Antibacterial antisense oligonucleotide and method
A method for enhancing, by at least 10 fold, the antibacterial activity of an antisense oligonucleotide composed of morpholino subunits linked by phosphorus-containing intersubunit linkages. The method includes one or both of: conjugating an arginine-rich carrier to a 3′ or 5′ end of the oligonucleotide and modifying the oligonucleotide to contain 20%-50% intersubunit linkages that are positively charged at physiological pH. Also disclosed is an antisense oligonucleotide having enhanced antibacterial activity by virtue of one or both modifications.
1. An antisense oligomer for use in treating a bacterial infection in a mammalian host, comprising a substantially uncharged antisense oligonucleotide 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, having between 10-20 bases and a targeting sequence of at least 10 contiguous bases complementary to a target region of the infecting bacteria's mRNA for acyl carrier protein (acpP),
where the target region contains the translational start codon of the bacterial mRNA, or a sequence that is within 20 bases, in a downstream direction, of the translational start codon, and where the oligonucleotide binds to the mRNA to form a heteroduplex having a T, of at least 50° C., thereby to inhibit replication of the bacteria.
2. The antisense oligomer of claim 1 , wherein the targeting sequence is selected from SEQ ID NO: 62-71.
3. The antisense oligomer of claim 1 , wherein the targeting sequence is selected from SEQ ID NO: 63, 66, or 67.
4. The antisense oligomer of claim 1 , wherein the targeting sequence is SEQ ID NO: 63.
5. The antisense oligomer of claim 1 , wherein the targeting sequence is SEQ ID NO: 66.
6. The antisense oligomer of claim 1 , wherein the targeting sequence is SEQ ID NO: 67.
7. The antisense oligomer of claim 1 , wherein the morpholino subunits in the oligonucleotide are joined by phosphorodiamidate linkages, in accordance with the structure:
where Y 1 ═O, Z═O, P j 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.
8. The antisense oligomer of claim 7 , wherein the targeting sequence is selected from SEQ ID NO: 62-71.
9. The antisense oligomer of claim 7 , wherein the targeting sequence is selected from SEQ ID NO: 63, 66, or 67.
10. The antisense oligomer of claim 7 , wherein the targeting sequence is SEQ ID NO: 63.
11. The antisense oligomer of claim 7 , wherein the targeting sequence is SEQ ID NO: 66.
12. The antisense oligomer of claim 7 , wherein the targeting sequence is SEQ ID NO: 67.
13. An antisense oligomer for use in treating a bacterial infection in a mammalian host, comprising a substantially uncharged antisense oligonucleotide 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, having 11 bases and a targeting sequence of at least 10 contiguous bases complementary to a target region of the infecting bacteria's mRNA for gyrase A subunit (gyrA),
wherein the target sequence contains or is within 20 bases of, in a downstream direction, the translational start codon.
14. The antisense oligomer of claim 13 , wherein the morpholino subunits in the oligonucleotide are joined by phosphorodiamidate linkages, in accordance with the structure:
where Y 1 ═O, Z═O, P j 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.