U.S. patent application number 10/430196 was filed with the patent office on 2003-10-16 for antisense oligonucleotide compositions and methods for the modulation of activating protein 1.
Invention is credited to Baker, Brenda, Dean, Nicholas M., McKay, Robert, Miraglia, Loren.
Application Number | 20030194738 10/430196 |
Document ID | / |
Family ID | 25273801 |
Filed Date | 2003-10-16 |
United States Patent
Application |
20030194738 |
Kind Code |
A1 |
Dean, Nicholas M. ; et
al. |
October 16, 2003 |
Antisense oligonucleotide compositions and methods for the
modulation of activating protein 1
Abstract
Compositions and methods for the treatment and diagnosis of
diseases or disorders amenable to treatment through modulation of
Activating Protein 1 (AP-1) expression are provided. In accordance
with various embodiments of the present invention, oligonucleotides
are provided which are specifically hybridizable with c-fos or
c-jun, the genes encoding c-Fos or c-Jun, respectively. In a
preferred embodiment, a method of modulating the metastasis of
malignant tumors via modulation of one or more of the AP-1 subunits
is provided; this method can be effected using the oligonucleotides
of the invention or any other agent which modulates AP-1 or
AP-1-mediated transcription.
Inventors: |
Dean, Nicholas M.;
(Encinitas, CA) ; McKay, Robert; (San Diego,
CA) ; Miraglia, Loren; (Encinitas, CA) ;
Baker, Brenda; (Carlsbad, CA) |
Correspondence
Address: |
Licata & Tyrrell P.C.
66 E. Main Street
Marlton
NJ
08053
US
|
Family ID: |
25273801 |
Appl. No.: |
10/430196 |
Filed: |
May 5, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10430196 |
May 5, 2003 |
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09923517 |
Aug 7, 2001 |
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09923517 |
Aug 7, 2001 |
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09364416 |
Jul 30, 1999 |
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6312900 |
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09364416 |
Jul 30, 1999 |
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08837201 |
Apr 14, 1997 |
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5985558 |
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Current U.S.
Class: |
435/6.13 ;
435/375; 514/44A; 536/23.2 |
Current CPC
Class: |
C12N 2310/321 20130101;
C12N 2310/335 20130101; C12N 15/1135 20130101; C12N 2310/321
20130101; C12N 2310/334 20130101; C12N 2310/322 20130101; C12N
2310/321 20130101; C12N 2310/321 20130101; C12N 2310/315 20130101;
C12N 2310/3181 20130101; C12N 2310/33 20130101; A61K 38/00
20130101; C12N 2310/3525 20130101; C12N 2310/3527 20130101; C12N
2310/3521 20130101 |
Class at
Publication: |
435/6 ; 435/375;
514/44; 536/23.2 |
International
Class: |
C12Q 001/68; C07H
021/04; A61K 048/00 |
Claims
What is claimed is:
1. An oligonucleotide comprising 8 to 30 nucleotides connected by
covalent linkages, wherein said oligonucleotide has a sequence
specifically hybridizable with a nucleic acid encoding a c-Fos
protein and said oligonucleotide modulates the expression of said
c-Fos protein.
2. The oligonucleotide of claim 1, wherein at least one of said
covalent linkages is a modified covalent linkage.
3. The oligonucleotide of claim 2, wherein said modified covalent
linkage is selected from the group consisting of a phosphorothioate
linkage, a phosphotriester linkage, a methyl phosphonate linkage, a
methylene(methylimino) linkage, a morpholino linkage, an amide
linkage, a polyamide linkage, a short chain alkyl intersugar
linkage, a cycloalkyl intersugar linkage, a short chain
heteroatomic intersugar linkage and a heterocyclic intersugar
linkage.
4. The oligonucleotide of claim 1, wherein at least one of said
nucleotides has a modified sugar moiety.
5. The oligonucleotide of claim 4, wherein said modified sugar
moiety is a modification at the 2' position of any nucleotide, the
3' position of the 3' terminal nucleotide or the 5' position of the
5' terminal oligonucleotide.
6. The oligonucleotide of claim 5, wherein said modification is
selected from the group consisting of a substitution of an azido
group for a 3' hydroxyl group and a substitution of a hydrogen atom
for a 3' or 5' hydroxyl group.
7. The oligonucleotide of claim 5, wherein said modification is a
substitution or addition at the 2' position of a moiety selected
from the group consisting of --OH, --SH, --SCH.sub.3, --F, --OCN,
--OCH.sub.3OCH.sub.3, --OCH.sub.3O(CH.sub.2).sub.nCH.sub.3,
--O(CH.sub.2).sub.nNH.sub.2 or --O(CH.sub.2).sub.nCH.sub.3 where n
is from 1 to about 10, a C.sub.1 to C.sub.10 lower alkyl group, an
alkoxyalkoxy group, a substituted lower alkyl group, a substituted
alkaryl group, a substituted aralkyl group, --Cl, --Br, --CN,
--CF.sub.3, --OCF.sub.3, an --O-alkyl group, an --S-alkyl group, an
N-alkyl group, an O-alkenyl group, an S-alkenyl group, an N-alkenyl
group, --SOCH.sub.3, --SO.sub.2CH.sub.3, --ONO.sub.2, --NO.sub.2,
--N.sub.3, --NH.sub.2, a heterocycloalkyl group, a
heterocycloalkaryl group, an aminoalkylamino group, a
polyalkylamino group, a substituted silyl group, an RNA cleaving
group, a reporter group, a DNA intercalating group, a group for
improving the pharmacokinetic properties of an oligonucleotide, a
group for improving the pharmacodynamic properties of an
oligonucleotide, a methoxyethoxy group and a methoxy group.
8. The oligonucleotide of claim 1, wherein at least one of said
nucleotides has a modified nucleobase.
9. The oligonucleotide of claim 8, wherein said modified nucleobase
is selected from the group consisting of hypoxanthine,
5-methylcytosine, 5-hydroxymethylcytosine, glycosyl
5-hydroxymethylcytosine, gentiobiosyl 5-hydroxymethylcytosine,
5-bromouracil, 5-hydroxymethyluracil, 6-methyladenine,
N.sup.6-(6-aminohexyl)adenine, 8-azaguanine, 7-deazaguanine and
2,6-diaminopurine.
10. The oligonucleotide of claim 1 wherein said nucleic acid is an
mRNA molecule encoding said c-Fos protein.
11. The oligonucleotide of claim 1 wherein said c-Fos protein is
human c-Fos.
12. The oligonucleotide of claim 11 wherein said sequence comprises
SEQ ID NO: 15, 17, 20 or 21.
13. An oligonucleotide comprising 8 to 30 nucleotides connected by
covalent linkages, wherein said oligonucleotide has a sequence
specifically hybridizable with a nucleic acid encoding a c-Fos
protein and said oligonucleotide modulates the expression of said
c-Fos protein, and wherein said oligonucleotide comprises at least
one lipophilic moiety which enhances the cellular uptake of said
oligonucleotide.
14. The oligonucleotide of claim 13 wherein said lipophilic moiety
is selected from the group consisting of a cholesterol moiety, a
cholesteryl moiety, cholic acid, a thioether, a thiocholesterol, an
aliphatic chain, a phospholipid, a polyamine chain, a polyethylene
glycol chain, adamantane acetic acid, a palmityl moiety, an
octadecylamine moiety and a hexylamino-carbonyl-oxycholesterol
moiety.
15. The oligonucleotide of claim 13 wherein at least one of said
linkages is selected from the group consisting of a
phosphorothioate linkage, a phosphodiester linkage, a
phosphotriester linkage, a methyl phosphonate linkage, a
methylene(methylimino) linkage, a morpholino linkage, an amide
linkage, a polyamide linkage, a short chain alkyl intersugar
linkage, a cycloalkyl intersugar linkage, a short chain
heteroatomic intersugar linkage and a heterocyclic intersugar
linkage.
16. The oligonucleotide of claim 13 wherein at least one of said
nucleotides has a modified sugar moiety.
17. The oligonucleotide of claim 16, wherein said modified sugar
moiety is a modification at the 2' position of any nucleotide, the
3' position of the 3' terminal nucleotide or the 5' position of the
5' terminal oligonucleotide.
18. The oligonucleotide of claim 17, wherein said modification is
selected from the group consisting of a substitution of an azido
group for a 3' hydroxyl group and a substitution of a hydrogen atom
for a 3' or 5' hydroxyl group.
19. The oligonucleotide of claim 17, wherein said modification is a
substitution or addition at the 2' position of a moiety selected
from the group consisting of --OH, --SH, --SCH.sub.3, --F, --OCN,
--OCH.sub.3OCH.sub.3, --OCH.sub.3O(CH.sub.2).sub.nCH.sub.3,
--O(CH.sub.2).sub.nNH.sub.2 or --O(CH.sub.2).sub.nCH.sub.3 where n
is from 1 to about 10, a C.sub.1 to C.sub.10 lower alkyl group, an
alkoxyalkoxy group, a substituted lower alkyl group, a substituted
alkaryl group, a substituted aralkyl group, --Cl, --Br, --CN,
--CF.sub.3, --OCF.sub.3, an --O-alkyl group, an --S-alkyl group, an
N-alkyl group, an O-alkenyl group, an S-alkenyl group, an N-alkenyl
group, --SOCH.sub.3, --SO.sub.2CH.sub.3, --ONO.sub.2, --NO.sub.2,
--N.sub.3, --NH.sub.2, a heterocycloalkyl group, a
heterocycloalkaryl group, an aminoalkylamino group, a
polyalkylamino group, a substituted silyl group, an RNA cleaving
group, a reporter group, a DNA intercalating group, a group for
improving the pharmacokinetic properties of an oligonucleotide, a
group for improving the pharmacodynamic properties of an
oligonucleotide, a methoxyethoxy group and a methoxy group.
20. The oligonucleotide of claim 13, wherein at least one of said
nucleotides has a modified nucleobase.
21. The oligonucleotide of claim 20, wherein said modified
nucleobase is selected from the group consisting of hypoxanthine,
5-methylcytosine, 5-hydroxymethylcytosine, glycosyl
5-hydroxymethylcytosine, gentiobiosyl 5-hydroxymethylcytosine,
5-bromouracil, 5-hydroxymethyluracil, 6-methyladenine,
N.sup.6-(6-aminohexyl)adenine, 8-azaguanine, 7-deazaguanine and
2,6-diaminopurine.
22. The oligonucleotide of claim 13 wherein said nucleic acid is an
mRNA molecule encoding said c-Fos protein.
23. The oligonucleotide of claim 13 wherein said c-Fos protein is
human c-Fos.
24. The oligonucleotide of claim 23 wherein said sequence comprises
SEQ ID NO: 15, 17, 20 or 21.
25. A pharmaceutical composition comprising an oligonucleotide of
claim 1 and a pharmaceutically acceptable carrier.
26. An oligonucleotide comprising 8 to 30 nucleotides connected by
covalent linkages, wherein said oligonucleotide has a sequence
specifically hybridizable with a nucleic acid encoding a c-Jun
protein and said oligonucleotide modulates the expression of said
c-Jun protein.
27. The oligonucleotide of claim 26, wherein at least one of said
covalent linkages is a modified covalent linkage.
28. The oligonucleotide of claim 27, wherein said modified covalent
linkage is selected from the group consisting of a phosphorothioate
linkage, a phosphotriester linkage, a methyl phosphonate linkage, a
methylene(methylimino) linkage, a morpholino linkage, an amide
linkage, a polyamide linkage, a short chain alkyl intersugar
linkage, a cycloalkyl intersugar linkage, a short chain
heteroatomic intersugar linkage and a heterocyclic intersugar
linkage.
29. The oligonucleotide of claim 26, wherein at least one of said
nucleotides has a modified sugar moiety.
30. The oligonucleotide of claim 29, wherein said modified sugar
moiety is a modification at the 2' position of any nucleotide, the
3' position of the 3' terminal nucleotide or the 5' position of the
5' terminal oligonucleotide.
31. The oligonucleotide of claim 30, wherein said modification is
selected from the group consisting of a substitution of an azido
group for a 3' hydroxyl group and a substitution of a hydrogen atom
for a 3' or 5' hydroxyl group.
32. The oligonucleotide of claim 30, wherein said modification is a
substitution or addition at the 2' position of a moiety selected
from the group consisting of --OH, --SH, --SCH.sub.3, --F, --OCN,
--OCH.sub.3OCH.sub.3, --OCH.sub.3O(CH.sub.2).sub.nCH.sub.3,
--O(CH.sub.2).sub.nNH.sub.2 or --O(CH.sub.2).sub.nCH.sub.3 where n
is from 1 to about 10, a C.sub.1 to C.sub.10 lower alkyl group, an
alkoxyalkoxy group, a substituted lower alkyl group, a substituted
alkaryl group, a substituted aralkyl group, --Cl, --Br, --CN,
--CF.sub.3, --OCF.sub.3, an --O-alkyl group, an --S-alkyl group, an
N-alkyl group, an O-alkenyl group, an S-alkenyl group, an N-alkenyl
group, --SOCH.sub.3, --SO.sub.2CH.sub.3, --ONO.sub.2, --NO.sub.2,
--N.sub.3, --NH.sub.2, a heterocycloalkyl group, a
heterocycloalkaryl group, an aminoalkylamino group, a
polyalkylamino group, a substituted silyl group, an RNA cleaving
group, a reporter group, a DNA intercalating group, a group for
improving the pharmacokinetic properties of an oligonucleotide, a
group for improving the pharmacodynamic properties of an
oligonucleotide, a methoxyethoxy group and a methoxy group.
33. The oligonucleotide of claim 26, wherein at least one of said
nucleotides has a modified nucleobase.
34. The oligonucleotide of claim 33, wherein said modified
nucleobase is selected from the group consisting of hypoxanthine,
5-methylcytosine, 5-hydroxymethylcytosine, glycosyl
5-hydroxymethylcytosine, gentiobiosyl 5-hydroxymethylcytosine,
5-bromouracil, 5-hydroxymethyluracil, 6-methyladenine,
N.sup.6-(6-aminohexyl)adenine, 8-azaguanine, 7-deazaguanine and
2,6-diaminopurine.
35. The oligonucleotide of claim 26 wherein said nucleic acid is an
mRNA molecule encoding said c-Jun protein.
36. The oligonucleotide of claim 26 wherein said c-Jun protein is
human c-Jun.
37. The oligonucleotide of claim 26 wherein said sequence comprises
SEQ ID NO: 3, 4, 5, 6, 7, 8 or 9.
38. An oligonucleotide comprising 8 to 30 nucleotides connected by
covalent linkages, wherein said oligonucleotide has a sequence
specifically hybridizable with a nucleic acid encoding a c-Jun
protein and said oligonucleotide modulates the expression of said
c-Jun protein, and wherein said oligonucleotide comprises at least
one lipophilic moiety which enhances the cellular uptake of said
oligonucleotide.
39. The oligonucleotide of claim 38 wherein said lipophilic moiety
is selected from the group consisting of a cholesterol moiety, a
cholesteryl moiety, cholic acid, a thioether, a thiocholesterol, an
aliphatic chain, a phospholipid, a polyamine chain, a polyethylene
glycol chain, adamantane acetic acid, a palmityl moiety, an
octadecylamine moiety and a hexylamino-carbonyl-oxycholesterol
moiety.
40. The oligonucleotide of claim 38 wherein at least one of said
linkages is selected from the group consisting of a
phosphorothioate linkage, a phosphodiester linkage, a
phosphotriester linkage, a methyl phosphonate linkage, a
methylene(methylimino) linkage, a morpholino linkage, an amide
linkage, a polyamide linkage, a short chain alkyl intersugar
linkage, a cycloalkyl intersugar linkage, a short chain
heteroatomic intersugar linkage and a heterocyclic intersugar
linkage.
41. The oligonucleotide of claim 38 wherein at least one of said
nucleotides has a modified sugar moiety.
42. The oligonucleotide of claim 41, wherein said modified sugar
moiety is a modification at the 2' position of any nucleotide, the
3' position of the 3' terminal nucleotide or the 5' position of the
5' terminal oligonucleotide.
43. The oligonucleotide of claim 42, wherein said modification is
selected from the group consisting of a substitution of an azido
group for a 3' hydroxyl group and a substitution of a hydrogen atom
for a 3' or 5' hydroxyl group.
44. The oligonucleotide of claim 42, wherein said modification is a
substitution or addition at the 2' position of a moiety selected
from the group consisting of --OH, --SH, --SCH.sub.3, --F, --OCN,
--OCH.sub.3OCH.sub.3, --OCH.sub.3O(CH.sub.2).sub.nCH.sub.3,
--O(CH.sub.2).sub.nNH.sub.2 or --O(CH.sub.2).sub.nCH.sub.3 where n
is from 1 to about 10, a C.sub.1 to C.sub.10 lower alkyl group, an
alkoxyalkoxy group, a substituted lower alkyl group, a substituted
alkaryl group, a substituted aralkyl group, --Cl, --Br, --CN,
--CF.sub.3, --OCF.sub.3, an --O-alkyl group, an --S-alkyl group, an
N-alkyl group, an O-alkenyl group, an S-alkenyl group, an N-alkenyl
group, --SOCH.sub.3, --SO.sub.2CH.sub.3, --ONO.sub.2, --NO.sub.2,
--N.sub.3, --NH.sub.2, a heterocycloalkyl group, a
heterocycloalkaryl group, an aminoalkylamino group, a
polyalkylamino group, a substituted silyl group, an RNA cleaving
group, a reporter group, a DNA intercalating group, a group for
improving the pharmacokinetic properties of an oligonucleotide, a
group for improving the pharmacodynamic properties of an
oligonucleotide, a methoxyethoxy group and a methoxy group.
45. The oligonucleotide of claim 38, wherein at least one of said
nucleotides has a modified nucleobase.
46. The oligonucleotide of claim 45, wherein said modified
nucleobase is selected from the group consisting of hypoxanthine,
5-methylcytosine, 5-hydroxymethylcytosine, glycosyl
5-hydroxymethylcytosine, gentiobiosyl 5-hydroxymethylcytosine,
5-bromouracil, 5-hydroxymethyluracil, 6-methyladenine,
N.sup.6-(6-aminohexyl)adenine, 8-azaguanine, 7-deazaguanine and
2,6-diaminopurine.
47. The oligonucleotide of claim 38 wherein said nucleic acid is an
mRNA molecule encoding said c-Jun protein.
48. The oligonucleotide of claim 38 wherein said c-Jun protein is
human c-Jun.
49. The oligonucleotide of claim 38 wherein said sequence comprises
SEQ ID NO: 3, 4, 5, 6, 7, 8 or 9.
50. A pharmaceutical composition comprising the oligonucleotide of
claim 26 and a pharmaceutically acceptable carrier.
51. A pharmaceutical composition comprising (a) a chemotherapeutic
agent; (b) the oligonucleotide of claim 1; and (c) a
pharmaceutically acceptable carrier.
52. A pharmaceutical composition comprising (a) a chemotherapeutic
agent; (b) the oligonucleotide of claim 26; and (c) a
pharmaceutically acceptable carrier.
53. A pharmaceutical composition comprising (a) an oligonucleotide
comprising 8 to 30 nucleotides connected by covalent linkages,
wherein said oligonucleotide has a sequence specifically
hybridizable with a nucleic acid encoding a c-Fos protein and said
oligonucleotide modulates the expression of said c-Fos protein; (b)
an oligonucleotide comprising 8 to 30 nucleotides connected by
covalent linkages, wherein said oligonucleotide has a sequence
specifically hybridizable with a nucleic acid encoding a c-Jun
protein and said oligonucleotide modulates the expression of said
c-Jun protein; and (c) a pharmaceutically acceptable carrier.
54. A pharmaceutical composition comprising (a) a chemotherapeutic
agent; (b) an oligonucleotide comprising 8 to 30 nucleotides
connected by covalent linkages, wherein said oligonucleotide has a
sequence specifically hybridizable with a nucleic acid encoding a
c-Fos protein and said oligonucleotide modulates the expression of
said c-Fos protein; (c) an oligonucleotide comprising 8 to 30
nucleotides connected by covalent linkages, wherein said
oligonucleotide has a sequence specifically hybridizable with a
nucleic acid encoding a c-Jun protein and said oligonucleotide
modulates the expression of said c-Jun protein; and (d) a
pharmaceutically acceptable carrier.
