Antisense antiviral compound and method for treating influenza viral infection

Abstract

The invention provides antisense antiviral compounds and methods of their use and production in inhibition of growth of viruses of the Orthomyxoviridae family and in the treatment of a viral infection. The compounds are particularly useful in the treatment of influenza virus infection in a mammal. The antisense antiviral compounds are substantially uncharged, including partially positively charged, morpholino oligonucleotides having 1) a nuclease resistant backbone, 2) 12-40 nucleotide bases, and 3) a targeting sequence of at least 12 bases in length that hybridizes to a target region selected from the following: a) the 5′ or 3′ terminal 25 bases of the negative sense viral RNA segment of Influenzavirus A, Influenzavirus B and Influenzavirus C; b) the terminal 25 bases of the 3′ terminus of the positive sense cRNA and; and c) the 50 bases surrounding the AUG start codon of an influenza viral mRNA.

1 . An antiviral compound comprising an oligonucleotide analog having 
 a) a nuclease-resistant backbone,    b) 12-40 nucleotide bases, and    c) a targeting sequence of at least 12 bases in length that hybridizes to a target region selected from the following: 
 i) the 5′ or 3′ terminal 25 bases of a negative sense viral RNA segment of Influenzavirus A, Influenzavirus B and Influenzavirus C,  
 ii) the terminal 25 bases of the 3′ terminus of a positive sense cRNA of Influenzavirus A, Influenzavirus B and Influenzavirus C, and  
 iii) the 50 bases surrounding the AUG start codon of an influenza viral mRNA,  
   wherein said oligonucleotide analog further has:    a) the capability of being actively taken up by mammalian host cells, and    b) the ability to form a heteroduplex structure with the viral target region, wherein said heteroduplex structure is: 
 i) composed of the positive or negative sense strand of the virus and the oligonucleotide compound, and  
 ii) characterized by a Tm of dissociation of at least 45° C.  
   
     
     
         2 . (canceled)  
     
     
         3 . The compound of  claim 1 , wherein the oligonucleotide analog is composed of morpholino subunits linked by phosphorous-containing intersubunit linkages that join a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit.  
     
     
         4 . The compound of  claim 3 , wherein the morpholino subunits are joined by phosphorodiamidate linkages in accordance with the structure:  
       
         
           
           
               
               
           
         
         where Y 1 ═O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X is alkyl, alkoxy, thioalkoxy, or alkyl amino.  
       
     
     
         5 . The compound of  claim 3 , in which at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH.  
     
     
         6 . The composition of  claim 5 , wherein said morpholino subunits are linked by phosphorodiamidate linkages, in accordance with the structure:  
       
         
           
           
               
               
           
         
         where Y 1 ═O, Z=O, Pj is a purine or pyrimidine base-pairing moiety effective to bind, by base-specific hydrogen bonding, to a base in a polynucleotide, and X for the uncharged linkages is alkyl, alkoxy, thioalkoxy, or an alkyl amino of the form wherein NR 2 , where each R is independently hydrogen or methyl, and for the positively charged linkages, X is 1-piperazine.  
       
     
     
         7 . The compound of  claim 1 , wherein the oligonucleotide analog hybridizes to a sequence selected from the group consisting of SEQ ID NOs:1-9.  
     
     
         8 . The compound of  claim 1 , wherein the viral target region comprises SEQ ID NO:3 or SEQ ID NO:5.  
     
     
         9 . The compound of  claim 1 , wherein the antisense compound has at least 12 contiguous bases from one of the sequences selected from the group consisting of SEQ ID NOs:10-24.  
     
     
         10 . The compound of  claim 1 , wherein the targeting sequence comprises SEQ ID NO:12 or SEQ ID NO:13.  
     
     
         11 . The compound of  claim 1 , wherein the oligonucleotide analog is conjugated to an arginine-rich polypeptide that enhances the uptake of the compound into host cells.  
     
     
         12 . The compound of  claim 10 , wherein the arginine-rich polypeptide is selected from the group consisting of SEQ ID NOs:25-30.