Antibacterial antisense oligonucleotide and method

• US 10,144,762 B2
• Filed: 10/14/2016
• Issued: 12/04/2018
• Est. Priority Date: 07/13/2005
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Sarepta Therapeutics, Inc.
• Inventors: Weller, Dwight D., Geller, Bruce L., Iversen, Patrick L., Tilley, Lucas D., Hassinger, Jed N.
• Primary Examiner(s): McGarry, Sean
• Application Number:
  US15/293,961
• Publication Number:
  US 20170247410A1
• Time in Patent Office: 781 Days
• Field of Search: 544386
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 31/04 Antibacterial agents
• CPC Class Codes: C07D 295/205 Radicals derived from carbonic acid
• CPC Class Codes: C07D 413/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 473/34 attached in position 6, e.g. adenine
• CPC Class Codes: C07F 9/65583 each of the hetero rings containing nitrogen as ring hetero atom
• CPC Class Codes: C07K 7/02 Linear peptides containing at least one abnormal peptide link
• CPC Class Codes: C08F 112/08 Styrene
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 2310/11 Antisense
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2320/50 Methods for regulating/modulating their activity

Abstract

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.

Claims

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:

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: