Targeting opposite strand replication intermediates of single-stranded viruses by rnai

Abstract

The invention relates to methods and compositions for modulating viral replication through double-stranded RNA-mediated gene silencing (RNAi), wherein the antiviral methods and compositions preferentially target opposite strand replication intermediates of single-stranded RNA viruses.

1 . A method of inhibiting the replication of a single-stranded RNA virus that has infected a vertebrate cell comprising administering to said vertebrate cell one or more dsRNA effector molecules comprising an effector sequence of at least 19 contiguous nucleotides from a reverse complement to an opposite strand replication intermediate of said single-stranded virus, wherein said effector sequence selectively targets said opposite strand replication intermediate of said single-stranded virus. 
     
     
         2 . The method of  claim 1 , wherein the single-stranded RNA virus is a plus strand virus. 
     
     
         3 . The method of  claim 2 , wherein the single-stranded plus strand virus is selected from the group consisting of the human coronaviruses, flaviviruses including West Nile encephalitis virus, Japan encephalitis virus, Murray Valley encephalitis virus, St. Louis encephalitis virus, yellow fever virus, hepatitis C virus, Dengue fever virus, Rubella virus, caliciviruses, hepatitis E virus, polio virus, rhinovirus, hepatitis A virus, coxsakie virus, Venezuelan equine encephalitis virus and foot and mouth disease virus. 
     
     
         4 . The method of  claim 3 , wherein the plus strand virus is a hepatitis C virus. 
     
     
         5 . The method of  claim 1  wherein the vertebrate cell is a human cell. 
     
     
         6 . The method of  claim 1 , wherein the effector sequence is about 19 to about 27 nucleotides in length. 
     
     
         7 . The method of any preceding claim, wherein said effector sequence selectively targets said opposite strand replication intermediate by preferentially associating with RISC. 
     
     
         8 . The method of any preceding claim, wherein at least one of the one or more dsRNA effector molecules comprises one or more sequences selected from: a reverse complement to the conserved 5′ non-translated region of the minus strand replication intermediate of the hepatitis C virus or the reverse complement to the conserved 3′ non-translated region of the minus strand replication intermediate of the hepatitis C virus. 
     
     
         9 . The method of  claim 8 , wherein the dsRNA effector molecule comprises a sequence selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; wherein U is substituted for T. 
     
     
         10 . The method of  claim 1 , wherein the single-stranded virus is a minus strand virus. 
     
     
         11 . The method of  claim 10 , wherein the single-stranded minus strand virus is selected from the group consisting of influenza virus, Ebola virus, Marburg virus, respiratory syncitial virus, parainfluenza virus, measles virus, mumps virus, rabies virus, and vesicular stomatotis virus. 
     
     
         12 . The method of any preceding claim, wherein the one or more dsRNA effector molecules are administered by expression from one or more expression constructs delivered to the vertebrate cell. 
     
     
         13 . The method of  claim 12 , wherein said one or more expression constructs further comprise one or more promoters selected from an RNA polymerase II promoter and an RNA polymerase III promoter, wherein said one or more promoters are positioned within the construct to drive the expression of said dsRNA effector molecules. 
     
     
         14 . The method of  claim 12 , wherein at least one of the expression constructs comprises at least two different RNA polymerase III promoters. 
     
     
         15 . The method of  claim 12 , wherein at least one of the expression constructs comprises at least three RNA polymerase III promoters, wherein said at least three RNA polymerase III promoters may be the same or different. 
     
     
         16 . The method of  claim 12 , wherein at least one of the expression constructs comprises at least two different dsRNA effector molecules. 
     
     
         17 . The method of  claim 16 , wherein the different dsRNA effector molecules are selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; wherein U is substituted for T. 
     
     
         18 . The method of  claim 12 , wherein at least one of the one or more dsRNA effector molecules is a dshairpin construct. 
     
     
         19 . The method of  claim 18 , wherein said dshairpin is selected from a single stem dshairpin, a bi-finger dshairpin and a multi-finger dahairpin. 
     
