U.S. patent application number 10/872106 was filed with the patent office on 2005-03-10 for gapped oligomeric compounds having linked bicyclic sugar moieties at the termini.
Invention is credited to Migawa, Michael T., Swayze, Eric E., Wyrzykiewicz, Tadeusz Krzysztof.
Application Number | 20050053981 10/872106 |
Document ID | / |
Family ID | 34229435 |
Filed Date | 2005-03-10 |
United States Patent
Application |
20050053981 |
Kind Code |
A1 |
Swayze, Eric E. ; et
al. |
March 10, 2005 |
Gapped oligomeric compounds having linked bicyclic sugar moieties
at the termini
Abstract
The present invention relates to bicyclic nucleosides and
oligomeric compounds comprising at least one such nucleoside. These
oligomeric compounds typically have enhanced binding affinity and
nuclease resistance properties compared to unmodified oligomeric
compounds. The oligomeric compounds are useful, for example, for
investigative and therapeutic purposes.
Inventors: |
Swayze, Eric E.; (Carlsbad,
CA) ; Migawa, Michael T.; (San Marcos, CA) ;
Wyrzykiewicz, Tadeusz Krzysztof; (Carlsbad, CA) |
Correspondence
Address: |
COZEN O'CONNOR, P.C.
1900 MARKET STREET
PHILADELPHIA
PA
19103-3508
US
|
Family ID: |
34229435 |
Appl. No.: |
10/872106 |
Filed: |
June 18, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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60501719 |
Sep 9, 2003 |
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60568039 |
May 3, 2004 |
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60568489 |
May 6, 2004 |
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Current U.S.
Class: |
435/6.12 ;
536/23.1; 536/25.32 |
Current CPC
Class: |
A61P 43/00 20180101;
C07H 21/04 20130101 |
Class at
Publication: |
435/006 ;
536/023.1; 536/025.32 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Claims
What is claimed is:
1. An oligomeric compound comprising the structure:
T.sub.1-(Nu.sub.1-L.sub.1).sub.n1-(Nu.sub.2-L.sub.2).sub.n2-(Nu.sub.3-L.s-
ub.3).sub.n3-T.sub.2 wherein: each Nu.sub.1 and Nu.sub.3 is,
independently, a high affinity modified nucleoside, wherein at
least one of Nu.sub.1 and Nu.sub.3 is a bicyclic sugar modified
nucleoside comprising a 4'-CH.sub.2--O-2' bridge or a
4'-(CH.sub.2).sub.2--O-2' bridge; each Nu.sub.2 is a 2'-deoxy
nucleoside; each L.sub.1, L.sub.2 and L.sub.3 is, independently, an
internucleoside linking group; each T.sub.1 and T.sub.2 is,
independently, H, a hydroxy protecting group, an optionally linked
conjugate group, or a covalent attachment to a solid support
medium; n1 is from 1 to about 6; n2 is from 11 to about 18; and n3
is from 2 to about 6.
2. The oligomeric compound of claim 1 wherein each of the high
affinity modified nucleosides is, independently, a bicyclic sugar
modified nucleoside, a 2'-O--(CH.sub.2).sub.2--O--CH.sub.3 modified
nucleoside, a 2'-F modified nucleoside, or a
2'-O--CH.sub.2--C(.dbd.O)--NR.sub.1R.sub.2 modified nucleoside,
where each R.sub.1 and R.sub.2 is, independently, H, a nitrogen
protecting group, substituted or unsubstituted C.sub.1-C.sub.10
alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl,
substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein the
substitution is OR.sub.3, SR.sub.3, NH.sub.3.sup.+,
NR.sub.3R.sub.4, guanidino or acyl, wherein the acyl is acid amide
or an ester, or R.sub.1 and R.sub.2, together, are a nitrogen
protecting group, or are joined in a ring structure that optionally
includes an additional heteroatom selected from N and O.
3. The oligomeric compound of claim 2 wherein each of the bicyclic
sugar modified nucleosides independently has a 4'-CH.sub.2--O-2'
bridge or a 4'-(CH.sub.2).sub.2--O-2' bridge.
4. The oligomeric compound of claim 2 wherein each R.sub.1 and
R.sub.2 each is, independently, H, a nitrogen protecting group, or
C.sub.1-C.sub.10 alkyl.
5. The oligomeric compound of claim 1 wherein T.sub.1 is H or a
hydroxyl protecting group.
6. The oligomeric compound of claim 1 wherein T.sub.2 is H or a
hydroxyl protecting group.
7. The oligomeric compound of claim 1 wherein each of the hydroxyl
protecting groups is, independently, 4,4'-dimethoxytrityl,
monomethoxytrityl, 9-phenylxanthen-9-yl,
9-(p-methoxyphenyl)xanthen-9-yl, t-butyl, t-butoxymethyl,
methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,
1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,
2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,
p,p-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,
trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetyl,
chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl,
p-phenylbenzoyl, mesyl, tosyl, 4,4',4"-tris-(benzyloxy)trityl,
4,4',4"-tris-(4,5-dichlorophthalimido)tri- tyl,
4,4',4"-tris(levulinyloxy)trityl,
3-(imidazolylmethyl)-4,4'-dimethoxy- trityl, 4-decyloxytrityl,
4-hexadecyloxytrityl, 9-(4-octadecyloxyphenyl)xa- nthene-9-yl,
1,1-bis-(4-methoxyphenyl)-1'-pyrenyl methyl,
p-phenylazophenyloxycarbonyl, 9-fluorenylmethoxycarbonyl,
2,4-dinitrophenylethoxycarb onyl, 4-(methylthiomethoxy)butyryl,
2-(methylthiomethoxymethyl)-benzoyl,
2-(isopropyl-thiomethoxymethyl)benzo- yl,
2-(2,4-dinitrobenzenesulphenyloxymethyl)benzoyl, or levulinyl
group.
8. The oligomeric compound of claim 1 wherein one of T.sub.1 and
T.sub.2 is a covalent attachment to a support medium.
9. The oligomeric compound of claim 8 wherein the support medium is
a controlled pore glass, oxalyl-controlled pore glass,
silica-containing particles, polymers of polystyrene, copolymers of
polystyrene, copolymers of styrene and divinylbenzene, copolymers
of dimethylacrylamide and N,N'-bisacryloylethylenediamine, soluble
support medium, or PEPS.
10. The oligomeric compound of claim 1 wherein each L.sub.1,
L.sub.2 and L.sub.3 is, independently, phosphodiester,
phosphorothioate, chiral phosphorothioate, phosphorodithioate,
phosphotriester, aminoalkylphosphotriester, methyl phosphonate,
alkyl phosphonate, 5'-alkylene phosphonate, chiral phosphonate,
phosphinate, phosphoramidate, 3'-amino phosphoramidate,
aminoalkylphosphoramidate, thionophosphoramidate,
thionoalkylphosphonate, thionoalkylphosphotriester- ,
selenophosphate, or boranophosphate.
11. The oligomeric compound of claim 10 wherein each L.sub.1,
L.sub.2 and L.sub.3 is, independently, a phosphodiester or a
phosphorothioate internucleoside linking group.
12. The oligomeric compound of claim 10 wherein each L.sub.1,
L.sub.2 and L.sub.3 is a phosphodiester internucleoside linking
group.
13. The oligomeric compound of claim 1 wherein each L.sub.1,
L.sub.2 and L.sub.3 is, independently, siloxane, sulfide,
sulfoxide, sulfone, formacetyl, thioformacetyl, methylene
formacetyl, thioformacetyl, sulfamate, methyleneimino,
methylenehydrazino, sulfonate, sulfonamide, or amide.
14. The oligomeric compound of claim 13 wherein each of the
internucleoside linking groups is, independently,
--CH.sub.2--NH-O--CH.su- b.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- or
--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--, or
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2--.
15. The oligomeric compound of claim 1 wherein each nucleoside
comprises a heterocyclic base moiety that is, independently,
adenine, guanine, thymine, cytosine, uracil, 5-methylcytosine,
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine,
5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo
cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil,
8-substituted adenines and guanines, 5-substituted uracils and
cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine,
8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, or
3-deazaadenine.
16. The oligomeric compound of claim 1 wherein n1 is from 1 to
about 5.
17. The oligomeric compound of claim 1 wherein n1 is from 1 to
about 3.
18. The oligomeric compound of claim 1 wherein n1 is from 2 to
about 3.
19. The oligomeric compound of claim 1 wherein n3 is from 2 to
about 5.
20. The oligomeric compound of claim 1 wherein n3 is from 2 to
about 3.
21. The oligomeric compound of claim 1 wherein n2 is from 12 to
about 18.
22. The oligomeric compound of claim 1 wherein n2 is from 12 to
about 16.
23. The oligomeric compound of claim 1 wherein n2 is from 14 to
about 16.
24. The oligomeric compound of claim 1 wherein the total of n1, n2
and n3 is from 14 to about 30.
25. The oligomeric compound of claim 1 wherein the total of n1, n2
and n3 is from 14 to 24.
26. The oligomeric compound of claim 1 wherein the total of n1, n2
and n3 is from 14 to 21.
27. The oligomeric compound of claim 1 wherein the total of n1, n2
and n3 is from 16 to 21.
28. The oligomeric compound of claim 1 wherein n1 is from 1 to
about 3, n2 is 12 or 13 and n3 is 2 or 3.
29. The oligomeric compound of claim 1 wherein n1 is 3, n2 is 12
and n3 is 3.
30. The oligomeric compound of claim 1 wherein n1 is from 1 to
about 3, n2 is 14 or 15 and n3 is 2 or 3.
31. The oligomeric compound of claim 1 wherein n1 is 2, n2 is 14
and n3 is 2.
32. The oligomeric compound of claim 1 wherein n1 is from 1 to
about 3, n2 is 16 or 17 and n3 is 2 or 3.
33. The oligomeric compound of claim 1 wherein n1 is 2, n2 is 16
and n3 is 2.
34. The oligomeric compound of claim 1 wherein at least one
Nu.sub.1 nucleoside and at least one Nu.sub.3 nucleside is an LNA
or ENA.
35. The oligomeric compound of claim 1 wherein at least one of the
5'-most or 3'-most terminal affinity modified nucleosides is an LNA
or ENA.
36. A method of inhibiting gene expression comprising contacting
one or more cells, a tissue, or an animal with an oligomeric
compound of claim 1.
37. An oligomeric compound comprsing the structure: 23wherein: each
Bx is a heterocyclic base moiety; each X is, independently, O or S;
T.sub.1 and T.sub.2 are each, independently, H, a hydroxy
protecting group, an optionally linked conjugate group, or a
covalent attachment to a solid support medium; each m is,
independently, 1 or 2; na is from 1 to about 6; nb is from 11 to
about 18; and nc is from 2 to about 6.
38. The oligomeric compound of claim 37 wherein each m is 1.
39. The oligomeric compound of claim 37 wherein each m is 2.
40. The oligomeric compound of claim 37 wherein at least one of
T.sub.1 and T.sub.2 is H or a hydroxyl protecting group.
41. The oligomeric compound of claim 40 wherein each of the
hydroxyl protecting groups is, independently, 4,4'-dimethoxytrityl,
monomethoxytrityl, 9-phenylxanthen-9-yl,
9-(p-methoxyphenyl)xanthen-9-yl, t-butyl, t-butoxymethyl,
methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,
1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,
2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,
p,p-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,
trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetyl,
chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl,
p-phenylbenzoyl, mesyl, tosyl, 4,4',4"-tris-(benzyloxy)trityl,
4,4',4"-tris-(4,5-dichlorophthalimido)tri- tyl,
4,4',4"-tris(levulinyloxy)trityl,
3-(imidazolylmethyl)-4,4'-dimethoxy- trityl, 4-decyloxytrityl,
4-hexadecyloxytrityl, 9-(4-octadecyloxyphenyl)xa- nthene-9-yl,
1,1-bis-(4-methoxyphenyl)-1'-pyrenyl methyl,
p-phenylazophenyloxycarbonyl, 9-fluorenylmethoxycarbonyl,
2,4-dinitrophenylethoxycarb onyl, 4-(methylthiomethoxy)butyryl,
2-(methylthiomethoxymethyl)-benzoyl,
2-(isopropyl-thiomethoxymethyl)benzo- yl,
2-(2,4-dinitrobenzenesulphenyloxymethyl)benzoyl, or levulinyl
group.
42. The oligomeric compound of claim 37 wherein one of T.sub.1 and
T.sub.2 is a covalent attachment to a support medium.
43. The oligomeric compound of claim 42 wherein the support medium
is a controlled pore glass, oxalyl-controlled pore glass,
silica-containing particles, polymers of polystyrene, copolymers of
polystyrene, copolymers of styrene and divinylbenzene, copolymers
of dimethylacrylamide and N,N'-bisacryloylethylenediamine, soluble
support medium, or PEPS.
44. The oligomeric compound of claim 37 wherein each L.sub.1,
L.sub.2 and L.sub.3 is, independently, a phosphodiester or a
phosphorothioate internucleoside linking group.
45. The oligomeric compound of claim 44 wherein each L.sub.1,
L.sub.2 and L.sub.3 is a phosphodiester internucleoside linking
group.
46. The oligomeric compound of claim 37 wherein each Bx is,
independently, adenine, guanine, thymine, cytosine, uracil,
5-methylcytosine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine,
2-aminoadenine, alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine, 2-thiocytosine, 5-halouracil,
5-halocytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo
uracil, 6-azo cytosine, 6-azo thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-substituted adenines and guanines, 5-substituted
uracils and cytosines, 7-methylguanine, 7-methyladenine,
8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-deazaadenine,
3-deazaguanine, or 3-deazaadenine.
47. The oligomeric compound of claim 37 wherein na is from 1 to
about 5.
48. The oligomeric compound of claim 37 wherein na is from 1 to
about 3.
49. The oligomeric compound of claim 37 wherein na is from 2 to
about 3.
50. The oligomeric compound of claim 37 wherein nc is from 2 to
about 5.
51. The oligomeric compound of claim 37 wherein nc is from 2 to
about 3.
52. The oligomeric compound of claim 37 wherein nb is from 12 to
about 18.
53. The oligomeric compound of claim 37 wherein nb is from 12 to
about 16.
54. The oligomeric compound of claim 37 wherein nb is from 14 to
about 16.
55. The oligomeric compound of claim 37 wherein the total of na, nb
and nc is from 14 to about 30.
56. The oligomeric compound of claim 37 wherein the total of na, nb
and nc is from 14 to 24.