55. A method of modulating the expression of a c-Fos protein in
cells or tissues comprising contacting said cells or tissues with
an oligonucleotide comprising 8 to 30 nucleotides connected by
covalent linkages, wherein said oligonucleotide has a sequence
specifically hybridizable with a nucleic acid encoding a c-Fos
protein and said oligonucleotide modulates the expression of said
c-Fos protein.
56. The method of claim 55 wherein said modulating comprises
inhibiting the expression of said c-Fos protein.
57. A method of modulating the expression of a c-Jun protein in
cells or tissues comprising contacting said cells or tissues with
an oligonucleotide comprising 8 to 30 nucleotides connected by
covalent linkages, wherein said oligonucleotide has a sequence
specifically hybridizable with a nucleic acid encoding a c-Jun
protein and said oligonucleotide modulates the expression of said
c-Jun protein.
58. The method of claim 57 wherein said modulating comprises
inhibiting the expression of said c-Jun protein.
59. A method of inhibiting tumor growth in an animal comprising
administering to said animal a therapeutically effective amount of
an oligonucleotide comprising 8 to 30 nucleotides connected by
covalent linkages, wherein said oligonucleotide has a sequence
specifically hybridizable with a nucleic acid encoding a c-Fos
protein and said oligonucleotide modulates the expression of said
c-Fos protein.
60. A method of inhibiting tumor growth in an animal comprising
administering to said animal a therapeutically effective amount of
an oligonucleotide comprising 8 to 30 nucleotides connected by
covalent linkages, wherein said oligonucleotide has a sequence
specifically hybridizable with a nucleic acid encoding a c-Jun
protein and said oligonucleotide modulates the expression of said
c-Jun protein.
61. A method of inhibiting tumor growth in an animal comprising
administering to said animal a therapeutically effective amount of
an oligonucleotide comprising 8 to 30 nucleotides connected by
covalent linkages, wherein said oligonucleotide modulates the
expression of one or more matrix metalloproteinases.
62. A method of modulating cell cycle progression in cultured cells
or the cells of an animal comprising administering to said cells an
effective amount of an oligonucleotide comprising 8 to 30
nucleotides connected by covalent linkages, wherein said
oligonucleotide has a sequence specifically hybridizable with a
nucleic acid encoding a c-Fos protein and said oligonucleotide
modulates the expression of said c-Fos protein.
63. A method of modulating cell cycle progression in cultured cells
or the cells of an animal comprising administering to said cells an
effective amount of an oligonucleotide comprising 8 to 30
nucleotides connected by covalent linkages, wherein said
oligonucleotide has a sequence specifically hybridizable with a
nucleic acid encoding a c-Jun protein and said oligonucleotide
modulates the expression of said c-Jun protein.
64. A prodrug of the oligonucleotide of claim 1.
65. A pharmaceutical composition comprising the oligonucleotide
prodrug of claim 64 and a pharmaceutically acceptable carrier.
66. A prodrug of the oligonucleotide of claim 25.
67. A pharmaceutical composition comprising the oligonucleotide
prodrug of claim 66 and a pharmaceutically acceptable carrier.
68. A method of treating an animal having, suspected of having or
prone to having a hyperproliferative disease comprising
administering to said animal a therapeutically effective amount of
a pharmaceutical composition comprising an oligonucleotide
comprising 8 to 30 nucleotides connected by covalent linkages,
wherein said oligonucleotide has a sequence specifically
hybridizable with a nucleic acid encoding a c-Fos protein and said
oligonucleotide modulates the expression of said c-Fos protein and
a pharmaceutically acceptable carrier.
69. A method of treating an animal having, suspected of having or
prone to having a hyperproliferative disease comprising
administering to said animal a therapeutically effective amount of
a pharmaceutical composition comprising an oligonucleotide
comprising 8 to 30 nucleotides connected by covalent linkages,
wherein said oligonucleotide has a sequence specifically
hybridizable with a nucleic acid encoding a c-Jun protein and said
oligonucleotide modulates the expression of said c-Jun protein and
a pharmaceutically acceptable carrier.
70. A method of treating-an animal having, suspected of having or
prone to having a hyperproliferative disease comprising
administering to said animal a therapeutically effective amount of
a pharmaceutical composition comprising (a) a chemotherapeutic
agent; (b) an oligonucleotide comprising 8 to 30 nucleotides
connected by covalent linkages, wherein said oligonucleotide has a
sequence specifically hybridizable with a nucleic acid encoding a
c-Fos protein and said oligonucleotide modulates the expression of
said c-Fos protein; and (c) a pharmaceutically acceptable
carrier.
71. A method of treating an animal having, suspected of having or
prone to having a hyperproliferative disease comprising
administering to said animal a therapeutically effective amount of
a pharmaceutical composition comprising (a) a chemotherapeutic
agent; (b) an oligonucleotide comprising 8 to 30 nucleotides
connected by covalent linkages, wherein said oligonucleotide has a
sequence specifically hybridizable with a nucleic acid encoding a
c-Jun protein and said oligonucleotide modulates the expression of
said c-Jun protein; and (c) a pharmaceutically acceptable
carrier.
72. A method of treating an animal having, suspected of having or
prone to having a hyperproliferative disease comprising
administering to said animal a therapeutically effective amount of
a pharmaceutical composition comprising (a) an oligonucleotide
comprising 8 to 30 nucleotides connected by covalent linkages,
wherein said oligonucleotide has a sequence specifically
hybridizable with a nucleic acid encoding a c-Fos protein and said
oligonucleotide modulates the expression of said c-Fos protein; (b)
an oligonucleotide comprising 8 to 30 nucleotides connected by
covalent linkages, wherein said oligonucleotide has a sequence
specifically hybridizable with a nucleic acid encoding a c-Jun
protein and said oligonucleotide modulates the expression of said
c-Jun protein; and (c) a pharmaceutically acceptable carrier.
73. A method of treating an animal having, suspected of having or
prone to having a hyperproliferative disease comprising
administering to said animal a therapeutically effective amount of
a pharmaceutical composition comprising (a) a chemotherapeutic
agent; (b) an oligonucleotide comprising 8 to 30 nucleotides
connected by covalent linkages, wherein said oligonucleotide has a
sequence specifically hybridizable with a nucleic acid encoding a
c-Fos protein and said oligonucleotide modulates the expression of
said c-Fos protein; (c) an oligonucleotide comprising 8 to 30
nucleotides connected by covalent linkages, wherein said
oligonucleotide has a sequence specifically hybridizable with a
nucleic acid encoding a c-Jun protein and said oligonucleotide
modulates the expression of said c-Jun protein; and (d) a
pharmaceutically acceptable carrier.
74. A method of treating an animal having, suspected of having or
prone to having a hyperproliferative disease comprising
administering to said animal a therapeutically effective amount of
a pharmaceutical composition comprising the oligonucleotide prodrug
comprising an oligonucleotide comprising 8 to 30 nucleotides
connected by covalent linkages, wherein said oligonucleotide has a
sequence specifically hybridizable with a nucleic acid encoding a
c-Fos protein and said oligonucleotide modulates the expression of
said c-Fos protein and a pharmaceutically acceptable carrier.
75. A method of treating an animal having, suspected of having or
prone to having a hyperproliferative disease comprising
administering to said animal a therapeutically effective amount of
a pharmaceutical composition comprising the oligonucleotide prodrug
comprising an oligonucleotide comprising 8 to 30 nucleotides
connected by covalent linkages, wherein said oligonucleotide has a
sequence specifically hybridizable with a nucleic acid encoding a
c-Fos protein and said oligonucleotide modulates the expression of
said c-Fos protein and a pharmaceutically acceptable carrier.
Description
INTRODUCTION
[0001] This application is a continuation of U.S. patent
application Ser. No. 09/923,517 filed Aug. 7, 2001, which is a
divisional of U.S. patent application Ser. No. 09/364,416 filed
Jul. 30, 1999, which is a continuation of U.S. patent application
Ser. No. 08/837,201 filed Apr. 14, 1997, now issued as U.S. Pat.
No. 5,985,558.
FIELD OF THE INVENTION
[0002] The present invention provides compositions and methods for
modulating levels of the c-fos and c-jun genes, which encode the
c-Fos and c-Jun subunits of AP-1, respectively. In vivo, AP-1, or
transcription factor activating protein 1, is a heterogenous
mixture of heterodimers of several related protein subunits
including, in addition to c-Fos and c-Jun, FosB, Fra-1, Fra-2,
c-Jun, JunB, JunD, etc. (The FOS and JUN Families of Proteins,
Angel and Herrlich, eds., CRC Press, Boca Raton, Fla., 1994). AP-1
has been implicated in abnormal cell proliferation and tumor
formation, events that thus might be controlled by modulating the
expression of c-fos and/or c-jun. The invention is further directed
to therapeutic, diagnostic, and research based reagents and methods
for evaluating and treating disease states or disorders which
result from and/or respond positively to modulation of one or more
AP-1 subunits. Such disease states and disorders include those
involving the hyperproliferation of cells such as, e.g., a tumor
(neoplasm) or malignant cancer. Inhibition of AP-1-mediated
hyperproliferation of cells, and corresponding prophylactic,
palliative and therapeutic effects result from treatment with the
oligonucleotides of the invention.
BACKGROUND OF THE INVENTION
[0003] Transcription factors play a central role in the expression
of specific genes upon stimulation by extracellular signals,
thereby regulating a complex array of biological processes. Members
of the family of transcription factors termed AP-1 (activating
protein-1) alter gene expression in response to growth factors,
cytokines, tumor promoters, carcinogens and increased expression of
certain oncogenes. Growth factors and cytokines exert their
function by binding to specific cell surface receptors. Receptor
occupancy triggers a signal transduction cascade to the nucleus. In
this cascade, transcription factors such as AP-1 execute long term
responses to the extracellular factors by modulating gene
expression. Such changes in cellular gene expression lead to DNA
synthesis, and eventually the formation of differentiated
derivatives (Angel and Karin, Biochim. Biophys. Acta, 1991, 1072,
129).
[0004] In general terms, AP-1 denotes one member of a family of
related heterodimeric transcription factor complexes found in
eukaryotic cells or viruses. However, as used herein, "AP-1"
specifically refers to the heterodimer formed of c-Fos and c-Jun
(Angel and Herrlich, Chapter 1, and Schuermann, Chapter 2 in: The
FOS and JUN Families of Proteins, Angel and Herrlich, eds., pp.
3-35, CRC Press, Boca Raton, Fla., 1994; Bohmann et al., Science,
1987, 238, 1386; Angel et al., Nature, 1988, 332, 166). These two
proteins are products of the c-fos and c-jun proto-oncogenes,
respectively. Repression of expression of either c-fos or c-jun, or
of both proto-oncogenes, and the resultant inhibition of the
formation of c-Fos and c-Jun proteins, is desirable for the
inhibition of cell proliferation, tumor formation and tumor
growth.
[0005] The phosphorylation of proteins plays a key role in the
transduction of extracellular signals into the cell.
Mitogen-activated protein (MAP) kinases, enzymes which effect such
phosphorylations are targets for the action of growth factors,
hormones, and other agents involved in cellular metabolism,
proliferation and differentiation (Cobb et al., J. Biol. Chem.,
1995, 270, 14843). MAP kinases are themselves activated by
phosphorylation catalyzed by, e.g., receptor tyrosine kinases, G
protein-coupled receptors, protein kinase C (PKC), and the
apparently MAP kinase dedicated kinases MEK1 and MEK2. MAP kinases
include, but are not limited to, ERK1, ERK2, two isoforms of ERK3,
ERK4 (ERK stands for "extracellular signal-regulated protein
kinase), Jun N-terminal kinases/stress-activated protein kinases
(JNKs/SAPKs), p38/HOG1, p57 MAP kinases, MKK3 (MAP kinase kinase 3)
and MKK4 (MAP kinase kinase 4, also known as SAPK/ERK kinase (SEK)
or JNK kinase (JNKK)) (Cobb et al., J. Biol. Chem., 1995, 270,
14843, and references cited therein). In general, MAP kinases are
involved in a variety of signal transduction pathways (sometimes
overlapping and sometimes parallel) that function to convey
extracellular stimuli to protooncogene products to modulate
cellular proliferation and/or differentiation.
[0006] One of the signal transduction pathways involves the MAP
kinases Jun kinase 1 and Jun kinase 2 which are responsible for the
phosphorylation of specific sites (Serine 63 and Serine 73) on
c-Jun. Phosphorylation of these sites potentiates the ability of
AP-1 to activate transcription (Binetruy et al., Nature, 1991, 351,
122; Smeal et al., Nature, 1991, 354, 494). At least one human
leukemia oncogene has been shown to enhance Jun N-terminal Kinase
(JNK) function (Raitano et al., Proc. Natl. Acad. Sci. (USA), 1995,
92, 11746), thus indirectly demonstrating a role for AP-1 in
cellular hyperproliferation and tumorigenesis. Cellular
hyperproliferation in an animal can have several outcomes.
Hyperproliferating cells might be attacked and killed by the
animal's immune system before a tumor can form. Tumors are abnormal
growths resulting from the hyperproliferation of cells. Cells that
proliferate to excess but stay put form benign tumors, which can
typically be removed by local surgery. In contrast, malignant
tumors or cancers comprise cells that are capable of undergoing
metastasis, i.e., a process by which hyperproliferative cells
spread to, and secure themselves within, other parts of the body
via the circulatory or lymphatic system (see, generally, Chapter 16
In: Molecular Biology of the Cell, Alberts et al., eds., pp.
891-950, Garland Publishing, Inc., New York, 1983). Using the
oligonucleotides of the invention, it has surprisingly been
discovered that several genes encoding enzymes required for
metastasis are positively regulated by AP-1. Accordingly,
inhibition of expression of c-fos and/or c-jun serves as a means to
modulate the metastasis of malignant tumors. A method of modulating
one or more metastatic events using the oligonucleotides of the
invention is thus herein provided.
RELEVANT ART
[0007] Soprano et al. (Ann. N. Y. Acad. Sci., 1992, 660, 231) have
used antisense oligodeoxynucleotides targeted to c-jun mRNA to
study their ability to inhibit DNA synthesis and cell division.
[0008] Liu et al. (Ann. Neurol., 1994, 36, 566) describe the
suppression of c-fos by intraventricular infusion of an antisense
oligodeoxynucleotide targeted to c-fos mRNA.
[0009] Chen et al. (Cancer Lett., 1994, 85, 119) describe
repression of c-jun expression by antisense oligodeoxynucleotides
resulting in the inhibition of cell proliferation in E5a
transformed cells.
[0010] Gillardon et al. describe the topical application of c-fos
antisense oligodeoxynucleotides to the rat spinal cord (Eur. J.
Neurosci., 1994, 6, 880) ultraviolet (UV)-exposed rat eyes (British
J. Ophthal., 1995, 79, 277) and UV-irradiated rat skin
(Carcinogenesis, 1995, 16, 1853).
[0011] U.S. Pat. No. 5,602,156, which issued Feb. 11, 1997, to Kohn
et al., discloses non-oligonucleotide compositions and methods for
inhibiting the expression of two metalloproteinases, MMP-1 and
MMP-2.
[0012] International Publication Number WO 95/02051, published Jan.
19, 1995, discloses antisense oligonucleotides targeted to the mRNA
of c-fos and c-jun.
[0013] International Publication Number WO 95/03323, published Feb.
2, 1995, discloses antisense nucleic acids which are complementary
to the polynucleotide encoding a polypeptide which is capable of
phosphorylating the c-jun N-terminal activation domain. Also
provided are methods for treating a cell proliferative disorder
associated with said polypeptide.
[0014] International Publication Number WO 95/03324, published Feb.
2, 1995, describes a polypeptide which phosphorylates the c-jun
N-terminal activation domain. This publication also discloses a
polynucleotide sequence encoding the polypeptide.
[0015] To date, there are no known therapeutic agents which
effectively inhibit gene expression of c-fos and/or c-jun.
Furthermore, there are to date no known therapeutic agents that
modulate the metastasis of malignant cells. The compositions and
methods of the invention overcome these limitations. Further
objectives of the invention are apparent from the present
disclosure.
SUMMARY OF THE INVENTION
[0016] In accordance with the present invention, oligonucleotides
are provided which specifically hybridize with nucleic acids
encoding c-Fos or c-Jun. Certain oligonucleotides of the invention
are designed to bind either directly to mRNA transcribed from, or
to a selected DNA portion of, the respective gene, thereby
modulating the amount of protein translated from a c-fos or c-jun
mRNA and/or the amount of mRNA transcribed from a c-fos or c-jun
gene, respectively. Such modulation can, in turn, effect the
modulation of enzymes and cellular processes involved in the
metastasis of malignant cells.
[0017] Oligonucleotides may comprise nucleotide sequences
sufficient in identity and number to effect specific hybridization
with a particular nucleic acid. Such noligonucleotides are commonly
described as "antisense." Antisense oligonucleotides are commonly
used as research reagents, diagnostic aids, and therapeutic
agents.
[0018] It has been discovered that the c-fos and c-jun genes,
encoding the c-Fos and c-Jun proteins, respectively, are
particularly amenable to this approach. As a consequence of the
association between cellular proliferation and AP-1 (the
heterodimer of c-Fos and c-Jun) expression, modulation of the
expression of c-fos and/or c-jun leads to modulation of AP-1, and,
accordingly, modulation of cellular proliferation. Such modulation
is desirable for treating or modulating various hyperproliferative
disorders or diseases, such as various cancers. Such inhibition is
further desirable for preventing or modulating the development of
such diseases or disorders in an animal suspected of being, or
known to be, prone to such diseases or disorders. If desired,
modulation of one subunit can be combined with modulation of the
subunit of AP-1 in order to achieve a requisite degree of effect
upon AP-1-mediated transcription.
[0019] Methods of modulating the expression of c-Fos or c-Jun
proteins comprising contacting animals with oligonucleotides
specifically hybridizable with a c-fos or c-jun gene, respectively,
are herein provided. These methods are believed to be useful both
therapeutically and diagnostically as a consequence of the
association between AP-1 expression and cellular proliferation.
These methods are also useful as tools, for example, in the
detection and determination of the role of AP-1 protein expression
in various cell functions and physiological processes and
conditions, and for the diagnosis of conditions associated with
such expression and activation.
[0020] The present invention also comprises methods of inhibiting
AP-1-mediated transcriptional activation using the oligonucleotides
of the invention. Methods of treating conditions in which abnormal
or excessive AP-1-mediated transcriptional activation and cellular
proliferation occur are also provided. These methods employ the
oligonucleotides of the invention and are believed to be useful
both therapeutically and as clinical research and diagnostic tools.
The oligonucleotides of the present invention may also be used for
research purposes. Thus, the specific hybridization exhibited by
the oligonucleotides of the present invention may be used for
assays, purifications, cellular product preparations and in other
methodologies which may be appreciated by persons of ordinary skill
in the art.
[0021] Methods comprising contacting animals with oligonucleotides
specifically hybridizable with nucleic acids encoding c-Fos or
c-Jun proteins are herein provided. Such methods can be used to
modulate or detect the expression of c-fos or c-jun genes and are
thus believed to be useful both therapeutically and
diagnostically.
[0022] The methods disclosed herein are also useful, for example,
as clinical research tools in the detection and determination of
the role of AP-1-mediated gene expression in various immune system
functions and physiological processes and conditions, and for the
diagnosis of conditions associated with their expression. The
specific hybridization exhibited by the oligonucleotides of the
present invention may be used for assays, purifications, cellular
product preparations and in other methodologies which may be
appreciated by persons of ordinary skill in the art. For example,
because the oligonucleotides of this invention specifically
hybridize to nucleic acids encoding c-Fos or c-Jun, sandwich and
other assays can easily be constructed to exploit this fact.
