Oligonucleotide analogs having cationic intersubunit linkages

• US 20090088562A1
• Filed: 05/10/2007
• Published: 04/02/2009
• Est. Priority Date: 05/10/2006
• Status: Active Grant

• Current Assignee: Avi Biopharma, Inc.
• Original Assignee: Avi Biopharma, Inc.
• Inventors: Weller, Dwight D., Hassinger, Jed N.
• Granted Patent: US 7,943,762 B2
• Time in Patent Office: Days
• US Class Current: 536/24.500
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 15/1132 against retroviridae, e.g. HIV
• CPC Class Codes: C12N 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 15/1137 against enzymes viral enzymes C12N15/1131; receptors C12N15/1138
• CPC Class Codes: C12N 15/1138 against receptors or cell surface proteins
• CPC Class Codes: C12N 2310/3145 with the nitrogen in 3' or 5'-position
• CPC Class Codes: C12N 2310/3233 Morpholino-type ring
• CPC Class Codes: C12N 2310/351 Conjugate
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2320/51 modulating the chemical stability, e.g. nuclease-resistance
• CPC Class Codes: C12N 2330/30 chemically synthesised

Abstract

Morpholino oligomers containing both uncharged and cationic intersubunit linkages are provided. The oligomers are oligonucleotide analogs containing predetermined sequences of base-pairing moieties. The presence of the cationic intersubunit linkages in the oligomers, typically at a level of about 10-50% of total linkages, provides enhanced antisense activity, in various antisense applications, relative to the corresponding uncharged oligomers. Also provided are such oligomers conjugated to peptide transporter moieties, where the transporters are preferably composed of arginine subunits, or arginine dimers, alternating with neutral amino acid subunits.

Claims

1. An oligomer comprising a sequence of morpholino subunits, each supporting a base-pairing moiety, such that said oligomer can bind in a sequence-specific manner to a target nucleic acid,

wherein said subunits are joined by phosphorus-containing intersubunit linkages having the structure:

2. The oligomer of claim 1, wherein said morpholino subunits have the structure:

3. The oligomer of claim 1, wherein all of the cationic linkages in the oligomer are of the same type.

4. The oligomer of claim 3, wherein all of the cationic linkages in the oligomer are of type (b1).

5. The oligomer of claim 3, wherein all of the cationic linkages in the oligomer are of type (b2).

6. The oligomer of claim 4, wherein all of the cationic linkages in the oligomer are of type (b3).

7. The oligomer of claim 1, wherein 5% to 50% of the linkages in the oligomer are cationic linkages.

8. The oligomer of claim 7, wherein 10% to 35% of the linkages in the oligomer are cationic linkages.

9. The oligomer of claim 1, wherein each of R¹ and R², in linkages of type (a), is methyl.

10. The oligomer of claim 1, wherein said oligomer includes at least two consecutive linkages of type (a).

11. The oligomer of claim 1, having a length of about 10 to 40 subunits.

12. The oligomer of claim 1, having a length of about 15 to 25 subunits.

13. The oligomer of claim 1, wherein said cationic linkages are 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.

14. The oligomer of claim 13, wherein said cationic linkages are of type (b1), where each R¹ is H, R⁴ is an electron pair, and R³ is selected from C(═

NH)NH₂ and C(O)-L-NHC(═

NH)NH₂.

15. The oligomer of claim 14, wherein R³ is C(O)-L-NHC(═

NH)NH2, and L is a hydrocarbon.

16. The oligomer of claim 15, wherein L has the structure —

(CH₂)_n—

, where n is 1 to 12.

17. The oligomer of claim 16, wherein n is 1 to 6.

18. The oligomer of claim 1, wherein said cationic linkages are of type (b1), where each R is H, and each of R³ and R⁴ is independently H or CH₃.

19. The oligomer of claim 18, wherein each of R³ and R⁴ is H.

20. The oligomer of claim 1, wherein said cationic linkages are of type (b2), where L is a linker up to 12 atoms in length having bonds selected from alkyl and alkylamino.

21. The oligomer of claim 1, wherein said oligomer contains no linkages of type (b1) in which each of R, R³ and R⁴ is H.

22. The oligomer of claim 1, further comprising, conjugated to a terminus of the oligomer, a peptide transport moiety, comprising 6 to 16 amino acids and composed of subsequences selected from the group consisting of (X′

Y′

X′

), (X′

Y′

), (X′

Z′

), and (X′

Z′

Z′

),

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)_n—

NH—

, where n is 1 to 7 and each R is independently H or methyl; and

(c) each Z′

subunit independently represents an α

-amino acid having a neutral aralkyl side chain.

23. The oligomer of claim 22, wherein the peptide comprises a sequence consisting of at least two repeats of a single subsequence selected from (X′

Y′

X′

), (X′

Y′

), (X′

Z′

), and (X′

Z′

Z′

).

24. The oligomer of claim 23, wherein the peptide comprises a sequence consisting of at least three repeats of a single subsequence selected from (X′

Y′

X′

), (X′

Y′

), (X′

Z′

), and (X′

Z′

Z′

).

25. The oligomer of claim 23, wherein the peptide comprises a sequence represented by one of (X′

Y′

X′

)_p, (X′

Y′

)_m, and (X′

Z′

Z′

)_p, where p is 2 to 5 and m is 2 to 8.

26. The oligomer of claim 22, wherein for each X′

, the side chain moiety is guanidyl.

27. The oligomer of claim 22, wherein each Y′

is —

CO—

(CH₂)_n—

NH—

, where n is 1 to 7.

28. The oligomer of claim 27, wherein n is selected from 2 and 5, such that Y′

is selected from a β

-alanine subunit and a 6-aminohexanoic acid subunit.

29. The oligomer of claim 22, where each Z′

is phenylalanine.

30. The oligomer of claim 22, wherein the conjugated peptide is linked to a terminus of the oligomer via a linker Ahx-B, where Ahx is a 6-aminohexanoic acid subunit and B is a β

-alanine subunit.

31. A method of enhancing the antisense activity of an oligomeric nucleic acid analog having a sequence of morpholino subunits, each supporting a base-pairing moiety, wherein said subunits are connected by intersubunit linkages, the method comprising

modifying at least one intersubunit linkage to contain a pendant cationic group, wherein said pendant cationic group includes a nitrogen atom that can bear a positive charge at physiological pH.

32. The method of claim 31, wherein said intersubunit linkages are phosphorus-containing intersubunit linkages, and said modifying comprises providing at least one intersubunit linkage having the structure:

33. The method of claim 32, wherein said morpholino subunits have the structure:

34. The method of claim 33, wherein all of the cationic linkages in the oligomer are of the same type.

35. The method of claim 31, wherein 5% to 50% of the intersubunit linkages are so modified.

36. The method of claim 34, wherein said modified linkages are of type (b1).

37. The method of claim 34, wherein said modified linkages are of type (b2).

38. The method of claim 34, wherein said modified linkages are of type (b3).

39. The method of claim 36, wherein said modified linkages are of type (b1), where each R is H, R⁴ is an electron pair, and R³ is C(O)-L-NHC(═

NH)NH₂.

40. The method of claim 36, wherein said modified linkages are of type (b1), where each of R, R³ and R⁴ is H.

41. The method of claim 37, wherein said cationic linkages are of type (b2), where L is a linker up to 12 atoms in length having bonds selected from alkyl and alkylamino.

42. The method of claim 31, wherein said oligomer contains no linkages of type (b1) in which each of R, R³ and R⁴ is H.