57. The oligomeric compound of claim 37 wherein the total of na, nb
and nc is from 14 to 21.
58. The oligomeric compound of claim 37 wherein the total of na, nb
and nc is from 16 to 21.
59. The oligomeric compound of claim 37 wherein na is from 1 to
about 3, nb is 12 or 13 and nc is 2 or 3.
60. The oligomeric compound of claim 37 wherein na is 3, nb is 12
and nc is 3.
61. The oligomeric compound of claim 37 wherein na is from 1 to
about 3, nb is 14 or 15 and nc is 2 or 3.
62. The oligomeric compound of claim 37 wherein na is 2, nb is 14
and nc is 2.
63. The oligomeric compound of claim 37 wherein na is from 1 to
about 3, nb is 16 or 17 and nc is 2 or 3.
64. The oligomeric compound of claim 37 wherein na is 2, nb is 16
and nc is 2.
65. A method of inhibiting gene expression comprising contacting
one or more cells, a tissue, or an animal with an oligomeric
compound of claim 37.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application Ser. No. 60/501,719 filed Sep. 9, 2003, U.S.
provisional application Ser. No. 60/568,039 filed May 3, 2004, and
U.S. provisional application Ser. No. 60/568,489 filed May 6, 2004,
each of which is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to nucleoside compositions
comprising novel bicyclic sugar moieties and oligomeric compounds
comprising at least one such nucleoside. The oligomeric compounds
of the present invention typically will have enhanced binding
affinity properties compared to unmodified oligomeric compounds.
The oligomeric compounds are useful, for example, for investigative
and therapeutic purposes.
BACKGROUND OF THE INVENTION
[0003] Nearly all disease states in multicellular organisms involve
the action of proteins. Classic therapeutic approaches have focused
on the interaction of proteins with other molecules in efforts to
moderate the proteins' disease-causing or disease-potentiating
activities. In newer therapeutic approaches, modulation of the
production of proteins has been sought. A general object of some
current therapeutic approaches is to interfere with or otherwise
modulate gene expression.
[0004] One method for inhibiting the expression of specific genes
involves the use of oligonucleotides, particularly oligonucleotides
that are complementary to a specific target messenger RNA (mRNA)
sequence. Due to promising research results in recent years,
oligonucleotides and oligonucleotide analogs are now accepted as
therapeutic agents holding great promise for therapeutic and
diagnostic methods.
[0005] Oligonucleotides and their analogs can be designed to have
particular properties. A number of chemical modifications have been
introduced into oligomeric compounds to increase their usefulness
as therapeutic agents. Such modifications include those designed to
increase binding affinity to a target strand, to increase cell
penetration, to stabilize against nucleases and other enzymes that
degrade or interfere with the structure or activity of the
oligonucleotide, to provide a mode of disruption (terminating
event) once the oligonucleotide is bound to a target, and to
improve the pharmacokinetic properties of the oligonucleotide.
[0006] One group of bicyclic nucleoside compounds having bicyclic
sugar moieties that are conformationally locked is locked nucleic
acids or LNA (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630;
U.S. Pat. No. 6,268,490). These compounds are also referred to in
the literature as bicyclic nucleotide analogs (International Patent
Application WO 98/39352), but this term is also applicable to a
genus of compounds that includes other analogs in addition to LNAs.
LNAs have been used in numerous studies where ribonucleoside mimics
are desired. Such modified nucleosides mimic the 3'-endo sugar
conformation of native ribonucleosides with the advantage of having
enhanced binding affinity and increased resistance to nucleases.
LNAs are discussed more thouroughly below.
[0007] One group has added an additional methlene group to the LNA
2',4'-bridging group (e.g. 4'-CH.sub.2--CH.sub.2--O-2' (ENA),
Kaneko et al., U.S. Patent Application Publication No.: U.S.
2002/0147332, also see Japanese Patent Application HEI-11-33863,
Feb. 12, 1999; U.S. Patent Application Publication Nos.
2003/0207841 and 2002/0147332).
[0008] Another publication reports a large genus of nucleosides
having a variety of bicyclic sugar moieties with the various
bridges creating a bicyclic sugar having a variety of
configurations and chemical composition (U.S. Patent Application
Publication No.: US 2002/0068708).
[0009] Despite these advances, a need exists in the art for the
development of means to improve the binding affinity and nuclease
resistance properties of oligomeric compounds.
BRIEF SUMMARY OF THE INVENTION
[0010] The present invention provides oligomeric compounds
comprising the structure
T.sub.1-(Nu.sub.1-L.sub.1).sub.n1-(Nu.sub.2-L.sub.2).sub.n2-(Nu-
.sub.3-L.sub.3).sub.n3-T.sub.2 wherein each Nu.sub.1 and Nu.sub.3
is, independently, a high affinity modified nucleoside, wherein at
least one nucleoside of Nu.sub.1 and/or at least one nucleoside of
Nu.sub.3 is a bicyclic sugar modified nucleoside comprising a
4'-CH.sub.2--O-2' bridge or a 4'-(CH.sub.2).sub.2--O-2' bridge.
Each Nu.sub.2 is a 2'-deoxy nucleoside and each L.sub.1, L.sub.2
and L.sub.3 is, independently, an internucleoside linking group.
Each T.sub.1 and T.sub.2 is, independently, H, a hydroxyl
protecting group, an optionally linked conjugate group, or a
covalent attachment to a solid support medium. n1 is from 1 to
about 6, n2 is from 11 to about 18, and n3 is from 2 to about
6.
[0011] In some embodiments, each of the high affinity modified
nucleosides is, independently, a bicyclic sugar modified
nucleoside, a 2'-O--(CH.sub.2).sub.2--O--CH.sub.3 modified
nucleoside, a 2'-F modified nucleoside, or a
2'-O--CH.sub.2--C(.dbd.O)--NR.sub.1R.sub.2 modified nucleoside,
where each R.sub.1 and R.sub.2 is, independently, H, a nitrogen
protecting group, substituted or unsubstituted C.sub.1-C.sub.10
alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl,
substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein the
substitution is OR.sub.3, SR.sub.3, NH.sub.3.sup.+,
NR.sub.3R.sub.4, guanidino or acyl, wherein the acyl is acid amide
or an ester, or R.sub.1 and R.sub.2, together, are a nitrogen
protecting group, or are joined in a ring structure that optionally
includes an additional heteroatom selected from N and O.
[0012] In some embodiments, each of the hydroxyl protecting groups
is, independently, 4,4'-dimethoxytrityl, monomethoxytrityl,
9-phenylxanthen-9-yl, 9-(p-methoxyphenyl)xanthen-9-yl, t-butyl,
t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,
1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,
2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,
p,p-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,
trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetyl,
chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl,
p-phenylbenzoyl, mesyl, tosyl, 4,4',4"-tris-(benzyloxy)trityl,
4,4',4"-tris-(4,5-dichlorophthalimido)tri- tyl,
4,4',4"-tris(levulinyloxy)trityl,
3-(imidazolylmethyl)-4,4'-dimethoxy- trityl, 4-decyloxytrityl,
4-hexadecyloxytrityl, 9-(4-octadecyloxyphenyl)xa- nthene-9-yl,
1,1-bis-(4-methoxyphenyl)-1'-pyrenyl methyl,
p-phenylazophenyloxycarbonyl, 9-fluorenylmethoxycarbonyl,
2,4-dinitrophenylethoxycarb onyl, 4-(methylthiomethoxy)butyryl,
2-(methylthiomethoxymethyl)-benzoyl,
2-(isopropyl-thiomethoxymethyl)benzo- yl,
2-(2,4-dinitrobenzenesulphenyloxymethyl)benzoyl, or levulinyl
group.
[0013] In some embodiments, the support medium is a controlled pore
glass, oxalyl-controlled pore glass, silica-containing particles,
polymers of polystyrene, copolymers of polystyrene, copolymers of
styrene and divinylbenzene, copolymers of dimethylacrylamide and
N,N'-bisacryloylethylenediamine, soluble support medium, or
PEPS.
[0014] In some embodiments, each L.sub.1, L.sub.2 and L.sub.3 is,
independently, phosphodiester, phosphorothioate, chiral
phosphorothioate, phosphorodithioate, phosphotriester,
aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate,
5'-alkylene phosphonate, chiral phosphonate, phosphinate,
phosphoramidate, 3'-amino phosphoramidate,
aminoalkylphosphoramidate, thionophosphoramidate,
thionoalkylphosphonate, thionoalkylphosphotriester,
selenophosphate, boranophosphate, siloxane, sulfide, sulfoxide,
sulfone, formacetyl, thioformacetyl, methylene formacetyl,
thioformacetyl, sulfamate, methyleneimino, methylenehydrazino,
sulfonate, sulfonamide, or amide. In some embodiments, each of the
internucleoside linking groups is, independently,
--CH.sub.2--NH--O--CH.sub.2--, --CH.sub.2--N(CH.sub.3)--O--
-CH.sub.2-- or --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--, or
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2--.
[0015] In some embodiments, each nucleoside comprises a
heterocyclic base moiety that is, independently, adenine, guanine,
thymine, cytosine, uracil, 5-methylcytosine, 5-hydroxy-methyl
cytosine, xanthine, hypoxanthine, 2-aminoadenine, alkyl derivatives
of adenine and guanine, 2-thiouracil, 2-thiothymine,
2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl uracil,
5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine,
5-uracil (pseudouracil), 4-thiouracil, 8-substituted adenines and
guanines, 5-substituted uracils and cytosines, 7-methylguanine,
7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine,
7-deazaadenine, 3-deazaguanine, or 3-deazaadenine.
[0016] In some embodiments, n1 is from 1 to about 5, or from 1 to
about 3, or from 2 to about 3. In some embodiments, n3 is from 2 to
about 5, or from 2 to about 3. In some embodiments, n2 is from 12
to about 18, or from 12 to about 16, or from 14 to about 16. In
some embodiments, the total of n1, n2 and n3 is from 14 to about
30, or from 14 to 24, or from 14 to 21, or from 16 to 21. In some
embodiments, n1 is from 1 to about 3, n2 is 12 or 13, and n3 is 2
or 3; or n1 is 3, n2 is 12 and n3 is 3; or n1 is from 1 to about 3,
n2 is 14 or 15 and n3 is 2 or 3; or n1 is 2, n2 is 14 and n3 is 2;
or n1 is from 1 to about 3, n2 is 16 or 17 and n3 is 2 or 3; or n1
is 2, n2 is 16 and n3 is 2.
[0017] In some embodiments, at least one Nu.sub.1 nucleoside and at
least one Nu.sub.3 nucleside is an LNA or ENA. In some embodiments,
at least one of the 5'-most or 3'-most terminal affinity modified
nucleosides is an LNA or ENA.
[0018] The present inventin also provides oligomeric compounds
comprsing the structure: 1
[0019] wherein each Bx is a heterocyclic base moiety; each X is,
independently, O or S; T.sub.1 and T.sub.2 are each, independently,
H, a hydroxy protecting group, an optionally linked conjugate
group, or a covalent attachment to a solid support medium; each m
is, independently, 1 or 2; na is from 1 to about 6; nb is from 11
to about 18; and nc is from 2 to about 6.
[0020] In some embodiments, each m is 1 or 2. In some embodiments,
at least one of T.sub.1 and T.sub.2 is H or a hydroxyl protecting
group, such as, 4,4'-dimethoxytrityl, monomethoxytrityl,
9-phenylxanthen-9-yl, 9-(p-methoxyphenyl)xanthen-9-yl, t-butyl,
t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,
1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,
2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,
p,p-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,
trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetyl,
chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl,
p-phenylbenzoyl, mesyl, tosyl, 4,4',4"-tris-(benzyloxy)trityl,
4,4',4"-tris-(4,5-dichlorophthalimido)trityl,
4,4',4"-tris(levulinyloxy)t- rityl,
3-(imidazolylmethyl)-4,4'-dimethoxytrityl, 4-decyloxytrityl,
4-hexadecyloxytrityl, 9-(4-octadecyloxyphenyl)xanthene-9-yl,
1,1-bis-(4-methoxyphenyl)-1'-pyrenyl methyl,
p-phenylazophenyloxycarbonyl- , 9-fluorenylmethoxycarbonyl,
2,4-dinitrophenylethoxycarb onyl, 4-(methylthiomethoxy)butyryl,
2-(methylthiomethoxymethyl)-benzoyl,
2-(isopropylthiomethoxymethyl)benzoyl,
2-(2,4-dinitrobenzenesulphenyl-oxy- methyl)benzoyl, or levulinyl
group.
[0021] In some embodiments, the support medium is a controlled pore
glass, oxalyl-controlled pore glass, silica-containing particles,
polymers of polystyrene, copolymers of polystyrene, copolymers of
styrene and divinylbenzene, copolymers of dimethylacrylamide and
N,N'-bisacryloylethylenediamine, soluble support medium, or
PEPS.
[0022] In some embodiments, each Bx is, independently, adenine,
guanine, thymine, cytosine, uracil, 5-methylcytosine,
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine,
5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo
cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil,
8-substituted adenines and guanines, 5-substituted uracils and
cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine,
8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, or
3-deazaadenine.
[0023] In some embodiments, na is from 1 to about 5, or from 1 to
about 3, or from 2 to about 3. In some embodiments, nc is from 2 to
about 5, or from 2 to about 3. In some embodiments, nb is from 12
to about 18, or from 12 to about 16, or from 14 to about 16. In
some embodiments, the total of na, nb and nc is from 14 to about
30, or from 14 to 24, or from 14 to 21, or from 16 to 21. In some
embodiments, na is from 1 to about 3, nb is 12 or 13 and nc is 2 or
3; or na is 3, nb is 12 and nc is 3; or na is from 1 to about 3, nb
is 14 or 15 and nc is 2 or 3; or na is 2, nb is 14 and nc is 2; or
na is from 1 to about 3, nb is 16 or 17 and nc is 2 or 3; or na is
2, nb is 16 and nc is 2.
[0024] The present invention also provides methods of inhibiting
gene expression comprising contacting one or more cells, a tissue,
or an animal with any oligomeric compound described herein.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The present invention provides bicyclic nucleoside analogs
and oligomeric compounds having at least one of these bicyclic
nucleoside analogs. Each of the bicyclic nucleoside analogs has a
bridge between the 2' and 4'-positions of the ribofuranose sugar
moiety. Oligomeric compounds having at least one of these bicyclic
nucleoside analogs will be useful in the modulation of gene
expression. More specifically, oligomeric compounds of the
invention will modulate gene expression by hybridizing to a nucleic
acid target resulting in loss of normal function of the target
nucleic acid.
