Inhibition of PCSK9 through RNAI
The invention relates to various PCSK9 RNAi constructs with gene silencing activities, and uses thereof. The construct has a double-stranded region of 19-49 nucleotides, preferably 25, 26, or 27 nucleotides, and preferably blunt-ended. The construct has selective minimal modifications to confer an optimal balance of biological activity, toxicity, stability, and target gene specificity. The sense strand may be modified such that the construct is not cleaved by Dicer or other RNAse III, and the entire length of the antisense strand is loaded into RISC In addition, the antisense strand may also be modified by 2′-O-methyl groups at the 2nd 5′-end nucleotide to greatly reduce off-target silencing. The constructs of the invention largely avoid the interferon response and sequence-independent apoptosis in mammalian cells, exhibits better serum stability, and enhanced target specificity.
1. An RNAi construct for inhibiting expression of a PCSK9 gene, comprising a double-stranded RNA (dsRNA) of 25-27 base pairs in length, the dsRNA comprising: (1) a sense strand having a 5′-end and a 3′-end, wherein the sense strand comprises SEQ ID NO: 65, and wherein the sense strand comprises 12-14 and 10-12 consecutive 2′-modified ribose sugars at the 5′-end and the 3′-end nucleotides, respectively, and (2) an antisense strand having a 5′-end and a 3′-end, which hybridizes to the sense strand.
2. A composition comprising the RNAi construct of claim 1 , and a pharmaceutically acceptable carrier or diluent.
3. A method for inhibiting the expression of a PCSK9 gene in a mammalian cell, comprising contacting the mammalian cell with the RNAi construct of claim 1 .
4. The method of claim 3 , wherein the mammalian cell is contacted in the presence of a delivery agent.
5. The method of claim 4 , wherein said delivery reagent comprises a lipid.
6. The method of claim 5 , wherein said lipid is a cationic lipid.
7. The method of claim 4 , wherein the delivery reagent is a liposome.
8. The method of claim 4 , wherein said delivery reagent comprises beta-glucan, chitosan, and/or PEI.
9. A method of inhibiting expression of a PCSK9 gene with an RNAi construct of claim 1 , wherein the RNAi construct mediates guide sequence-dependent reduction in PCSK9 expression.
10. The RNAi construct of claim 1 , wherein the RNAi construct comprises one or more hydrophobic modifications.
11. The RNAi construct of claim 1 , wherein the double stranded RNA (dsRNA) is 25 or 26 nucleotides in length.