Antisense antibacterial method and compound

• US 20070049542A1
• Filed: 05/10/2006
• Published: 03/01/2007
• Est. Priority Date: 07/02/2004
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Weller, Dwight, Deere, Jesse, Geller, Bruce, Iversen, Patrick
• Granted Patent: US 7,625,873 B2
• Time in Patent Office: Days
• US Class Current: 514/44.A
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: C07F 9/65583 each of the hetero rings containing nitrogen as ring hetero atom
• CPC Class Codes: C07F 9/65613 containing the ring system (X = CH2, O, S, NH) optionally with an additional double bond and/or substituents, e.g. cephalosporins and analogs
• CPC Class Codes: C12N 15/113 Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing when used in plants C12N15/8218
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 2310/11 Antisense
• CPC Class Codes: C12N 2310/3145 with the nitrogen in 3' or 5'-position
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring

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.

Claims

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:

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:

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:

b 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:

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:

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.