[0026] As used herein, the term "target nucleic acid" or "nucleic
acid target" is used for convenience to encompass any nucleic acid
capable of being targeted including without limitation DNA, RNA
(including pre-mRNA and mRNA or portions thereof) transcribed from
such DNA, and also cDNA derived from such RNA. In some embodiment
of the invention, the target nucleic acid is a messenger RNA that
is degraded by a mechanism involving a nuclease such as or RNaseH.
The hybridization of an oligomeric compound of this invention with
its target nucleic acid is generally referred to as "antisense."
Consequently, one mechanism believed to be included in the practice
of some embodiments of the invention is referred to herein as
"antisense inhibition." Such antisense inhibition is typically
based upon hydrogen bonding-based hybridization of oligonucleotide
strands or segments such that at least one strand or segment is
cleaved, degraded, or otherwise rendered inoperable. In this
regard, it is presently suitable to target specific nucleic acid
molecules and their functions for such antisense inhibition.
[0027] The functions of DNA to be interfered include, but are not
limited to, replication and transcription. Replication and
transcription, for example, can be from an endogenous cellular
template, a vector, a plasmid construct or otherwise. The functions
of RNA to be interfered with include, but are not limited to,
functions such as translocation of the RNA to a site of protein
translation, translocation of the RNA to sites within the cell
which are distant from the site of RNA synthesis, translation of
protein from the RNA, splicing of the RNA to yield one or more RNA
species, and catalytic activity or complex formation involving the
RNA which may be engaged in or facilitated by the RNA.
[0028] In the context of the present invention, "modulation" and
"modulation of expression" mean either an increase (stimulation) or
a decrease (inhibition) in the amount or levels of a nucleic acid
molecule encoding the gene, e.g., DNA or RNA. Inhibition is often
the desired form of modulation of expression and mRNA is often a
suitable target nucleic acid.
[0029] Compounds of the Invention
[0030] In the context of the present invention, the term
"oligomeric compound" refers to a polymeric structure capable of
hybridizing a region of a nucleic acid molecule. This term includes
oligonucleotides, oligonucleosides, oligonucleotide analogs,
oligonucleotide mimetics and combinations of these. Oligomeric
compounds routinely prepared linearly but can be joined or
otherwise prepared to be circular and may also include branching.
Oligomeric compounds can hybridized to form double stranded
compounds which can be blunt ended or may include overhangs. In
general an oligomeric compound comprises a backbone of linked
momeric subunits where each linked momeric subunit is directly or
indirectly attached to a heterocyclic base moiety. The linkages
joining the monomeric subunits, the sugar moieties or surrogates
and the heterocyclic base moieties can be independently modified
giving rise to a plurality of motifs for the resulting oligomeric
compounds including hemimers, gapmers and chimeras.
[0031] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base moiety. The two most common classes of such
heterocyclic bases are purines and pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to either the 2', 3' or 5' hydroxyl moiety of
the sugar. In forming oligonucleotides, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. The respective ends of this linear
polymeric structure can be joined to form a circular structure by
hybridization or by formation of a covalent bond, however, open
linear structures are generally suitable. Within the
oligonucleotide structure, the phosphate groups are commonly
referred to as forming the internucleoside linkages of the
oligonucleotide. The normal internucleoside linkage of RNA and DNA
is a 3' to 5' phosphodiester linkage.
[0032] In the context of this invention, the term "oligonucleotide"
refers to an oligomer or polymer of ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA). This term includes oligonucleotides
composed of naturally-occurring nucleobases, sugars and covalent
internucleoside linkages. The term "oligonucleotide analog" refers
to oligonucleotides that have one or more non-naturally occurring
portions which function in a similar manner to oligonulceotides.
Such non-naturally occurring oligonucleotides are often desired
over the naturally occurring forms because of desirable properties
such as, for example, enhanced cellular uptake, enhanced affinity
for nucleic acid target and increased stability in the presence of
nucleases.
[0033] In the context of this invention, the term "oligonucleoside"
refers to nucleosides that are joined by internucleoside linkages
that do not have phosphorus atoms. Internucleoside linkages of this
type include short chain alkyl, cycloalkyl, mixed heteroatom alkyl,
mixed heteroatom cycloalkyl, one or more short chain heteroatomic
and one or more short chain heterocyclic. These internucleoside
linkages include, but are not limited to, siloxane, sulfide,
sulfoxide, sulfone, acetyl, formacetyl, thioformacetyl, methylene
formacetyl, thioformacetyl, alkeneyl, sulfamate; methyleneimino,
methylenehydrazino, sulfonate, sulfonamide, amide and others having
mixed N, O, S and CH.sub.2 component parts.
[0034] Representative United States patents that teach the
preparation of the above oligonucleosides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608;
5,646,269 and 5,677,439.
[0035] Further included in the present invention are oligomeric
compounds such as antisense oligomeric compounds, antisense
oligonucleotides, ribozymes, external guide sequence (EGS)
oligonucleotides, alternate splicers, primers, probes, and other
oligomeric compounds which hybridize to at least a portion of the
target nucleic acid. As such, these oligomeric compounds may be
introduced in the form of single-stranded, double-stranded,
circular or hairpin oligomeric compounds and may contain structural
elements such as internal or terminal bulges or loops. Once
introduced to a system, the oligomeric compounds of the invention
may elicit the action of one or more enzymes or structural proteins
to effect modification of the target nucleic acid.
[0036] One non-limiting example of such an enzyme is RNAse H, a
cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. It is known in the art that single-stranded antisense
oligomeric compounds which are "DNA-like" elicit RNAse H.
Activation of RNase H, therefore, results in cleavage of the RNA
target, thereby greatly enhancing the efficiency of
oligonucleotide-mediated inhibition of gene expression. Similar
roles have been postulated for other ribonucleases such as those in
the RNase III and ribonuclease L family of enzymes.
[0037] While one form of antisense oligomeric compound is a
single-stranded antisense oligonucleotide, in many species the
introduction of double-stranded structures, such as double-stranded
RNA (dsRNA) molecules, has been shown to induce potent and specific
antisense-mediated reduction of the function of a gene or its
associated gene products. This phenomenon occurs in both plants and
animals and is believed to have an evolutionary connection to viral
defense and transposon silencing.
[0038] In addition to the modifications described above, the
nucleosides of the oligomeric compounds of the invention can have a
variety of other modification so long as these other modifications
either alone or in combination with other nucleosides enhance one
or more of the desired properties described above. Thus, for
nucleotides that are incorporated into oligonucleotides of the
invention, these nucleotides can have sugar portions that
correspond to naturally-occurring sugars or modified sugars.
Representative modified sugars include carbocyclic or acyclic
sugars, sugars having substituent groups at one or more of their
2', 3' or 4' positions and sugars having substituents in place of
one or more hydrogen atoms of the sugar. Additional nucleosides
amenable to the present invention having altered base moieties and
or altered sugar moieties are disclosed in U.S. Pat. No. 3,687,808
and PCT application PCT/US89/02323.
[0039] Altered base moieties or altered sugar moieties also include
other modifications consistent with the spirit of this invention.
Such oligonucleotides are best described as being structurally
distinguishable from, yet functionally interchangeable with,
naturally occurring or synthetic wild type oligonucleotides. All
such oligonucleotides are comprehended by this invention so long as
they function effectively to mimic the structure of a desired RNA
or DNA strand. A class of representative base modifications include
tricyclic cytosine analog, termed "G clamp" (Lin et al., J. Am.
Chem. Soc., 1998, 120, 8531). This analog makes four hydrogen bonds
to a complementary guanine (G) within a helix by simultaneously
recognizing the Watson-Crick and Hoogsteen faces of the targeted G.
This G clamp modification when incorporated into phosphorothioate
oligonucleotides, dramatically enhances antisense potencies in cell
culture. The oligonucleotides of the invention also can include
phenoxazine-substituted bases of the type disclosed by Flanagan et
al., Nat. Biotechnol., 1999, 17, 48-52.
[0040] The present invention provides oligomeric compounds
comprising the structure:
T.sub.1-(Nu.sub.1-L.sub.1).sub.n1-(Nu.sub.2-L.sub.2).sub.n2-(Nu.sub.3-L.su-
b.3).sub.n3-T.sub.2
[0041] wherein:
[0042] each Nu.sub.1 and Nu.sub.3 is, independently, a high
affinity modified nucleoside, wherein at least one of Nu.sub.1 and
Nu.sub.3 is a bicyclic sugar modified nucleoside comprising a
4'-CH.sub.2--O-2' bridge or a 4'-(CH.sub.2).sub.2--O-2' bridge;
[0043] each Nu.sub.2 is a 2'-deoxy nucleoside;
[0044] each L.sub.1, L.sub.2 and L.sub.3 is, independently, an
internucleoside linking group;
[0045] each T.sub.1 and T.sub.2 is, independently, H, a hydroxy
protecting group, an optionally linked conjugate group, or a
covalent attachment to a solid support medium;
[0046] n1 is from 1 to about 6;
[0047] n2 is from 11 to about 18; and
[0048] n3 is from 2 to about 6.
[0049] In some embodiments, each of the high affinity modified
nucleosides is, independently, a bicyclic sugar modified
nucleoside, a 2'-O--(CH.sub.2).sub.2--O--CH.sub.3 modified
nucleoside, a 2'-F modified nucleoside, or a
2'-O--CH.sub.2--C(.dbd.O)--NR.sub.1R.sub.2 modified nucleoside,
where each R.sub.1 and R.sub.2 is, independently, H, a nitrogen
protecting group, substituted or unsubstituted C.sub.1-C.sub.10
alkyl, substituted or unsubstituted C.sub.2-C.sub.10 alkenyl,
substituted or unsubstituted C.sub.2-C.sub.10 alkynyl, wherein the
substitution is OR.sub.3, SR.sub.3, NH.sub.3.sup.+,
NR.sub.3R.sub.4, guanidino or acyl, wherein the acyl is acid amide
or an ester, or R.sub.1 and R.sub.2, together, are a nitrogen
protecting group, or are joined in a ring structure that optionally
includes an additional heteroatom selected from N and O. In some
embodiments, each R.sub.1 and R.sub.2 each is, independently, H, a
nitrogen protecting group, or C.sub.1-C.sub.10 alkyl. In some
embodiments, T.sub.1 is H or a hydroxyl protecting group. In some
embodiments, T.sub.2 is H or a hydroxyl protecting group.
[0050] In some embodiments, each of the hydroxyl protecting groups
is, independently, 4,4'-dimethoxytrityl, monomethoxytrityl,
9-phenylxanthen-9-yl, 9-(p-methoxyphenyl)xanthen-9-yl, t-butyl,
t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,
1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,
2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,
p,p-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,
trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetyl,
chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl,
p-phenylbenzoyl, mesyl, tosyl, 4,4',4"-tris-(benzyloxy)trityl,
4,4',4"-tris-(4,5-dichlorophthalimido)tri- tyl,
4,4',4"-tris(levulinyloxy)trityl,
3-(imidazolylmethyl)-4,4'-dimethoxy- trityl, 4-decyloxytrityl,
4-hexadecyloxytrityl, 9-(4-octadecyloxyphenyl)xa- nthene-9-yl,
1,1-bis-(4-methoxyphenyl)-1'-pyrenyl methyl,
p-phenylazophenyloxycarbonyl, 9-fluorenylmethoxycarbonyl,
2,4-dinitrophenylethoxycarb onyl, 4-(methylthiomethoxy)butyryl,
2-(methylthiomethoxymethyl)-benzoyl,
2-(isopropyl-thiomethoxymethyl)benzo- yl,
2-(2,4-dinitrobenzenesulphenyloxymethyl)benzoyl, or levulinyl
group.
[0051] In some embodiments, one of T.sub.1 and T.sub.2 is a
covalent attachment to a support medium, such as, for example, a
controlled pore glass, oxalyl-controlled pore glass,
silica-containing particles, polymers of polystyrene, copolymers of
polystyrene, copolymers of styrene and divinylbenzene, copolymers
of dimethylacrylamide and N,N'-bisacryloyl-ethylenediamine, soluble
support medium, or PEPS.
[0052] In some embodiments, each L.sub.1, L.sub.2 and L.sub.3 is,
independently, phosphodiester, phosphorothioate, chiral
phosphorothioate, phosphorodithioate, phosphotriester,
aminoalkylphosphotriester, methyl phosphonate, alkyl phosphonate,
5'-alkylene phosphonate, chiral phosphonate, phosphinate,
phosphoramidate, 3'-amino phosphoramidate,
aminoalkylphosphoramidate, thionophosphoramidate,
thionoalkylphosphonate, thionoalkylphosphotriester,
selenophosphate, or boranophosphate. In some embodiments, each
L.sub.1, L.sub.2 and L.sub.3 is, independently, a phosphodiester or
a phosphorothioate internucleoside linking group. In some
embodiments, each L.sub.1, L.sub.2 and L.sub.3 is a phosphodiester
internucleoside linking group. In some embodiments, each L.sub.1,
L.sub.2 and L.sub.3 is, independently, siloxane, sulfide,
sulfoxide, sulfone, formacetyl, thioformacetyl, methylene
formacetyl, thioformacetyl, sulfamate, methyleneimino,
methylenehydrazino, sulfonate, sulfonamide, or amide. In some
embodiments, each of the internucleoside linking groups is,
independently, --CH.sub.2--NH--O--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)- --O--CH.sub.2-- or
--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--, or
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2--.
[0053] In some embodiments, each nucleoside comprises a
heterocyclic base moiety that is, independently, adenine, guanine,
thymine, cytosine, uracil, 5-methylcytosine, 5-hydroxy-methyl
cytosine, xanthine, hypoxanthine, 2-aminoadenine, alkyl derivatives
of adenine and guanine, 2-thiouracil, 2-thiothymine,
2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl uracil,
5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine,
5-uracil (pseudouracil), 4-thiouracil, 8-substituted adenines and
guanines, 5-substituted uracils and cytosines, 7-methylguanine,
7-methyladenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine,
7-deazaadenine, 3-deazaguanine, or 3-deazaadenine.
[0054] In some embodiments, n1 is from 1 to about 5, or from 1 to
about 3, or from 2 to about 3. In some embodiments, n3 is from 2 to
about 5, or from 2 to about 3. In some embodiments, n2 is from 12
to about 18, or from 12 to about 16, or from 14 to about 16. In
some embodiments, the total of n1, n2 and n3 is from 14 to about
30; or the total of n1, n2 and n3 is from 14 to 24; or the total of
n1, n2 and n3 is from 14 to 21; or the total of n1, n2 and n3 is
from 16 to 21. In some embodiments, n1 is from 1 to about 3, n2 is
12 or 13 and n3 is 2 or 3; or n1 is 3, n2 is 12 and n3 is 3; or n1
is from 1 to about 3, n2 is 14 or 15 and n3 is 2 or 3; or n1 is 2,
n2 is 14 and n3 is 2; or n1 is from 1 to about 3, n2 is 16 or 17
and n3 is 2 or 3; or n1 is 2, n2 is 16 and n3 is 2.
[0055] In some embodiments, at least one Nu.sub.1 nucleoside and at
least one Nu.sub.3 nucleside is an LNA or ENA. In some embodiments,
at least one of the 5'-most or 3'-most terminal affinity modified
nucleosides is an LNA or ENA. Thus, the terminal most 5' and/or 3'
nucleoside can, independently, be either LNA or ENA.
[0056] The present invention also provides oligomeric compounds
comprsing the structure: 2
[0057] wherein:
[0058] each Bx is a heterocyclic base moiety;
[0059] each X is, independently, O or S;
[0060] T.sub.1 and T.sub.2 are each, independently, H, a hydroxy
protecting group, an optionally linked conjugate group, or a
covalent attachment to a solid support medium;
[0061] each m is, independently, 1 or 2;
[0062] na is from 1 to about 6;
[0063] nb is from 11 to about 18; and
[0064] nc is from 2 to about 6.
[0065] In some embodiments, each m is 1 or 2. In some embodiments,
at least one of T.sub.1 and T.sub.2 is H or a hydroxyl protecting
group. In some embodiments, each of the hydroxyl protecting groups
is, independently, 4,4'-dimethoxytrityl, monomethoxytrityl,
9-phenylxanthen-9-yl, 9-(p-methoxyphenyl)xanthen-9-yl, t-butyl,
t-butoxymethyl, methoxymethyl, tetrahydropyranyl, 1-ethoxyethyl,
1-(2-chloroethoxy)ethyl, 2-trimethylsilylethyl, p-chlorophenyl,
2,4-dinitrophenyl, benzyl, 2,6-dichlorobenzyl, diphenylmethyl,
p,p-dinitrobenzhydryl, p-nitrobenzyl, triphenylmethyl,
trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,
t-butyldiphenylsilyl, triphenylsilyl, benzoylformate, acetyl,
chloroacetyl, trichloroacetyl, trifluoroacetyl, pivaloyl, benzoyl,
p-phenylbenzoyl, mesyl, tosyl, 4,4',4"-tris-(benzyloxy)trityl,
4,4',4"-tris-(4,5-dichlorophthalimido)tri- tyl,
4,4',4"-tris(levulinyloxy)trityl,
3-(imidazolylmethyl)-4,4'-dimethoxy- trityl, 4-decyloxytrityl,
4-hexadecyloxytrityl, 9-(4-octadecyloxyphenyl)xa- nthene-9-yl,
1,1-bis-(4-methoxyphenyl)-1'-pyrenyl methyl,
p-phenylazophenyloxycarbonyl, 9-fluorenylmethoxycarbonyl,
2,4-dinitrophenylethoxycarb onyl, 4-(methylthiomethoxy)butyryl,
2-(methylthiomethoxymethyl)-benzoyl,
2-(isopropylthiomethoxymethyl)benzoy- l,
2-(2,4-dinitrobenzenesulphenyl-oxymethyl)benzoyl, or levulinyl
group.
[0066] In some embodiments, one of T.sub.1 and T.sub.2 is a
covalent attachment to a support medium such as, for example, a
controlled pore glass, oxalyl-controlled pore glass,
silica-containing particles, polymers of polystyrene, copolymers of
polystyrene, copolymers of styrene and divinylbenzene, copolymers
of dimethylacrylamide and N,N'-bisacryloyl-ethylenediamine, soluble
support medium, or PEPS.
[0067] In some embodiments, each L.sub.1, L.sub.2 and L.sub.3 is,
independently, a phosphodiester or a phosphorothioate
internucleoside linking group. In some embodiments, each L.sub.1,
L.sub.2 and L.sub.3 is a phosphodiester internucleoside linking
group.
[0068] In some embodiments, each Bx is, independently, adenine,
guanine, thymine, cytosine, uracil, 5-methylcytosine,
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
alkyl derivatives of adenine and guanine, 2-thiouracil,
2-thiothymine, 2-thiocytosine, 5-halouracil, 5-halocytosine,
5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo
cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil,
8-substituted adenines and guanines, 5-substituted uracils and
cytosines, 7-methylguanine, 7-methyladenine, 8-azaguanine,
8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, or
3-deazaadenine.
[0069] In some embodiments, n1 is from 1 to about 5 (e.g., 1, 2, 3,
4, 5), or from 1 to about 3 (e.g., 1, 2, 3), or from 2 to about 3.
In some embodiments, n3 is from 2 to about 5 (e.g., 2, 3, 4, 5), or
from 2 to about 3. In some embodiments, n2 is from 12 to about 18
(e.g., 12, 13, 14, 15, 16, 17, 18), or from 12 to about 16 (e.g.,
12, 13, 14, 15, 16), or from 14 to about 16 (e.g., 14, 15, 16). In
some embodiments, the total of n1, n2 and n3 is from 14 to about 30
(e.g., 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28,
29, 30); or the total of n1, n2 and n3 is from 14 to 24 (e.g., 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24); or the total of n1, n2 and
n3 is from 14 to 21 (e.g., 14, 15, 16, 17, 18, 19, 20, 21); or the
total of n1, n2 and n3 is from 16 to 21 (e.g., 16, 17, 18, 19, 20,
21). In some embodiments, n1 is from 1 to about 3, n2 is 12 or 13
and n3 is 2 or 3; or n1 is 3, n2 is 12 and n3 is 3; or n1 is from 1
to about 3, n2 is 14 or 15 and n3 is 2 or 3; or n1 is 2, n2 is 14
and n3 is 2; or n1 is from 1 to about 3, n2 is 16 or 17 and n3 is 2
or 3; or n1 is 2, n2 is 16 and n3 is 2.
[0070] Oligomer Synthesis
[0071] Oligomerization of modified and unmodified nucleosides is
performed according to literature procedures for DNA (Protocols for
Oligonucleotides and Analogs, Ed. Agrawal (1993), Humana Press)
and/or RNA (Scaringe, Methods (2001), 23, 206-217. Gait et al.,
Applications of Chemically synthesized RNA in RNA: Protein
Interactions, Ed. Smith (1998), 1-36. Gallo et al., Tetrahedron
(2001), 57, 5707-5713) synthesis as appropriate. In addition
specific protocols for the synthesis of oligomeric compounds of the
invention are illustrated in the examples below.
[0072] The oligomeric compounds 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 well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives.
[0073] The present invention is also useful for the preparation of
oligomeric compounds incorporating at least one 2'-O-protected
nucleoside. After incorporation and appropriate deprotection the
2'-O-protected nucleoside will be converted to a ribonucleoside at
the position of incorporation. The number and position of the
2-ribonucleoside units in the final oligomeric compound can vary
from one at any site or the strategy can be used to prepare up to a
full 2'-OH modified oligomeric compound. All 2'-O-protecting groups
amenable to the synthesis of oligomeric compounds are included in
the present invention. In general a protected nucleoside is
attached to a solid support by for example a succinate linker. Then
the oligonucleotide is elongated by repeated cycles of deprotecting
the 5'-terminal hydroxyl group, coupling of a further nucleoside
unit, capping and oxidation (alternatively sulfurization). In a
more frequently used method of synthesis the completed
oligonucleotide is cleaved from the solid support with the removal
of phosphate protecting groups and exocyclic amino protecting
groups by treatment with an ammonia solution. Then a further
deprotection step is normally required for the more specialized
protecting groups used for the protection of 2'-hydroxyl groups
which will give the fully deprotected oligonucleotide.
[0074] A large number of 2'-O-protecting groups have been used for
the synthesis of oligoribonucleotides but over the years more
effective groups have been discovered. The key to an effective
2'-O-protecting group is that it is capable of selectively being
introduced at the 2'-O-position and that it can be removed easily
after synthesis without the formation of unwanted side products.
The protecting group also needs to be inert to the normal
deprotecting, coupling, and capping steps required for
oligoribonucleotide synthesis. Some of the protecting groups used
initially for oligoribonucleotide synthesis included
tetrahydropyran-1-yl and 4-methoxytetrahydropyran-4-yl. These two
groups are not compatible with all 5'-O-protecting groups so
modified versions were used with 5'-DMT groups such as
1-(2-fluorophenyl)-4-methoxypiperidi- n-4-yl (Fpmp). Reese has
identified a number of piperidine derivatives (like Fpmp) that are
useful in the synthesis of oligoribonucleotides including
1-((chloro-4-methyl)phenyl)-4'-methoxypiperidin-4-yl (Reese et al.,
Tetrahedron Lett., 1986, 27, 2291). Another approach was to replace
the standard 5'-DMT (dimethoxytrityl) group with protecting groups
that were removed under non-acidic conditions such as levulinyl and
9-fluorenylmethoxycarbonyl. Such groups enable the use of acid
labile 2'-protecting groups for oligoribonucleotide synthesis.
Another more widely used protecting group initially used for the
synthesis of oligoribonucleotides was the t-butyldimethylsilyl
group (Ogilvie et al., Tetrahedron Lett., 1974, 2861; Hakimelahi et
al., Tetrahedron Lett., 1981, (22), 2543; and Jones et al., J.
Chem. Soc. Perkin I., 2762). The 2'-O-protecting groups can require
special reagents for their removal such as for example the
t-butyldimethylsilyl group is normally removed after all other
cleaving/deprotecting steps by treatment of the oligomeric compound
with tetrabutylammonium fluoride (TBAF).
[0075] One group of researchers examined a number of
2'-O-protecting groups (Pitsch, Chimia, 2001, (55), 320-324.) The
group examined fluoride labile and photolabile protecting groups
that are removed using moderate conditions. One photolabile group
that was examined was the (2-(nitrobenzyl)oxy)methyl (nbm)
protecting group (Schwartz et al., Bioorg. Med. Chem. Lett., 1992,
2, 1019). Other groups examined included a number structurally
related formaldehyde acetal-derived, 2'-O-protecting groups. Also
prepared were a number of related protecting groups for preparing
2'-O-alkylated nucleoside phosphoramidites including
2'-O-((triisopropylsilyl)oxy)methyl
(2'-O--CH.sub.2--O-Si(iPr).sub.3, TOM). One 2'-O-protecting group
that was prepared to be used orthogonally to the TOM group was
2'-O-((R)-1-(2-nitrophenyl)ethyloxy)methyl) ((R)-mnbm).
[0076] Another strategy using a fluoride labile 5'-O-protecting
group (non-acid labile) and an acid labile 2'-O-protecting group
has been reported (Scaringe, Stephen A., Methods, 2001, 23,
206-217). A number of possible silyl ethers were examined for
5'-O-protection and a number of acetals and orthoesters were
examined for 2'-O-protection. The protection scheme that gave the
best results was 5'-O-silyl ether-2'-ACE
(5'-O-bis(trimethylsiloxy)cyclododecyloxysilyl ether
(DOD)-2'-O-bis(2-acetoxyethoxy)methyl (ACE). This approach uses a
modified phosphoramidite synthesis approach in that some different
reagents are required that are not routinely used for RNA/DNA
synthesis.
[0077] Although a lot of research has focused on the synthesis of
oligoribonucleotides the main RNA synthesis strategies that are
presently being used commercially include
5'-O-DMT-2'-O-t-butyldimethylsilyl (TBDMS), 5'-O-DMT-2'-O-(1
(2-fluorophenyl)-4-methoxypiperidin-4-yl) (FPMP),
2'-O-((triisopropylsilyl)oxy)methyl (2'-O--CH.sub.2--O-Si(iPr).su-
b.3 (TOM), and the 5'-O-silyl ether-2'-ACE
(5'-O-bis(trimethylsiloxy)cyclo- dodecyloxysilyl ether
(DOD)-2'-O-bis(2-acetoxyethoxy)methyl (ACE). A current list of some
of the major companies currently offering RNA products include
Pierce Nucleic Acid Technologies, Dharmacon Research Inc., Ameri
Biotechnologies Inc., and Integrated DNA Technologies, Inc. One
company, Princeton Separations, is marketing an RNA synthesis
activator advertised to reduce coupling times especially with TOM
and TBDMS chemistries. Such an activator would also be amenable to
the present invention.
[0078] The primary groups being used for commercial RNA synthesis
are:
[0079] TBDMS=5'-O-DMT-2'-O-t-butyldimethylsilyl;
[0080] TOM=2'-O-((triisopropylsilyl)oxy)methyl;
[0081] DOD/ACE=(5'-O-bis(trimethylsiloxy)cyclododecyloxysilyl
ether-2'-O-bis(2-acetoxyethoxy)methyl
[0082] FPMP=5'-O-DMT-2'-O-(1
(2-fluorophenyl)-4-methoxypiperidin-4-yl).
[0083] All of the aforementioned RNA synthesis strategies are
amenable to the present invention. Strategies that would be a
hybrid of the above e.g. using a 5'-protecting group from one
strategy with a 2'-O-protecting from another strategy is also
amenable to the present invention.
[0084] The preparation of ribonucleotides and oligomeric compounds
having at least one ribonucleoside incorporated and all the
possible configurations falling in between these two extremes are
encompassed by the present invention. The corresponding oligomeric
comounds can be hybridized to further oligomeric compounds
including oligoribonucleotides having regions of complementarity to
form double-stranded (duplexed) oligomeric compounds. Such double
stranded oligonucleotide moieties have been shown in the art to
modulate target expression and regulate translation as well as RNA
processsing via an antisense mechanism. Moreover, the
double-stranded moieties may be subject to chemical modifications
(Fire et al., Nature, 1998, 391, 806-811; Timmons and Fire, Nature
1998, 395, 854; Timmons et al., Gene, 2001, 263, 103-112; Tabara et
al., Science, 1998, 282, 430-431; Montgomery et al., Proc. Natl.
Acad. Sci. USA, 1998, 95, 15502-15507; Tuschl et al., Genes Dev.,
1999, 13, 3191-3197; Elbashir et al., Nature, 2001, 411, 494-498;
Elbashir et al., Genes Dev. 2001, 15, 188-200). For example, such
double-stranded moieties have been shown to inhibit the target by
the classical hybridization of antisense strand of the duplex to
the target, thereby triggering enzymatic degradation of the target
(Tijsterman et al., Science, 2002, 295, 694-697).
