Methods and compositions for the specific inhibition of gene expression by double-stranded RNA
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 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.
2. The formulation 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 formulation of claim 1 , wherein each of said first and said second strands has a length of 27 nucleotides.
4. The formulation 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.
5. The formulation of claim 4 , wherein the cleavage facilitates incorporation of the second oligonucleotide strand into RISC.
6. The formulation of claim 1 , wherein said formulation is for a route of administration selected from the group consisting of intravenous injection, intramuscular injection, intraperitoneal injection, infusion, subcutaneous injection, transdermal, aerosol, rectal, vaginal, topical, oral, and inhaled delivery.
7. The formulation of claim 1 , 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 mammalian cell.
8. The formulation of claim 1 , wherein said effective amount is administered to a mammalian subject in need thereof, and wherein said effective amount is a dosage selected from the group consisting of 1 microgram to 5 milligrams per kilogram of said mammalian subject per day, 100 micrograms to 0.5 milligrams per kilogram of said mammalian subject per day, 0.001 to 0.25 milligrams per kilogram of said mammalian subject per day, 0.01 to 20 micrograms per kilogram of said mammalian subject per day, 0.01 to 10 micrograms per kilogram of said mammalian subject per day, 0.10 to 5 micrograms per kilogram of said mammalian subject per day, and 0.10 to 2.5 micrograms per kilogram of said mammalian subject per day.