Oligonucleotide compound and method for treating nidovirus infections

Abstract

A method and oligonucleotide compound for inhibiting replication of a nidovirus in virus-infected animal cells are disclosed. The compound (i) has a nuclease-resistant backbone, (ii) is capable of uptake by the infected cells, (iii) contains between 8-25 nucleotide bases, and (iv) has a sequence capable of disrupting base pairing between the transcriptional regulatory sequences in the 5′ leader region of the positive-strand viral genome and negative-strand 3′ subgenomic region. In practicing the method, infected cells are exposed to the compound in an amount effective to inhibit viral replication.

It is claimed: 
     
         1 . An oligonucleotide compound, which is composed of morpholino subunits and uncharged, phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, for use in inhibiting replication of a nidovirus in host cells, characterized by:
 (i) a nuclease-resistant backbone, 
 (ii) capable of uptake by virus-infected host cells, 
 (iii) containing between 8-25 nucleotide bases, 
 (iv) having a sequence that is complementary to at least 12 contiguous bases contained in SEQ ID NO: 1; and 
 (v) capable of forming with SEQ ID NO: 1, a heteroduplex structure characterized by a Tm of dissociation of at least 45° C. 
 
     
     
         2 . The compound of  claim 1 , 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, amino or alkyl amino, including dialkylamino. 
       
     
     
         3 . The compound of  claim 2 , in which at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH. 
     
     
         4 . The compound of  claim 3 , wherein said 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 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. 
       
     
     
         5 . The compound of  claim 1 , which contains a sequence selected from the group consisting of SEQ ID NOS: 20 and 21. 
     
     
         6 . The compound of  claim 1 , which further includes an Arg-rich peptide conjugated to the oligonucleotide. 
     
     
         7 . The compound of  claim 6 , wherein the peptide is SEQ ID NO: 47. 
     
     
         8 . An oligonucleotide compound, which is composed of morpholino subunits and uncharged, phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit, for use in inhibiting replication of a nidovirus in host cells, characterized by:
 (i) a nuclease-resistant backbone, 
 (ii) capable of uptake by virus-infected host cells, 
 (iii) containing between 8-25 nucleotide bases, 
 (iv) having a sequence that is complementary to at least 12 contiguous bases contained in SEQ ID NO: 3; and 
 (v) capable of forming with SEQ ID NO: 3, a heteroduplex structure characterized by a Tm of dissociation of at least 45° C. 
 
     
     
         9 . The compound of  claim 8 , 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, amino or alkyl amino, including dialkylamino. 
       
     
     
         10 . The compound of  claim 9 , in which at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH. 
     
     
         11 . The compound of  claim 10 , wherein said 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 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. 
       
     
     
         12 . The compound of  claim 8 , which contains a sequence selected from the group consisting of SEQ ID NOS: 22 and 23. 
     
     
         13 . The compound of  claim 8 , which further includes an Arg-rich peptide conjugated to the oligonucleotide. 
     
     
         14 . The compound of  claim 13 , wherein the peptide is SEQ ID NO: 47.