[0085] The methods of preparing oligomeric compounds of the present
invention can also be applied in the areas of drug discovery and
target validation. The present invention comprehends the use of the
oligomeric compounds and suitable targets identified herein in drug
discovery efforts to elucidate relationships that exist between
proteins and a disease state, phenotype, or condition. These
methods include detecting or modulating a target peptide comprising
contacting a sample, tissue, cell, or organism with the oligomeric
compounds of the present invention, measuring the nucleic acid or
protein level of the target and/or a related phenotypic or chemical
endpoint at some time after treatment, and optionally comparing the
measured value to a non-treated sample or sample treated with a
further oligomeric compound of the invention. These methods can
also be performed in parallel or in combination with other
experiments to determine the function of unknown genes for the
process of target validation or to determine the validity of a
particular gene product as a target for treatment or prevention of
a particular disease, condition, or phenotype.
[0086] Effect of nucleoside modifications on RNAi activity is
evaluated according to existing literature (Elbashir et al., Nature
(2001), 411, 494-498; Nishikura et al., Cell (2001), 107, 415-416;
and Bass et al., Cell (2000), 101, 235-238.)
[0087] A number of chemical functional groups can be introduced
into compounds of the invention in a blocked form and subsequently
deblocked to form a final, desired compound. Such as groups
directly or indirectly attached at the heterocyclic bases, the
internucleoside linkages and the sugar substituent groups at the
2', 3' and 5'-positions. Protecting groups can be selected to block
functional groups located in a growing oligomeric compound during
iterative oligonucleotide synthesis while other positions can be
selectively deblocked as needed. In general, a blocking group
renders a chemical functionality of a larger molecule inert to
specific reaction conditions and can later be removed from such
functionality without substantially damaging the remainder of the
molecule (Greene and Wuts, Protective Groups in Organic Synthesis,
3rd ed, John Wiley & Sons, New York, 1999). For example, the
nitrogen atom of amino groups can be blocked as phthalimido groups,
as 9-fluorenylmethoxycarbonyl (FMOC) groups, and with
triphenylmethylsulfenyl, t-BOC or benzyl groups. Carboxyl groups
can be blocked as acetyl groups. Representative hydroxyl protecting
groups are described by Beaucage et al., Tetrahedron 1992, 48,
2223. Suitable hydroxyl protecting groups are acid-labile, such as
the trityl, monomethoxytrityl, dimethoxytrityl, trimethoxytrityl,
9-phenylxanthine-9-yl (Pixyl) and 9-(p-methoxyphenyl)xanthine-9-yl
(MOX).
[0088] Chemical functional groups can also be "blocked" by
including them in a precursor form. Thus, an azido group can be
used considered as a "blocked" form of an amine since the azido
group is easily converted to the amine. Further representative
protecting groups utilized in oligonucleotide synthesis are
discussed in Agrawal, et al., Protocols for Oligonucleotide
Conjugates, Eds, Humana Press; New Jersey, 1994; Vol. 26 pp. 1
72.
[0089] Examples of hydroxyl protecting groups include, but are not
limited to, t-butyl, t-butoxymethyl, methoxymethyl,
tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl,
2-trimethylsilylethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl,
2,6-dichlorobenzyl, diphenylmethyl, p,=-dinitrobenzhydryl,
p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl,
t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,
benzoylformate, acetate, chloroacetate, trichloroacetate,
trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate,
9-fluorenylmethyl carbonate, mesylate and tosylate.
[0090] Amino-protecting groups stable to acid treatment are
selectively removed with base treatment, and are used to make
reactive amino groups selectively available for substitution.
Examples of such groups are the Fmoc (E. Atherton and R. C.
Sheppard in The Peptides, S. Udenfriend, J. Meienhofer, Eds.,
Academic Press, Orlando, 1987, volume 9, p.1), and various
substituted sulfonylethyl carbamates exemplified by the Nsc group
(Samukov et al., Tetrahedron Lett, 1994, 35:7821; Verhart and
Tesser, Rec. Trav. Chim. Pays-Bas, 1987, 107, 621).
[0091] Additional amino-protecting groups include but are not
limited to, carbamate-protecting groups, such as
2-trimethylsilylethoxycarbonyl (Teoc),
1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl
(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl
(Fmoc), and benzyloxycarbonyl (Cbz); amide-protecting groups, such
as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;
sulfonamide-protecting groups, such as 2-nitrobenzenesulfonyl; and
imine- and cyclic imide-protecting groups, such as phthalimido and
dithiasuccinoyl.
[0092] The current method of choice for the preparation of
oligomeric compounds utilizes support media. Support media is used
for attachment of a first nucleoside or other synthon which is then
iteratively elongated to give a final oligomeric compound or other
polymer such as a polypeptide. Support media can be selected to be
insoluble or have variable solubility in different solvents to
allow the growing support bound polymer to be either in or out of
solution as desired. Traditional support media such as solid
supports are for the most part insoluble and are routinely placed
in a reaction vessel while reagents and solvents react and or wash
the growing chain until cleavage the final polymeric compound. More
recent approaches have introduced soluble supports including
soluble polymer supports to allow precipitating and dissolving the
iteratively synthesized product at desired points in the synthesis
(Gravert et al., Chem. Rev., 1997, 97, 489-510).
[0093] The term support media is intended to include all forms of
support known to the art skilled for the synthesis of oligomeric
compounds and related compounds such as peptides. Some
representative support media that are amenable to the methods of
the present invention include but are not limited to the following:
controlled pore glass (CPG); oxalyl-controlled pore glass (see,
e.g., Alul, et al., Nucleic Acids Research 1991, 19, 1527);
silica-containing particles, such as porous glass beads and silica
gel such as that formed by the reaction of
trichloro-(3-(4-chloromethyl)phenyl)propylsilane and porous glass
beads (see Parr and Grohmann, Angew. Chem. Internal. Ed. 1972, 11,
314, sold under the trademark "PORASIL E" by Waters Associates,
Framingham, Mass., USA); the mono ester of
1,4-dihydroxymethylbenzene and silica (see Bayer and Jung,
Tetrahedron Lett., 1970, 4503, sold under the trademark "BIOPAK" by
Waters Associates); TENTAGEL (see, e.g., Wright, et al.,
Tetrahedron Letters 1993, 34, 3373); cross-linked
styrene/divinylbenzene copolymer beaded matrix or POROS, a
copolymer of polystyrene/divinylbenze- ne (available from
Perceptive Biosystems); soluble support media, polyethylene glycol
PEG's (see Bonora et al., Organic Process Research &
Development, 2000, 4, 225-231).
[0094] Further support media amenable to the present invention
include without limitation PEPS support a polyethylene (PE) film
with pendant long-chain polystyrene (PS) grafts (molecular weight
on the order of 106, (see Berg, et al., J. Am. Chem. Soc., 1989,
111, 8024 and International Patent Application WO 90/02749). The
loading capacity of the film is as high as that of a beaded matrix
with the additional flexibility to accomodate multiple syntheses
simultaneously. The PEPS film may be fashioned in the form of
discrete, labeled sheets, each serving as an individual
compartment. During all the identical steps of the synthetic
cycles, the sheets are kept together in a single reaction vessel to
permit concurrent preparation of a multitude of peptides at a rate
close to that of a single peptide by conventional methods. Also,
experiments with other geometries of the PEPS polymer such as, for
example, non-woven felt, knitted net, sticks or microwellplates
have not indicated any limitations of the synthetic efficacy.
[0095] Further support media amenable to the present invention
include without limitation particles based upon copolymers of
dimethylacrylamide cross-linked with
N,N'-bisacryloylethylenediamine, including a known amount of
N-tertbutoxycarbonyl-beta-alanyl-N'-acryloylhexamethylenediamin- e.
Several spacer molecules are typically added via the beta alanyl
group, followed thereafter by the amino acid residue subunits.
Also, the beta alanyl-containing monomer can be replaced with an
acryloyl safcosine monomer during polymerization to form resin
beads. The polymerization is followed by reaction of the beads with
ethylenediamine to form resin particles that contain primary amines
as the covalently linked functionality. The polyacrylamide-based
supports are relatively more hydrophilic than are the
polystyrene-based supports and are usually used with polar aprotic
solvents including dimethylformamide, dimethylacetamide,
N-methylpyrrolidone and the like (see Atherton, et al., J. Am.
Chem. Soc., 1975, 97, 6584, Bioorg. Chem. 1979, 8, 351, and J. C.
S. Perkin 1538 (1981)).
[0096] Further support media amenable to the present invention
include without limitation a composite of a resin and another
material that is also substantially inert to the organic synthesis
reaction conditions employed. One exemplary composite (see Scott,
et al., J. Chrom. Sci., 1971, 9, 577) utilizes glass particles
coated with a hydrophobic, cross-linked styrene polymer containing
reactive chloromethyl groups, and is supplied by Northgate
Laboratories, Inc., of Hamden, Conn., USA. Another exemplary
composite contains a core of fluorinated ethylene polymer onto
which has been grafted polystyrene (see Kent and Merrifield, Israel
J. Chem. 1978, 17, 243 and van Rietschoten in Peptides 1974, Y.
Wolman, Ed., Wiley and Sons, New York, 1975, pp. 113-116).
Contiguous solid supports other than PEPS, such as cotton sheets
(Lebl and Eichler, Peptide Res. 1989, 2, 232) and
hydroxypropylacrylate-coated polypropylene membranes (Daniels, et
al., Tetrahedron Lett. 1989, 4345). Acrylic acid-grafted
polyethylene-rods and 96-microtiter wells to immobilize the growing
peptide chains and to perform the compartmentalized synthesis.
(Geysen, et al., Proc. Natl. Acad. Sci. USA, 1984, 81, 3998). A
"tea bag" containing traditionally-used polymer beads. (Houghten,
Proc. Natl. Acad. Sci. USA, 1985, 82, 5131). Simultaneous use of
two different supports with different densities (Tregear, Chemistry
and Biology of Peptides, J. Meienhofer, ed., Ann Arbor Sci. Publ.,
Ann Arbor, 1972 pp. 175-178). Combining of reaction vessels via a
manifold (Gorman, Anal. Biochem., 1984, 136, 397). Multicolumn
solid-phase synthesis (e.g., Krchnak, et al., Int. J. Peptide
Protein Res., 1989, 33, 209), and Holm and Meldal, in "Proceedings
of the 20th European Peptide Symposium", G. Jung and E. Bayer,
eds., Walter de Gruyter & Co., Berlin, 1989 pp. 208-210).
Cellulose paper (Eichler, et al., Collect. Czech. Chem. Commun.,
1989, 54, 1746). Support mediated synthesis of peptides have also
been reported (see, Synthetic Peptides: A User's Guide, Gregory A.
Grant, Ed. Oxford University Press 1992; U.S. Pat. Nos. 4,415,732;
4,458,066; 4,500,707; 4,668,777; 4,973,679; 5,132,418; 4,725,677
and Re-34,069.)
[0097] Support bound oligonucleotide synthesis relies on sequential
addition of nucleotides to one end of a growing chain. Typically, a
first nucleoside (having protecting groups on any exocyclic amine
functionalities present) is attached to an appropriate glass bead
support and activated phosphite compounds (typically nucleotide
phosphoramidites, also bearing appropriate protecting groups) are
added stepwise to elongate the growing oligonucleotide. Additional
methods for solid-phase synthesis may be found in Caruthers U.S.
Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679;
and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re.
34,069.
[0098] Commercially available equipment routinely used for the
support media based synthesis of oligomeric compounds and related
compounds 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. Suitable solid phase techniques, including automated
synthesis techniques, are described in F. Eckstein (ed.),
Oligonucleotides and Analogues, a Practical Approach, Oxford
University Press, New York (1991).
[0099] Oligomer and Monomer Modifications
[0100] As is known in the art, a nucleoside is a base-sugar
combination. The base portion of the nucleoside is normally a
heterocyclic base. The two most common classes of such heterocyclic
bases are the purines and the pyrimidines. Nucleotides are
nucleosides that further include a phosphate group covalently
linked to the sugar portion of the nucleoside. For those
nucleosides that include a pentofuranosyl sugar, the phosphate
group can be linked to either the 2', 3' or 5' hydroxyl moiety of
the sugar. In forming oligonucleotides, the phosphate groups
covalently link adjacent nucleosides to one another to form a
linear polymeric compound. In turn, the respective ends of this
linear polymeric compound can be further joined to form a circular
compound, however, linear compounds are generally suitable. In
addition, linear compounds may have internal nucleobase
complementarity and may therefore fold in a manner as to produce a
fully or partially double-stranded compound. Within
oligonucleotides, the phosphate groups are commonly referred to as
forming the internucleoside linkage or in conjunction with the
sugar ring the backbone of the oligonucleotide. The normal
internucleoside linkage that makes up the backbone of RNA and DNA
is a 3' to 5' phosphodiester linkage.
[0101] Modified Internucleoside Linkages
[0102] Specific examples of suitable oligomeric compounds useful in
this invention include oligonucleotides containing modified e.g.
non-naturally occurring internucleoside linkages. As defined in
this specification, oligonucleotides having modified
internucleoside linkages include internucleoside linkages that
retain a phosphorus atom and internucleoside linkages that do not
have a phosphorus atom. For the purposes of this specification, and
as sometimes referenced in the art, modified oligonucleotides that
do not have a phosphorus atom in their internucleoside backbone can
also be considered to be oligonucleosides.
[0103] In the C. elegans system, modification of the
internucleotide linkage (phosphorothioate) did not significantly
interfere with RNAi activity. Based on this observation, it is
suggested that certain oligomeric compounds of the invention can
also have one or more modified internucleoside linkages. A suitable
phosphorus containing modified internucleoside linkage is the
phosphorothioate internucleoside linkage.
[0104] Suitable modified oligonucleotide backbones containing a
phosphorus atom therein include, for example, phosphorothioates,
chiral phosphorothioates, phosphorodithioates, phosphotriesters,
aminoalkylphosphotriesters, methyl and other alkyl phosphonates
including 3'-alkylene phosphonates, 5'-alkylene phosphonates and
chiral phosphonates, phosphinates, phosphoramidates including
3'-amino phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, selenophosphates and boranophosphates
having normal 3'-5' linkages, 2'-5' linked analogs of these, and
those having inverted polarity wherein one or more internucleotide
linkages is a 3' to 3', 5' to 5' or 2' to 2' linkage. Suitable
oligonucleotides having inverted polarity comprise a single 3' to
3' linkage at the 3'-most internucleotide linkage i.e. a single
inverted nucleoside residue which may be abasic (the nucleobase is
missing or has a hydroxyl group in place thereof). Various salts,
mixed salts and free acid forms are also included.
