 to 2.5 micrograms per kilogram of said subject per day.
23. A mammalian cell containing an isolated double stranded nucleic acid, wherein said isolated double stranded nucleic acid comprises a first oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus and a second oligonucleotide strand comprising ribonucleotides and having a 5′ terminus and a 3′ terminus and wherein said double stranded nucleic acid comprises blunt ends, wherein each of said first and said second strands consists of the same number of nucleotide residues and is 25-30 nucleotides, wherein the ultimate and penultimate residues of said 3′ terminus of said first strand and the ultimate and penultimate residues of said 5′ terminus of said second strand form one or two mismatched base pairs, and wherein said second oligonucleotide strand is sufficiently complementary to a target mRNA along at least 19 nucleotides of said second oligonucleotide strand length to reduce target gene expression when said double stranded nucleic acid is introduced into a mammalian cell.