2'-substituted oligomeric compounds and compositions for use in gene modulations

• US 20040171033A1
• Filed: 11/04/2003
• Published: 09/02/2004
• Est. Priority Date: 06/06/1996
• Status: Active Grant

• Current Assignee: ISIS Pharmaceuticals Incorporated
• Original Assignee: ISIS Pharmaceuticals Incorporated
• Inventors: Bhat, Balkrishen, Swayze, Eric E., Baker, Brenda F., Prakash, Thazha P., Manoharan, Muthiah, Crooke, Stanley T., Eldrup, Anne B., Griffey, Richard
• Granted Patent: US 7,919,612 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 35/00 Antineoplastic agents
• CPC Class Codes: A61P 43/00 Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• 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 15/113 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 15/1135 against oncogenes or tumor suppressor genes
• CPC Class Codes: C12N 2310/311 Phosphotriesters
• CPC Class Codes: C12N 2310/312 Phosphonates
• CPC Class Codes: C12N 2310/314 Phosphoramidates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/316 Phosphonothioates
• CPC Class Codes: C12N 2310/318 where the PO2 is completely replaced, e.g. MMI or formacetal
• CPC Class Codes: C12N 2310/3181 Peptide nucleic acid, PNA
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3341 5-Methylcytosine
• CPC Class Codes: C12N 2310/335 Modified T or U
• CPC Class Codes: C12N 2310/341 Gapmers, i.e. of the type ===---===
• CPC Class Codes: C12N 2310/346 : having a combination of backbone and sugar modifications
• CPC Class Codes: C12N 2310/3521 : Methyl
• CPC Class Codes: C12N 2310/3525 : MOE, methoxyethoxy
• CPC Class Codes: C12N 2310/3527 : Other alkyl chain
• CPC Class Codes: C12N 9/22 : Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113
• CPC Class Codes: Y02A 50/30 : Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
• CPC Class Codes: Y02P 20/582 : Recycling of unreacted starting or intermediate materials

Abstract

Compositions comprising first and second oligomers are provided wherein at least a portion of the first oligomer is capable of hybridizing with at least a portion of the second oligomer, at least a portion of the first oligomer is complementary to and capable of hybridizing to a selected target nucleic acid, and at least one of the first or second oligomers includes a modified sugar and/or backbone modification. In some embodiments the modification is a 2′ substituent group on a sugar moiety that is not H or OH. Oligomer/protein compositions are also provided comprising an oligomer complementary to and capable of hybridizing to a selected target nucleic acid and at least one protein comprising at least a portion of an RNA-induced silencing complex (RISC), wherein at least one nucleotide of the oligomer has a modified sugar and/or backbone modification.

Claims

1. A composition comprising a first oligomer and a second oligomer, wherein:

at least a portion of said first oligomer is capable of hybridizing with at least a portion of said second oligomer,

at least a portion of said first oligomer is complementary to and capable of hybridizing with a selected target nucleic acid,

at least one of said first or said second oligomers includes at least one sugar moiety having a 2′

substituent group that is not H or OH.

2. The composition of claim 1 wherein said first and said second oligomers are a complementary pair of siRNA oligomers.

3. The composition of claim 1 wherein said first and said second oligomers are an antisense/sense pair of oligomers.

4. The composition of claim 1 wherein each of said first and second oligomers has 10 to 40 nucleotides.

5. The composition of claim 1 wherein each of said first and second oligomers has 18 to 30 nucleotides.

6. The composition of claim 1 wherein each of said first and second oligomers has 21 to 24 nucleotides.

7. The composition of claim 1 wherein said first oligomer is an antisense oligomer.

8. The composition of claim 7 wherein said second oligomer comprises a sense oligomer.

9. The composition of claim 7 wherein said second oligomer has a plurality of ribose nucleotide units.

10. The composition of claim 1 wherein said first oligomer includes said 2′

substituent group.