[0105] Representative U.S. patents that teach the preparation of
the above phosphorus-containing linkages include, but are not
limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899;
5,721,218; 5,672,697 and 5,625,050.
[0106] In some embodiments of the invention, oligomeric compounds
have one or more phosphorothioate and/or heteroatom internucleoside
linkages, in particular
--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-- (wherein the native
phosphodiester internucleotide linkage is represented as
--O--P(.dbd.O)(OH)--O--CH.sub.2--). The MMI type internucleoside
linkages are disclosed in the above referenced U.S. Pat. No.
5,489,677. Suitable amide internucleoside linkages are disclosed in
the above referenced U.S. Pat. No. 5,602,240.
[0107] Suitable modified oligonucleotide backbones that do not
include a phosphorus atom therein have backbones that are formed by
short chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; riboacetyl backbones; alkene containing backbones;
sulfamate backbones; methyleneimino and methylenehydrazino
backbones; sulfonate and sulfonamide backbones; amide backbones;
and others having mixed N, O, S and CH.sub.2 component parts.
[0108] Representative United States patents that teach the
preparation of the above oligonucleosides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608;
5,646,269 and 5,677,439.
[0109] In some embodiments, the nucleoside components of the
oligomeric compounds are connected to each other by optionally
protected phosphorothioate internucleoside linkages. Representative
protecting groups for phosphorus containing internucleoside
linkages such as phosphite, phosphodiester and phosphorothioate
linages include .beta.-cyanoethyl, diphenylsilylethyl,
.delta.-cyanobutenyl, cyano p-xylyl (CPX),
N-methyl-N-trifluoroacetyl ethyl (META), acetoxy phenoxy ethyl
(APE) and butene-4-yl groups. See for example U.S. Pat. Nos.
4,725,677 and Re. 34,069 (.beta.-cyanoethyl); Beaucage, S. L. and
Iyer, R. P., Tetrahedron, 49 No. 10, pp. 1925-1963 (1993);
Beaucage, S. L. and Iyer, R. P., Tetrahedron, 49 No. 46, pp.
10441-10488 (1993); Beaucage, S. L. and Iyer, R. P., Tetrahedron,
48 No. 12, pp. 2223-2311 (1992).
[0110] Oligomer Mimetics (Oligonucleotide Mimics)
[0111] Another suitable group of oligomeric compounds amenable to
the present invention includes oligonucleotide mimetics. The term
mimetic as it is applied to oligonucleotides is intended to include
oligomeric compounds wherein only the furanose ring or both the
furanose ring and the internucleotide linkage are replaced with
novel groups, replacement of only the furanose ring is also
referred to in the art as being a sugar surrogate. The heterocyclic
base moiety or a modified heterocyclic base moiety is maintained
for hybridization with an appropriate target nucleic acid. One such
oligomeric compound, an oligonucleotide mimetic that has been shown
to have excellent hybridization properties, is referred to as a
peptide nucleic acid (PNA). In PNA oligomeric compounds, the
sugar-backbone of an oligonucleotide is replaced with an amide
containing backbone, in particular an aminoethylglycine backbone.
The nucleobases are retained and are bound directly or indirectly
to aza nitrogen atoms of the amide portion of the backbone.
Representative United States patents that teach the preparation of
PNA oligomeric compounds include, but are not limited to, U.S. Pat.
Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of PNA
oligomeric compounds can be found in Nielsen et al., Science, 1991,
254, 1497-1500.
[0112] One oligonucleotide mimetic that has been reported to have
excellent hybridization properties, is peptide nucleic acids (PNA).
The backbone in PNA compounds is two or more linked
aminoethylglycine units which gives PNA an amide containing
backbone. The heterocyclic base moieties are bound directly or
indirectly to aza nitrogen atoms of the amide portion of the
backbone. Representative United States patents that teach the
preparation of PNA compounds include, but are not limited to, U.S.
Pat. Nos. 5,539,082; 5,714,331; and 5,719,262. Further teaching of
PNA compounds can be found in Nielsen et al., Science, 1991, 254,
1497-1500.
[0113] PNA has been modified to incorporate numerous modifications
since the basic PNA structure was first prepared. The basic
structure is shown below: 3
[0114] wherein
[0115] Bx is a heterocyclic base moiety;
[0116] T.sub.4 is hydrogen, an amino protecting group,
--C(O)R.sub.5, substituted or unsubstituted C.sub.1-C.sub.12 alkyl,
substituted or unsubstituted C.sub.2-C.sub.12 alkenyl, substituted
or unsubstituted C.sub.2-C.sub.12 alkynyl, alkylsulfonyl,
arylsulfonyl, a chemical functional group, a reporter group, a
conjugate group, a D or L .alpha.-amino acid linked via the
.alpha.-carboxyl group or optionally through the .omega.-carboxyl
group when the amino acid is aspartic acid or glutamic acid or a
peptide derived from D, L or mixed D and L amino acids linked
through a carboxyl group, wherein the substituent groups are
selected from hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl,
nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and
alkynyl;
[0117] T.sub.5 is --OH, --N(Z.sub.1)Z.sub.2, R.sub.5, D or L
.alpha.-amino acid linked via the .alpha.-amino group or optionally
through the c-amino group when the amino acid is lysine or
ornithine or a peptide derived from D, L or mixed D and L amino
acids linked through an amino group, a chemical functional group, a
reporter group or a conjugate group;
[0118] Z.sub.1 is hydrogen, C.sub.1-C.sub.6 alkyl, or an amino
protecting group;
[0119] Z.sub.2 is hydrogen, C.sub.1-C.sub.6 alkyl, an amino
protecting group, --C(.dbd.O)--(CH.sub.2).sub.n-J-Z.sub.3, a D or L
.alpha.-amino acid linked via the .alpha.-carboxyl group or
optionally through the .omega.-carboxyl group when the amino acid
is aspartic acid or glutamic acid or a peptide derived from D, L or
mixed D and L amino acids linked through a carboxyl group;
[0120] Z.sub.3 is hydrogen, an amino protecting group,
--C.sub.1-C.sub.6 alkyl, --C(.dbd.O)--CH.sub.3, benzyl, benzoyl, or
--(CH.sub.2).sub.n--N(H- )Z.sub.1;
[0121] each J is O, S or NH;
[0122] R.sub.5 is a carbonyl protecting group; and
[0123] n is from 2 to about 50.
[0124] Another class of oligonucleotide mimetic that has been
studied is based on linked morpholino units (morpholino nucleic
acid) having heterocyclic bases attached to the morpholino ring. A
number of linking groups have been reported that link the
morpholino monomeric units in a morpholino nucleic acid. One class
of linking groups have been selected to give a non-ionic oligomeric
compound. The non-ionic morpholino-based oligomeric compounds are
less likely to have undesired interactions with cellular proteins.
Morpholino-based oligomeric compounds are non-ionic mimics of
oligonucleotides which are less likely to form undesired
interactions with cellular proteins (Dwaine A. Braasch and David R.
Corey, Biochemistry, 2002, 41(14), 4503-4510). Morpholino-based
oligomeric compounds are disclosed in U.S. Pat. No. 5,034,506,
issued Jul. 23, 1991. The morpholino class of oligomeric compounds
have been prepared having a variety of different linking groups
joining the monomeric subunits.
[0125] Morpholino nucleic acids have been prepared having a variety
of different linking groups (L.sub.2) joining the monomeric
subunits. The basic formula is shown below: 4
[0126] wherein:
[0127] T.sub.1 is hydroxyl or a protected hydroxyl;
[0128] T.sub.5 is hydrogen or a phosphate or phosphate
derivative;
[0129] L.sub.2 is a linking group; and
[0130] n is from 2 to about 50.
[0131] A further class of oligonucleotide mimetic is referred to as
cyclohexenyl nucleic acids (CeNA). The furanose ring normally
present in an DNA/RNA molecule is replaced with a cyclohenyl ring.
CeNA DMT protected phosphoramidite monomers have been prepared and
used for oligomeric compound synthesis following classical
phosphoramidite chemistry. Fully modified CeNA oligomeric compounds
and oligonucleotides having specific positions modified with CeNA
have been prepared and studied (see Wang et al., J. Am. Chem. Soc.,
2000, 122, 8595-8602). In general the incorporation of CeNA
monomers into a DNA chain increases its stability of a DNA/RNA
hybrid. CeNA oligoadenylates formed complexes with RNA and DNA
complements with similar stability to the native complexes. The
study of incorporating CeNA structures into natural nucleic acid
structures was shown by NMR and circular dichroism to proceed with
easy conformational adaptation. Furthermore the incorporation of
CeNA into a sequence targeting RNA was stable to serum and able to
activate E. Coli RNase resulting in cleavage of the target RNA
strand.
[0132] The general formula of CeNA is shown below: 5
[0133] wherein:
[0134] each Bx is a heterocyclic base moiety;
[0135] T.sub.1 is hydroxyl or a protected hydroxyl; and
[0136] T2 is hydroxyl or a protected hydroxyl.
[0137] Another class of oligonucleotide mimetic (anhydrohexitol
nucleic acid) can be prepared from one or more anhydrohexitol
nucleosides (see, Wouters and Herdewijn, Bioorg. Med. Chem. Lett.,
1999, 9, 1563-1566) and would have the general formula: 6
[0138] Another modification includes Locked Nucleic Acids (LNAs) in
which the 2'-hydroxyl group is linked to the 4' carbon atom of the
sugar ring thereby forming a 2'-C,4'-C-oxymethylene linkage thereby
forming a bicyclic sugar moiety. The linkage can be a methylene
(--CH.sub.2--).sub.n group bridging the 2' oxygen atom and the 4'
carbon atom wherein n is 1 for LNA (or 2 for ENA, Singh et al.,
Chem. Commun., 1998, 4, 455-456). LNA and LNA analogs display very
high duplex thermal stabilities with complementary DNA and RNA (Tm
=+3 to +10 C), stability towards 3'-exonucleolytic degradation and
good solubility properties. The basic structure of LNA showing the
bicyclic ring system is shown below: 7
[0139] The conformations of LNAs determined by 2D NMR spectroscopy
have shown that the locked orientation of the LNA nucleotides, both
in single-stranded LNA and in duplexes, constrains the phosphate
backbone in such a way as to introduce a higher population of the
N-type conformation (Petersen et al., J. Mol. Recognit., 2000, 13,
44-53). These conformations are associated with improved stacking
of the nucleobases (Wengel et al., Nucleosides Nucleotides, 1999,
18, 1365-1370).
[0140] LNA has been shown to form exceedingly stable LNA:LNA
duplexes (Koshkin et al., J. Am. Chem. Soc., 1998, 120,
13252-13253). LNA:LNA hybridization was shown to be the most
thermally stable nucleic acid type duplex system, and the
RNA-mimicking character of LNA was established at the duplex level.
Introduction of 3 LNA monomers (T or A) significantly increased
melting points (Tm=+15/+11) toward DNA complements. The
universality of LNA-mediated hybridization has been stressed by the
formation of exceedingly stable LNA:LNA duplexes. The RNA-mimicking
of LNA was reflected with regard to the N-type conformational
restriction of the monomers and to the secondary structure of the
LNA:RNA duplex.
[0141] LNAs also form duplexes with complementary DNA, RNA or LNA
with high thermal affinities. Circular dichroism (CD) spectra show
that duplexes involving fully modified LNA (esp. LNA:RNA)
structurally resemble an A-form RNA:RNA duplex. Nuclear magnetic
resonance (NMR) examination of an LNA:DNA duplex confirmed the
3'-endo conformation of an LNA monomer. Recognition of
double-stranded DNA has also been demonstrated suggesting strand
invasion by LNA. Studies of mismatched sequences show that LNAs
obey the Watson-Crick base pairing rules with generally improved
selectivity compared to the corresponding unmodified reference
strands.
[0142] Novel types of LNA-oligomeric compounds, as well as the
LNAs, are useful in a wide range of diagnostic and therapeutic
applications. Among these are antisense applications, PCR
applications, strand-displacement oligomers, substrates for nucleic
acid polymerases and generally as nucleotide based drugs.
[0143] Potent and nontoxic antisense oligonucleotides containing
LNAs have been described (Wahlestedt et al., Proc. Natl. Acad. Sci.
U.S.A., 2000, 97, 5633-5638.) The authors have demonstrated that
LNAs confer several desired properties to antisense agents. LNA/DNA
copolymers were not degraded readily in blood serum and cell
extracts. LNA/DNA copolymers exhibited potent antisense activity in
assay systems as disparate as G-protein-coupled receptor signaling
in living rat brain and detection of reporter genes in Escherichia
coli. Lipofectin-mediated efficient delivery of LNA into living
human breast cancer cells has also been accomplished.
[0144] The synthesis and preparation of the LNA monomers adenine,
cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along
with their oligomerization, and nucleic acid recognition properties
have been described (Koshkin et al., Tetrahedron, 1998, 54,
3607-3630). LNAs and preparation thereof are also described in WO
98/39352 and WO 99/14226.
[0145] The first analogs of LNA, phosphorothioate-LNA and
2'-thio-LNAs, have also been prepared (Kumar et al., Bioorg. Med.
Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside
analogs containing oligodeoxyribonucleotide duplexes as substrates
for nucleic acid polymerases has also been described (Wengel et
al., PCT International Application WO 98-DK393 19980914).
Furthermore, synthesis of 2'-amino-LNA, a novel conformationally
restricted high-affinity oligonucleotide analog with a handle has
been described in the art (Singh et al., J. Org. Chem., 1998, 63,
10035-10039). In addition, 2'-Amino- and 2`-methylamino-LNA`s have
been prepared and the thermal stability of their duplexes with
complementary RNA and DNA strands has been previously reported.
[0146] One group has added an additional methlene group to the LNA
2',4'-bridging group (e.g. 4'-CH.sub.2--CH.sub.2--O-2' (ENA),
Kaneko et al., United States Patent Application Publication No.: US
2002/0147332, also see Japanese Patent Application HEI-11-33863,
Feb. 12, 1999).