Detection of specific hybridization of an oligonucleotide of the
invention with a nucleic acid encoding a c-Fos or c-Jun protein
present in a sample can routinely be accomplished. Such detection
may include detectably labeling an oligonucleotide of the invention
by enzyme conjugation, radiolabeling or any other suitable
detection system. A number of assays may be formulated employing
the present invention, which assays will commonly comprise
contacting a tissue or cell sample with a detectably labeled
oligonucleotide of the present invention under conditions selected
to permit hybridization and measuring such hybridization by
detection of the label, as is appreciated by those of ordinary
skill in the art.
DETAILED DESCRIPTION OF THE INVENTION
[0023] The present invention employs oligonucleotides for use in
antisense inhibition of the function of RNA and DNA encoding a
c-Fos protein or a c-Jun protein. The present invention also
employs oligonucleotides which are designed to be specifically
hybridizable to DNA or messenger RNA (mRNA) encoding such proteins
and ultimately modulating the amount of such proteins transcribed
from their respective genes. Such hybridization with mRNA
interferes with the normal role of mRNA and causes a modulation of
its function in cells. The functions of mRNA to be interfered with
include all vital functions such as translocation of the RNA to the
site for protein translation, actual translation of protein from
the RNA, splicing of the RNA to yield one or more mRNA species, and
possibly even independent catalytic activity which may be engaged
in by the RNA. The overall effect of such interference with mRNA
function is modulation of the expression of a c-Fos protein or a
c-Jun protein. In the context of this invention, "modulation" means
either an increase (stimulation) or a decrease (inhibition) in the
expression of a gene. In the context of the present invention,
inhibition is the preferred form of modulation of gene
expression.
[0024] In the context of this invention, the term "oligonucleotide"
refers to an oligomer or polymer of ribonucleic acid or
deoxyribonucleic acid. This term includes oligonucleotides composed
of naturally-occurring nucleobases, sugars and covalent intersugar
(backbone) linkages as well as oligonucleotides having
non-naturally-occurring portions which function similarly. Such
modified or substituted oligonucleotides are often preferred over
native forms because of desirable properties such as, for example,
enhanced cellular uptake, enhanced binding to target and increased
stability in the presence of nucleases.
[0025] An oligonucleotide is a polymer of a repeating unit
generically known as a nucleotide. An unmodified (naturally
occurring) nucleotide has three components: (1) a nitrogenous base
linked by one of its nitrogen atoms to (2) a 5-carbon cyclic sugar
and (3) a phosphate, esterified to carbon 5 of the sugar. When
incorporated into an oligonucleotide chain, the phosphate of a
first nucleotide is also esterified to carbon 3 of the sugar of a
second, adjacent nucleotide. The "backbone" of an unmodified
oligonucleotide consists of (2) and (3), that is, sugars linked
together by phosphodiester linkages between the carbon 5 (5')
position of the sugar of a first nucleotide and the carbon 3 (3')
position of a second, adjacent nucleotide. A "nucleoside" is the
combination of (1) a nucleobase and (2) a sugar in the absence of
(3) a phosphate moiety (Kornberg, A., DNA Replication, pp. 4-7, W.
H. Freeman & Co., San Francisco, 1980). The backbone of an
oligonucleotide positions a series of bases in a specific order;
the written representation of this series of bases, which is
conventionally written in 5' to 3' order, is known as a nucleotide
sequence. The oligonucleotides in accordance with this invention
preferably comprise from about 8 to about 30 nucleotides. It is
more preferred that such oligonucleotides comprise from about 15 to
25 nucleotides.
[0026] Oligonucleotides may comprise nucleotide sequences
sufficient in identity and number to effect specific hybridization
with a particular nucleic acid. Such oligonucleotides which
specifically hybridize to a portion of the sense strand of a gene
are commonly described as "antisense." Antisense oligonucleotides
are commonly used as research reagents, diagnostic aids, and
therapeutic agents. For example, antisense oligonucleotides, which
are able to inhibit gene expression with exquisite specificity, are
often used by those of ordinary skill to elucidate the function of
particular genes, for example to distinguish between the functions
of various members of a biological pathway. This specific
inhibitory effect has, therefore, been harnessed by those skilled
in the art for research uses.
[0027] The specificity and sensitivity of oligonucleotides is also
harnessed by those of skill in the art for therapeutic uses. For
example, the following U.S. patents demonstrate palliative,
therapeutic and other methods utilizing antisense oligonucleotides.
U.S. Pat. No. 5,135,917 provides antisense oligonucleotides that
inhibit human interleukin-1 receptor expression. U.S. Pat. No.
5,098,890 is directed to antisense oligonucleotides complementary
to the c-myb oncogene and antisense oligonucleotide therapies for
certain cancerous conditions. U.S. Pat. No. 5,087,617 provides
methods for treating cancer patients with antisense
oligonucleotides. U.S. Pat. No. 5,166,195 provides oligonucleotide
inhibitors of Human Immunodeficiency Virus (HIV). U.S. Pat. No.
5,004,810 provides oligomers capable of hybridizing to herpes
simplex virus Vmw65 mRNA and inhibiting replication. U.S. Pat. No.
5,194,428 provides antisense oligonucleotides having antiviral
activity against influenzavirus. U.S. Pat. No. 4,806,463 provides
antisense oligonucleotides and methods using them to inhibit
HTLV-III replication. U.S. Pat. No. 5,286,717 provides
oligonucleotides having a complementary base sequence to a portion
of an oncogene. U.S. Pat. No. 5,276,019 and U.S. Pat. No. 5,264,423
are directed to phosphorothioate oligonucleotide analogs used to
prevent replication of foreign nucleic acids in cells. U.S. Pat.
No. 4,689,320 is directed to antisense oligonucleotides as
antiviral agents specific to cytomegalovirus (CMV). U.S. Pat. No.
5,098,890 provides oligonucleotides complementary to at least a
portion of the mRNA transcript of the human c-myb gene. U.S. Pat.
No. 5,242,906 provides antisense oligonucleotides useful in the
treatment of latent Epstein-Barr virus (EBV) infections.
[0028] It is preferred to target specific genes for antisense
attack. "Targeting" an oligonucleotide to the associated nucleic
acid, in the context of this invention, is a multistep process. The
process usually begins with the identification of a nucleic acid
sequence whose function is to be modulated. This may be, for
example, a cellular gene (or mRNA transcribed from the gene) whose
expression is associated with a particular disorder or disease
state, or a foreign nucleic acid from an infectious agent. In the
present invention, the target is a cellular gene (c-fos or c-jun)
encoding a subunit of AP-1, for which modulation is desired in
certain instances. The targeting process also includes
determination of a region (or regions) within this gene for the
oligonucleotide interaction to occur such that the desired effect,
either detection or modulation of expression of the protein, will
result. Once the target region have been identified,
oligonucleotides are chosen which are sufficiently complementary to
the target, i.e., hybridize sufficiently well and with sufficient
specificity to give the desired effect.
[0029] There are many regions of a gene that may be targeted for
antisense modulation: the region of the 5' Cap, a specialized
structure that at least partially mediates ribosome binding; the 5'
untranslated (noncoding) region (hereinafter, the "5'-UTR"); the
translation initiation codon region (hereinafter, the "tIR"); the
open reading frame (hereinafter, the "ORF"); the translation
termination codon region (hereinafter, the "tTR"); and the 3'
untranslated (noncoding) region (hereinafter, the "3'-UTR"), which
has at its 3' end a "poly A" tail. As is known in the art, these
regions are arranged in a typical messenger RNA molecule in the
following order (left to right, 5' to 3'): 5' Cap, 5'-UTR, tIR,
ORF, tTR, 3'-UTR, poly A tail. As is also known in the art,
although some eukaryotic transcripts are directly translated, many
ORFs contain one or more sequences, known as "introns," which are
excised from a transcript before it is translated; the expressed
(unexcised) portions of the ORF are referred to as "exons" (Alberts
et al., Molecular Biology of the Cell, pp. 331-332 and 411-415,
Garland Publishing Inc., New York, 1983). Furthermore, because many
eukaryotic ORFs are a thousand nucleotides or more in length, it is
often convenient to subdivide the ORF into, e.g., the 5' ORF
region, the central ORF region, and the 3' ORF region. In some
instances, an ORF contains one or more sites that may be targeted
due to some functional significance in vivo. Examples of the latter
types of sites include intragenic stem-loop structures (see, e.g.,
U.S. Pat. No. 5,512,438) and, in unprocessed mRNA molecules,
intron/exon splice sites. Within the context of the present
invention, one preferred intragenic site is the region encompassing
the translation initiation codon of the open reading frame (ORF) of
the gene. Because, as is known in the art, the translation
initiation codon is typically 5'-AUG (in transcribed mRNA
molecules; 5'-ATG in the corresponding DNA molecule), the
translation initiation codon is also referred to as the "AUG
codon," the "start codon" or the "AUG start codon." A minority of
genes have a translation initiation codon having the RNA sequence
5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and 5'-CUG have been
shown to function in vivo. Furthermore, 5'-UUU functions as a
translation initiation codon in vitro (Brigstock et al., Growth
Factors, 1990, 4, 45; Gelbert et al., Somat. Cell. Mol. Genet.,
1990, 16, 173; Gold and Stormo, Chapter 78 in: Escherichia coli and
Salmonella typhimurium: Cellular and Molecular Biology, Vol. 2, p.
1303, Neidhardt et al., eds., American Society for Microbiology,
Washington, D.C., 1987). Thus, the terms "translation initiation
codon" and "start codon" can encompass many codon sequences, even
though the initiator amino acid in each instance is typically
methionine (in eukaryotes) or formylmethionine (prokaryotes). It is
also known in the art that eukaryotic and prokaryotic genes may
have two or more alternative start codons, any one of which may be
preferentially utilized for translation initiation in a particular
cell type or tissue, or under a particular set of conditions, in
order to generate related polypeptides having different amino
terminal sequences (Markussen et al., Development, 1995, 121, 3723;
Gao et al., Cancer Res., 1995, 55, 743; McDermott et al., Gene,
1992, 117, 193; Perri et al., J. Biol. Chem., 1991, 266, 12536;
French et al., J. Virol., 1989, 63, 3270; Pushpa-Rekha et al., J.
Biol. Chem., 1995, 270, 26993; Monaco et al., J. Biol. Chem., 1994,
269, 347; DeVirgilio et al., Yeast, 1992, 8, 1043; Kanagasundaram
et al., Biochim. Biophys. Acta, 1992, 1171, 198; Olsen et al., Mol.
Endocrinol., 1991, 5, 1246; Saul et al., Appl. Environ. Microbiol.,
1990, 56, 3117; Yaoita et al., Proc. Natl. Acad. Sci. USA, 1990,
87, 7090; Rogers et al., EMBO J., 1990, 9, 2273). In the context of
the invention, "start codon" and "translation initiation codon"
refer to the codon or codons that are used in vivo to initiate
translation of an mRNA molecule transcribed from a gene encoding a
c-Fos or c-Jun protein, regardless of the sequence(s) of such
codons. It is also known in the art that a translation termination
codon (or "stop codon") of a gene may have one of three sequences,
i.e., 5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences
are 5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start
codon region" and "translation initiation region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to
about 50 contiguous nucleotides in either direction (i.e., 5' or
3') from a translation initiation codon. Similarly, the terms "stop
codon region" and "translation termination region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to
about 50 contiguous nucleotides in either direction (i.e., 5' or
3') from a translation termination codon.
[0030] In the context of this invention, the term "oligonucleotide"
includes oligonucleotides composed of naturally-occurring
nucleobases, sugars and covalent intersugar (backbone) linkages as
well as oligonucleotides having non-naturally-occurring portions
which function similarly. Such modified or substituted
oligonucleotides may be preferred over native forms because of
desirable properties such as, for example, enhanced cellular
uptake, enhanced binding to target and increased stability in the
presence of nucleases.
[0031] Specific examples of some preferred modified
oligonucleotides envisioned for this invention include those
containing phosphorothioates, phosphotriesters, methyl
phosphonates, short chain alkyl or cycloalkyl intersugar linkages
or short chain heteroatomic or heterocyclic intersugar linkages.
Most preferred are oligonucleotides with phosphorothioates and
those with CH.sub.2--NH--O--CH.sub.2,
CH.sub.2--N(CH.sub.3)--O--CH.sub.2 [known as a
methylene(methylimino) or MMI backbone],
CH.sub.2--O--N(CH.sub.3)--CH.sub.2,
CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2 and
O--N(CH.sub.3)--CH.sub.2- --CH.sub.2 backbones, wherein the native
phosphodiester backbone is represented as O--P--O--CH.sub.2). Also
preferred are oligonucleotides having morpholino backbone
structures (Summerton and Weller, U.S. Pat. No. 5,034,506). Further
preferred are oligonucleotides with NR--C(*)--CH.sub.2--CH.sub.2,
CH.sub.2--NR--C(*)--CH.sub.2, CH.sub.2--CH.sub.2--NR--C(*),
C(*)--NR--CH.sub.2--CH.sub.2 and CH.sub.2--C(*)--NR--CH.sub.2
backbones, wherein "*" represents O or S (known as amide backbones;
DeMesmaeker et al., WO 92/20823, published Nov. 26, 1992). In other
preferred embodiments, such as the peptide nucleic acid (PNA)
backbone, the phosphodiester backbone of the oligonucleotide is
replaced with a polyamide backbone, the nucleobases being bound
directly or indirectly to the aza nitrogen atoms of the polyamide
backbone (Nielsen et al., Science, 1991, 254, 1497; U.S. Pat. No.
5,539,082).
[0032] The oligonucleotides of the invention may additionally or
alternatively include nucleobase modifications or substitutions. As
used herein, "unmodified" or "natural" nucleobases include adenine
(A), guanine (G), thymine (T), cytosine (C) and uracil (U).
Modified nucleobases include nucleobases found only infrequently or
transiently in natural nucleic acids, e.g., hypoxanthine,
6-methyladenine, 5-methylcytosine, 5-hydroxymethylcytosine (HMC),
glycosyl HMC and gentiobiosyl HMC, as well synthetic nucleobases,
e.g., 2-aminoadenine, 2-thiouracil, 2-thiothymine, 5-bromouracil,
5-hydroxymethyluracil, 8-azaguanine, 7-deazaguanine,
N.sup.6(6-aminohexyl)adenine and 2,6-diaminopurine (Kornberg, A.,
DNA Replication, pp. 75-77, W. H. Freeman & Co., San Francisco,
1980; Gebeyehu, G., et al., Nucleic Acids Res., 1987, 15,
4513).
[0033] The oligonucleotides of the invention may additionally or
alternatively comprise substitutions of the sugar portion of the
individual nucleotides. For example, oligonucleotides may also have
sugar mimetics such as cyclobutyls in place of the pentofuranosyl
group. Other preferred modified oligonucleotides may contain one or
more substituted sugar moieties comprising one of the following at
the 2' position: OH, SH, SCH.sub.3, F, OCN, OCH.sub.3OCH.sub.3,
OCH.sub.3O(CH.sub.2).sub.nCH.s- ub.3, O(CH.sub.2).sub.nNH.sub.2 or
O(CH.sub.2).sub.nCH.sub.3 where n is from 1 to about 10; C.sub.1 to
C.sub.10 lower alkyl, alkoxyalkoxy, substituted lower alkyl,
alkaryl or aralkyl; Cl; Br; CN; CF.sub.3; OCF.sub.3; O--, S--, or
N-alkyl; O--, S--, or N-alkenyl; SOCH.sub.3; SO.sub.2CH.sub.3;
ONO.sub.2; NO.sub.2; N.sub.3; NH.sub.2; heterocycloalkyl;
heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted
silyl; an RNA cleaving group; a reporter group; an intercalator; a
group for improving the pharmacokinetic properties of an
oligonucleotide; or a group for improving the pharmacodynamic
properties of an oligonucleotide and other substituents having
similar properties. A preferred modification, particularly for
orally deliverable pharmaceutical compositions, is 2'-methoxyethoxy
[2'-O--CH.sub.2CH.sub.2O- CH.sub.3, also known as
2'-O-(2-methoxyethyl)] (Martin et al., Helv. Chim. Acta, 1995, 78,
486). Other preferred modifications include 2'-methoxy
(2'-O--CH.sub.3), 2'-propoxy (2'-OCH.sub.2CH.sub.2CH.sub.3) and
2'-fluoro (2'-F).
[0034] Similar modifications may also be made at other positions on
the oligonucleotide, particularly the 3' position of the sugar on
the 3' terminal nucleotide and the 5' position of 5' terminal
nucleotide. The 5' and 3' termini of an oligonucleotide may also be
modified to serve as points of chemical conjugation of, e.g.,
lipophilic moieties (see immediately subsequent paragraph),
intercalating agents (Kuyavin et al., WO 96/32496, published Oct.
17, 1996; Nguyen et al., U.S. Pat. No. 4,835,263, issued May 30,
1989) or hydroxyalkyl groups (Helene et al., WO 96/34008, published
Oct. 31, 1996).
[0035] Other positions within an oligonucleotide of the invention
can be used to chemically link thereto one or more effector groups
to form an oligonucleotide conjugate. An "effector group" is a
chemical moiety that is capable of carrying out a particular
chemical or biological function. Examples of such effector groups
include, but are not limited to, an RNA cleaving group, a reporter
group, an intercalator, a group for improving the pharmacokinetic
properties of an oligonucleotide, or a group for improving the
pharmacodynamic properties of an oligonucleotide and other
substituents having similar properties. A variety of chemical
linkers may be used to conjugate an effector group to an
oligonucleotide of the invention. As an example, U.S. Pat. No.
5,578,718 to Cook et al. discloses methods of attaching an
alkylthio linker, which may be further derivatized to include
additional groups, to ribofuranosyl positions, nucleosidic base
positions, or on internucleoside linkages. Additional methods of
conjugating oligonucleotides to various effector groups are known
in the art; see, e.g., Protocols for Oligonucleotide Conjugates
(Methods in Molecular Biology, Volume 26) Agrawal, S., ed., Humana
Press, Totowa, N.J., 1994.
[0036] Another preferred additional or alternative modification of
the oligonucleotides of the invention involves chemically linking
to the oligonucleotide one or more lipophilic moieties which
enhance the cellular uptake of the oligonucleotide. Such lipophilic
moieties may be linked to an oligonucleotide at several different
positions on the oligonucleotide. Some preferred positions include
the 3' position of the sugar of the 3' terminal nucleotide, the 5'
position of the sugar of the 5' terminal nucleotide, and the 2'
position of the sugar of any nucleotide. The N.sup.6 position of a
purine nucleobase may also be utilized to link a lipophilic moiety
to an oligonucleotide of the invention (Gebeyehu et al., Nucleic
Acids Res., 1987, 15, 4513). Such lipophilic moieties include but
are not limited to a cholesteryl moiety (Letsinger et al., Proc.
Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et
al., Bioorg. Med. Chem. Let., 1994, 4, 1053), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,
1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl
residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 111; Kabanov
et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie,
1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate
(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al.,
Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene
glycol chain (Manoharan et al., Nucleosides & Nucleotides,
1995, 14, 969), or adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et
al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923). Oligonucleotides comprising
lipophilic moieties, and methods for preparing such
oligonucleotides, are disclosed in U.S. Pat. Nos. 5,138,045,
5,218,105 and 5,459,255, the contents of which are hereby
incorporated by reference.
[0037] The oligonucleotides of the invention may additionally or
alternatively be prepared to be delivered in a "prodrug" form. The
term "prodrug" indicates a therapeutic agent that is prepared in an
inactive form that is converted to an active form (i.e., drug)
within the body or cells thereof by the action of endogenous
enzymes or other chemicals and/or conditions. In particular,
prodrug versions of the oligonucleotides of the invention are
prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives
according to the methods disclosed in WO 93/24510 to Gosselin et
al., published Dec. 9, 1993.
