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.

1 . A method of inhibiting replication of a nidovirus in virus-infected animal cells, comprising 
 (a) exposing the cells to an oligonucleotide compound (i) having a nuclease-resistant backbone, (ii) capable of uptake by the cells, (iii) containing between 8-25 nucleotide bases, and (iv) having a sequence complementary to at least 8 bases contained in:    (1) a sequence in a 5′ leader sequence of the nidovirus′ positive-strand genomic RNA consisting of each sequence of which includes an internal transcription regulatory sequence;    (b) by said exposing, forming within said cells a based-paired heteroduplex structure composed of one of (1) the virus positive-strand genomic RNA and the oligonucleotide compound, and (2) the negative-strand 3′ subgenomic region of the virus and the oligonucleotides compound, said structure being characterized by    (1 ) a of dissociation of at least 45° C., and    (2) disrupted base pairing between the transcriptional regulatory sequences in the 5′ leader region of the positive-strand viral genome and negative-strand 3′ subgenomic region,    where the amount of compound to which the cells are exposed is sufficient inhibit nidovirus replication in the virus-infected cells.    
     
     
         2 . The method of  claim 1 , wherein said oligonucleotide compound to which the cells are exposed 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.  
     
     
         3 . The method of  claim 2 , wherein the morpholino subunits in the conjugate to which the cells are exposed 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.  
     
     
         4 . The method of  claim 2 , 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 method 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.  
     
     
         6 - 7 . (canceled)  
     
     
         8 . The method of claim  7 , wherein the oligonucleotide analog to which the cells are exposed contains a sequence selected from the group consisting of.  
     
     
         9 - 17 . (canceled)  
     
     
         18 . The method of  claim 1 , for treating a nidovirus infection in a human or veterinary-animal subject, wherein said exposing includes administering the oligonucotide compound orally to the subject.  
     
     
         19 . The method of  claim 1 , for treating a nidovirus infection in a human or veterinary animal subject, wherein said exposing includes administering the oligonucleotide compound to the subject, and which further includes monitoring a body fluid for the appearance of a heteroduplex composed of the oligonucleotide complementary portion of the viral genome in positive- or negative-strand form.  
     
     
         20 . An oligonucleotide compound for use in inhibiting replication of a nidovirus in human cells, characterized by: 
 (i) a nuclease-resistant backbone,    (ii) capable of uptake by virus-infected human cells,    (iii) containing between 8-25 nucleotide bases,    (iv) having a sequence that is complementary to at least 8 bases contained in one of:    (1) a sequence in a 5′ leader sequence of the nidovirus' positive-strand genomic RNA selected from the group consisting of SEQ ID NOS: 1-9, each sequence of which includes an internal transcription regulatory sequence; and,    (2) a sequence in a negative-strand 3′ subgenomic region of the virus selected from the group consisting of SEQ ID NOS: 10-18, each sequence of which includes an internal transcriptional regulatory sequence that is substantially complementary to the corresponding transcriptional regulatory sequence contained within SEQ ID NOS: 1-9, respectively; and    (v) capable of forming with the nidovirus (1) positive-strand genomic RNA or (2) the negative-strand 3′ subgenomic region, a heteroduplex structure characterized by (1) a Tm of dissociation of at least 45° C., and (2) a disrupted base pairing between the transcriptional regulatory sequences in the 5′ leader region of the positive-strand viral genome and negative-strand 3′ subgenomic region.    
     
     
         21 . The compound of  claim 20 , 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.  
     
     
         22 . The compound of  claim 21 , 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.  
     
     
         23 . The compound of  claim 21 , in which at least 2 and no more than half of the total number of intersubunit linkages are positively charged at physiological pH.  
     
     
         24 . The compound of  claim 23 , 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.  
     
     
         25 . The compound of  claim 20 , which has a sequence complementary to at least 8 bases contained in the 5′ leader sequence of the nidovirus' positive-strand genomic RNA selected from the group consisting of SEQ ID NOS: 1-9.  
     
     
         26 . The compound of  claim 25 , which has a sequence complementary to at least a portion of the transcriptional regulatory sequence contained within one of the sequences SEQ ID NOS: 1-9.  
     
     
         27 . The compound of  claim 26 , which has a sequence selected from the group consisting of SEQ ID NOS: 19-34.  
     
     
         28 . The compound of  claim 27 , for use in inhibiting replication of human SARS virus, which contains a sequence selected from the group consisting of SEQ ID NOS: 25 and 26.  
     
     
         29 . The compound of  claim 27 , for use in inhibiting replication of human coronavius-229E or human coronavirus-OC43, which contains the sequence selected from the group consisting of SEQ ID NOS: 21 and 22, for the coronavirus-229E and contains the sequence SEQ ID NOS: 23 for the coronavisus-OC42.  
     
     
         30 . The compound of  claim 27 , for use in inhibiting replication of feline coronavirus, which contains the SEQ ID NO: 19.  
     
     
         31 . The compound of  claim 20 , which has a sequence complementary to at least 8 bases contained in the negative-strand 3′ subgenomic region of the virus selected from the group consisting of SEQ ID NOS: 10-18.  
     
     
         32 . The compound of  claim 31 , which has a sequence complementary to at least a portion of the transcriptional regulatory sequence contained within one of the sequences SEQ ID NOS: 10-18.  
     
     
         33 . The compound of  claim 32 , which contains a sequence selected from the group consisting of SEQ ID NOS: 35-50.  
     
     
         34 . The compound of  claim 33 , for use in inhibiting replication of human SARS virus, which contains a sequence selected from the group consisting of SEQ ID NOS: 41 and 42.  
     
     
         35 . The compound of  claim 33 , for use in inhibiting replication of human coronavius-229E or human coronavirus-OC43, which contains the sequence selected from the group consisting of SEQ ID NOS: 37 and 38, for the coronavirus-229E and contains the sequence SEQ ID NOS: 39 for the coronavisus-OC42.  
     
     
         36 . The compound of  claim 33 , for use in inhibiting replication of feline coronavirus, which contains the SEQ ID NO: 35.