Inhibition of pcsk9 through rnai

Abstract

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 guide sequence that hybridizes to a target sequence on an mRNA of the PCSK9 gene and inhibits the expression of the PCSK9 gene through an RNA interference mechanism, wherein said target sequence is selected from the group consisting of: SEQ ID NOS: 53 and 63. 
     
     
         2 - 34 . (canceled) 
     
     
         35 . The RNAi construct of  claim 1 , comprising a double-stranded RNA (dsRNA) construct of 25 base pairs in length, said dsRNA comprising:
 (1) a sense strand having a 5′-end and a 3′-end, said sense strand corresponding to the target sequence, and,   (2) said guide sequence having a 5′-end and a 3′-end, and hybridizing to said sense strand under physiological condition of a cell or under high stringency hybridization condition.   
     
     
         36 . The RNAi construct of  claim 35 , wherein said construct is blunt-ended. 
     
     
         37 . The RNAi construct of  claim 35 , wherein said sense strand comprises one or more consecutive 2′-modified ribose sugar at each of said 5′- and 3′-ends of said sense strand. 
     
     
         38 . The RNAi construct of  claim 37 , wherein said sense strand comprises 12 and 10 consecutive 2′-modified ribose sugars at the 5′-end and the 3′-end nucleotides, respectively. 
     
     
         39 . The RNAi construct of  claim 37 , wherein said sense strand comprises 4 consecutive 2′-modified ribose sugars at both ends. 
     
     
         40 . The RNAi construct of  claim 37 , wherein said 2′-modified ribose sugars are 2′-O-alkyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, 2′-H (deoxyribonucleotides), or combination thereof. 
     
     
         41 . The RNAi construct of  claim 35 , wherein said guide sequence further comprises a 2′-modified ribose sugar. 
     
     
         42 . The RNAi construct of  claim 41 , wherein the 2′-modified ribose sugar is 2′-O-alkyl nucleotides, 2′-deoxy-2′-fluoro nucleotides, 2′-deoxy nucleotides, 2′-H (deoxyribonucleotides), or combination thereof. 
     
     
         43 . The RNAi construct of  claim 41 , wherein said guide sequence comprises a 2′-modification at the 2 nd  nucleotide from the 5′-end of the guide sequence. 
     
     
         44 . The RNAi construct of  claim 41 , wherein said guide sequence comprises four consecutive 2′-modification at the 3′-most end. 
     
     
         45 - 61 . (canceled) 
     
     
         62 . A composition comprising the RNAi construct of  claim 35 , and a pharmaceutically acceptable carrier or diluent. 
     
     
         63 . 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 35 . 
     
     
         64 - 65 . (canceled) 
     
     
         66 . The method of  claim 63 , wherein the mammalian cell is contacted in the presence of a delivery agent. 
     
     
         67 . The method of  claim 66 , wherein said delivery reagent comprises a lipid. 
     
     
         68 . The method of  claim 67 , wherein said lipid is a cationic lipid. 
     
     
         69 . The method of  claim 66 , wherein the delivery reagent is a liposome. 
     
     
         70 . The method of  claim 66 , wherein said delivery reagent comprises beta-glucan, chitosan, and/or PEI. 
     
     
         71 . A method of treating a patient for a disease characterized by overexpression of a PCSK9 gene, comprising administering to the patient an RNAi construct of  claim 35 , wherein the RNAi construct mediates guide sequence-dependent reduction in PCSK9 expression. 
     
     
         72 . A method of inhibiting expression of a PCSK9 gene with an RNAi construct of  claim 35 , wherein the RNAi construct mediates guide sequence-dependent reduction in PCSK9 expression.