[0038] The present invention also includes oligonucleotides that
are substantially chirally pure with regard to particular positions
within the oligonucleotides. Examples of substantially chirally
pure oligonucleotides include, but are not limited to, those having
phosphorothioate linkages that are at least 75% Sp or Rp (Cook et
al., U.S. Pat. No. 5,587,361, issued Dec. 24, 1996) and those
having substantially chirally pure (Sp or Rp) alkylphosphonate,
phosphoamidate or phosphotriester linkages (Cook, U.S. Pat. No.
5,212,295, issued May 18, 1993; Cook, U.S. Pat. No. 5,521,302,
issued May 28, 1996).
[0039] The present invention also includes oligonucleotides which
are chimeric oligonucleotides. "Chimeric" oligonucleotides or
"chimeras," in the context of this invention, are oligonucleotides
which contain two or more chemically distinct regions, each made up
of at least one nucleotide. These oligonucleotides typically
contain at least one region wherein the oligonucleotide is modified
so as to confer upon the oligonucleotide increased resistance to
nuclease degradation, increased cellular uptake, and/or increased
binding affinity for the target nucleic acid. An additional region
of the oligonucleotide may serve as a substrate for enzymes capable
of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H
is a cellular endonuclease which cleaves the RNA strand of an
RNA:DNA duplex. Activation of RNase H, therefore, results in
cleavage of the RNA target, thereby greatly enhancing the
efficiency of antisense inhibition of gene expression. Cleavage of
the RNA target can be routinely detected by gel electrophoresis
and, if necessary, associated nucleic acid hybridization techniques
known in the art. By way of example, such "chimeras" may be
"gapmers," i.e., oligonucleotides in which a central portion (the
"gap") of the oligonucieotide serves as a substrate for, e.g.,
RNase H, and the 5' and 3' portions (the "wings") are modified in
such a fashion so as to have greater affinity for the target RNA
molecule but are unable to support nuclease activity (e.g.,
2'-fluoro- or 2'-methoxyethoxy substituted). Other chimeras include
"wingmers," that is, oligonucleotides in which the 5' portion of
the oligonucleotide serves as a substrate for, e.g., RNase H,
whereas the 3' portion is modified in such a fashion so as to have
greater affinity for the target RNA molecule but is unable to
support nuclease activity (e.g., 2'-fluoro- or 2'-methoxyethoxy
substituted), or vice-versa.
[0040] The oligonucleotides used in accordance with this invention
may be conveniently and routinely made through the well-known
technique of solid phase synthesis. Equipment for such synthesis is
sold by several vendors including, for example, Applied Biosystems
(Foster City, Calif.). Any other means for such synthesis known in
the art may additionally or alternatively be employed. It is also
known to use similar techniques to prepare other oligonucleotides
such as the phosphorothioates and alkylated derivatives. Teachings
regarding the synthesis of particular modified oligonucleotides are
hereby incorporated by reference from the following U.S. patents or
pending patent applications, each of which is commonly assigned
with this application: U.S. Pat. Nos. 5,138,045 and 5,218,105,
drawn to polyamine conjugated oligonucleotides; U.S. Pat. No.
5,212,295, drawn to monomers for the preparation of
oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos.
5,378,825 and 5,541,307, drawn to oligonucleotides having modified
backbones; U.S. Pat. No. 5,386,023, drawn to backbone modified
oligonucleotides and the preparation thereof through reductive
coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases
based on the 3-deazapurine ring system and methods of synthesis
thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases
based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to
processes for preparing oligonucleotides having chiral phosphorus
linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids;
U.S. Pat. No. 5,554,746, drawn to oligonucleotides having b-lactam
backbones; U.S. Pat. No. 5,571,902, drawn to methods and materials
for the synthesis of oligonucleotides; U.S. Pat. No. 5,578,718,
drawn to nucleosides having alkylthio groups, wherein such groups
may be used as linkers to other moieties, attached at any of a
variety of positions of the nucleoside; and U.S. Pat. No.
5,587,361, drawn to oligonucleotides having phosphorothioate
linkages of high chiral purity.
[0041] The oligonucleotides of the present invention can be
utilized as therapeutic compounds, diagnostic tools and as research
reagents and kits. The term "therapeutic uses" is intended to
encompass prophylactic, palliative and curative uses wherein the
oligonucleotides of the invention are contacted with animal cells
either in vivo or ex vivo. When contacted with animal cells ex
vivo, a therapeutic use includes incorporating such cells into an
animal after treatment with one or more oligonucleotides of the
invention.
[0042] For therapeutic uses, an animal suspected of having a
disease or disorder which can be treated or prevented by modulating
the expression or activity of a c-Fos or c-Jun protein is, for
example, treated by administering oligonucleotides in accordance
with this invention. The oligonucleotides of the invention can be
utilized in pharmaceutical compositions by adding an effective
amount of an oligonucleotide to a suitable pharmaceutically
acceptable diluent or carrier. Workers in the field have identified
antisense, triplex and other oligonucleotide compositions which are
capable of modulating expression of genes implicated in viral,
fungal and metabolic diseases. Antisense oligonucleotides have been
safely administered to humans and several clinical trials are
presently underway. It is thus established that oligonucleotides
can be useful therapeutic instrumentalities that can be configured
to be useful in treatment regimes for treatment of cells, tissues
and animals, especially humans.
[0043] The oligonucleotides of the present invention can be further
used to detect the presence of c-fos- or c-jun-specific nucleic
acids in a cell or tissue sample. For example, radiolabeled
oligonucleotides can be prepared by .sup.32P labeling at the 5' end
with polynucleotide kinase (Sambrook et al., Molecular Cloning. A
Laboratory Manual, Vol. 2, p. 10.59, Cold Spring Harbor Laboratory
Press, 1989). Radiolabeled oligonucleotides are then contacted with
cell or tissue samples suspected of containing c-fos or c-jun
message RNAs (and thus c-Fos or c-Jun proteins), and the samples
are washed to remove unbound oligonucleotide. Radioactivity
remaining in the sample indicates the presence of bound
oligonucleotide, which in turn indicates the presence of nucleic
acids complementary to the oligonucleotide, and can be quantitated
using a scintillation counter or other routine means. Expression of
nucleic acids encoding these proteins is thus detected.
[0044] Radiolabeled oligonucleotides of the present invention can
also be used to perform autoradiography of tissues to determine the
localization, distribution and quantitation of c-Fos or c-Jun
proteins for research, diagnostic or therapeutic purposes. In such
studies, tissue sections are treated with radiolabeled
oligonucleotide and washed as described above, then exposed to
photographic emulsion according to routine autoradiography
procedures. The emulsion, when developed, yields an image of silver
grains over the regions expressing a c-Fos or c-Jun gene.
Quantitation of the silver grains permits detection of the
expression of mRNA molecules encoding these proteins and permits
targeting of oligonucleotides to these areas.
[0045] Analogous assays for fluorescent detection of expression of
c-fos or c-jun nucleic acids can be developed using
oligonucleotides of the present invention which are conjugated with
fluorescein or other fluorescent tags instead of radiolabeling.
Such conjugations are routinely accomplished during solid phase
synthesis using fluorescently-labeled amidites or controlled pore
glass (CPG) columns. Fluorescein-labeled amidites and CPG are
available from, e.g., Glen Research (Sterling, Va.).
[0046] The present invention employs oligonucleotides targeted to
nucleic acids encoding c-Fos or c-Jun proteins and oligonucleotides
targeted to nucleic acids encoding such proteins. Kits for
detecting the presence or absence of expression of a c-Fos and/or
c-Jun protein may also be prepared. Such kits include an
oligonucleotide targeted to an appropriate gene, i.e., a gene
encoding a c-Fos or c-Jun protein. Appropriate kit and assay
formats, such as, e.g., "sandwich" assays, are known in the art and
can easily be adapted for use with the oligonucleotides of the
invention. Hybridization of the oligonucleotides of the invention
with a nucleic acid encoding a c-Fos or c-Jun protein can be
detected by means known in the art. Such means may include
conjugation of an enzyme to the oligonucleotide, radiolabelling of
the oligonucleotide or any other suitable detection systems. Kits
for detecting the presence or absence of a c-Fos or c-Jun protein
may also be prepared.
[0047] In the context of this invention, "hybridization" means
hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed
Hoogsteen hydrogen bonding, between complementary nucleotides. For
example, adenine and thymine are complementary nucleobases which
pair through the formation of hydrogen bonds. "Complementary," as
used herein, refers to the capacity for precise pairing between two
nucleotides. For example, if a nucleotide at a certain position of
an oligonucleotide is capable of hydrogen bonding with a nucleotide
at the same position of a DNA or RNA molecule, then the
oligonucleotide and the DNA or RNA are considered to be
complementary to each other at that position. The oligonucleotide
and the DNA or RNA are complementary to each other when a
sufficient number of corresponding positions in each molecule are
occupied by nucleotides which can hydrogen bond with each other.
Thus, "specifically hybridizable" and "complementary" are terms
which are used to indicate a sufficient degree of complementarity
or precise pairing such that stable and specific binding occurs
between the oligonucleotide and the DNA or RNA target. It is
understood in the art that an oligonucleotide need not be 100%
complementary to its target DNA sequence to be specifically
hybridizable. An oligonucleotide is specifically hybridizable when
binding of the oligonucleotide to the target DNA or RNA molecule
interferes with the normal function of the target DNA or RNA to
cause a decrease or loss of function, and there is a sufficient
degree of complementarity to avoid non-specific binding of the
oligonucleotide to non-target sequences under conditions in which
specific binding is desired, i.e., under physiological conditions
in the case of in vivo assays or therapeutic treatment, or in the
case of in vitro assays, under conditions in which the assays are
performed.
[0048] The formulation of therapeutic compositions and their
subsequent administration is believed to be within the skill of
those in the art. In general, for therapeutics, a patient in need
of such therapy is administered an oligonucleotide in accordance
with the invention, commonly in a pharmaceutically acceptable
carrier, in doses ranging from 0.01 ug to 100 g per kg of body
weight depending on the age of the patient and the severity of the
disorder or disease state being treated. Further, the treatment
regimen may last for a period of time which will vary depending
upon the nature of the particular disease or disorder, its severity
and the overall condition of the patient, and may extend from once
daily to once every 20 years. Following treatment, the patient is
monitored for changes in his/her condition and for alleviation of
the symptoms of the disorder or disease state. The dosage of the
oligonucleotide may either be increased in the event the patient
does not respond significantly to current dosage levels, or the
dose may be decreased if an alleviation of the symptoms of the
disorder or disease state is observed, or if the disorder or
disease state has been abated.
[0049] In some cases it may be more effective to treat a patient
with an oligonucleotide of the invention in conjunction with other
traditional therapeutic modalities in order to increase the
efficacy of a treatment regimen. In the context of the invention,
the term "treatment regimen" is meant to encompass therapeutic,
palliative and prophylactic modalities. For example, a patient may
be treated with conventional chemotherapeutic agents, particularly
those used for tumor and cancer treatment. Examples of such
chemotherapeutic agents include but are not limited to
daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,
idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,
cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,
mitomycin C, actinomycin D, mithramycin, prednisone,
hydroxyprogesterone, testosterone, tamoxifen, dacarbazine,
procarbazine, hexamethylmelamine, pentamethylmelamine,
mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,
nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,
6-thioguanine, cytarabine (CA), 5-azacytidine, hydroxyurea,
deoxycoformycin, 4-hydroxyperoxycyclophosphor- amide,
5-fluorouracil (5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate
(MTX), colchicine, vincristine, vinblastine, etoposide,
trimetrexate, teniposide, cisplatin and diethylstilbestrol (DES).
See, generally, The Merck Manual of Diagnosis and Therapy, 15th
Ed., pp. 1206-1228, Berkow et al., eds., Rahay, N.J., 1987). When
used with the compounds of the invention, such chemotherapeutic
agents may be used individually (e.g., 5-FU and oligonucleotide),
sequentially (e.g., 5-FU and oligonucleotide for a period of time
followed by MTX and oligonucleotide), or in combination with one or
more other such chemotherapeutic agents (e.g., 5-FU, MTX and
oligonucleotide, or 5-FU, radiotherapy and oligonucleotide).
[0050] In another preferred embodiment of the invention, a first
antisense oligonucleotide targeted to c-fos is used in combination
with a second antisense oligonucleotide targeted to c-jun in order
to modulate AP-1 molecules to a more extensive degree than can be
achieved when either oligonucleotide used individually.
[0051] Following successful treatment, it may be desirable to have
the patient undergo maintenance therapy to prevent the recurrence
of the disease state, wherein the oligonucleotide is administered
in maintenance doses, ranging from 0.01 ug to 100 g per kg of body
weight, once or more daily, to once every 20 years. In the case of
in individual known or suspected of being prone to a neoplastic or
malignant condition, prophylactic effects may be achieved by
administration of preventative doses, ranging from 0.01 ug to 100 g
per kg of body weight, once or more daily, to once every 20
years.
[0052] The pharmaceutical compositions of the present invention may
be administered in a number of ways depending upon whether local or
systemic treatment is desired and upon the area to be treated.
Within the context of the invention, "administration" indicates the
topical (including ophthalmic, vaginal, rectal, intranasal,
transdermal), oral or parenteral contacting of an oligonucleotide,
or pharmaceutical composition comprising an oligonucleotide, with
cells, tissues or organs of a mammal including a human. Parenteral
administration includes intravenous drip; subcutaneous,
intraperitoneal, intravitreal or intramuscular injection; and
intrathecal or intraventricular administration as herein
described.
[0053] Formulations for topical administration may include
transdermal patches, ointments, lotions, creams, gels, drops,
suppositories, sprays, liquids and powders. Conventional
pharmaceutical carriers, nucleic acid carriers, aqueous, powder or
oily bases, thickeners and the like may be necessary or desirable
in certain instances. Topical administration also includes the
delivery of oligonucleotides into the epidermis of a mammal by
electroporation (Zewert et al., WO 96/39531, published Dec. 12,
1996).
[0054] Compositions for oral administration include powders or
granules, suspensions or solutions in water or non-aqueous media,
capsules, sachets or tablets. Thickeners, flavoring agents,
diluents, emulsifiers, dispersing aids or binders may be
desirable.
[0055] Intravitreal injection, for the direct delivery of drug to
the vitreous humor of a mammalian eye, is described in U.S. Pat.
No. 5,595,978, which issued Jan. 21, 1997, and which is assigned to
the same assignee as the instant application, the contents of which
are hereby incorporated by reference.
[0056] Intraluminal drug administration, for the direct delivery of
drug to an isolated portion of a tubular organ or tissue (e.g.,
such as an artery, vein, ureter or urethra), may be desired for the
treatment of patients with diseases or conditions afflicting the
lumen of such organs or tissues. To effect this mode of
oligonucleotide administration, a catheter or cannula is surgically
introduced by appropriate means. For example, for treatment of the
left common carotid artery, a cannula is inserted thereinto via the
external carotid artery. After isolation of a portion of the
tubular organ or tissue for which treatment is sought, a
composition comprising the oligonucleotides of the invention is
infused through the cannula or catheter into the isolated segment.
After incubation for from about 1 to about 120 minutes, during
which the oligonucleotide is taken up by cells of the interior
lumen of the vessel, the infusion cannula or catheter is removed
and flow within the tubular organ or tissue is restored by removal
of the ligatures which effected the isolation of a segment thereof
(Morishita et al., Proc. Natl. Acad. Sci. (U.S.A.), 1993, 90,
8474). Antisense oligonucleotides may also be combined with a
biocompatible matrix or carrier, such as a hydrogel material, and
applied directly to vascular tissue in vivo (Rosenberg et al., U.S.
Pat. No. 5,593,974, issued Jan. 14, 1997).
[0057] Intraventricular drug administration, for the direct
delivery of drug to the brain of a patient, may be desired for the
treatment of patients with diseases or conditions afflicting the
brain. To effect this mode of oligonucleotide administration, a
silicon catheter is surgically introduced into a ventricle of the
brain of a human patient, and is connected to a subcutaneous
infusion pump (Medtronic Inc., Minneapolis, Minn.) that has been
surgically implanted in the abdominal region (Zimm et al., Cancer
Research, 1984, 44, 1698; Shaw, Cancer, 1993, 72(11 Suppl.), 3416).
The pump is used to inject the oligonucleotides and allows precise
dosage adjustments and variation in dosage schedules with the aid
of an external programming device. The reservoir capacity of the
pump is 18-20 mL and infusion rates may range from 0.1 mL/h to 1
mL/h. Depending on the frequency of administration, ranging from
daily to monthly, and the dose of drug to be administered, ranging
from 0.01 .mu.g to 100 g per kg of body weight, the pump reservoir
may be refilled at 3-10 week intervals. Refilling of the pump is
accomplished by percutaneous puncture of the self-sealing septum of
the pump.
[0058] Intrathecal drug administration for the introduction of drug
into the spinal column of a patient may be desired for the
treatment of patients with diseases of the central nervous system.
To effect this route of oligonucleotide administration, a silicon
catheter is surgically implanted into the L3-4 lumbar spinal
interspace of a human patient, and is connected to a subcutaneous
infusion pump which has been surgically implanted in the upper
abdominal region (Luer and Hatton, The Annals of Pharmacotherapy,
1993, 27, 912; Ettinger et al., 1978, Cancer, 41, 1270, 1978; Yaida
et al., Regul. Pept., 1995, 59, 193). The pump is used to inject
the oligonucleotides and allows precise dosage adjustments and
variations in dose schedules with the aid of an external
programming device. The reservoir capacity of the pump is 18-20 mL,
and infusion rates may vary from 0.1 mL/h to 1 mL/h. Depending on
the frequency of drug administration, ranging from daily to
monthly, and dosage of drug to be administered, ranging from 0.01
.mu.g to 100 g per kg of body weight, the pump reservoir may be
refilled at 3-10 week intervals. Refilling of the pump is
accomplished by a single percutaneous puncture to the self-sealing
septum of the pump. The distribution, stability and
pharmacokinetics of oligonucleotides within the central nervous
system may be followed according to known methods (Whitesell et
al., Proc. Natl. Acad. Sci. (USA), 1993, 90, 4665).
[0059] To effect delivery of oligonucleotides to areas other than
the brain or spinal column via this method, the silicon catheter is
configured to connect the subcutaneous infusion pump to, e.g., the
hepatic artery, for delivery to the liver (Kemeny et al., Cancer,
1993, 71:1964). Infusion pumps may also be used to effect systemic
delivery of oligonucleotides (Ewel et al., Cancer Research, 1992,
52:3005; Rubenstein et al., J. Surg. Oncol., 1996, 62:194).
[0060] Regardless of the method by which the oligonucleotides of
the invention are introduced into a patient, colloidal dispersion
systems may be used as delivery vehicles to enhance the in vivo
stability of the oligonucleotides and/or to target the
oligonucleotides to a particular organ, tissue or cell type.
Colloidal dispersion systems include, but are not limited to,
macromolecule complexes, nanocapsules, microspheres, beads and
lipid-based systems including oil-in-water emulsions, micelles,
mixed micelles and liposomes. A preferred colloidal dispersion
system is a plurality of liposomes, artificial membrane vesicles
which may be used as cellular delivery vehicles for bioactive
agents in vitro and in vivo (Mannino et al., Biotechniques, 1988,
6, 682; Blume and Cevc, Biochem. et Biophys. Acta, 1990, 1029, 91;
Lappalainen et al., Antiviral Res., 1994, 23, 119; Chonn and
Cullis, Current Op. Biotech., 1995, 6, 698). It has been shown that
large unilamellar vesicles (LUV), which range in size from 0.2-0.4
.mu.m, can encapsulate a substantial percentage of an aqueous
buffer containing large macromolecules. RNA, DNA and intact virions
can be encapsulated within the aqueous interior and delivered to
brain cells in a biologically active form (Fraley et al., Trends
Biochem. Sci., 1981, 6, 77). The composition of the liposome is
usually a combination of lipids, particularly phospholipids, in
particular, high phase transition temperature phospholipids,
usually in combination with one or more steroids, particularly
cholesterol. Examples of lipids useful in liposome production
include phosphatidyl compounds, such as phosphatidylglycerol,
phosphatidylcholine, phosphatidylserine, phosphatidylethanolamine,
sphingolipids, cerebrosides and gangliosides. Particularly useful
are diacyl phosphatidylglycerols, where the lipid moiety contains
from 14-18 carbon atoms, particularly from 16-18 carbon atoms, and
is saturated (lacking double bonds within the 14-18 carbon atom
chain). Illustrative phospholipids include phosphatidylcholine,
dipalmitoylphosphatidylcholine and
distearoylphosphatidylcholine.