11. The composition of claim 1 wherein the 2′

substituent group is halogen, amino, trifluoroalkyl, trifluoroalkoxy, azido, aminooxy, alkyl, alkenyl, alkynyl, O—

, S—

, or N(R*)-alkyl;

O—

, S—

, or N(R*)-alkenyl;

O—

, S—

or N(R*)-alkynyl;

O—

, S—

or N-aryl, O—

, S—

, or N(R*)-aralkyl;

wherein said alkyl, alkenyl, alkynyl, aryl and aralkyl may be substituted or unsubstituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₂ to C₁₀ alkynyl, C₅-C₂₀ aryl or C₆-C₂₀ aralkyl; and

said substituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₂ to C₁₀ alkynyl, C₅-C₂₀ aryl or C₆-C₂₀ aralkyl comprising substitution with hydroxy, alkoxy, thioalkoxy, phthalimido, halogen, amino, keto, carboxyl, nitro, nitroso, cyano, aryl, haloalkyl, haloalkoxy, imidazole, azido, hydrazino, aminooxy, isocyanato, sulfoxide, sulfone, disulfide, silyl, heterocycle, carbocycle, an intercalator, a reporter group, a conjugate, a polyamine, a polyamide, a polyalkylene glycol, or a polyether of the formula (—

O-alkyl)_m, where m is 1 to about 10; and

R* is hydrogen, or a protecting group.

12. The composition of claim 11 wherein the 2′

substituent group is a halogen atom.

13. The composition of claim 12 wherein the halogen atom is F.

14. The composition of claim 11 wherein the 2′

substituent group is a —

O-alkyl substituent.

15. The composition of claim 14 wherein the —

O-alkyl is —

O—

CH₃.

16. The composition of claim 11 wherein the 2′

substituent group is —

OCH₂CH₂OCH₃.

17. The composition of claim 11 wherein the 2′

substituent group is (N,N-dimethylaminoethyl)oxyethyl.

18. The composition of claim 1 wherein the 2′

substituent group is —

O—

R²⁶-thio-R²⁶ or —

C—

R²⁶-thio-R²⁶, wherein R²⁶ is alkyl, allyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, or heterocyclic aryl.

19. The composition of claim 1 wherein the 2′

substituent is cyano, fluoromethyl, thioalkoxyl, fluoroalkoxyl, alkylsulfinyl, alkylsulfonyl, allyloxy or alkeneoxy.

20. The composition of claim 1 wherein the 2′

substituent is alkylsulfinyl or alkylsulfonyl.

21. The composition of claim 1 wherein the 2′

substituent is -thioalkoxyl substituent.

22. The composition of claim 1 wherein the 2′

substituent is of the formula —

X—

Y, wherein;

X is O, S, NR²⁷, or CR²⁷ wherein each R²⁷ is independently H or C_1-6 alkyl; and

Y is a drug moiety, a substituted or unsubstituted C_1-20 alkyl, a substituted or unsubstituted C_2-20 alkenyl, or substituted or unsubstituted C_6-20 aryl.

23. The composition of claim 1 wherein the 2′

substituent is of the formula —

O-G1-G2-G3, wherein;

G1 is alkyl, alkenyl, or alkynyl;

G2 is an aryl; and

G3 is an imidazole or a bis-imidazole moiety.

24. The composition of claim 1 wherein the 2′

substituent is of the formula —

C(X)—

N(R²⁹)(R³⁰) wherein;

R²⁹ and R³⁰, independently, are H, R³³, R³⁴, an amine protecting group or have the formula R³³—

N(R³¹)(R³²), C(X)—

R³³, C(X)—

R³⁴—

R³³, C(X)—

Q-R³⁴—

R³³, or C(X)—

Q-R³³;

R³¹ and R³², independently, are H, R³³, R³⁴, an amine protecting group or have formula C(X)—

R³³, C(X)—

R³⁴—

R³³, C(X)—

Q-R³⁴—

R³³, or C(X)—

Q-R³³;

R₃₃ is a steroid molecule, biotin, dinitrophenyl, a fluorescein dye, a lipophilic molecule, a reporter enzyme, a peptide, a protein, includes folic acid, or has formula —

Q—

(CH₂CH₂—

Q—

)_x-R³⁵;

R³⁴ is alkyl having from 1 to about 10 carbon atoms;

X is O or S;

each Q is, independently, is NH, O, or S;

R³⁵ is H, R³⁴, C(O)OH, C(O)OR³⁴, C(O)R⁴¹, R³⁴—

N₃, or R³⁴—

NH₂;

R⁴¹ is Cl, Br, I, SO₂R⁴² or has structure;

m is 2 or 7; and

R⁴² alkyl having 1 to about 10 carbon atoms.

