Modified small interfering RNA molecules and methods of use
View Patent ↗The present invention provides double-stranded RNA molecules that mediate RNA interference in target cells, preferably hepatic cells. The invention also provides double-stranded RNA (dsRNA) molecules that are modified to be resistant to nuclease degradation, which inactivates a virus, and more specifically, hepatitis C virus (HCV). The invention also provides a method of using these modified RNA molecules to inactivate virus in mammalian cells and a method of making modified small interfering RNAs (siRNAs) using human Dicer. The invention provides modified RNA molecules that are modified to include a dsRNA or siRNA wherein one or more of the pyrimidines in the RNA molecule are modified to include 2′-Fluorine. The invention also provides dsRNA or siRNA in which all pyrimidines are modified to include a 2′-Fluorine. The invention provides that the 2′-Fluorine dsRNA or siRNA molecule is further modified to include a two base deoxynucleotide “TT” sequence at the 3′ end of the molecule.
1. A method for inhibiting Hepatitis B Virus (HBV) replication in a patient, the method comprising the steps of:
(a) administering to said patient a composition comprising a modified double-stranded RNA (dsRNA) or modified small interfering RNA (siRNA) in an amount effective to mediate RNA interference and to inhibit HBV replication, wherein the modified dsRNA or modified siRNA comprises a first strand and a second strand, wherein the first and second strand are each no more than about 30 ribonucleotides in length and wherein the first or second strand targets HBV; and wherein the modified dsRNA or modified siRNA is cholesterol-labeled, and
(b) administering to said patient a cholesterol-lowering drug,
wherein steps (a) and (b) can be performed simultaneously or in any order, and
(c) wherein the cholesterol-lowering drug reduces the level of competing cholesterol in the serum, allowing more efficient uptake of the cholesterol-labeled modified dsRNA or modified siRNA by hepatocytes.
2. The method of claim 1 , wherein said cholesterol-lowering drug is a statin, resin, nicotinic acid, gemfibrozil or clofibrate.
3. The method of claim 1 , wherein said cholesterol-lowering drug is a statin.
4. A method for inhibiting Hepatitis B Virus (HBV) replication in a patient, the method comprising the steps of:
(a) administering to said patient a composition comprising a modified double-stranded RNA (dsRNA) or modified small interfering RNA (siRNA) in an amount effective to mediate RNA interference and to inhibit HBV replication, wherein the modified dsRNA or modified siRNA comprises a first strand and a second strand, wherein the first and second strand are each no more than about 30 ribonucleotides in length and wherein the first or second strand targets HBV; and wherein the modified dsRNA or modified siRNA is cholesterol-labeled, and
(b) administering to said patient a cholesterol-lowering drug,
wherein steps (a) and (b) are performed simultaneously, and
(c) wherein the cholesterol-lowering drug reduces the level of competing cholesterol in the serum, allowing more efficient uptake of the cholesterol-labeled modified dsRNA or modified siRNA by hepatocytes.
5. The method of claim 4 , wherein said cholesterol-lowering drug is a statin, resin, nicotinic acid, gemfibrozil or clofibrate.
6. The method of claim 4 , wherein said cholesterol-lowering drug is a statin.