[0061] The targeting of colloidal dispersion systems, including
liposomes, can be either passive or active. Passive targeting
utilizes the natural tendency of liposomes to distribute to cells
of the reticuloendothelial system in organs that contain sinusoidal
capillaries. Active targeting, by contrast, involves modification
of the liposome by coupling thereto a specific ligand such as a
viral protein coat (Morishita et al., Proc. Natl. Acad. Sci.
(U.S.A.), 1993, 90, 8474), monoclonal antibody (or a suitable
binding portion thereof), sugar, glycolipid or protein (or a
suitable oligopeptide fragment thereof), or by changing the
composition and/or size of the liposome in order to achieve
distribution to organs and cell types other than the naturally
occurring sites of localization. The surface of the targeted
colloidal dispersion system can be modified in a variety of ways.
In the case of a liposomal targeted delivery system, lipid groups
can be incorporated into the lipid bilayer of the liposome in order
to maintain the targeting ligand in close association with the
lipid bilayer. Various linking groups can be used for joining the
lipid chains to the targeting ligand. The targeting ligand, which
binds a specific cell surface molecule found predominantly on cells
to which delivery of the oligonucleotides of the invention is
desired, may be, for example, (1) a hormone, growth factor or a
suitable oligopeptide fragment thereof which is bound by a specific
cellular receptor predominantly expressed by cells to which
delivery is desired or (2) a polyclonal or monoclonal antibody, or
a suitable fragment thereof (e.g., Fab; F(ab').sub.2) which
specifically binds an antigenic epitope found predominantly on
targeted cells. Two or more bioactive agents (e.g., an
oligonucleotide and a conventional drug; two oligonucleotides) can
be combined within, and delivered by, a single liposome. It is also
possible to add agents to colloidal dispersion systems which
enhance the intercellular stability and/or targeting of the
contents thereof.
[0062] Compositions for parenteral, intrathecal or intraventricular
administration, or colloidal dispersion systems, may include
sterile aqueous solutions which may also contain buffers, diluents
and other suitable additives. Dosing is dependent on severity and
responsiveness of the disease state to be treated, with the course
of treatment lasting from several days to several months, or until
a cure is effected or a diminution of the disease state is
achieved. Optimal dosing schedules can be calculated from
measurements of drug accumulation in the body of the patient.
Persons of ordinary skill can easily determine optimum dosages,
dosing methodologies and repetition rates. Optimum dosages may vary
depending on the relative potency of individual oligonucleotides,
and can generally be estimated based on EC.sub.50s found to be
effective in in vitro and in vivo animal models. In general, dosage
is from 0.01 .mu.g to 100 g per kg of body weight, and may be given
once or more daily, weekly, monthly or yearly, or even once every 2
to 20 years.
[0063] The following examples illustrate the invention and are not
intended to limit the same. Those skilled in the art will
recognize, or be able to ascertain through routine experimentation,
numerous equivalents to the specific substances and procedures
described herein. Such equivalents are considered to be within the
scope of the present invention.
EXAMPLES
Example 1
[0064] Chemical Synthesis and Nucleotide Sequence of
Oligonucleotides
[0065] General Synthetic Techniques: Oligonucleotides were
synthesized on an automated DNA synthesizer using standard
phosphoramidite chemistry with oxidation using iodine.
.beta.-Cyanoethyldiisopropyl phosphoramidites were purchased from
Applied Biosystems (Foster City, Calif.). For phosphorothioate
oligonucleotides, the standard oxidation bottle was replaced by a
0.2 M solution of 3H-1,2-benzodithiole-3-one-1,1- -dioxide in
acetonitrile for the stepwise thiation of the phosphite
linkages.
[0066] The synthesis of 2'-O-methyl- (a.k.a. 2'-methoxy-)
phosphorothioate oligonucleotides was according to the procedures
set forth above substituting 2'-O-methyl
.beta.-cyanoethyldiisopropyl phosphoramidites (Chemgenes, Needham,
Mass.) for standard phosphoramidites and increasing the wait cycle
after the pulse delivery of tetrazole and base to 360 seconds.
[0067] Similarly, 2'-O-propyl- (a.k.a 2'-propoxy-) phosphorothioate
oligonucleotides were prepared by slight modifications of this
procedure and essentially according to procedures disclosed in U.S.
patent application Ser. No. 08/383,666, filed Feb. 3, 1995, which
is assigned to the same assignee as the instant application and
which is incorporated by reference herein.
[0068] The 2'-fluoro-phosphorothioate oligonucleotides of the
invention were synthesized using
5'-dimethoxytrityl-3'-phosphoramidites and prepared as disclosed in
U.S. patent application Ser. No. 08/383,666, filed Feb. 3, 1995,
and U.S. Pat. No. 5,459,255, which issued Oct. 8, 1996, both of
which are assigned to the same assignee as the instant application
and which are incorporated by reference herein. The
2'-fluoro-oligonucleotides were prepared using phosphoramidite
chemistry and a slight modification of the standard DNA synthesis
protocol (i.e., deprotection was effected using methanolic ammonia
at room temperature).
[0069] The 2'-methoxyethoxy oligonucleotides were synthesized
essentially according to the methods of Martin et al. (Helv. Chim.
Acta, 1995, 78, 486). For ease of synthesis, the 3' nucleotide of
the 2'-methoxyethoxy oligonucleotides was a deoxynucleotide, and
2'-O--CH.sub.2CH.sub.2OCH.sub- .3-cytosines were 5-methyl
cytosines, which were synthesized according to the procedures
described below.
[0070] PNA antisense analogs were prepared essentially as described
in U.S. Pat. Nos. 5,539,082 and 5,539,083, both of which (1) issued
Jul. 23, 1996, (2) are assigned to the same assignee as the instant
application and (3) are incorporated by reference herein.
[0071] Oligonucleotides comprising 2,6-diaminopurine were prepared
essentially as described in U.S. Pat. No. 5,506,351 which issued
Apr. 9, 1996, is assigned to the same assignee as the instant
application and which is incorporated by reference herein.
oligonucleotides comprising 2,6-diaminopurine can also be prepared
by enzymatic means (Bailly et al., Proc. Natl. Acad. Sci. U.S.A.,
1996, 93:13623).
[0072] After cleavage from the controlled pore glass (CPG) column
(Applied Biosystems) and deblocking in concentrated ammonium
hydroxide, at 55.degree. C. for 18 hours, the oligonucleotides were
purified by precipitation 2.times. from 0.5 M NaCl with 2.5 volumes
of ethanol. Analytical gel electrophoresis was accomplished in 20%
acrylamide, 8 M urea and 45 mM Tris-borate buffer (pH 7).
[0073] Synthesis of 5-Methyl Cytosine Monomers:
[0074] 2,2'-Anhydro[1-(.beta.-D-arabinofuranosyl)-5-methyluridine]:
5-Methyluridine (ribosylthymine, commercially available through
Yamasa, Choshi, Japan) (72.0 g, 0.279 M), diphenylcarbonate (90.0
g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) were added to
DMF (300 mL). The mixture was heated to reflux, with stirring,
allowing the evolved carbon dioxide gas to be released in a
controlled manner. After 1 hour, the slightly darkened solution was
concentrated under reduced pressure. The resulting syrup was poured
into diethylether (2.5 L), with stirring. The product formed a gum.
The ether was decanted and the residue was dissolved in a minimum
amount of methanol (ca. 400 mL). The solution was poured into fresh
ether (2.5 L) to yield a stiff gum. The ether was decanted and the
gum was dried in a vacuum oven (60.degree. C. at 1 mm Hg for 24 h)
to give a solid which was crushed to a light tan powder (57 g, 85%
crude yield). The material was used as is for further
reactions.
[0075] 2'-O-Methoxyethyl-5-methyluridine:
2,2'-Anhydro-5-methyluridine (195 g, 0.81 M),
tris(2-methoxyethyl)borate (231 g, 0.98 M) and 2-methoxyethanol
(1.2 L) were added to a 2 L stainless steel pressure vessel and
placed in a pre-heated oil bath at 160.degree. C. After heating for
48 hours at 155-160.degree. C., the vessel was opened and the
solution evaporated to dryness and triturated with MeOH (200 mL).
The residue was suspended in hot acetone (1 L). The insoluble salts
were filtered, washed with acetone (150 mL) and the filtrate
evaporated. The residue (280 g) was dissolved in CH.sub.3CN (600
mL) and evaporated. A silica gel column (3 kg) was packed in
CH.sub.2Cl.sub.2/acetone/MeOH (20:5:3) containing 0.5% Et.sub.3NH.
The residue was dissolved in CH.sub.2Cl.sub.2 (250 mL) and adsorbed
onto silica (150 g) prior to loading onto the column. The product
was eluted with the packing solvent to give 160 g (63%) of
product.
[0076] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine:
2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506 M) was
co-evaporated with pyridine (250 mL) and the dried residue
dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the mixture stirred at
room temperature for one hour. A second aliquot of dimethoxytrityl
chloride (94.3 g, 0.278 M) was added and the reaction stirred for
an additional one hour. Methanol (170 mL) was then added to stop
the reaction. HPLC showed the presence of approximately 70%
product. The solvent was evaporated and triturated with CH.sub.3CN
(200 mL) The residue was dissolved in CHCl.sub.3 (1.5 L) and
extracted with 2.times.500 mL of saturated NaHCO.sub.3 and
2.times.500 mL of saturated NaCl. The organic phase was dried over
Na.sub.2SO.sub.4, filtered and evaporated. 275 g of residue was
obtained. The residue was purified on a 3.5 kg silica gel column,
packed and eluted with EtOAc/Hexane/Acetone (5:5:1) containing 0.5%
Et.sub.3NH. The pure fractions were evaporated to give 164 g of
product. Approximately 20 g additional was obtained from the impure
fractions to give a total yield of 183 g (57%).
[0077]
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine:
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g,
0.167 M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562
mL of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL,
0.258 M) were combined and stirred at room temperature for 24
hours. The reaction was monitored by tlc by first quenching the tlc
sample with the addition of MeOH. Upon completion of the reaction,
as judged by tlc, MeOH (50 mL) was added and the mixture evaporated
at 35.degree. C. The residue was dissolved in CHCl.sub.3 (800 mL)
and extracted with 2.times.200 mL of saturated sodium bicarbonate
and 2.times.200 mL of saturated NaCl. The water layers were back
extracted with 200 mL of CHCl.sub.3. The combined organics were
dried with sodium sulfate and evaporated to give 122 g of residue
(approximately 90% product). The residue was purified on a 3.5 kg
silica gel column and eluted using EtOAc/Hexane (4:1). Pure product
fractions were evaporated to yield 96 g (84%).
[0078]
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triaz-
oleuridine: A first solution was prepared by dissolving
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
(96 g, 0.144 M) in CH.sub.3CN (700 mL) and set aside. Triethylamine
(189 mL, 1.44 M) was added to a solution of triazole (90 g, 1.3 M)
in CH.sub.3CN (1 L), cooled to -5.degree. C. and stirred for 0.5 h
using an overhead stirrer. POCl.sub.3 was added dropwise, over a 30
minute period, to the stirred solution maintained at 0-10.degree.
C., and the resulting mixture stirred for an additional 2 hours.
The first solution was added dropwise, over a 45 minute period, to
the later solution. The resulting reaction mixture was stored
overnight in a cold room. Salts were filtered from the reaction
mixture and the solution was evaporated. The residue was dissolved
in EtOAc (1 L) and the insoluble solids were removed by filtration.
The filtrate was washed with 1.times.300 mL of NaHCO.sub.3 and
2.times.300 mL of saturated NaCl, dried over sodium sulfate and
evaporated. The residue was triturated with EtOAc to give the title
compound.
[0079] 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine: A
solution of
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxy-trityl-5-methyl-4-triazol-
euridine (103 g, 0.141 M) in dioxane (500 mL) and NH.sub.4OH (30
mL) was stirred at room temperature for 2 hours. The dioxane
solution was evaporated and the residue azeotroped with MeOH
(2.times.200 mL). The residue was dissolved in MeOH (300 mL) and
transferred to a 2 liter stainless steel pressure vessel. Methanol
(400 mL) saturated with NH.sub.3 gas was added and the vessel
heated to 100.degree. C. for 2 hours (thin layer chromatography,
tlc, showed complete conversion). The vessel contents were
evaporated to dryness and the residue was dissolved in EtOAc (500
mL) and washed once with saturated NaCl (200 mL). The organics were
dried over sodium sulfate and the solvent was evaporated to give 85
g (95%) of the title compound.
[0080]
N.sup.4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcyti-
dine: 2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85
g, 0.134 M) was dissolved in DMF (800 mL) and benzoic anhydride
(37.2 g, 0.165 M) was added with stirring. After stirring for 3
hours, tlc showed the reaction to be approximately 95% complete.
The solvent was evaporated and the residue azeotroped with MeOH
(200 mL). The residue was dissolved in CHCl.sub.3 (700 mL) and
extracted with saturated NaHCO.sub.3 (2.times.300 mL) and saturated
NaCl (2.times.300 mL), dried over MgSO.sub.4 and evaporated to give
a residue (96 g). The residue was chromatographed on a 1.5 kg
silica column using EtOAc/Hexane (1:1) containing 0.5% Et.sub.3NH
as the eluting solvent. The pure product fractions were evaporated
to give 90 g (90%) of the title compound.
[0081]
N.sup.4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcyti-
dine-3'-amidite:
N.sup.4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5--
methylcytidine (74 g, 0.10 M) was dissolved in CH.sub.2Cl.sub.2 (1
L). Tetrazole diisopropylamine (7.1 g) and
2-cyanoethoxy-tetra(isopropyl)phos- phite (40.5 mL, 0.123 M) were
added with stirring, under a nitrogen atmosphere. The resulting
mixture was stirred for 20 hours at room temperature (tlc showed
the reaction to be 95% complete). The reaction mixture was
extracted with saturated NaHCO.sub.3 (1.times.300 mL) and saturated
NaCl (3.times.300 mL). The aqueous washes were back-extracted with
CH.sub.2Cl.sub.2 (300 mL), and the extracts were combined, dried
over MgSO.sub.4 and concentrated. The residue obtained was
chromatographed on a 1.5 kg silica column using
EtOAc.backslash.Hexane (3:1) as the eluting solvent. The pure
fractions were combined to give 90.6 g (87%) of the title
compound.
[0082] Nucleotide Sequences: Table 1 shows the sequence and
activity of each of the oligonucleotides evaluated for inhibition
of c-jun mRNA expression, and Table 2 shows the sequence and
activity of the oligonucleotides evaluated for inhibition of c-fos
mRNA expression. Oligonucleotide activities were evaluated as
described infra in Example 2 et seq. For the nucleotide sequence of
the human c-jun gene, see Hattori et al., Proc. Natl. Acad. Sci.
U.S.A., 1988, 85:9148, and Genbank accession No. J04111
("HUMJUNA"). The nucleotide sequence of the human c-fos gene is
described by Van Straaten et al., Proc. Natl. Acad. Sci. U.S.A.,
1983, 80:3183, and in Genbank accession No. K00650 ("HUMFOS").
1TABLE 1 Phosphorothioate Oligonucleotides Targeted to Human c-jun
TARGET ISIS # SEQUENCE SEQ ID NO: REGION % CONTROL* 10570
GCC-ACA-CTC-AGT-GCA-ACT-CT 1 5' Cap 62 10571
CGC-ACC-TCC-ACT-CCC-GCC-TC 2 5'-UTR 100 10572
ACC-AGC-CCG-GGA-GCC-ACA-GG 3 5'-UTR 39 10578
GCT-GCG-CCG-CCG-ACG-TGA-CG 4 ORF 37 10579
CGC-CCC-GCC-GCC-GCT-GCT-CA 5 ORF 41 10580
GTG-TCT-CGC-CGG-GCA-TCT-CG 6 ORF 19 10581
CCC-CCG-ACC-GTC-TCT-CTT-CA 7 tTR 24 10582
TCA-GCC-CCC-GAC-GGT-CTC-TC 8 3'-UTR 17 10583
TGC-CCC-TCA-GCC-CCC-GAC-GG 9 3'-UTR 20 13305 TGC-GGG-TGA-GTG-GTA-G
118 ORF N.D.** 10582 Controls: 10582 TCA-GCC-CCC-GAC-GGT-CTC-TC 8
3'-UTR 11562 GAG-AGA-CCG-TCG-GGC-GCT-GA 29 sense control 11563
CAC-CTC-CAC-GCG-CTT-CTG-GC 30 scrambled control 11564
TCG-GCA-CCT-GAA-GGA-CTT-TC 31 mismatch control *Contro1 is TPA
induction, at 1 hour, in A549 cells. **N.D., not determined.
[0083]
2TABLE 2 Phosphorothioate Oligonucleotides Targeted to Human c-fos
TARGET ISIS # SEQUENCE SEQ ID NO: REGION % CONTROL* 10628
TGC-TCG-CTG-CAC-ATG-CGG-TT 10 5' Cap 79 10629
CGG-TCA-CTG-CTC-GTT-CGC-TG 11 5'-UTR 72 10630
CAT-CGT-GGC-GGT-TAG-GCA-AA 12 tIR 91 10631
GAG-AAC-ATC-ATC-GTG-GCG-GT 13 tIR 118 10632
ACC-GTG-GGA-ATG-AAG-TTG-GC 14 ORF 63 10633
AGC-TCC-CTC-CTC-CGG-TTG-CG 15 ORF 24 10634
TTG-CAG-GCA-GGT-CGG-TGA-GC 16 ORF 42 10635
TGG-CAC-GGA-GCG-GGC-TGT-CT 17 ORF 12 10636
TGC-TGC-TGC-CCT-TGC-GGT-GG 18 ORF 42 10637
CCT-CAC-AGG-GCC-AGC-AGC-GT 19 tTR 35 10638
GGT-GCC-GGC-TGC-CTC-CCC-TT 20 3'-UTR 22 10639
AAG-TCC-TTG-AGG-CCC-ACA-GC 21 3'-UTR 9 10640
CCC-CTC-CAG-CAG-CTA-CCC-TT 22 3'-UTR 87 10641
TCC-CGT-CCC-CAG-AAG-CAG-TA 23 3'-UTR 68 10642
CGC-GCC-CGG-CCT-GAA-AAT-TT 24 3'-UTR 87 10643
CCT-GCC-TCG-GCC-TCC-CAA-AG 25 3'-UTR 39 10644
CCC-CCA-CTT-CCG-CCC-ACT-AT 26 3'-UTR 104 10645
TGG-TGC-CTG-CGT-GAT-ACT-CG 27 3'-UTR 56 10646
CCC-TCC-CAG-GCT-CAA-GTC-AT 28 3'-UTR 100 10639 Controls: 10639
AAG-TCC-TTG-AGG-CCC-ACA-GC 21 3'-UTR 11184
GCT-GTG-GGC-CTC-AAG-GAC-TT 32 sense control 11185
ATG-TGC-TAG-ATG-CGC-AAA-GT 33 mismatch control 11186
ACG-TCC-GAT-TCC-GAG-CGC-AA 34 scrambled control 11187
CAG-TGG-CCA-TCA-AAC-CCG-TG 35 scrambled control *Control is TPA
induction, at 1 hour, in A549 cells.