25. The composition of claim 1 wherein the 2′

substituent is an aminoalkoxy or an imidazolylalkoxy substituent.

26. The composition of claim 1 wherein the 2′

substituent is of the formula;

27. The composition of claim 1 wherein the 2′

substituent is of the formula;

28. The composition of claim 1 wherein the 2′

substituent is of the formula;

29. The composition of claim 1 wherein the 2′

substituent is of the formula;

—

X—

(CR⁷¹R⁷²)_n—

O—

SO₂—

O⁻

Y⁺

wherein;

X is a O, S, or N;

R⁷¹ and R⁷² are each, independently, H, alkyl, aryl, O-alkyl, O-aryl, carboxylic acid, amide, ester, halogen, trifluoromethyl, or amine;

n is an integer from about 2 to about 10; and

Y is H, Li, Na, K, Cs or an amine.

30. The composition of claim 1 wherein the 2′

substituent is of the formula;

31. The composition of claim 1 wherein the 2′

substituent is of the formula —

OR⁵² wherein R⁵² is

32. The composition of claim 1 wherein the 2′

substituent is of the formula;

33. The composition of claim 1 including at least one sugar moiety comprising two 2′

substituent groups Y¹ and Y².

34. The composition of claim 33 wherein:

Y¹ and Y² are each independently hydrogen;

hydroxyl;

halogen;

C_2-4 alkenyl, C_2-4 alkynyl, or C_1-4 alkyl;

C_1-10 alkoxy;

C_2-6 alkenyloxy;

C_1-4 alkylthio;

C_1-8 alkylcarbonyloxy;

aryloxycarbonyl;

azido;

amino;

C_1-4 alkylamino;

di(C_1-4 alkyl)amino;

or Y³; and

Y³ is a conjugate molecule or a reporter molecule.

35. The composition of claim 34 wherein:

yl is C_2-4 alkenyl, C_2-4 alkynyl, or C_1-4 alkyl; and

Y² is hydrogen, fluorine, hydroxy, C_1-10 alkoxy, or C_1-10 alkyl.

75. A pharmaceutical composition comprising the composition of claim 1 and a pharmaceutically acceptable carrier.

78. A method of modulating the expression of a target nucleic acid in a cell comprising contacting said cell with a composition of claim 1.

81. A method of treating or preventing a disease or disorder associated with a target nucleic acid comprising administering to an animal having or predisposed to said disease or disorder a therapeutically effective amount of a composition of claim 1.

36. A composition comprising an oligomer complementary to and capable of hybridizing to a selected target nucleic acid and at least one protein, said protein comprising at least a portion of a RNA-induced silencing complex (RISC), wherein:

said oligomer includes includes at least one sugar moiety having a 2′

substituent group that is not H or OH.

37. The composition of claim 36 wherein said oligomer is an antisense oligomer.

38. The composition of claim 36 wherein said oligomer has 10 to 40 nucleotides.

39. The composition of claim 36 wherein said oligomer has 18 to 30 nucleotides.

40. The composition of claim 36 wherein said oligomer has 21 to 24 nucleotides.

41. The composition of claim 38 further including a further oligomer, wherein said further oligomer is complementary to said oligomer.

42. The composition of claim 41 wherein said further oligomer is a sense oligomer.

43. The composition of claim 41 wherein said further oligomer is an oligomer having a plurality of ribose nucleotide units.

