Antisense antibacterial method and compound

• US 9,534,220 B2
• Filed: 11/19/2012
• Issued: 01/03/2017
• Est. Priority Date: 07/02/2004
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Sarepta Therapeutics, Inc.
• Inventors: Geller, Bruce L., Deere, Jesse D., Iversen, Patrick L.
• Primary Examiner(s): ZARA, JANE J
• Application Number:
  US13/680,790
• Publication Number:
  US 20130289091A1
• Time in Patent Office: 1,506 Days
• Field of Search: 435/6, 435/91.1, 435/91.31, 435/455, 435/458, 435/6.1, 435/6.13, 435/6.15, 435/471, 514/44, 536/23.1, 536/24.5, 536/24.32, 544/81
• US Class Current: 1/1
• 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

of 10 to 12 bases in length having a targeting sequence of at least 10 bases that are complementary to a target sequence containing or within 10 bases downstream 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, wherein the antisense oligonucleotide compound

is a morpholino antisense oligonucleotide.

2. The method of claim 1, wherein the morpholino subunits of the antisense oligonucleotide compound are joined by phosphorus-containing linkages, in accordance with the structure:

3. The method of claim 1, wherein the targeting sequence is complementary to a target sequence containing the translational start codon of the bacterial mRNA.

4. The method of claim 1, wherein the targeting sequence is complementary to a target sequence that is within 10 bases downstream of the translational start codon of the bacterial mRNA.

5. The method of claim 1, wherein the antisense oligonucleotide compound contains 11 bases, and has a nucleic acid sequence that is completely complementary to the mRNA target sequence.

6. The method of claim 1, for use in inhibiting a bacterial infection in a mammalian subject, wherein said exposing includes administering the antisense oligonucleotide compound in a therapeutically effective amount.

7. The method of claim 6, further comprising the step of treating the subject by administration of a non-antisense compound having antibacterial activity.

17. The method of claim 1, wherein the bacterial mRNA encodes the ftsZ protein, and the 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, 19, 22, 27, 30, 33, 38, 41, 44, 47, 50, 53, and 56.

18. The method of claim 1, wherein the bacterial mRNA encodes the acpP protein, and the 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, 17, 23, 28, 31, 34, 36, 39, 42, 45, 48, 51, 54, and 57.

19. The method of claim 1, wherein the bacterial mRNA encodes the gyrA protein, and the 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, 20, 24, 29, 32, 35, 37, 40, 43, 46, 49, 52, 55, and 58.

20. The method of claim 2, wherein the phosphorus-containing linkages are phosphorodiamidate linkages.

21. The method of claim 20, wherein each X is NR₂ wherein each R is CH₃.

22. The method of claim 2, wherein up to one of every five phosphorus-containing linkages is positively charged, wherein X is 1-piperazine.

8. A substantially uncharged antisense oligonucleotide compound

of 10 to 12 bases in length having a targeting sequence of at least 10 bases that are complementary to a target sequence containing or within 10 bases downstream 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, wherein the antisense oligonucleotide compound

is a morpholino antisense oligonucleotide.

9. The antisense oligonucleotide compound of claim 8, wherein the bacterial mRNA encodes the ftsZ protein, and the 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, 19, 22, 27, 30, 33, 38, 41, 44, 47, 50, 53, and 56.

10. The antisense oligonucleotide compound of claim 8, wherein the bacterial mRNA encodes the acpP protein, and the 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, 17, 23, 28, 31, 34, 36, 39, 42, 45, 48, 51, 54, and 57.

11. The antisense oligonucleotide compound of claim 8, wherein the bacterial mRNA encodes the gyrA protein, and the 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, 20, 24, 29, 32, 35, 37, 40, 43, 46, 49, 52, 55, and 58.

12. The antisense oligonucleotide compound of claim 8, wherein the morpholino subunits of the antisense oligonucleotide compound are joined by phosphorus-containing linkages, in accordance with the structure:

13. The antisense oligonucleotide compound of claim 8, wherein the targeting sequence is complementary to a target sequence containing the translational start codon of the bacterial mRNA.

14. The antisense oligonucleotide compound of claim 8, wherein the targeting sequence is complementary to a target sequence that is within 10 bases downstream of the translational start codon of the bacterial mRNA.

15. The antisense oligonucleotide compound of claim 8, which contains 11 bases, and has a nucleic acid sequence that is completely complementary to the mRNA target sequence.

16. The antisense oligonucleotide compound of claim 8, which contains 10 bases, and has a nucleic acid sequence that is completely complementary to the mRNA target sequence.

23. The antisense oligonucleotide compound of claim 12, wherein the phosphorus-containing linkages are phosphorodiamidate linkages.

24. The antisense oligonucleotide compound of claim 23, wherein each X is NR₂ wherein each R is CH₃.

25. The antisense oligonucleotide compound of claim 12, wherein up to one of every five phosphorus-containing linkages is positively charged, wherein X is 1-piperazine.

26. An antisense oligonucleotide of 11 nucleotide bases for inhibiting the growth of pathogenic bacterial cells comprising a base sequence of CTTCGATAGTG (SEQ ID NO:

109) that is completely complementary to a bacterial mRNA that encodes the acyl carrier protein (acpP), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide.

27. The antisense oligonucleotide of claim 26, wherein the antisense oligonucleotide is conjugated to a polyethylene glycol moiety.

28. An antisense oligonucleotide of 10 nucleotide bases for inhibiting the growth of pathogenic bacterial cells comprising a base sequence of TTCGATAGTG (SEQ ID NO:

110) that is completely complementary to a bacterial mRNA that encodes the acyl carrier protein (acpP), wherein the antisense oligonucleotide is a morpholino antisense oligonucleotide.

29. The antisense oligonucleotide of claim 28, wherein the antisense oligonucleotide is conjugated to a polyethylene glycol moiety.