Example 2
[0084] Screening for Oligonucleotides that Modulate mRNA Expression
of the AP-1 Subunits c-fos and c-jun
[0085] In order to evaluate the activity of potential c-fos and
c-jun modulating oligonucleotides, A549 cells were grown in T-75
flasks until 80-90% confluent. (Cell line A549 is available from,
inter alia, the American Type Culture Collection, Rockville, Md.,
as ATCC No. CCL-185.) At this time, the cells were washed twice
with 10 mL of media (DMEM), followed by the addition of 5 mL of
DMEM containing 20 .mu.g/mL of LIPOFECTIN.TM. (i.e., DOTMA/DOPE
(N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trie- thylammonium
chloride/dioleoylphosphatidyl ethanolamine)). The oligonucleotides
were added from a 10 .mu.M stock solution to a final concentration
of 400 nM, and the two solutions were mixed by swirling the flasks.
After 4 hours at 37.degree. C., the medium was replaced with DMEM
containing 10% serum. At this point, 1 .mu.M
12-O-tetradecanoylphorbol 13-acetate (TPA) was added to induce
expression of c-fos and c-jun. Cells were extracted in guanidinium
one hour later, and the c-fos and c-jun mRNA expression was
determined by Northern blotting. Probes for human c-jun and c-fos
were PCR products prepared using primers based on the published
sequences thereof (respectively, Hattori et al., Proc. Natl. Acad.
Sci. U.S.A., 1988, 85:9148, and Van Straaten et al., Proc. Natl.
Acad. Sci. U.S.A., 1983, 80:3183).
[0086] As described in Table 1, for inhibiting c-jun mRNA
expression, ISIS 10580, 10581, 10582 and 10583 were most active
(81%, 76%, 83% and 80% inhibition, respectively). Treatment of
cells with these oligonucleotides reduced c-jun expression to 19%,
24%, 17% and 20% (81%, 76%, 83% and 80% inhibition), respectively,
of the level seen in control experiments (100% expression, 0%
inhibition). These oligonucleotides effect significant inhibition
of c-jun and are therefor preferred. Basal levels of c-jun mRNA are
typically about 30% of the control value; ISIS 10572, 10578 and
10579 reduce c-jun levels to near basal levels (39%, 37% and 41%,
respectively) and are thus also preferred.
[0087] As described in Table 2, the oligonucleotides most effective
in reducing c-fos mRNA expression are ISIS 10633, 10635, 10638 and
10639. Treatment of cells with these oligonucleotides reduced c-fos
expression to 24%, 12%, 22% and 9% (76%, 88%, 78% and 91%
inhibition), respectively, of the level seen in control experiments
(100% expression, 0% inhibition); basal levels of c-fos mRNA are
typically about 3% of the control value. These oligonucleotides
effect significant inhibition of c-fos and are therefor
preferred.
Example 3
[0088] Dose Response and Specificity of Oligonucleotides Targeted
to AP-1 Subunits
[0089] Dose-response experiments were performed at different
oligonucleotide concentrations to determine the potency (i.e.,
ability to decrease expression of the appropriate mRNA target) of
the most active compounds identified in the initial screen (Tables
3 and 4). The decreases in target mRNA expression effected by ISIS
10582 (c-jun) and ISIS 10639 (c-fos) are dose-dependent, as shown
in Tables 3 and 4, respectively.
3TABLE 3 Dose-Response to Oligonucleotides Targeted to c-jun
OLIGONUCLEOTIDE c-jun mRNA LEVELS TREATMENT CONCENTRATION (%
CONTROL) None (basal level) -- 31 Control* -- 100 TPA + ISIS 10582
50 nM 72 TPA + ISIS 10582 100 nM 45.5 TPA + ISiS 10582 200 nM 29.5
TPA + ISIS 10582 400 nM 16 *Control is TPA induction, at 1 hour, in
A549 cells.
[0090]
4TABLE 4 Dose-Response to Oligonucleotides Targeted to c-fos
OLIGONUCLEOTIDE c-fos mRNA LEVELS TREATMENT CONCENTRATION (%
CONTROL) None (basal level) -- 3 Control* -- 100 TEA +ISIS 10639 50
nM 64 TEA +ISiS 10639 100 nM 46 TEA +ISIS 10639 200 nM 20.5 TEA
+ISIS 10639 400 nM 9 *Control is TPA induction, at 1 hour, in A549
cells.
[0091] The specificity of the oligonucleotide-mediated inhibition
of c-fos and c-jun mRNA expression was further examined by
determining the effects of scrambled, 6- or 7-base mismatch and
sense control versions of the most active oligonucleotides, ISIS
10582 (c-jun) and ISIS 10639 (c-fos). As can be seen in Table 5,
ISIS 10582 exhibited potent and specific inhibition of c-jun mRNA
expression, but ISIS 11562 (sense version of ISIS 10582; SEQ ID
NO:29), ISIS 11564 (6 base pair mismatch version of ISIS 10639; SEQ
ID NO:31) and ISIS 11563 (scrambled version of ISIS 10639; SEQ ID
NO:30) had no detectable effect on c-jun mRNA levels during TPA
induction (the sequences of ISIS 11562-11564 are given in Table
1).
[0092] As can further be seen in Table 5, ISIS 10639 exhibited
potent and specific inhibition of c-fos mRNA expression, but ISIS
11184 (sense version of ISIS 10639; SEQ ID NO:32), ISIS 11185 (7
base pair mismatch version of ISIS 10639; SEQ ID NO:33) and ISIS
11186 (scrambled version of ISIS 10639; SEQ ID NO:34) had no
detectable effect on c-fos mRNA levels during TPA induction (the
sequences of ISIS 11184-11186 are given in Table 2). Finally, it
can also be seen from Table 5 that neither active oligonucleotide
has any detectable effect on mRNA levels of the other active
oligonucleotide's target. That is, ISIS 10639, targeted to c-fos,
has no detectable effect on c-jun levels; similarly, ISIS 10582,
targeted to c-jun, has no detectable effect on c-fos levels.
5TABLE 5 Specificity of c-fos and c-jun Oligonucleotides Treatment
c-fos c-jun Basal 5 23 TPA - no oligo 100 100 10639: c-fos active 9
97 11184: c-fos sense 84 91 11185: c-fos mismatch 93 98 11186:
c-fos scrambled 98 99 10582: c-jun active 91 4 11562: c-jun sense
89 87 11563: c-jun scrambled 99 93 11564: c-jun mismatch 99 71
[0093] These results demonstrate that ISIS 10582 effects potent and
specific modulation (i.e., inhibition) of c-jun mRNA levels and
that ISIS 10639 effects potent and specific modulation of c fos
mRNA levels.
Example 4
[0094] Effect of Oligonucleotides Targeted to an AP-1 Subunit on
Human Tumor Growth in Nude Mice
[0095] In order to evaluate the in vivo activity of c-fos
oligonucleotides, 25 mg of tumor fragments of A549 tumors were
implanted subcutaneously in nude mice (n=6). ISIS 10639 was
administered daily, i.v., for three weeks. The oligonucleotide
dosage was 25 mg/kg. Tumor size was recorded weekly, and the
results are shown in Table 6. A substantial reduction in tumor
growth rate was obtained upon treatment with ISIS 10639. By day 34,
saline-treated tumors were 0.56.+-.0.12 g by weight, while tumors
treated with ISIS 10639 were 0.31.+-.0.1 g by weight.
6TABLE 6 Response of Transplanted Tumors in Mice to
Oligonucleotides Targeted to c-jun Mean Tumor Treatment/Time Weight
(g) Std. Dev. Std. Error Saline: Day 17 0.113 0.033 0.014 Day 20
0.177 0.045 0.019 Day 27 0.272 0.086 0.035 Day 34 0.560 0.293 0.120
ISIS 10639: Day 17 0.105 0.035 0.014 Day 20 0.138 0.074 0.030 Day
27 0.225 0.070 0.028 Day 34 0.310 0.104 0.042
Example 5
[0096] Effect of Oligonucleotides on Protein Levels of AP-1
Subunits
[0097] The ability of the c-fos active oligonucleotide ISIS 10639
to reduce expression of the c-Fos protein was examined as follows.
A549 cells were treated with oligonucleotides as in Examples 2-3,
except that induction of c-Fos was effected by treatment of cells
with TPA (1 uM) for three hours. At this time, whole cell protein
was extracted in SDS (sodium dodecyl sulfate) buffer. Samples of
extracts were electrophoresed, transferred to nitrocellulose
filters which were immunoblotted using a c-Fos-specific antibody
(Santa Cruz AB, Santa Cruz, Calif.). The results (Table 7)
demonstrate that treatment of cells with the c-fos antisense
oligonucleotide results in basal levels of c-Fos protein.
7TABLE 7 Effect of c-fos Oligonucleotides on c-Fos Protein Levels
Treatment c-Fos Basal 21 TPA - no oligo 100 10639: c-fos active 19
11184: c-fos sense 97 11185: c-fos mismatch 91 11186: c-fos
scrambled 99
Example 6
[0098] Modified Oligonucleotides and PNA Antisense Analogs to Human
AP-1 Subunits
[0099] Once oligonucleotides that modulate c-Fos or c-Jun are
identified, derivative or modified oligonucleotides having the same
sequence thereas are prepared. In order to evaluate the effect of
chemical modifications to oligonucleotides to c-fos and c-jun, the
modified oligonucleotides described in Tables 8 and 9 were
prepared. The effect of the c-fos-targeted oligonucleotides on
c-fos RNA levels were evaluated as described in Examples 2-3. The
results (Table 10) demonstrate that some enhancement of c-fos
modulation can be achieved by the use modifications such as, e.g.,
2'-fluoro (ISIS 11200). Other modified oligonucleotides of the
invention are evaluated in like fashion. In order to evaluate the
effect of PNA antisense analogs, the PNA analogs of the invention
are introduced into appropriate cell lines by microinjection
according to the method of Hanvey et al. (Science, 1992, 258:1481).
Intracellular delivery of PNA analogs is confirmed by the use of a
fluorescently tagged PNA antisense analog conjugate such as, e.g.,
ISIS 14240.
8TABLE 8 Additional Oligonucleotides and PNA Antisense Analogs
Targeted to Human AP-1 Subunits SEQ Oligonucleotide Sequence
(5'->3') ID ISIS # Target and Chemical Modifications* NO: C-JUN:
10570 & Derivatives: 10570 c-jun, 5' cap
G.sup.SC.sup.SC.sup.SA.sup.SC.sup-
.SA.sup.SC.sup.ST.sup.SC.sup.SA.sup.SG.sup.ST.sup.SG.sup.SC.sup.SA.sup.SA.-
sup.SC.sup.ST.sup.SC.sup.ST P=S 1 13306 c-jun, 5' cap
C.sup.SA.sup.SC.sup.ST.sup.SC.sup.SA.sup.SG.sup.ST.sup.SG.sup.SC.sup.SA.s-
up.SA.sup.SC.sup.ST.sup.SC.sup.ST P=S 36 13297 c-jun, 5' cap
C.sup.SA.sup.SC.sup.ST.sup.SC.sup.SA.sup.SG.sup.ST.sup.SG.sup.SC.sup.-
SA.sup.SA.sup.SC.sup.ST.sup.SChu ST P=S/2'MO 36 12166 c-jun, 5' cap
C.sup.NA.sup.NC.sup.NT.sup.NC.sup.NA.sup.NG.sup.NT.sup.NG.s-
up.NC.sup.NA.sup.NA.sup.NC.sup.NT.sup.NC.sup.NT.sup.N PNA (N) 36
13699 c-jun, 5' cap C.sup.NA.sup.NC.sup.NT.sup.KC.sup.NA.sup.NG.su-
p.NT.sup.KG.sup.NC.sup.NA.sup.NA.sup.NC.sup.NT.sup.KC.sup.NT.sup.K
PNA (N)/Lys (K) 36 10579 & Derivatives: 10579 c-jun, ORF
C.sup.SG.sup.SC.sup.SC.sup.SC.sup.SC.sup.SG.sup.SC.sup.SC.sup.SG.sup.-
SC.sup.SC.sup.SG.sup.SC.sup.ST.sup.SG.sup.SC.sup.ST.sup.SC.sup.SA
P=S 5 11567 c-jun, ORF Chu SG.sup.SC.sup.SC.sup.SC.sup.SC.sup.SG.-
sup.SC.sup.SC.sup.SG.sup.SC.sup.SC.sup.SG.sup.SC.sup.ST.sup.SG.sup.SC.sup.-
ST.sup.SC.sup.SA 2'F 5 10571 & Derivatives: 10571 c-jun, 5'-UTR
C.sup.SG.sup.SC.sup.SA.sup.SC.sup.SC.sup.ST.sup.SC.sup.SC.s-
up.SA.sup.SC.sup.ST.sup.SC.sup.SC.sup.SC.sup.SG.sup.SC.sup.SC.sup.ST.sup.S-
C P=S 2 11568 c-jun, 5'-UTR C.sup.SG.sup.SC.sup.SA.sup.SC-
.sup.SC.sup.ST.sup.SC.sup.SC.sup.SA.sup.SC.sup.ST.sup.SC.sup.SC.sup.SC.sup-
.SG.sup.SC.sup.SC.sup.ST.sup.SC 2'F 2 13307 c-jun, 5'-UTR
C.sup.SC.sup.ST.sup.SC.sup.SC.sup.SA.sup.SC.sup.ST.sup.SC.sup.SC.sup.SC.s-
up.SG.sup.SC.sup.SC.sup.ST.sup.SC P=S 37 13296 c-jun, 5'-UTR
C.sup.SC.sup.ST.sup.SC.sup.SC.sup.SA.sup.SC.sup.ST.sup.SC.sup.SC.s-
up.SC.sup.SG.sup.SC.sup.SC.sup.ST.sup.SC P=S/2'MO 37 12167 c-jun,
5'-UTR C.sup.NC.sup.NT.sup.NC.sup.NC.sup.NA.sup.NC.sup.NT.sup.NC.s-
up.NC.sup.NC.sup.NG.sup.NC.sup.NC.sup.NT.sup.NC.sup.N PNA (N) 37
13698 c-jun, 5'-UTR C.sup.NC.sup.NT.sup.KC.sup.NC.sup.NA.sup.NC.su-
p.NT.sup.KC.sup.NC.sup.NC.sup.NG.sup.NC.sup.NC.sup.NT.sup.KC.sup.N
PNA (N)/Lys (K) 37 10582 & Derivatives: 10582 c-jun, 3'-UTR
T.sup.SC.sup.SA.sup.SG.sup.SC.sup.SC.sup.SC.sup.SC.sup.SC.sup.SG.s-
up.SA.sup.SC.sup.SG.sup.SG.sup.ST.sup.SC.sup.ST.sup.SC.sup.ST.sup.SC
P=S 8 11569 c-jun, 3'-UTR T.sup.SC.sup.SA.sup.SG.sup.SC.sup.SC-
.sup.SC.sup.SC.sup.SC.sup.SG.sup.SA.sup.SC.sup.SG.sup.SG.sup.ST.sup.SC.sup-
.ST.sup.SC.sup.ST.sup.SC 2'F 8 11537 c-jun, 3'-UTR
T.sup.SC.sup.SA.sup.SG.sup.SC.sup.SC.sup.SC.sup.SC.sup.SC.sup.SG.sup.SA.s-
up.SC.sup.SG.sup.SG.sup.ST.sup.SC.sup.ST.sup.SC.sup.ST.sup.SC
2'propoxy- 8 14314 c-jun, 3'-UTR
T.sup.SC.sup.SA.sup.SG.sup.SC.sup.SC.-
sup.SC.sup.SC.sup.SC.sup.SG.sup.SA.sup.SC.sup.SG.sup.SG.sup.ST.sup.SC.sup.-
ST.sup.SC.sup.ST.sup.SC 2'ME 8 C-FOS: 10639 & Derivatives:
10639 c-fos, 3'-UTR A.sup.SA.sup.SG.sup.ST.sup.SC.su-
p.SC.sup.ST.sup.ST.sup.SG.sup.SA.sup.SG.sup.SG.sup.SC.sup.SC.sup.SC.sup.SA-
.sup.SC.sup.SA.sup.SG.sup.SC P=S 21 11200 c-fos, 3'-UTR
A.sup.SA.sup.SG.sup.ST.sup.SC.sup.SC.sup.ST.sup.ST.sup.SG.sup.SA.sup.SG.s-
up.SG.sup.SC.sup.SC.sup.SChu SA.sup.SC.sup.SA.sup.SG.sup.SC 2'MO 21
11538 c-fos, 3'-UTR A.sup.SA.sup.SG.sup.ST.sup.SC.sup.SC.sup.ST.-
sup.ST.sup.SG.sup.SA.sup.SG.sup.SG.sup.SC.sup.SC.sup.SC.sup.SA.sup.SC.sup.-
SA.sup.SG.sup.SC 2'propoxy- 21 14315 c-fos, 3'-UTR
A.sup.SA.sup.SG.sup.ST.sup.SC.sup.SC.sup.ST.sup.ST.sup.SG.sup.SA.sup.SG.s-
up.SG.sup.SC.sup.SC.sup.SC.sup.SA.sup.SC.sup.SA.sup.SG.sup.SC 2'ME
21 13298 & Derivatives: 13298 c-fos, ORF
T.sup.SG.sup.SC.sup.SG.sup.SG.sup.SG.sup.ST.sup.SG.sup.SA.sup.SG.sup.ST.s-
up.SG.sup.SG.sup.ST.sup.SA.sup.SG 2'ME 120 12165 c-fos, ORF
T.sup.NG.sup.NC.sup.NG.sup.NG.sup.NG.sup.NT.sup.NG.sup.NA.sup.NG.sup.-
NT.sup.NG.sup.NG.sup.NT.sup.NA.sup.NG.sup.N PNA (N) 120 13646
c-fos, ORF
T.sup.NG.sup.NC.sup.NG.sup.NG.sup.NG.sup.NT.sup.KG.sup.N-
A.sup.NG.sup.NT.sup.KG.sup.NG.sup.NT.sup.KA.sup.NG.sup.N PNA
(N)/Lys (K) 120 10628 & Derivatives: 10628 c-fos, 5' cap
T.sup.SG.sup.SC.sup.ST.sup.SC.sup.SG.sup.SC.sup.ST.sup.SG.sup.SC.sup.SA.s-
up.SG.sup.SA.sup.ST.sup.SG.sup.SC.sup.SG.sup.SG.sup.ST.sup.ST P=S 1
13302 c-fos, 5' cap C.sup.SG.sup.SC.sup.ST.sup.SG.sup.SC.sup.SA-
.sup.SG.sup.SA.sup.ST.sup.SG.sup.SC.sup.SG.sup.SG.sup.ST.sup.ST P=S
121 13301 c-fos, 5' cap C.sup.SG.sup.SC.sup.ST.sup.SG.sup.SC.sup-
.SA.sup.SG.sup.SA.sup.ST.sup.SG.sup.SC.sup.SG.sup.SG.sup.ST.sup.ST
P=S, 2'MO 121 12162 c-fos, 5' cap C.sup.NG.sup.NC.sup.NT.sup.N-
G.sup.NC.sup.NA.sup.NG.sup.NA.sup.NT.sup.NG.sup.NC.sup.NG.sup.NG.sup.NT.su-
p.NT PNA (N) 121 13643 c-fos, 5' cap
C.sup.NG.sup.NC.sup.NT.sup.KG.sup.NC.sup.NA.sup.NG.sup.NA.sup.NT.sup.KG.s-
up.NC.sup.NG.sup.NG.sup.NT.sup.KT PNA (N)/Lys (K) 121 13303 &
Derivatives: 13303 c-fos, 5'-UTR C.sup.SC.sup.SG.sup.SC.s-
up.SC.sup.SG.sup.SG.sup.SC.sup.ST.sup.SC.sup.SA.sup.SG.sup.ST.sup.SC.sup.S-
T.sup.ST P=S 122 13300 c-fos, 5'-UTR
C.sup.SC.sup.SG.sup.SC.sup.SC.sup.SG.sup.SG.sup.SC.sup.ST.sup.SC.sup.SA.s-
up.SG.sup.ST.sup.SC.sup.ST.sup.ST P=S, 2'MO 122 12163 c-fos, 5'-UTR
C.sup.NC.sup.NG.sup.NC.sup.NC.sup.NG.sup.NG.sup.NC.sup.NT.s-
up.NC.sup.NA.sup.NG.sup.NT.sup.NC.sup.NT.sup.NT.sup.N PNA (N) 122
13644 c-fos, 5'-UTR C.sup.NC.sup.NG.sup.NC.sup.NC.sup.NG.sup.NG.su-
p.NC.sup.NT.sup.KC.sup.NG.sup.NT.sup.KC.sup.NT.sup.KT.sup.K PNA
(N)/Lys (K) 122 13304 & Derivatives: 13304 c-fos, tIR
C.sup.SA.sup.ST.sup.SC.sup.SG.sup.ST.sup.SG.sup.SG.sup.SC.sup.SG.sup.SG.s-
up.ST.sup.ST.sup.SA.sup.SG.sup.SG P=S 123 13299 c-fos, tIR
C.sup.SA.sup.ST.sup.SC.sup.SG.sup.ST.sup.SG.sup.SG.sup.SC.sup.SG.sup.-
SG.sup.ST.sup.ST.sup.SA.sup.SG.sup.SG P=S, 2'MO 123 12164 c-fos,
tIR C.sup.NA.sup.NT.sup.NC.sup.NG.sup.NT.sup.NG.sup.NG.sup.NC.sup.-
NG.sup.NG.sup.NT.sup.NT.sup.NA.sup.NG.sup.NG.sup.N PNA (N) 123
13700 c-fos, tIR
C.sup.KA.sup.NT.sup.KC.sup.KG.sup.NT.sup.KG.sup.NG.s-
up.NC.sup.KG.sup.NG.sup.NT.sup.KT.sup.KA.sup.NG.sup.NG.sup.K PNA
(N)/Lys (K) 123 14240 c-fos, tIR C.sup.KA.sup.NT.sup.KC.sup.KG.su-
p.NT.sup.KG.sup.NG.sup.NC.sup.KG.sup.NG.sup.NT.sup.KT.sup.KA.sup.NG.sup.NG-
.sup.K PNA (N)/Lys (K)/5'FITC 123 *Phosphorothioate linkages are
indicated by ".sup.S" and "P = S"; emboldened residues indicate the
additional indicated modifications: 2'F = 2'-fluoro-; 2'propoxy =
2'-propoxy-; 2'MO = 2'-methoxy-; 2'ME = 2'-methoxyethoxy-; PNA =
peptide (polyamide) nucleic acid backbone having a side chain
corresponding to that of either glycine (N) or D-Lys (K); 5'FITC =
5'-fluorescein isothiocyanate.