     
         20 . A method of inhibiting replication of a single-stranded RNA virus in a vertebrate cell infected with said virus, comprising administering to the cell nucleic acid molecules comprising;
 a) an at least 19 contiguous nucleotide sequence which is a reverse complement to an opposite strand replication intermediate of the single-stranded RNA virus, and   b) an at least 19 contiguous nucleotide sequence which is a reverse complement to a genomic strand of the single-stranded RNA virus.   
     
     
         21 . The method of  claim 20 , wherein at least one of the nucleic acids molecules administered to the cell comprise dsRNA molecules. 
     
     
         22 . The method of  claim 20 , wherein the single-stranded RNA virus is hepatitis C and the cell is a human cell. 
     
     
         23 . The method of  claim 20 , wherein nucleotide sequence a and nucleotide sequence b are complementary sequences. 
     
     
         24 . The method of  claim 20 , wherein nucleotide sequence a and nucleotide b are not complementary sequences. 
     
     
         25 . The method of  claim 20 , wherein nucleotide sequence a and nucleotide sequence b each comprise an at least 19 contiguous nucleotide sequence selected from the group consisting of sequences which are the reverse complement to the conserved 5′ nontranslated region or the conserved 3′ nontranslated region of the minus strand of the hepatitis C virus or the plus strand of the hepatitis C virus. 
     
     
         26 . The method of  claim 20 , wherein nucleotide sequence a and nucleotide sequence b are administered by expression from one or more expression constructs delivered to the vertebrate cell. 
     
     
         27 . The method of  claim 26 , wherein said one or more expression constructs comprise one of more promoters selected from an RNA polymerase II promoter and an RNA polymerase III promoter. 
     
     
         28 . The method of  claim 27 , comprising at least two different RNA polymerase III promoters. 
     
     
         29 . The method of  claim 27 , comprising at least three RNA polymerase III promoters. 
     
     
         30 . A double-stranded RNA effector molecule comprising an at least 19 contiguous nucleotide effector sequence which is a reverse complement to an opposite strand replication intermediate of a single-stranded RNA virus. 
     
     
         31 . A double-stranded RNA effector molecule of  claim 30 , further comprising a second sequence which is the reverse complement of the effector sequence, and wherein the effector sequence preferentially associates with the RISC relative to the second sequence. 
     
     
         32 . A double-stranded RNA effector molecule of  claim 31 , which has a lower thermal stability (Tm) at the terminus comprising the 5′ end of the effector sequence compared to the terminus comprising the 3′ end of the second sequence. 
     
     
         33 . A double-stranded RNA effector molecule of  claim 30 , wherein the single-stranded RNA virus is a plus strand virus. 
     
     
         34 . A double-stranded RNA effector molecule of  claim 30 , wherein said effector sequence has an A or U at position 1 of the 5′ end of said reverse complement. 
     
     
         35 . The double-stranded RNA effector molecule of  claim 30 , wherein said plus strand virus is hepatitis C virus. 
     
     
         36 . The double-stranded RNA effector molecule of  claim 30 , wherein the double-stranded RNA effector molecule comprises an effector sequence which is a reverse complement of a conserved 5′ non-translated region or a conserved 3′ non-translated region of the minus strand replication intermediate of the hepatitis C virus. 
     
     
         37 . The double-stranded RNA effector molecule of  claim 36 , wherein the double-stranded RNA effector molecule comprises an effector sequence selected from SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, and SEQ ID NO:23; wherein U is substituted for T. 
     
     
         38 . A method of treating an infection of a vertebrate cell by a single-stranded RNA virus comprising administering to said vertebrate cell one or more dsRNA effector molecules comprising an effector sequence of at least 19 contiguous nucleotides from a reverse complement to an opposite strand replication intermediate of said single-stranded virus, wherein said effector sequence selectively targets said opposite strand replication intermediate of said single-stranded virus. 
     
     
         39 . The method of  claim 38 , wherein said vertebrate cell is a human cell and said single-stranded virus is hepatitis C virus.