[0147] Further oligonucleotide mimetics have been prepared to
incude bicyclic and tricyclic nucleoside analogs having the
formulas (amidite monomers shown): 8
[0148] (see Steffens et al., Helv. Chim. Acta, 1997, 80, 2426-2439;
Steffens et al., J. Am. Chem. Soc., 1999, 121, 3249-3255; and
Renneberg et al., J. Am. Chem. Soc., 2002, 124, 5993-6002). These
modified nucleoside analogs have been oligomerized using the
phosphoramidite approach and the resulting oligomeric compounds
containing tricyclic nucleoside analogs have shown increased
thermal stabilities (Tm's) when hybridized to DNA, RNA and itself.
Oligomreric compounds containing bicyclic nucleoside analogs have
shown thermal stabilities approaching that of DNA duplexes.
[0149] Another class of oligonucleotide mimetic is referred to as
phosphonomonoester nucleic acids incorporate a phosphorus group in
a backbone the backbone. This class of olignucleotide mimetic is
reported to have useful physical and biological and pharmacological
properties in the areas of inhibiting gene expression (antisense
oligonucleotides, ribozymes, sense oligonucleotides and
triplex-forming oligonucleotides), as probes for the detection of
nucleic acids and as auxiliaries for use in molecular biology.
[0150] The general formula (for definitions of Markush variables
see: U.S. Pat. Nos. 5,874,553 and 6,127,346) is shown below. 9
[0151] Another oligonucleotide mimetic has been reported wherein
the furanosyl ring has been replaced by a cyclobutyl moiety.
[0152] Modified Sugars
[0153] Oligomeric compounds of the invention may also contain one
or more substituted sugar moieties. Suiotable oligomeric compounds
comprise a sugar substituent group selected from: OH; F; O-, S-, or
N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or
O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may be
substituted or unsubstituted C.sub.1 to C.sub.12 alkyl or C.sub.2
to C.sub.12 alkenyl and alkynyl. Particularly suitable are
O((CH.sub.2).sub.nO).sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub- .3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON((CH.sub.2).sub.nCH.su- b.3).sub.2, where n and
m are from 1 to about 10. Other suitable oligonucleotides comprise
a sugar substituent group selected from: C.sub.1 to C.sub.12 lower
alkyl, substituted lower alkyl, alkenyl, alkynyl, alkaryl, aralkyl,
O-alkaryl or O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3,
OCF.sub.3, 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. One modification
includes 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also
known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.
Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy group. Another
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylamino-ethoxyethoxy (also known in the art as
2'-O-dimethyl-amino-ethoxy-ethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.3).sub.2.
[0154] Other sugar substituent groups include methoxy
(--O--CH.sub.3), aminopropoxy
(--OCH.sub.2CH.sub.2CH.sub.2NH.sub.2), allyl
(--CH.sub.2--CH.dbd.CH.sub.2), --O-allyl
(--O--CH.sub.2--CH.dbd.CH.sub.2) and fluoro (F). 2'-Sugar
substituent groups may be in the arabino (up) position or ribo
(down) position. One 2'-arabino modification is 2'-F. Similar
modifications may also be made at other positions on the oligomeric
compoiund, particularly the 3' position of the sugar on the 3'
terminal nucleoside or in 2'-5' linked oligonucleotides and the 5'
position of 5' terminal nucleotide. Oligomeric compounds may also
have sugar mimetics such as cyclobutyl moieties in place of the
pentofuranosyl sugar. Representative United States patents that
teach the preparation of such modified sugar structures include,
but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;
5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;
5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; 5,792,747;
and 5,700,920, certain of which are commonly owned with the instant
application.
[0155] Further representative sugar substituent groups include
groups of formula I.sub.a or II.sub.a: 10
[0156] wherein:
[0157] R.sub.b is O, S or NH;
[0158] R.sub.d is a single bond, O, S or C(.dbd.O);
[0159] R.sub.e is C.sub.1-C.sub.12 alkyl, N(R.sub.k)(R.sub.m),
N(R.sub.k)(R.sub.n), N.dbd.C(R.sub.p)(R.sub.q),
N.dbd.C(R.sub.p)(R.sub.r) or has formula III.sub.a; 11
[0160] R.sub.p and R.sub.q are each independently hydrogen or
C.sub.1-C.sub.12 alkyl;
[0161] R.sub.r is --R.sub.x--R.sub.y;
[0162] each R.sub.s, R.sub.t, R.sub.u and R.sub.v is,
independently, hydrogen, C(O)R.sub.w, substituted or unsubstituted
C.sub.1-C.sub.12 alkyl, substituted or unsubstituted
C.sub.2-C.sub.12 alkenyl, substituted or unsubstituted
C.sub.2-C.sub.12 alkynyl, alkylsulfonyl, arylsulfonyl, a chemical
functional group or a conjugate group, wherein the substituent
groups are selected from hydroxyl, amino, alkoxy, carboxy, benzyl,
phenyl, nitro, thiol, thioalkoxy, halogen, alkyl, aryl, alkenyl and
alkynyl;
[0163] or optionally, R.sub.u and R.sub.v, together form a
phthalimido moiety with the nitrogen atom to which they are
attached;
[0164] each R.sub.w is, independently, substituted or unsubstituted
C.sub.1-C.sub.12alkyl, trifluoromethyl, cyanoethyloxy, methoxy,
ethoxy, t-butoxy, allyloxy, 9-fluorenylmethoxy,
2-(trimethylsilyl)-ethoxy, 2,2,2-trichloroethoxy, benzyloxy,
butyryl, iso-butyryl, phenyl or aryl;
[0165] R.sub.k is hydrogen, a nitrogen protecting group or
--R.sub.x--R.sub.y;
[0166] R.sub.p is hydrogen, a nitrogen protecting group or
--R.sub.x--R.sub.y;
[0167] R.sub.x is a bond or a linking moiety;
[0168] R.sub.y is a chemical functional group, a conjugate group or
a solid support medium;
[0169] each R.sub.m and R.sub.n is, independently, H, a nitrogen
protecting group, substituted or unsubstituted C.sub.1-C.sub.12
alkyl, substituted or unsubstituted C.sub.2-C.sub.12 alkenyl,
substituted or unsubstituted C.sub.2-C.sub.12 alkynyl, wherein the
substituent groups are selected from hydroxyl, amino, alkoxy,
carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, halogen, alkyl,
aryl, alkenyl, alkynyl; NH.sub.3.sup.+, N(R.sub.u)(R.sub.v)
guanidino and acyl where said acyl is an acid amide or an
ester;
[0170] or R.sub.m and R.sub.n, together, are a nitrogen protecting
group, are joined in a ring structure that optionally includes an
additional heteroatom selected from N and O or are a chemical
functional group;
[0171] R.sub.i is OR.sub.z, SR.sub.z, or N(R.sub.z).sub.2;
[0172] each R.sub.z is, independently, H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 haloalkyl, C(.dbd.NH)N(H)R.sub.u,
C(.dbd.O)N(H)R.sub.u or OC(.dbd.O)N(H)R.sub.u;
[0173] R.sub.f, R.sub.g and R.sub.h comprise a ring system having
from about 4 to about 7 carbon atoms or having from about 3 to
about 6 carbon atoms and 1 or 2 heteroatoms wherein said
heteroatoms are selected from oxygen, nitrogen and sulfur and
wherein said ring system is aliphatic, unsaturated aliphatic,
aromatic, or saturated or unsaturated heterocyclic;
[0174] R.sub.j is alkyl or haloalkyl having 1 to about 10 carbon
atoms, alkenyl having 2 to about 10 carbon atoms, alkynyl having 2
to about 10 carbon atoms, aryl having 6 to about 14 carbon atoms,
N(R.sub.k)(R.sub.m)OR.sub.k, halo, SR.sub.k or CN;
[0175] m.sub.a is 1 to about 10;
[0176] each mb is, independently, 0 or 1;
[0177] mc is 0 or an integer from 1 to 10;
[0178] md is an integer from 1 to 10;
[0179] me is from 0, 1 or 2; and
[0180] provided that when mc is 0, md is greater than 1.
[0181] Representative substituents groups of Formula I are
disclosed in U.S. patent application Ser. No. 09/130,973, filed
Aug. 7, 1998, entitled "Capped 2'-Oxyethoxy Oligonucleotides."
[0182] Representative cyclic substituent groups of Formula II are
disclosed in U.S. patent application Ser. No. 09/123,108, filed
Jul. 27, 1998, entitled "RNA Targeted 2'-Oligomeric compounds that
are Conformationally Preorganized."
[0183] Particularly suitable sugar substituent groups include
O((CH.sub.2).sub.nO).sub.mCH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2 and
O(CH.sub.2).sub.nON((CH.sub.2).sub.nCH.sub- .3)).sub.2, where n and
m are from 1 to about 10.
[0184] Representative guanidino substituent groups that are shown
in formula III and IV are disclosed in co-owned U.S. patent
application Ser. No. 09/349,040, entitled "Functionalized
Oligomers" filed Jul. 7, 1999.
[0185] Representative acetamido substituent groups are disclosed in
U.S. Pat. No. 6,147,200.
[0186] Representative dimethylaminoethyloxyethyl substituent groups
are disclosed in International Patent Application PCT/US99/17895,
entitled "2'-O-Dimethylaminoethyloxy-ethyl-Oligomeric compounds",
filed Aug. 6, 1999.
[0187] Modified Nucleobases/Naturally Occurring Nucleobases
[0188] Oligomeric compounds may also include nucleobase (often
referred to in the art simply as "base" or "heterocyclic base
moiety") modifications or substitutions. As used herein,
"unmodified" or "natural" nucleobases include the purine bases
adenine (A) and guanine (G), and the pyrimidine bases thymine (T),
cytosine (C) and uracil (U). Modified nucleobases also referred
herein as heterocyclic base moieties include other synthetic and
natural nucleobases such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl (--C.ident.C--CH.sub.3) uracil and cytosine
and other alkynyl derivatives of pyrimidine bases, 6-azo uracil,
cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil,
8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other
8-substituted adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine
and 3-deazaguanine and 3-deazaadenine.
[0189] Heterocyclic base moieties may also include those in which
the purine or pyrimidine base is replaced with other heterocycles,
for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and
2-pyridone. Further nucleobases include those disclosed in U.S.
Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of
Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I.,
ed. John Wiley & Sons, 1990, those disclosed by Englisch et
al., Angewandte Chemie, International Edition, 1991, 30, 613, and
those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research
and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed.,
CRC Press, 1993. Certain of these nucleobases are particularly
useful for increasing the binding affinity of the oligomeric
compounds of the invention. These include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted
purines, including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C.
(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense
Research and Applications, CRC Press, Boca Raton, 1993, pp.
276-278) and are presently suitable base substitutions, even more
particularly when combined with 2'-O-methoxyethyl sugar
modifications.
[0190] In one aspect of the present invention oligomeric compounds
are prepared having polycyclic heterocyclic compounds in place of
one or more heterocyclic base moieties. A number of tricyclic
heterocyclic comounds have been previously reported. These
compounds are routinely used in antisense applications to increase
the binding properties of the modified strand to a target strand.
The most studied modifications are targeted to guanosines hence
they have been termed G-clamps or cytidine analogs. Many of these
polycyclic heterocyclic compounds have the general formula: 12
[0191] Representative cytosine analogs that make 3 hydrogen bonds
with a guanosine in a second strand include
1,3-diazaphenoxazine-2-one (R.sub.10.dbd.O,
R.sub.11--R.sub.14.dbd.H) (Kurchavov, et al., Nucleosides and
Nucleotides, 1997, 16, 1837-1846), 1,3-diazaphenothiazine-2-one
(R.sub.10.dbd.S, R.sub.11--R.sub.14.dbd.H), (Lin, K.-Y.; Jones, R.
J.; Matteucci, M. J. Am. Chem. Soc. 1995, 117, 3873-3874) and
6,7,8,9-tetrafluoro-1,3-diazaphenoxazine-2-one (R.sub.10.dbd.O,
R.sub.11--R.sub.14.dbd.F) (Wang, J.; Lin, K.-Y., Matteucci, M.
Tetrahedron Lett. 1998, 39, 8385-8388). Incorporated into
oligonucleotides these base modifications were shown to hybridize
with complementary guanine and the latter was also shown to
hybridize with adenine and to enhance helical thermal stability by
extended stacking interactions (also see U.S. Patent Application
entitled "Modified Peptide Nucleic Acids" filed May 24, 2002, Ser.
No. 10/155,920; and U.S. Patent Application entitled "Nuclease
Resistant Chimeric Oligonucleotides" filed May 24, 2002, Ser. No.
10/013,295).
[0192] Further helix-stabilizing properties have been observed when
a cytosine analog/substitute has an aminoethoxy moiety attached to
the rigid 1,3-diazaphenoxazine-2-one scaffold (R.sub.10.dbd.O,
R.sub.1.dbd.--O--(CH.sub.2).sub.2--NH.sub.2, R.sub.12-14.dbd.H)
(Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998, 120, 8531-8532).
Binding studies demonstrated that a single incorporation could
enhance the binding affinity of a model oligonucleotide to its
complementary target DNA or RNA with a .DELTA.T.sub.m of up to
18.degree. relative to 5-methyl cytosine (dC5.sup.me), which is the
highest known affinity enhancement for a single modification, yet.
On the other hand, the gain in helical stability does not
compromise the specificity of the oligonucleotides. The T.sub.m
data indicate an even greater discrimination between the perfect
match and mismatched sequences compared to dC5.sup.me. It was
suggested that the tethered amino group serves as an additional
hydrogen bond donor to interact with the Hoogsteen face, namely the
O6, of a complementary guanine thereby forming 4 hydrogen bonds.
This means that the increased affinity of G-clamp is mediated by
the combination of extended base stacking and additional specific
hydrogen bonding.
[0193] Further tricyclic heterocyclic compounds and methods of
using them that are amenable to the present invention are disclosed
in U.S. Pat. No. 6,028,183, which issued on May 22, 2000, and U.S.
Pat. No. 6,007,992, which issued on Dec. 28, 1999.
[0194] The enhanced binding affinity of the phenoxazine derivatives
together with their uncompromised sequence specificity makes them
valuable nucleobase analogs for the development of more potent
antisense-based drugs. In fact, promising data have been derived
from in vitro experiments demonstrating that heptanucleotides
containing phenoxazine substitutions are capable to activate
RNaseH, enhance cellular uptake and exhibit an increased antisense
activity (Lin et al., J. Am. Chem. Soc., 1998, 120, 8531-8532). The
activity enhancement was even more pronounced in case of G-clamp,
as a single substitution was shown to significantly improve the in
vitro potency of a 20mer 2'-deoxyphosphorothioate oligonucleotides
(Flanagan et al., Proc. Natl. Acad. Sci. USA, 1999, 96, 3513-3518).