[0100]
9TABLE 9 5-Methyl-Cytosine, Fully 2'-Methoxyethoxy-Oligonucleotides
Targeted to the 3'-UTR of Human c-fos ISIS SEQ ID NO. SEQUENCE NO.
15828
C.sup.OC.sup.OA.sup.OT.sup.OC.sup.OT.sup.OT.sup.OA.sup.OA.sup.OT.sup.OA.s-
up.OA.sup.OA.sup.OT.sup.OA.sup.OA.sup.OA.sup.OT.sup.OT.sup.OA.sup.OA.sup.O-
A.sup.OA.sup.OA.sup.OC.sup.OA.sup.OC.sup.OA.sup.OA.sup.OT 14660
A.sup.OA.sup.OA.sup.OT.sup.OA.sup.OA.sup.OA.sup.OT.sup.OT.sup.OA.-
sup.OA.sup.OA.sup.OA.sup.OA.sup.OC.sup.OA.sup.OC.sup.OA.sup.OA.sup.OT
2,6-A* 14659 A.sup.OA.sup.OT.sup.OT.sup.OA.sup.OA.sup.OA.-
sup.OA.sup.OA.sup.OC.sup.OA.sup.OC.sup.OA.sup.OA.sup.OT.sup.OA.sup.OA.sup.-
OA.sup.OA.sup.OC 2,6-A 15829 A.sup.OT.sup.OA.sup.OT.sup.OA-
.sup.OA.sup.OA.sup.OT.sup.OA.sup.OT.sup.OC.sup.OT.sup.OG.sup.OA.sup.OG.sup-
.OA.sup.OA.sup.OT.sup.OC.sup.OC 14662
A.sup.OT.sup.OA.sup.OT.sup.OA.sup.OA.sup.OA.sup.OT.sup.OA.sup.OT.sup.OC.s-
up.OT.sup.OG.sup.OA.sup.OG.sup.OA.sup.OA.sup.OT.sup.OC.sup.OC 2,6-A
14661 A.sup.OT.sup.OC.sup.OT.sup.OG.sup.OA.sup.OG.sup.OA.sup.OA.-
sup.OT.sup.OC.sup.OC.sup.OA.sup.OT.sup.OC.sup.OT.sup.OT.sup.OA.sup.OA.sup.-
OT 2,6-A 14663** A.sup.OA.sup.OA.sup.OT.sup.OA.sup.OT.sup.-
OA.sup.OA.sup.OA.sup.OT.sup.OA.sup.OT.sup.OC.sup.OT.sup.OG.sup.OA.sup.OG.s-
up.OA.sup.OA.sup.OT 2,6-A 14664 A.sup.OA.sup.OG.sup.OA.sup-
.OC.sup.OC.sup.O7.sup.OC.sup.OA.sup.OA.sup.OG.sup.O3.sup.OT.sup.OA.sup.OG.-
sup.OA.sup.OA.sup.OA.sup.OA.sup.OA *Emboldened residues indicate
2,6-A residues, i.e., those having 2,6-diaminopurine as a
nucleobase. **ISIS 14663 is a 2'-deoxy- rather than a
2'-methoxyethoxy-oligonucleotide.
[0101]
10TABLE 10 Effect of Modified c-fos Oligonucleotides on c-fos
Expression Treatment c-fos Basal 3 TPA - no oligo 100 10639: c-fos
active, P = S 9 11200: c-fos, 2'-fluoro- 5 11538: c-fos,
2'-propoxy- 15
Example 7
[0102] Oligonucleotides to Mouse AP-1 Subunits
[0103] Tables 11 and 12 show the sequences of oligonucleotides
designed to modulate mouse c-jun and c-fos mRNA expression,
respectively. For the nucleotide sequence of the mouse c-jun gene,
see Genbank accession No. J04115/MUSCJUN and Ryder et al., Proc.
Natl. Acad. Sci. U.S.A., 1988, 85:8464. The nucleotide sequence of
the mouse c-fos gene is described in Genbank accession No.
J00370/MUSFOS and by Van Beveren et al., Cell, 1983, 32:1241.
Oligonucleotide activities are evaluated as described infra in
Example 2 et seq. with the exception that mouse Swiss 3T3 cells
(available from, inter alia, the American Type Culture Collection,
Rockville, Md., as ATCC No. CCL-92) are used instead of human A549
cells. Due to the high degree of homology between human and murine
c-jun and c-fos nucleotide sequences (Van Straaten et al., Proc.
Natl. Acad. Sci. U.S.A., 1983, 80:3183), probes derived from the
human genes were used to detect murine messages.
11TABLE 11 Phosphorothioate Oligonucleotides Targeted to Mouse
c-jun 1 ISIS # SEQUENCE SEQ ID NO: TARGET REGION* 12292
CTC-GCC-CAA-CTT-CAG-CCG-CC 38 5'-UTR: 434-453 12293
CCA-GTC-CCA-GCA-ACA-GCG-GC 39 5'-UTR: 588-607 12294
GCA-ACA-GCG-CGC-CGG-GAA-GC 40 5'-UTR: 838-857 12295
CCG-GCG-ACG-CCA-GCT-TGA-GC 41 ORF: 1120-1139 12296
GGC-TGT-GCC-GCG-GAG-GTG-AC 42 ORF: 1304-1323 12297
CGC-CCC-ACC-GCC-GCT-GCT-CA 43 ORF: 1458-1477 12298
AGC-CCG-GCC-GCG-CCA-TAG-GA 44 ORF: 1481-1500 12299
CTG-CAC-CGG-GAT-CTG-TTG-GG 45 ORF: 1560-1579 12300
GGC-GGC-GTC-TCT-CCC-GGC-ATC-TC 46 ORF: 1625-1647 12301
TGG-AGG-CGG-CAA-TGC-GGT-TC 47 ORF: 1708-1727 12302
CCC-TGA-GCA-TGT-TGG-CCG-TG 48 ORF: 1813-1832 12303
CAA-AGC-CAG-GCG-CGC-CAC-GT 49 3'-UTR: 2096-2115 12304
TTG-AGA-GAG-GCA-GGC-CAG-GG 50 3'-UTR: 2388-2407 12305
TGG-ACT-TGT-GTG-TTG-CCG-GG 51 3'-UTR: 2807-2826 12306
TCC-ATG-GGT-CCC-TGC-TTT-GA 52 3'-UTR: 2999-3018 12321
TGG-TCG-CGC-GCG-GGC-ACA-GC 53 3'-UTR: 2166-2185 *Nucleotide
co-ordinates from Genbank accession No. J04115/MUSCJUN.
[0104]
12TABLE 12 Phosphorothioate Oligonucleotides Targeted to Mouse
c-fos ISIS # SEQUENCE SEQ ID NO: TARGET REGION* 11249
AGC-TCC-CTC-CTC-CGA-TTC-CG 54 ORF: 1905-1924 11250
GCT-CTG-TGA-CCA-TGG-GCC-CC 55 ORF: 2498-2517 12307
GAA-CCG-CCG-GCT-CTA-TCC-AG 56 5'-UTR: 164-183 12308
GCC-CCT-GCG-ACT-CAC-ACC-CC 57 ORF: 485-504 12309
TAA-GGC-TGC-TCT-GAC-CGC-GC 58 ORF: 541-560 12310
CGC-CCG-CAG-CAC-CCT-CCT-CC 59 ORF: 804-823 12311
CAG-GCG-CTG-CTC-CGG-AGT-CT 60 ORF: 868-887 12312
TCC-CTT-GAA-TTC-CGC-AGC-GC 61 ORF: 989-1008 12313
AGC-GGA-GGT-GAG-CGA-GGA-GG 62 ORF: 1136-1155 12314
CCC-CAG-CCC-ACA-AAG-GTC-CA 63 ORF: 1445-1464 12315
TGC-TCA-AGG-ACC-CTG-CGC-CC 64 ORF: 2001-2020 12316
GGG-AAG-CCA-AGG-TCA-TCG-GG 65 ORF: 2178-2197 12317
TGC-TGC-TGC-CCT-TTC-GGT-GG 66 ORF: 2630-2649 12318
CTG-GAT-GCC-GGC-TGC-CTT-GC 67 3'-UTR: 2716-2735 12319
CAG-CTC-GGG-CAG-TGG-CAC-GT 68 3'-UTR: 2736-2755 12320
GGA-ACA-CGC-TAT-TGC-CAG-GA 69 3'-UTR: 3138-3157 *Nucleotide
co-ordinates from Genbank accession No. J00370/MUSFOS.
Example 8
[0105] Oligonucleotides to Rat AP-1 Subunits
[0106] Tables 13 and 14 show the sequences of oligonucleotides
designed to modulate rat c-jun and c-fos mRNA expression,
respectively. For the nucleotide sequence of the rat c-jun gene,
see Genbank accession No. X17163/RSJUNAP1 and Saaki et al., Cancer
Res., 1989, 49:5633. The nucleotide sequence of the rat c-fos gene
is described in Genbank accession No. X06769/RNCFOSR and Curran et
al., Oncogene, 1987, 2:79. Oligonucleotide activities were
evaluated as described infra in Example 2 et seq. with the
exception that rat A10 cells (available from, inter alia, the
American Type Culture Collection, Rockville, Md., as ATCC No.
CRL-1476) were used instead of human A549 cells. Due to the high
degree of homology between human and rat c-jun and c-fos nucleotide
sequences, probes derived from the human genes were used to detect
the rat messages.
[0107] ISIS 12633 (SEQ ID NO:78), a 20-mer phosphorothioate
oligonucleotide complementary to a portion of the 3' UTR of rat
c-jun mRNA, was selected as an active modulator of c-jun for
further studies. Another preferred oligonucleotide targeted to rat
AP-1 subunits is ISIS 12635 (SEQ ID NO:80).
Example 9
[0108] Modified Oligonucleotides to Rat AP-1 Subunits
[0109] Tables 15 and 16 show the sequences and chemical
modifications of second generation oligonucleotides designed to
modulate mouse c-jun and c-fos mRNA expression. The activities of
these modified oligonucleotide are evaluated as described infra in
Example 8.
13TABLE 13 Phosphorothioate Oligonucleotides Targeted to Rat c-jun
ISIS # SEQUENCE SEQ ID NO: TARGET REGION* 12624
CGG-CGG-CGC-AGA-CCA-GTC-GT 70 5'-UTR: 2-21 12625
GCC-GCG-GGA-CCA-GCC-CCA-GC 119 5'-UTR: 35-54 12626
GGC-ATC-GTC-GTA-GAA-GGT-CG 71 5'-UTR: 20-39 12627
GGA-GGT-GCG-GCT-TCA-GAT-TG 72 ORF: 493-512 12623
CCC-TCC-TGC-TCG-TCG-GTC-AC 73 5'-UTR: 307-326 12629
ACT-GAC-TGG-TTG-TGC-CGC-GG 74 ORF: 747-766 12630
CGC-TGT-AGC-CGC-CGC-CGC-CG 75 ORF: 814-833 12631
CCT-TGA-TCC-GCT-CCT-GAG-AC 76 ORF: 1105-1124 12632
GCC-AGC-TCG-GAG-TTT-TGC-GC 77 ORF: 1226-1245 12633
TTT-TCT-TCC-ACT-GCC-CCT-CA 78 3'-UTR: 1375-1394 12634
CCC-TTG-GCT-TCA-GTA-CTC-GG 79 3'-UTR: 1451-1470 12635
CTT-CCC-ACT-CCA-GCA-CAT-TG 80 3'-UTR: 1509-1528 12636
GCA-CAG-CCC-GTT-CGC-AAA-GC 81 3'-UTR: 1584-1603 12637
AAT-GCA-GCA-GAG-AGG-TTG-GG 82 3'-UTR: 2089-2108 12638
GAC-GGG-AGG-GAC-TAC-AGG-CT 83 3'-UTR: 2168-2187 12639
TCT-GGA-CTT-GTG-GGT-TGC-TG 84 3'-UTR: 2240-2259 12640
TAA-ACG-ATC-ACA-GCG-CAT-GC 85 3'-UTR: 2375-2394 12628 controls:
12628 CCC-TCC-TGC-TCG-TCG-GTC-AC 73 5'-UTR 12893
GGG-AGG-ACG-AGC-AGC-CAG-TG 86 reverse sense control 12894
CCC-GGC-CTT-TTG-ACC-GCC-TC 87 scrambled control 12895
CCG-CCT-CCC-CGG-CCT-TTT-GA 88 scrambled control 12896
CCG-TCG-TGG-TCC-TCC-GTG-AC 89 mismatch control 12992
GTG-ACC-GAC-GAG-CAG-GAG-GG 90 sense control 12633 controls: 12633
TTT-TCT-TCC-ACT-GCC-CCT-CA 78 3'-UTR 12897
AAA-AGA-AGG-TGA-CGG-GGA-GT 91 reverse sense control 12898
TTC-TCT-TTT-AGC-CTC-CCC-CA 92 scrambled control 12899
TCC-CCC-ATT-CTC-TTT-TAG-CC 93 scrambled control 12900
TTA-TCA-TCG-ACA-GCG-CCA-CA 94 mismatch control 12993
TGA-GGG-GCA-GTG-GAA-GAA-AA 95 sense control *Nucleotide
co-ordinates from Genbank accession No. X17163/RSJUNAP1.
