Rnai inhibition of influenza virus replication

Abstract

The invention relates to compositions and methods for modulating the expression of influenza viral genes, and more particularly to the downregulation of influenza viral genes by chemically modified oligonucleotides.

1 . An iRNA agent comprising a sense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences of any one of the agents provided in Tables 1A-1H, agents numbered AL-DP-2241-AL-DP-8631, and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the antisense sequences of any one of the agents provided in Tables 1A-1H, agents numbered AL-DP-2241-AL-DP-8631.  
     
     
         2 . An iRNA agent including a sense strand, wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences of any one of the agents provided in Tables 1A-1H, agents numbered AL-DP-2241-AL-DP-8631, and an antisense strand wherein the antisense strand comprises at least 15 contiguous nucleotides of the antisense sequences of any one of the agents provided in Tables 1A-1H, agents numbered AL-DP-2241-AL-DP-8631, and wherein the iRNA agent reduces the expression of its respective target gene in Cos-7 cells engineered to express the respective target gene by more than 20%, 30%, 40%, 50%, 60%, 70%, or 80% compared to cells which have not been incubated with the iRNA agent.  
     
     
         3 . An iRNA agent comprising a sense strand and an antisense strand each comprising a sequence of at least 16, 17 or 18 nucleotides which is essentially identical to one of the sequences of any one of the agents provided in Tables 1A-1H, agents numbered AL-DP-2241-AL-DP-8631, except that not more than 1, 2 or 3 nucleotides per strand, respectively, have been substituted by other nucleotides (e.g. adenosine replaced by uracil), while essentially retaining the ability to reduce the amount of influenza A plaques formed in cells in a plaque forming assay.  
     
     
         4 . The iRNA agent of  claim 1 , wherein the antisense RNA strand is 30 or fewer nucleotides in length, and the duplex region of the iRNA agent is 15-30 nucleotide pairs in length.  
     
     
         5 . The iRNA agent of any of  claim 1 , comprising a modification that causes the iRNA agent to have increased stability in a biological sample.  
     
     
         6 . The iRNA agent of  claim 1 , comprising a phosphorothioate or a 2′-modified nucleotide.  
     
     
         7 . The iRNA agent of  claim 1 , comprising at least one 5′-uridine-adenine-3′ (5′-ua-3′) dinucleotide wherein the uridine is a 2′-modified nucleotide; at least one 5′-uridine-guanine-3′ (5′-ug-3′) dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide; at least one 5′-cytidine-adenine-3′ (5′-ca-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide; or at least one 5′-uridine-uridine-3′ (5′-uu-3′) dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide.  
     
     
         8 . The iRNA agent of  claim 6 , wherein the 2′-modification is selected from the group consisting of: 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-methyl, 2′-O-methoxyethyl (2′-O-MOE), 2′-O-aminopropyl (2′-O-AP), 2′-O-dimethylaminoethyl (2′-O-DMAOE), 2′-O-dimethylaminopropyl (2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl (2′-O-DMAEOE), and 2′-O—N-methylacetamido (2′-O-NMA).  
     
     
         9 . The iRNA agent of any of  claim 1 , comprising a nucleotide overhang having 1 to 4 unpaired nucleotides.  
     
     
         10 . The iRNA agent of  claim 9 , wherein the nucleotide overhang has 2 or 3 unpaired nucleotides.  
     
     
         11 . The iRNA agent of  claim 9 , wherein the nucleotide overhang is at the 3′-end of the antisense strand of the iRNA agent.  
     
     
         12 . The iRNA agent of  claim 1 , comprising a cholesterol moiety.  
     
     
         13 . The iRNA agent of  claim 12 , wherein the cholesterol moiety is conjugated to the 3′-end of the sense strand of the iRNA agent.  
     
     
         14 . The iRNA agent of  claim 1 , wherein the iRNA agent is targeted for uptake by cells of the lung.  
     
     
         15 . The iRNA agent of  claim 1 , wherein the iRNA agent comprises at least one non-natural nucleobase.  
     
     
         16 . The iRNA agent of  claim 15 , wherein the non-natural nucleobase is difluorotolyl, nitroindolyl, nitropyrrolyl, or nitroimidazolyl.  
     
     
         17 . The iRNA agent of  claim 15 , wherein the non-natural nucleobase is difluorotolyl.  
     
     
         18 . The iRNA agent of  claim 15 , wherein only one of the two oligonucleotide strands comprising the double-stranded oligonucleotide contains a non-natural nucleobase.  
     
     
         19 . The iRNA agent of  claim 15 , wherein both of the oligonucleotide strands comprising the double-stranded oligonucleotide independently contain a non-natural nucleobase.  
     
     
         20 . A method of treating a human subject having a pathological process mediated in part by the replication of influenza A virus, wherein the iRNA agent comprises a sense strand wherein the sense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the sense strand sequences any one of the agents provided in Tables 1A-1H, agents numbered AL-DP-2241-AL-DP-8631, and an antisense strand, wherein the antisense strand comprises at least 15 contiguous nucleotides that differ by no more than 1, 2, or 3 nucleotides from the antisense strand sequences of any one of the agents provided in Tables 1A-1H, agents numbered AL-DP-2241-AL-DP-8631.  
     
     
         21 . The method of  claim 20 , wherein the iRNA agent is administered in an amount sufficient to reduce the replication of influenza virus in a cell or tissue of the subject.  
     
     
         22 . The method of  claim 20 , wherein the subject is a human.  
     
     
         23 . A pharmaceutical composition, comprising: 
 a.) an iRNA agent of  claim 1;  and    b.) a pharmaceutically acceptable carrier.