44. The composition of claim 36 wherein the 2′

substituent group is halogen, amino, trifluoroalkyl, trifluoroalkoxy, azido, aminooxy, alkyl, alkenyl, alkynyl, O—

, S—

, or N(R*)-alkyl;

O—

, S—

, or N(R*)-alkenyl;

O—

, S—

or N(R*)-alkynyl;

O—

, S—

or N-aryl, O—

, S—

, or N(R*)-aralkyl;

wherein said alkyl, alkenyl, alkynyl, aryl and aralkyl may be substituted or unsubstituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₂ to C₁₀ alkynyl, C₅-C₂₀ aryl or C₆-C₂₀ aralkyl; and

said substituted C₁ to C₁₀ alkyl, C₂ to C₁₀ alkenyl, C₂ to C₁₀ alkynyl, C₅-C₂₀ aryl or C₆-C₂₀ aralkyl comprising substitution with hydroxy, alkoxy, thioalkoxy, phthalimido, halogen, amino, keto, carboxyl, nitro, nitroso, cyano, aryl, haloalkyl, haloalkoxy, imidazole, azido, hydrazino, aminooxy, isocyanato, sulfoxide, sulfone, disulfide, silyl, heterocycle, carbocycle, an intercalator, a reporter group, a conjugate, a polyamine, a polyamide, a polyalkylene glycol, or a polyether of the formula (—

O-alkyl)_m, where m is 1 to about 10; and

R* is hydrogen, or a protecting group.

45. The composition of claim 44 wherein the 2′

substituent group is a halogen atom.

46. The composition of claim 45 wherein the halogen atom is F.

47. The composition of claim 46 wherein the 2′

substituent group is a —

O-alkyl substituent.

48. The composition of claim 47 wherein the —

O-alkyl is —

O—

CH₃.

49. The composition of claim 44 wherein the 2′

substituent group is —

OCH₂CH₂OCH₃.

50. The composition of claim 44 wherein the 2′

substituent group is (N,N-dimethylaminoethyl)oxyethyl.

51. The composition of claim 36 wherein the 2′

substituent group is —

O—

R²⁶-thio-R²⁶ or —

C—

R²⁶-thio-R²⁶, wherein R²⁶ is alkyl, allyl, alkenyl, alkynyl, aryl, alkylaryl, carbocyclic aryl, or heterocyclic aryl.

52. The composition of claim 36 wherein the 2′

substituent is cyano, fluoromethyl, thioalkoxyl, fluoroalkoxyl, alkylsulfinyl, alkylsulfonyl, allyloxy or alkeneoxy.

53. The composition of claim 36 wherein the 2′

substituent is -alkylsulfinyl or alkylsulfonyl.

54. The composition of claim 36 wherein the 2′

substituent is -thioalkoxyl substituent.

55. The composition of claim 36 wherein the 2′

substituent is of the formula —

X—

Y, wherein;

X is O, S, NR²⁷, or CR²⁷ wherein each R²⁷ is independently H or C_1-6 alkyl; and

Y is a drug moiety, a substituted or unsubstituted C_1-20 alkyl, a substituted or unsubstituted C_2-20 alkenyl, or substituted or unsubstituted C_6-20 aryl.

56. The composition of claim 36 wherein the 2′

substituent is of the formula —

O-G1-G2-G3, wherein;

G1 is alkyl, alkenyl, or alkynyl;

G2 is an aryl; and

G3 is an imidazole or a bis-imidazole moiety.

57. The composition of claim 36 wherein the 2′

substituent is of the formula —

C(X)—

N(R²⁹)(R³⁰) wherein;

R²⁹ and R³⁰, independently, are H, R³³, R³⁴, an amine protecting group or have the formula R³³—

N(R³¹)(R³²), C(X)—

R³³, C(X)—

R³⁴—

R³³, C(X)—

Q-R³⁴—

R³³, or C(X)—

Q-R³³;

R³¹ and R³², independently, are H, R³³, R³⁴, an amine protecting group or have formula C(X)—

R³³, C(X)—

R³⁴—

R³³, C(X)—

Q-R³⁴—

R³³, or C(X)—

Q-R³³;

R₃₃ is a steroid molecule, biotin, dinitrophenyl, a fluorescein dye, a lipophilic molecule, a reporter enzyme, a peptide, a protein, includes folic acid, or has formula —

Q—

(CH₂CH₂—

Q—

)_x-R³⁵;

R³⁴ is alkyl having from 1 to about 10 carbon atoms;

X is O or S;

each Q is, independently, is NH, O, or S;

R³⁵ is H, R³⁴, C(O)OH, C(O)OR³⁴, C(O)R⁴¹, R³⁴—

N₃, or R³⁴—

NH₂;

R⁴¹ is Cl, Br, I, SO₂R⁴² or has structure;

m is 2 or 7; and

R⁴² alkyl having 1 to about 10 carbon atoms.