[0110]
14TABLE 14 Phosphorothioate Oligonucleotides Targeted to Rat c-fos
ISIS # SEQUENCE SEQ ID NO: TARGET REGION* 11124
GTT-CTC-GGC-TCC-GCC-GGC-TC 96 5'-UTR: 22-41 11245
CAT-CAT-GGT-CGT-GGT-TTG-GG 97 tIR: 122-140 12246
TCC-GCG-TTG-AAA-CCC-GAG-AA 98 ORF: 141-160 12247
TGG-GCT-GGT-GGA-GAT-GGC-TG 99 ORF: 328-347 12248
CGA-TGC-TCT-GCG-CTC-TGC-CG 100 ORF: 485-504 12251
TTC-GGT-GGG-CAG-CTG-CGC-AG 101 ORF: 1193-1212 12252
CAG-GGC-TAG-CAG-TGT-GGG-CG 102 tTR: 1255-1274 12253
CCA-GCT-CAG-TCA-GTG-CCG-GC 103 3'-UTR: 1299-1318 12254
TCT-ACG-GGA-ACC-CCT-CGA-GG 104 3'-UTR: 1348-1367 12255
CTC-CAT-GCG-GTT-GCT-TTT-GA 105 3'-UTR: 1518-1537 12256
CAG-GCC-TGG-CTC-ACA-TGC-TA 106 3'-UTR: 1576-1595 11254 controls:
11254 TCT-ACG-GGA-ACC-CCT-CGA-GG 104 3'-UTR 12698
AGA-TGC-CCT-TGG-GGA-GCT-CC 107 reverse sense control 12699
TGA-CTA-TAG-ACC-GCC-GCC-GG 108 scrambled control 12700
CCG-CCG-GTG-ACT-ATA-GAC-CG 109 scrambled control 12726
TCA-ACC-GGT-ACG-CCA-CGT-GG 110 mismatch control 12990
CCT-CGA-GGG-GTT-CCC-GTA-GA 111 sense control 11256 controls: 11256
CAG-GCC-TGG-CTC-ACA-TGC-TA 106 12701 GTC-CGG-ACC-CAG-TGT-ACG-AT 112
reverse sense control 12702 GCG-CAC-CGT-CAT-TAC-GTC-CA 113
scrambled control 12703 ACG-TCG-AGC-GCA-CCG-TCA-TT 114 scrambled
control 12729 CAC-GCG-TGC-CTG-ACT-TGG-TA 115 mismatch control 12991
TAG-CAT-GTG-AGC-CAG-GCC-TG 116 sense control *Nucleotide
co-ordinates from Genbank accession No. X06769/RNCFOSR (see also
Curran et al., Oncogene, 1987, 2:79)
[0111]
15TABLE 15 Additional Oligonucleotides Targeted to Rat c-jun
Oligonucleotide Sequence (5'->3') SEQ ID ISIS # Target and
Chemical Modifications* NO: 12633 & Derivatives: 12633 3'-UTR
T.sup.ST.sup.ST.sup.ST.sup.SC.sup.ST.su-
p.ST.sup.SC.sup.SC.sup.SA.sup.SC.sup.ST.sup.SG.sup.SC.sup.SC.sup.SC.sup.SC-
.sup.ST.sup.SC.sup.SA 78 13047 3'-UTR
T.sup.OT.sup.OT.sup.OT.sup.OC.sup.OT.sup.ST.sup.SC.sup.SC.sup.SA.sup.SC.s-
up.ST.sup.SG.sup.SC.sup.SC.sup.OC.sup.OC.sup.OT.sup.OC.sup.OA.sup.d
2'MO 78 13714 3'-UTR T.sup.OT.sup.OT.sup.OT.sup.OC.sup.OT.sup.-
ST.sup.SC.sup.SC.sup.SA.sup.SC.sup.ST.sup.SG.sup.SC.sup.SC.sup.OC.sup.OC.s-
up.OT.sup.OC.sup.OA.sup.d 2'ME 78 15707 3'-UTR
T.sup.ST.sup.ST.sup.ST.sup.SC.sup.OT.sup.OT.sup.OC.sup.OC.sup.OA.sup.OC.s-
up.OT.sup.OG.sup.OC.sup.OC.sup.OC.sup.SC.sup.ST.sup.SC.sup.SA.sup.d
2'ME 78 15708 3'-UTR T.sup.ST.sup.ST.sup.ST.sup.SC.sup.ST.sup.-
OT.sup.OC.sup.OC.sup.OA.sup.OC.sup.OT.sup.OG.sup.OC.sup.OC.sup.SC.sup.SC.s-
up.ST.sup.SC.sup.SA.sup.d 2'ME 78 13881 3'-UTR
T.sup.OT.sup.OT.sup.OT.sup.OC.sup.OT.sup.OT.sup.OC.sup.OC.sup.OA.sup.OC.s-
up.OT.sup.OG.sup.SC.sup.SC.sup.SC.sup.SC.sup.ST.sup.SC.sup.SA.sup.d
2'ME 78 13882 3'-UTR T.sup.ST.sup.ST.sup.ST.sup.SC.sup.ST.sup.-
ST.sup.SC.sup.SC.sup.OA.sup.OC.sup.OT.sup.OG.sup.OC.sup.OC.sup.OC.sup.OC.s-
up.OT.sup.OC.sup.OA.sup.d 2'ME 78 12898 (scrambled 12633) &
Derivatives: 12898 control T.sup.ST.sup.SC.sup.ST.sup.SC.sup.ST-
.sup.ST.sup.ST.sup.ST.sup.SA.sup.SG.sup.SC.sup.SC.sup.ST.sup.SC.sup.SC.sup-
.SC.sup.SC.sup.SC.sup.SA 92 13046 control
T.sup.OT.sup.OC.sup.OT.sup.OC.sup.OT.sup.ST.sup.ST.sup.ST.sup.SA.sup.SG.s-
up.SC.sup.SC.sup.ST.sup.SC.sup.OC.sup.OC.sup.OC.sup.OC.sup.OA.sup.d
2'MO 92 13715 control T.sup.OT.sup.OC.sup.OT.sup.OC.sup.OT.sup-
.ST.sup.ST.sup.ST.sup.SA.sup.SG.sup.SC.sup.SC.sup.ST.sup.SC.sup.OC.sup.OC.-
sup.OC.sup.OC.sup.OA.sup.d 2'ME 92 13912 control
T.sup.OT.sup.OC.sup.OT.sup.OC.sup.OT.sup.OT.sup.OT.sup.OT.sup.OA.sup.OG.s-
up.OC.sup.OC.sup.ST.sup.SC.sup.SC.sup.SC.sup.SC.sup.SC.sup.SA.sup.d
2'ME 92 13913 control T.sup.ST.sup.SC.sup.ST.sup.SC.sup.ST.sup-
.ST.sup.ST.sup.ST.sup.OA.sup.OG.sup.OC.sup.OC.sup.OT.sup.OC.sup.OC.sup.OC.-
sup.OC.sup.OC.sup.OA.sup.d 2'ME 92 15705 control
T.sup.ST.sup.SC.sup.ST.sup.SC.sup.OT.sup.OT.sup.OT.sup.OT.sup.OA.sup.OG.s-
up.OC.sup.OC.sup.OT.sup.OC.sup.OC.sup.SC.sup.SC.sup.SC.sup.SA.sup.d
2'ME 92 15706 control T.sup.ST.sup.SC.sup.ST.sup.SC.sup.OT.sup-
.OT.sup.OT.sup.OT.sup.OA.sup.OG.sup.OC.sup.OC.sup.OT.sup.OC.sup.OC.sup.OC.-
sup.SC.sup.SC.sup.SA.sup.d 2'ME 92 12628 & Derivatives: 12628
5'-UTR C.sup.SC.sup.SC.sup.ST.sup.SC.sup.SC.sup.ST.sup.SG.sup.-
SC.sup.ST.sup.SC.sup.SG.sup.ST.sup.SC.sup.SG.sup.SG.sup.ST.sup.SC.sup.SA.s-
up.SC 73 13049 5'-UTR C.sup.OC.sup.OC.sup.OT.sup.OC.sup.-
OC.sup.ST.sup.SG.sup.SC.sup.ST.sup.SC.sup.SG.sup.ST.sup.SC.sup.SG.sup.OG.s-
up.OT.sup.OC.sup.OAhu OC.sup.d 2'MO 73 13712 5'-UTR
C.sup.OC.sup.OC.sup.OT.sup.OC.sup.OC.sup.ST.sup.SG.sup.SC.sup.ST.sup.SC.s-
up.SG.sup.ST.sup.SC.sup.SG.sup.OG.sup.OT.sup.OC.sup.OA.sup.OC.sup.d
2'ME 73 13879 5'-UTR C.sup.OC.sup.OC.sup.OT.sup.OC.sup.OC.sup.-
OT.sup.OG.sup.OC.sup.OT.sup.OC.sup.OG.sup.ST.sup.SC.sup.SG.sup.SG.sup.ST.s-
up.SC.sup.SA.sup.SC.sup.d 2'ME 73 13880 5'-UTR
C.sup.SC.sup.SC.sup.ST.sup.SC.sup.SC.sup.ST.sup.SG.sup.SC.sup.OT.sup.OC.s-
up.OG.sup.OT.sup.OC.sup.OG.sup.OG.sup.OT.sup.OC.sup.OA.sup.OC.sup.d
2'ME 73 12894 (scrambled 12628) & Derivatives: 12894
C.sup.SC.sup.SC.sup.SG.sup.SG.sup.SC.sup.SC.sup.ST.sup.ST.sup.ST.sup.ST.s-
up.SG.sup.SA.sup.SC.sup.SC.sup.SG.sup.SC.sup.SC.sup.ST.sup.SC 87
13713 C.sup.OC.sup.OC.sup.OG.sup.OG.sup.OC.sup.SC.sup.ST.sup.ST.-
sup.ST.sup.ST.sup.SG.sup.SA.sup.SC.sup.SC.sup.OG.sup.OC.sup.OC.sup.OT.sup.-
OC.sup.d 2'ME 87 13048 C.sup.OC.sup.OC.sup.OG.sup.OG.sup.-
OC.sup.SC.sup.ST.sup.ST.sup.ST.sup.ST.sup.SG.sup.SA.sup.SC.sup.SC.sup.OG.s-
up.OC.sup.OC.sup.OT.sup.OC.sup.d 2'MO 87 12635 & Derivatives:
12635 C.sup.ST.sup.ST.sup.SC.sup.SC.sup.SC.sup.SA.s-
up.SC.sup.ST.sup.SC.sup.SC.sup.SA.sup.SG.sup.SC.sup.SA.sup.SC.sup.SA.sup.S-
T.sup.ST.sup.SG 80 15711 C.sup.ST.sup.ST.sup.SC.sup.SC.-
sup.SC.sup.SA.sup.SC.sup.ST.sup.SC.sup.SC.sup.SA.sup.SG.sup.SC.sup.SA.sup.-
SC.sup.SA.sup.ST.sup.ST.sup.SG 2'ME 80 15712
C.sup.ST.sup.ST.sup.SC.sup.SC.sup.SC.sup.SA.sup.SC.sup.ST.sup.SC.sup.SC.s-
up.SA.sup.SG.sup.SC.sup.SA.sup.SC.sup.SA.sup.ST.sup.ST.sup.SGhu d
2'ME 80 15709 (scrambled 12635) & Derivatives: 15709
T.sup.ST.sup.SC.sup.ST.sup.SC.sup.SA.sup.SC.sup.SC.sup.SC.sup.SA.sup.SC.s-
up.SC.sup.SA.sup.SC.sup.SG.sup.ST.sup.SA.sup.SC.sup.SGhu ST b 2'ME
117 15710 T.sup.ST.sup.SC.sup.ST.sup.SC.sup.OA.sup.OC.sup.OC.su-
p.OC.sup.OA.sup.OC.sup.OC.sup.OA.sup.OC.sup.OG.sup.OT.sup.SA.sup.SC.sup.SG-
.sup.ST 2'ME 117 *Phosphorothioate linkages are indicated by
".sup.S", whereas phosphodiester linkages are signified by
".sup.O"; emboldened residues comprise the additional indicated
modifications: 2'MO=2'-methoxy-; 2'ME=2'-methoxyethoxy-.
[0112]
16TABLE 16 Additional Oligonucleotides Targeted to Rat c-fos
Oligonucleotide Sequence (5'->3') SEQ ID ISIS # Target and
Chemical Modifications* NO: 11256 & Derivatives: 106 11256
3'-UTR C.sup.SA.sup.SG.sup.SG.sup.SC.sup.S-
C.sup.ST.sup.SG.sup.SG.sup.SC.sup.ST.sup.SC.sup.SA.sup.SC.sup.SA.sup.ST.su-
p.SG.sup.SC.sup.ST.sup.SA 106 13051 3'-UTR
C.sup.OA.sup.OG.sup.OG.sup.OC.sup.OC.sup.ST.sup.SG.sup.SG.sup.SC.sup.ST.s-
up.SC.sup.SA.sup.SC.sup.SA.sup.OT.sup.OG.sup.OC.sup.OT.sup.OA.sup.d
2'MO 106 13718 3'-UTR C.sup.SA.sup.SG.sup.SG.sup.SC.sup.SC.sup-
.ST.sup.SG.sup.SG.sup.SC.sup.ST.sup.SC.sup.SA.sup.SC.sup.SA.sup.ST.sup.SG.-
sup.SC.sup.ST.sup.SA.sup.d 2'ME 106 13877 3'-UTR
C.sup.OA.sup.OG.sup.OG.sup.OC.sup.OC.sup.OT.sup.OG.sup.OG.sup.OC.sup.OT.s-
up.OC.sup.OA.sup.SC.sup.SA.sup.ST.sup.SG.sup.SC.sup.ST.sup.SA.sup.d
2'ME 106 13878 3'-UTR C.sup.OA.sup.OG.sup.OG.sup.OC.sup.OC.sup-
.OT.sup.OG.sup.OG.sup.SC.sup.ST.sup.SC.sup.SA.sup.SC.sup.SA.sup.ST.sup.SG.-
sup.SC.sup.ST.sup.SA.sup.d 2'ME 106 12703 (Scrambled 11256) &
Derivatives: 12703 control A.sup.SC.sup.SG.sup.ST.sup.SC-
.sup.SG.sup.SA.sup.SG.sup.SC.sup.SG.sup.SC.sup.SA.sup.SC.sup.SC.sup.SG.sup-
.ST.sup.SC.sup.SA.sup.ST.sup.ST 114 13050 control
A.sup.OC.sup.OG.sup.OT.sup.OC.sup.OG.sup.SA.sup.SG.sup.SC.sup.SG.sup.SC.s-
up.SA.sup.SC.sup.SC.sup.SG.sup.OT.sup.OC.sup.OA.sup.OT.sup.OT.sup.d
2'MO 114 13719 control A.sup.SC.sup.SG.sup.ST.sup.SC.sup.SG.su-
p.SA.sup.SG.sup.SC.sup.SG.sup.SC.sup.SA.sup.SC.sup.SC.sup.SG.sup.ST.sup.SC-
.sup.SA.sup.ST.sup.ST.sup.d 2'ME 114 11254 & Derivatives: 11254
3'-UTR T.sup.SC.sup.ST.sup.SA.sup.SC.sup.SG.sup.SG.sup.SG.su-
p.SA.sup.SA.sup.SC.sup.SC.sup.SC.sup.SC.sup.ST.sup.SC.sup.SG.sup.SA.sup.SG-
.sup.SG 104 13053 3'-UTR T.sup.OC.sup.OT.sup.OA.sup.OC.s-
up.OG.sup.SG.sup.SG.sup.SA.sup.SA.sup.SC.sup.SC.sup.SC.sup.SC.sup.ST.sup.O-
C.sup.OG.sup.OA.sup.OG.sup.OG.sup.d 2'MO 104 13716 3'-UTR
T.sup.SC.sup.ST.sup.SA.sup.SC.sup.SG.sup.SG.sup.SG.sup.SA.sup.SA.sup.SC.s-
up.SC.sup.SC.sup.SC.sup.ST.sup.SC.sup.SG.sup.SA.sup.SG.sup.SG.sup.d
2'ME 104 13875 3'-UTR T.sup.OC.sup.OT.sup.OA.sup.OC.sup.OG.sup-
.OG.sup.OG.sup.OA.sup.OA.sup.OC.sup.OC.sup.OC.sup.SC.sup.ST.sup.SC.sup.SG.-
sup.SA.sup.SG.sup.SG.sup.d 2'ME 104 13876 3'-UTR
T.sup.SC.sup.ST.sup.SA.sup.SC.sup.SG.sup.SG.sup.SG.sup.SA.sup.OA.sup.OC.s-
up.OC.sup.OC.sup.OC.sup.OT.sup.OC.sup.OG.sup.OA.sup.OG.sup.OG.sup.d
2'ME 104 12700 (Scrambled 11254) & Derivatives: 12700 control
C.sup.SC.sup.SG.sup.SC.sup.SC.sup.SG.sup.SG.sup.ST.sup.SG.sup.SA.-
sup.SC.sup.ST.sup.SA.sup.ST.sup.SA.sup.SG.sup.SA.sup.SC.sup.SC.sup.SG
109 13052 control C.sup.OC.sup.OG.sup.OC.sup.OC.sup.OG.sup.S-
G.sup.ST.sup.SG.sup.SA.sup.SC.sup.ST.sup.SA.sup.ST.sup.SA.sup.OG.sup.OA.su-
p.OC.sup.OC.sup.OG.sup.d 2'MO 109 13717 control
C.sup.SC.sup.SG.sup.SC.sup.SC.sup.SG.sup.SG.sup.ST.sup.SG.sup.SA.sup.SC.s-
up.ST.sup.SA.sup.ST.sup.SA.sup.SG.sup.SA.sup.SC.sup.SC.sup.SG.sup.d
2'ME 109 *Phosphorothioate and phosphodiester linkages are
indicared by ".sup.S" and ".sup.O," respectively, whereas ".sup.d"
indicates a dideoxy (chain-terminating) residue; emboldened
residues comprise the additional indicated modifications: 2'MO,
2'-methoxy; 2'ME, 2'-methoxyethoxy-.
Example 11
[0113] Effect of Oligonucleotides Targeted to AP-1 Subunits on
PDGF-Induced Proliferation of Rat Aortic Smooth Muscle Cells
[0114] In order to evaluate the effect of AP-1 modulation on cell
cycle progression, the following study was performed. Cultured rat
aortic smooth muscle (RASM) cells are stimulated to proliferate
upon contact with platelet-derived growth factor (PDGF). Primary
RASM cells (passages 6-8) were synchronized by incubation for 48
hours in DMEM containing 0.1% FBS. The cells were treated for 4
hours with 200 nM ISIS 12633 (SEQ ID NO:78), a 20-mer
phosphorothioate oligonucleotide complementary to a portion of the
3' UTR of rat c-jun mRNA, or ISIS 12898 (SEQ ID NO:92), a scrambled
control of ISIS 12633. Cells were then contacted with PDGF (10
ng/ml) (R&D Systems, Minneapolis, Minn.), and cell cycle
progression was assessed by FACS analysis 24 hours later. At 2 and
6 hours after exposure to PDGF, c-jun mRNA levels were markedly
less in ISIS 12633-treated cells as compared to untreated cells or
cells treated with ISIS 12898. The decrease in c-jun mRNA levels
was associated with a significant decrease in the proportion of
cells in the G2/M interface at 24 hours. This result provides
evidence of the role of AP-1-mediated gene expression in cellular
proliferation and indicate that cell cycle progression can be
modulated by preventing expression of one or both of the genes
which encode a subunit of AP-1.
Example 12
[0115] Effect of Oligonucleotides Targeted to AP-1 Subunits on
Enzymes Involved in Metastasis
[0116] Patients having benign tumors, and primary malignant tumors
that have been detected early in the course of their development,
may often be successfully treated by the surgical removal of the
benign or primary tumor. If unchecked, however, cells from
malignant tumors are spread throughout a patient's body through the
processes of invasion and metastasis. Invasion refers to the
ability of cancer cells to detach from a primary site of attachment
and penetrate, e.g., an underlying basement membrane. Metastasis
indicates a sequence of events wherein (1) a cancer cell detaches
from its extracellular matrices, (2) the detached cancer cell
migrates to another portion of the patient's body, often via the
circulatory system, and (3) attaches to a distal and inappropriate
extracellular matrix, thereby created a focus from which a
secondary tumor can arise. Normal cells do not possess the ability
to invade or metastasize and/or undergo apoptosis (programmed cell
death) if such events occur (Ruoslahti, Sci. Amer., 1996, 275,
72).
[0117] The matrix metalloproteinases (MMPs) are a family of enzymes
which have the ability to degrade components of the extracellular
matrix (Birkedal-Hansen, Current Op. Biol., 1995, 7, 728). Many
members of the MMP family have been found to have elevated levels
of activity in human tumors as well as other disease states
(Stetler-Stevenson et al., Annu. Rev. Cell Biol., 1993, 9, 541;
Bernhard et al., Proc. Natl. Acad. Sci. (U.S.A.), 1994, 91, 4293).
In particular, one member of this family, matrix
metalloproteinase-9 (MMP-9), is often found to be expressed only in
tumors and other diseased tissues (Himeistein et al., Invasion
& Metastasis, 1994, 14, 246). Several studies have shown that
regulation of the MMP-9 gene may be controlled by the AP-1
transcription factor (Kerr et al., Science, 1988, 242, 1242; Kerr
et al., Cell, 1990, 61, 267; Gum et al., J. Biol. Chem., 1996, 271,
10672; Hua et al., Cancer Res., 1996, 56, 5279). In order to
determine whether MMP-9 expression can be influenced by AP-1
modulation, the following experiments were conducted on normal
human epidermal keratinocytes (NHEKs). Although NHEKs normally
express no detectable MMP-9, MMP-9 can be induced by a number of
stimuli, including TPA. ISIS 10582, an oligonucleotide targeted to
c-jun, was evaluated for its ability to modulate MMP-9 expression
according to the protocols described in Examples 2-3 with the
following exceptions: (1) NHEK cells were used instead of A549
cells, (2) the probe used, a PCR product prepared using the
published sequence of the MMP-9 gene (Huhtala et al., J. Biol.
Chem., 1991, 266:16485; Sato et al., Oncogene, 1993, 8:395), is
specific for MMP-9 rather than c-jun, and (3) the cells were
harvested 24 hours after TPA treatment for 4 hours. The results
(Table 17) demonstrate that ISIS 10582 is able to completely
inhibit the expression of MMP-9 after induction with TPA.
17TABLE 17 Effect of c-jun Oligonucleotide on MMP-9 Expression
Treatment MMP-9 Basal 4 TPA - no oligo 100 10582: c-jun active 6
11562: sense control 99 11563: scrambled control 95 11564: mismatch
control 89
[0118] These results demonstrate that c-Jun is required for
TPA-mediated induction of MMP-9, and indicate that oligonucleotides
targeted to AP-1 subunits can inhibit the expression of MMP family
members, thereby modulating the ability of cancer cells to invade
other tissues and/or metastasize to other sites in a patient's
body.
Example 13
[0119] Antagonism of Inducers Other than TPA by Oligonucleotides
Targeted to AP-1 Subunits
[0120] Inducing agents other than TPA function to raise AP-1 levels
in vivo. In order to assess the ability of oligonucleotides
targeted to AP-1 to antagonize the action of three such inducers,
A549 cells were treated and evaluated as in Examples 2 et seq. with
the exception that TNF-a, IL-1b or TGE-b (each at 10 ng/mi and all
from R&D Systems, Minneapolis, Minn.) were used in place of TPA
as inducers. The results (Table 18) demonstrate that ISTS 10582
(SEQ ID NO:8, targeted to human c-jun) effectively reduces
stimulation of c-Jun by TNF-a or IL-1b. In contrast, a scrambled
control oligonucleotide, ISIS 11563 (SEQ TD NO:30), did not reverse
the induction of c-Jun.
18TABLE 18 Effect of Oligonucleotides Targeted to c-Jun on
Induction by TNF-a, IL-lb or TGF-b ISIS ISIS Inducer Basal No Oligo
10582 11563 TNF-a 5 100 20 98 IL-1b 9 100 15 94 TQF-b 2 100 95
99
[0121]
Sequence CWU 1
1
* * * * *