Antisense antibacterial method and compound

Abstract

A method and antisense compound for inhibiting the growth of pathogenic bacterial cells are disclosed. The compound contains no more than 12 nucleotide bases and has a targeting nucleic acid sequence of no fewer than 10 bases in length that is complementary to a target sequence containing or within 10 bases, in a downstream direction, of the translational start codon of a bacterial mRNA that encodes a bacterial protein essential for bacterial replication. The compound binds to a target mRNA with a T m of between 50° to 60° C. The relatively short antisense compounds are substantially more active than conventional antisense compounds having a targeting base sequence of 15 or more bases.

1 . A method of inhibiting the growth of pathogenic bacterial cells, comprising
 exposing the bacterial cells to a growth-inhibiting amount of a substantially uncharged antisense oligonucleotide compound having:   (i) no more than 12 nucleotide bases,   (ii) a targeting nucleic acid sequence of no fewer than 10 bases in length that is complementary to a target sequence containing or within 10 bases, in a downstream direction, of the translational start codon of a bacterial mRNA that encodes a bacterial protein essential for bacterial replication; and   (iii) a T m , when hybridized with the target sequence, between 50° to 60° C.   
     
     
         2 . The method of  claim 1 , wherein said oligonucleotide compound to which the cells are exposed is composed of morpholino subunits and 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 oligonucleotide compound to which the cells are exposed is administered to the subject is 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 at least one and no more than about half of the intersubunit linkages are positively charged at physiological pH, and interspersed along the compound backbone. 
     
     
         5 . The method of  claim 4 , wherein the morpholino subunits in the oligonucleotide compound to which the cells are exposed is administered to the subject 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, in the uncharged linkages, X is alkyl, alkoxy, thioalkoxy, amino or alkyl amino, including dialkylamino, and in the charged linkages, X is 1-piperazine. 
       
     
     
         6 . The method of  claim 1 , wherein the oligonucleotide compound to which the cells are exposed has a targeting sequence that is complementary to a target sequence containing the translational start codon of the bacterial mRNA. 
     
     
         7 . The method of  claim 1 , wherein the oligonucleotide compound to which the cells are is exposed has a targeting sequence that is complementary to a target sequence that is within 10 bases, in a downstream direction, of the translational start codon of the bacterial mRNA. 
     
     
         8 . The method of  claim 1 , wherein the oligonucleotide compound to which the cells are exposed contains only 11 bases, and its nucleic acid sequence is completely complementary to the mRNA target sequence. 
     
     
         9 . The method of  claim 1 , for use in inhibiting a bacterial infection in a mammalian subject, wherein said exposing includes administering said compound in a therapeutically effective amount. 
     
     
         10 . The method of  claim 9 , which further includes treating the subject by administration of a non-antisense compound having antibacterial activity. 
     
     
         11 . An antibacterial compound comprising a substantially uncharged antisense oligonucleotide compound having:
 (i) no more than 12 nucleotide bases,   (ii a targeting nucleic acid sequence of no fewer than 10 bases in length that is complementary to a target sequence containing or within 10 bases, in a downstream direction, of the translational start codon of a bacterial mRNA that encodes whose targeting sequence is complementary to a target sequence containing or within 10 bases, in a downstream direction, of the translational start codon of a bacterial mRNA that encodes a bacterial protein selected from the group consisting of acyl carrier protein (acpP), gyrase A subunit (gyrA), and the cell division protein ftsZ; and   (iii) a T m , when hybridized with the target sequence, between 50° to 60° C.   
     
     
         12 . The compound of  claim 11 , wherein the bacterial mRNA encodes the ftsZ protein, and the compound targeting sequence is complementary to at least ten contiguous bases in a sequence selected from the group consisting of SEQ ID NOS: 1, 4, 7, 10, 13, 16, 17, 19, 22, 25, 28, 31, and 34. 
     
     
         13 . The compound of  claim 11 , wherein the bacterial mRNA encodes the acpP protein, and the compound targeting sequence is complementary to at least ten contiguous bases in a sequence selected from the group consisting of SEQ ID NOS: 2, 5, 8, 11, 14, 20, 23, 26, 29, 32, and 35. 
     
     
         14 . The compound of  claim 11 , wherein the bacterial mRNA encodes the gyrA protein, and the compound targeting sequence is complementary to at least ten contiguous bases in a sequence selected from the group consisting of SEQ ID NOS: 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, and 36. 
     
     
         15 . The compound of  claim 11 , which is composed of morpholino subunits and phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′ exocyclic carbon of an adjacent subunit. 
     
     
         16 . The compound of  claim 15 , wherein the morpholino subunits in the compound 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. 
       
     
     
         17 . The compound of  claim 15 , wherein at least one and no more than about half of the intersubunit linkages are positively charged at physiological pH, and interspersed along the compound backbone. 
     
     
         18 . The compound of  claim 17 , 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, in the uncharged linkages, X is alkyl, alkoxy, thioalkoxy, amino or alkyl amino, including dialkylamino, and in the charged linkages, X is 1-piperazine. 
       
     
     
         19 . The compound of  claim 11 , which has a targeting sequence that is complementary to a target sequence containing the translational start codon of the bacterial mRNA. 
     
     
         20 . The compound of  claim 11 , which is complementary to a target sequence that is within 10 bases, in a downstream direction, of the translational start codon of the bacterial mRNA. 
     
     
         21 . The compound of  claim 11 , which contains only 11 bases, and its nucleic acid sequence is completely complementary to the mRNA target sequence. 
     
     
         22 . The compound of  claim 11 , which contains only 10 bases, and its nucleic acid sequence is completely complementary to the mRNA target sequence.