Antibacterial antisense oligonucleotide and method

• US 20080194463A1
• Filed: 05/11/2007
• Published: 08/14/2008
• Est. Priority Date: 07/13/2005
• Status: Active Grant

• Current Assignee: Sarepta Therapeutics, Inc.
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Iversen, Patrick L., Weller, Dwight D., Hassinger, Jed N., Geller, Bruce L., Tilley, Lucas D.
• Granted Patent: US 8,067,571 B2
• Time in Patent Office: Days
• US Class Current: 514/1.1
• 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. A method for enhancing the antibacterial activity of 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, where the oligonucleotide contains between 10-20 bases and a targeting sequence of at least 10 contiguous bases complementary to a bacterial RNA target, and where binding of the oligonucleotide to the RNA target region is effective to inhibit growth of a infectious bacterium in a mammalian host, comprising one or both of steps (a) and (b);

(a) conjugating to the oligonucleotide, a carrier peptide (i) containing 8-14 amino acids composed of the subsequences selected from the group represented by XXY, XY, XZZ and XZ, and permutations of the subsequences, where (a) each X subunit independently represents arginine or an arginine analog, said analog being a cationic α

-amino acid comprising a side chain of the structure R1N═

C(NH2)R2, where R1 is H or R;

R2 is R, NH2, NHR, or NR2, where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen;

R1 and R2 may together form a ring; and

the side chain is linked to said amino acid via R1 or R2;

(b) each Y subunit independently represents a neutral linear amino acid —

C(O)—

(CHR)_m—

NH—

, where (i) m is 1 to 7 and each R is independently H or methyl, and (c) Z is an α

-amino acid having a neutral side chain selected from a substituted or unsubstituted aralkyl, and (ii) coupled to the oligonucleotide at the peptide'"'"'s C terminus, and

(b) modifying the oligonucleotide to contain 20%-50% intersubunit cationic linkages that are positively charged at physiological pH.

2. The method of claim 1, wherein the carrier peptide is represented by the sequence (RY′

R)_n and (RY′

)_n, where R is arginine and Y′

is a linear alkanoic acid having 2-7 carbon atoms in its backbone chain.

3. The method 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 method of claim 3, wherein the linker is Ahxβ

Ala, where Ahx is 6-aminohexanoic acid and Ala is β

-alanine.

5. The method of claim 1, wherein the peptide is represented by (RAhxR)₄, where Ahx is 6-amino hexanoic acid.

6. The method of claim 1, wherein the peptide is represented by (RAhx)₆, where Ahx is 6-amino hexanoic acid.

7. The method of claim 1, wherein said oligonucleotide has approximately equal-length 5′

, 3′ and

center regions, and the percentage of cationic linkages in said center region is greater than about 70%.

8. The method of claim 1, wherein step (a) of the method is effective to enhance the anti-bacterial activity of an uncharged oligonucleotide, as measured by inhibition in bacterial growth in vitro over an eight-hour period, by a factor of at least 10 relative to the measured inhibition of the uncharged oligonucleotide in the absence of the carrier peptide.

9. The method of claim 8, wherein step (a) and step (b) of the method are effective to enhance the anti-bacterial activity of an uncharged oligonucleotide, as measured by inhibition in bacterial growth in vitro over an eight-hour period, by a factor of at least 10 relative to the measured inhibition of the uncharged oligonucleotide in the absence of the carrier peptide.

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

11. The method of claim 10, wherein each of R¹ and R², in linkages of type (a), is methyl.

12. The method of claim 10, 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.

13. The method of claim 10, 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.

14. The method of claim 10, 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.

15. The method oligomer of claim 13, 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.

16. The method of claim 1, 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.

17. The method of claim 1, for treating a gram-negative bacterial infection, wherein the targeting sequence is 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).

18. The method of claim 1, for treating a gram-negative bacterial infection, wherein the targeting sequence is 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 gyrase A subunit (gyrA).

19. In an antisense oligonucleotide useful for treating a bacterial infection and 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, where the oligonucleotide contains between 10-20 bases and a targeting sequence of at least 10 contiguous bases complementary to a bacterial RNA target, and where binding of the oligonucleotide to the RNA target region is effective to inhibit growth of the infectious bacterium, an improvement that enhances the antibacterial active of the oligonucleotide at least 10-fold, comprising one or both of (a) and (b);

(a) conjugated to the oligonucleotide, a carrier peptide (i) containing 8-14 amino acids composed of the subsequences selected from the group represented by XXY, XY, XZZ and XZ, and permutations of the subsequences, where (a) each X subunit independently represents arginine or an arginine analog, said analog being a cationic α

-amino acid comprising a side chain of the structure R1N═

C(NH2)R2, where R1 is H or R;

R2 is R, NH2, NHR, or NR2, where R is lower alkyl or lower alkenyl and may further include oxygen or nitrogen;

R1 and R2 may together form a ring; and

the side chain is linked to said amino acid via R1 or R2;

(b) each Y subunit independently represents a neutral linear amino acid —

C(O)—

(CHR)m-NH—

, where (i) m is 1 to 7 and each R is independently H or methyl, and (c) Z is an α

-amino acid having a neutral side chain selected from a substituted or unsubstituted aralkyl, and (ii) coupled to the oligonucleotide at the peptide'"'"'s C terminus, and

(b) the presence in the oligonucleotide of 20%-50% intersubunit cationic linkages that are positively charged at physiological pH.

20. The improvement of claim 19, wherein the carrier peptide is represented by the sequence (RY′

R)_n and (RY′

)_n, where R is arginine and Y′

is a linear alkanoic acid having 2-7 carbon atoms in its backbone chain.