58. The composition of claim 36 wherein the 2′

substituent is an aminoalkoxy or an imidazolylalkoxy substituent.

59. The composition of claim 36 wherein the 2′

substituent is of the formula;

60. The composition of claim 36 wherein the 2′

substituent is of the formula;

61. The composition of claim 36 wherein the 2′

substituent is of the formula;

62. The composition of claim 36 wherein the 2′

substituent is of the formula;

—

X—

(CR⁷¹R⁷²)_n—

O—

SO₂—

O⁻

Y⁺

wherein;

X is a O, S, or N;

R⁷¹ and R⁷² are each, independently, H, alkyl, aryl, O-alkyl, O-aryl, carboxylic acid, amide, ester, halogen, trifluoromethyl, or amine;

n is an integer from about 2 to about 10; and

Y is H, Li, Na, K, Cs or an amine.

63. The composition of claim 36 wherein the 2′

substituent is of the formula;

64. The composition of claim 36 wherein the 2′

substituent is of the formula —

OR⁵² wherein R⁵² is

65. The composition of claim 36 wherein the 2′

substituent is of the formula;

66. The composition of claim 36 including at least one sugar moiety comprising two 2′

substituent groups Y¹ and Y².

67. The composition of claim 66 wherein:

Y¹ and Y² are each independently hydrogen;

hydroxyl;

halogen;

C_2-4 alkenyl, C_2-4 alkynyl, or C_1-4 alkyl;

C_1-10 alkoxy;

C_2-6 alkenyloxy;

C_1-4 alkylthio;

C_1-8 alkylcarbonyloxy;

aryloxycarbonyl;

azido;

amino;

C_1-4 alkylamino;

di(C_1-4 alkyl)amino;

or Y³; and

Y³ is a conjugate molecule or a reporter molecule.

68. The composition of claim 67 wherein:

Y¹ is C_2-4 alkenyl, C_2-4 alkynyl, or C_1-4 alkyl; and

Y² is hydrogen, fluorine, hydroxy, C_1-10 alkoxy, or C_1-10 alkyl.

76. A pharmaceutical composition comprising the composition of claim 36 and a pharmaceutically acceptable carrier.

79. A method of modulating the expression of a target nucleic acid in a cell comprising contacting said cell with a composition of claim 36.

82. A method of treating or preventing a disease or disorder associated with a target nucleic acid comprising administering to an animal having or predisposed to said disease or disorder a therapeutically effective amount of a composition of claim 36.

69. An oligomer having at least a first region and a second region, wherein:

said first region of said oligomer is complementary to and capable of hybridizing with said second region of said oligomer,

at least a portion of said oligomer is complementary to and capable of hybridizing to a selected target nucleic acid, and

said oligomer further includes at least one sugar moiety having a 2′

substituent group that is not H or OH.

70. The oligomer of claim 69 wherein each of said first and said second regions has at least 10 nucleotides.

71. The oligomer of claim 69 wherein said first regions in a 5′

to 3′

direction is complementary to said second region in a 3′

to 5′

direction.

72. The oligomer of claim 69 wherein said oligomer includes a hairpin structure.

73. The oligomer of claim 69 wherein said first region of said oligomer is spaced from said second region of said oligomer by a third region and where said third region comprises at least two nucleotides.

74. The oligomer of claim 69 wherein said first region of said oligomer is spaced from said second region of said oligomer by a third region and where said third region comprises a non-nucleotide region.

77. A pharmaceutical composition comprising the oligomeric compound of claim 69 and a pharmaceutically acceptable carrier.

80. A method of modulating the expression of a target nucleic acid in a cell comprising contacting said cell with an oligomeric compound of claim 69.

83. A method of treating or preventing a disease or disorder associated with a target nucleic acid comprising administering to an animal having or predisposed to said disease or disorder a therapeutically effective amount of a composition of claim 69.