Antibacterial antisense oligonucleotide and method

• US 8,536,147 B2
• Filed: 03/12/2010
• Issued: 09/17/2013
• 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:
  US12/723,413
• Publication Number:
  US 20100234281A1
• Time in Patent Office: 1,285 Days
• Field of Search: None
• US Class Current: 514/44.A
• 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 conjugate, comprising

(a) an antisense oligonucleotide composed of morpholino subunits linked by phosphorus-containing intersubunit linkages joining a morpholino nitrogen of one subunit to a 5′

exocyclic carbon of an adjacent subunit, wherein the oligonucleotide contains between 10-20 bases and a targeting sequence of at least 10 contiguous bases complementary to a target sequence containing or within 20 bases, in a downstream direction, of the translational start codon of a bacterial mRNA that encodes acyl carrier protein (acpP), wherein the target sequence is the Burkholderia mallei acpP sequence of SEQ ID NO;

54 or the Burkholderia pseudomallei acpP sequence of SEQ ID NO;

57, and

(b) a carrier peptide represented by the formula (RAhxR)₄ or (RFF)₃, wherein Ahx is aminohexanoic acid.

2. The conjugate of claim 1, wherein the oligonucleotide contains 10%-50% intersubunit cationic linkages.

3. The conjugate of claim 1, wherein the carrier peptide is linked at its C-terminus to the 5′

end of the oligonucleotide through a one- or two-amino acid linker.

4. The conjugate of claim 1, wherein the carrier peptide is linked at its C-terminus to the 3′

end of the oligonucleotide through a one- or two-amino acid linker.

5. The conjugate of claim 3, wherein the linker is Ahxβ

Ala, wherein Ahx is 6-amino hexanoic acid and β

Ala is β

-alanine.

6. The conjugate of claim 1, wherein the peptide is represented by the formula (RAhxR)₄.

7. The conjugate of claim 1, wherein the morpholino subunits in the oligonucleotide are joined by phosphorodiamidate linkages, in accordance with the structure:

8. The conjugate of claim 7, wherein each of R¹ and R², in linkages of type (a), is methyl.

9. The conjugate of claim 7, wherein at least one linkage is of type (b1), where each R is H, R⁴ is H, CH₃, or an electron pair, and R³ is selected from H, CH₃, C(═

NH)NH₂, and C(O)-L-NHC(═

NH)NH₂.

10. The conjugate of claim 7, wherein at least one linkage is of type (b1), where each R is H, R⁴ is an electron pair, and R³ is selected from C(═

NH)NH₂ and C(O)-L-NHC(═

NH)NH₂.

11. The conjugate of claim 7, wherein at least one linkage is of type (b1), where each R is H, R⁴ is an electron pair, and R³ is selected from C(═

NH)NH₂ and C(O)-L-NHC(═

NH)NH₂.

12. The conjugate of claim 10, wherein R³ is C(O)-L-NHC(NH)NH2, and L is a hydrocarbon having the structure —

(CH₂)_n—

, where n is 1 to 12.

13. The conjugate of claim 7, wherein at least one linkage is of type (b1), where each R is H, and each of R³ and R⁴ is independently H or CH₃.