Nevertheless, to optimize oligonucleotide design and to better
understand the impact of these heterocyclic modifications on the
biological activity, it is important to evaluate their effect on
the nuclease stability of the oligomers.
[0195] Further modified polycyclic heterocyclic compounds useful as
heterocyclcic bases are disclosed in but not limited to, the above
noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205;
5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,434,257;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,646,269;
5,750,692; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, and U.S.
patent application Ser. No. 09/996,292 filed Nov. 28, 2001.
[0196] Conjugates
[0197] Another substitution that can be appended to the oligomeric
compounds of the invention involves the linkage of one or more
moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the resulting oligomeric
compounds. In one embodiment such modified oligomeric compounds are
prepared by covalently attaching conjugate groups to functional
groups such as hydroxyl or amino groups. Conjugate groups of the
invention include intercalators, reporter molecules, polyamines,
polyamides, poly-ethylene glycols, polyethers, groups that enhance
the pharmacodynamic properties of oligomers, and groups that
enhance the pharmacokinetic properties of oligomers. Typical
conjugates groups include cholesterols, lipids, phospholipids,
biotin, phenazine, folate, phenanthridine, anthraquinone, acridine,
fluoresceins, rhodamines, coumarins, and dyes. Groups that enhance
the pharmacodynamic properties, in the context of this invention,
include groups that improve oligomer uptake, enhance oligomer
resistance to degradation, and/or strengthen sequence-specific
hybridization with RNA. Groups that enhance the pharmacokinetic
properties, in the context of this invention, include groups that
improve oligomer uptake, distribution, metabolism or excretion.
Representative conjugate groups are disclosed in International
Patent Application PCT/US92/09196, filed Oct. 23, 1992.
[0198] Conjugate moieties include but are not limited to lipid
moieties such as a cholesterol moiety (Letsinger et al., Proc.
Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan
et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether,
e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci.,
1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let.,
1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl.
Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,
dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,
1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,
327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), 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, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxychol- esterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937).
[0199] The oligomeric compounds of the invention may also be
conjugated to active drug substances, for example, aspirin,
warfarin, phenylbutazone, ibuprofen, suprofen, fenbufen,
ketoprofen, (S)-(+)-pranoprofen, carprofen, dansylsarcosine,
2,3,5-triiodobenzoic acid, flufenamic acid, folinic acid, a
benzothiadiazide, chlorothiazide, a diazepine, indomethicin, a
barbiturate, a cephalosporin, a sulfa drug, an antidiabetic, an
antibacterial or an antibiotic. Oligonucleotide-drug conjugates and
their preparation are described in U.S. patent application Ser. No.
09/334,130 (filed Jun. 15, 1999).
[0200] Representative United States patents that teach the
preparation of such oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928 and 5,688,941.
[0201] Chimeric Oligomeric Compounds
[0202] It is not necessary for all positions in a oligomeric
compound to be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
oligomeric compound or even at a single monomeric subunit such as a
nucleoside within a oligomeric compound. The present invention also
includes oligomeric compounds which are chimeric oligomeric
compounds. "Chimeric" oligomeric compounds or "chimeras," in the
context of this invention, are oligomeric compounds which contain
two or more chemically distinct regions, each made up of at least
one monomer unit, i.e., a nucleotide in the case of a nucleic acid
based oligomer.
[0203] Chimeric oligomeric compounds typically contain at least one
region modified so as to confer increased resistance to nuclease
degradation, increased cellular uptake, and/or increased binding
affinity for the target nucleic acid. An additional region of the
oligomeric compound 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
inhibition of gene expression. Consequently, comparable results can
often be obtained with shorter oligomeric compounds when chimeras
are used, compared to for example phosphorothioate
deoxyoligonucleotides hybridizing to the same target region.
Cleavage of the RNA target can be routinely detected by gel
electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
[0204] Chimeric oligomeric compounds of the invention may be formed
as composite structures of two or more oligonucleotides,
oligonucleotide analogs, oligonucleosides and/or oligonucleotide
mimetics as described above. Such oligomeric compounds have also
been referred to in the art as hybrids hemimers, gapmers or
inverted gapmers. Representative United States patents that teach
the preparation of such hybrid structures include, but are not
limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;
5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922.
[0205] 3'-endo Modifications
[0206] In one aspect of the present invention oligomeric compounds
include nucleosides synthetically modified to induce a 3'-endo
sugar conformation. A nucleoside can incorporate synthetic
modifications of the heterocyclic base, the sugar moiety or both to
induce a desired 3'-endo sugar conformation. These modified
nucleosides are used to mimic RNA like nucleosides so that
particular properties of an oligomeric compound can be enhanced
while maintaining the desirable 3'-endo conformational geometry.
There is an apparent preference for an RNA type duplex (A form
helix, predominantly 3'-endo) as a requirement (e.g. trigger) of
RNA interference which is supported in part by the fact that
duplexes composed of 2'-deoxy-2'-F-nucleosides appears efficient in
triggering RNAi response in the C. elegans system. Properties that
are enhanced by using more stable 3'-endo nucleosides include but
aren't limited to modulation of pharmacokinetic properties through
modification of protein binding, protein off-rate, absorption and
clearance; modulation of nuclease stability as well as chemical
stability; modulation of the binding affinity and specificity of
the oligomer (affinity and specificity for enzymes as well as for
complementary sequences); and increasing efficacy of RNA cleavage.
The present invention provides oligomeric triggers of RNAi having
one or more nucleosides modified in such a way as to favor a
C3'-endo type conformation. 13
[0207] Nucleoside conformation is influenced by various factors
including substitution at the 2', 3' or 4'-positions of the
pentofuranosyl sugar. Electronegative substituents generally prefer
the axial positions, while sterically demanding substituents
generally prefer the equatorial positions (Principles of Nucleic
Acid Structure, Wolfgang Sanger, 1984, Springer-Verlag.)
Modification of the 2' position to favor the 3'-endo conformation
can be achieved while maintaining the 2'-OH as a recognition
element, as illustrated in FIG. 2, below (Gallo et al., Tetrahedron
(2001), 57, 5707-5713. Harry-O'kuru et al., J. Org. Chem., (1997),
62(6), 1754-1759 and Tang et al., J. Org. Chem. (1999), 64,
747-754.) Alternatively, preference for the 3'-endo conformation
can be achieved by deletion of the 2'-OH as exemplified by
2'deoxy-2' F-nucleosides (Kawasaki et al., J. Med. Chem. (1993),
36, 831-841), which adopts the 3'-endo conformation positioning the
electronegative fluorine atom in the axial position. Other
modifications of the ribose ring, for example substitution at the
4'-position to give 4'-F modified nucleosides (Guillerm et al.,
Bioorganic and Medicinal Chemistry Letters (1995), 5, 1455-1460 and
Owen et al., J. Org. Chem. (1976), 41, 3010-3017), or for example
modification to yield methanocarba nucleoside analogs (Jacobson et
al., J. Med. Chem. Lett. (2000), 43, 2196-2203 and Lee et al.,
Bioorganic and Medicinal Chemistry Letters (2001), 11, 1333-1337)
also induce preference for the 3'-endo conformation. Along similar
lines, oligomeric triggers of RNAi response might be composed of
one or more nucleosides modified in such a way that conformation is
locked into a C3'-endo type conformation, i.e. Locked Nucleic Acid
(LNA, Singh et al, Chem. Commun. (1998), 4, 455-456), and ethylene
bridged Nucleic Acids (ENA, Morita et al, Bioorganic &
Medicinal Chemistry Letters (2002), 12, 73-76.) Examples of
modified nucleosides amenable to the present invention are shown
below in Table I. These examples are meant to be representative and
not exhaustive.
1TABLE I 14 15 16 17 18 19 20
[0208] A preferred conformation of modified nucleosides and their
oligomers can be estimated by various methods such as molecular
dynamics calculations, nuclear magnetic resonance spectroscopy and
CD measurements. Hence, modifications predicted to induce RNA like
conformations, A-form duplex geometry in an oligomeric context, are
selected for use in the modified oligoncleotides of the present
invention. The synthesis of numerous of the modified nucleosides
amenable to the present invention are known in the art (see for
example, Chemistry of Nucleosides and Nucleotides Vol 1-3, ed.
Leroy B. Townsend, 1988, Plenum press., and the examples section
below.) Nucleosides known to be inhibitors/substrates for RNA
dependent RNA polymerases (for example HCV NS5B
[0209] In one aspect, the present invention is directed to
oligonucleotides that are prepared having enhanced properties
compared to native RNA against nucleic acid targets. A target is
identified and an oligonucleotide is selected having an effective
length and sequence that is complementary to a portion of the
target sequence. Each nucleoside of the selected sequence is
scrutinized for possible enhancing modifications. Another
modification would be the replacement of one or more RNA
nucleosides with nucleosides that have the same 3'-endo
conformational geometry. Such modifications can enhance chemical
and nuclease stability relative to native RNA while at the same
time being much cheaper and easier to synthesize and/or incorporate
into an oligonulceotide. The selected sequence can be further
divided into regions and the nucleosides of each region evaluated
for enhancing modifications that can be the result of a chimeric
configuration. Consideration is also given to the 5' and 3'-termini
as there are often advantageous modifications that can be made to
one or more of the terminal nucleosides. The oligomeric compounds
of the present invention include at least one 5'-modified phosphate
group on a single strand or on at least one 5'-position of a double
stranded sequence or sequences. Further modifications are also
considered such as internucleoside linkages, conjugate groups,
substitute sugars or bases, substitution of one or more nucleosides
with nucleoside mimetics and any other modification that can
enhance the selected sequence for its intended target.
[0210] The terms used to describe the conformational geometry of
homoduplex nucleic acids are "A Form" for RNA and "B Form" for DNA.
The respective conformational geometry for RNA and DNA duplexes was
determined from X-ray diffraction analysis of nucleic acid fibers
(Arnott and Hukins, Biochem. Biophys. Res. Comm., 1970, 47, 1504.)
In general, RNA:RNA duplexes are more stable and have higher
melting temperatures (Tm's) than DNA:DNA duplexes (Sanger et al.,
Principles of Nucleic Acid Structure, 1984, Springer-Verlag; New
York, N.Y.; Lesnik et al., Biochemistry, 1995, 34, 10807-10815;
Conte et al., Nucleic Acids Res., 1997, 25, 2627-2634). The
increased stability of RNA has been attributed to several
structural features, most notably the improved base stacking
interactions that result from an A-form geometry (Searle et al.,
Nucleic Acids Res., 1993, 21, 2051-2056). The presence of the 2'
hydroxyl in RNA biases the sugar toward a C3' endo pucker, i.e.,
also designated as Northern pucker, which causes the duplex to
favor the A-form geometry. In addition, the 2' hydroxyl groups of
RNA can form a network of water mediated hydrogen bonds that help
stabilize the RNA duplex (Egli et al., Biochemistry, 1996, 35,
8489-8494). On the other hand, deoxy nucleic acids prefer a C2'
endo sugar pucker, i.e., also known as Southern pucker, which is
thought to impart a less stable B-form geometry (Sanger, W. (1984)
Principles of Nucleic Acid Structure, Springer-Verlag, New York,
N.Y.). As used herein, B-form geometry is inclusive of both
C2'-endo pucker and 04'-endo pucker. This is consistent with
Berger, et. al., Nucleic Acids Research, 1998, 26, 2473-2480, who
pointed out that in considering the furanose conformations which
give rise to B-form duplexes consideration should also be given to
a 04'-endo pucker contribution.
[0211] DNA:RNA hybrid duplexes, however, are usually less stable
than pure RNA:RNA duplexes, and depending on their sequence may be
either more or less stable than DNA:DNA duplexes (Searle et al.,
Nucleic Acids Res., 1993, 21, 2051-2056). The structure of a hybrid
duplex is intermediate between A- and B-form geometries, which may
result in poor stacking interactions (Lane et al., Eur. J.
Biochem., 1993, 215, 297-306; Fedoroff et al., J. Mol. Biol., 1993,
233, 509-523; Gonzalez et al., Biochemistry, 1995, 34, 4969-4982;
Horton et al., J. Mol. Biol., 1996, 264, 521-533). The stability of
the duplex formed between a target RNA and a synthetic sequence is
central to therapies such as but not limited to antisense and RNA
interference as these mechanisms require the binding of a synthetic
oligonucleotide strand to an RNA target strand. In the case of
antisense, effective inhibition of the mRNA requires that the
antisense DNA have a very high binding affinity with the mRNA.
Otherwise the desired interaction between the synthetic
oligonucleotide strand and target mRNA strand will occur
infrequently, resulting in decreased efficacyl
[0212] One routinely used method of modifying the sugar puckering
is the substitution of the sugar at the 2'-position with a
substituent group that influences the sugar geometry. The influence
on ring conformation is dependant on the nature of the substituent
at the 2'-position. A number of different substituents have been
studied to determine their sugar puckering effect. For example,
2'-halogens have been studied showing that the 2'-fluoro derivative
exhibits the largest population (65%) of the C3'-endo form, and the
2'-iodo exhibits the lowest population (7%). The populations of
adenosine (2'-OH) versus deoxyadenosine (2'-H) are 36% and 19%,
respectively. Furthermore, the effect of the 2'-fluoro group of
adenosine dimers
(2'-deoxy-2'-fluoroadenosine-2'-deoxy-2'-fluoro-adenosin- e) is
further correlated to the stabilization of the stacked
conformation.
[0213] As expected, the relative duplex stability can be enhanced
by replacement of 2'-OH groups with 2'-F groups thereby increasing
the C3'-endo population. It is assumed that the highly polar nature
of the 2'-F bond and the extreme preference for C3'-endo puckering
may stabilize the stacked conformation in an A-form duplex. Data
from UV hypochromicity, circular dichroism, and .sup.1H NMR also
indicate that the degree of stacking decreases as the
electronegativity of the halo substituent decreases. Furthermore,
steric bulk at the 2'-position of the sugar moiety is better
accommodated in an A-form duplex than a B-form duplex. Thus, a
2'-substituent on the 3'-terminus of a dinucleoside monophosphate
is thought to exert a number of effects on the stacking
conformation: steric repulsion, furanose puckering preference,
electrostatic repulsion, hydrophobic attraction, and hydrogen
bonding capabilities. These substituent effects are thought to be
determined by the molecular size, electronegativity, and
hydrophobicity of the substituent. Melting temperatures of
complementary strands is also increased with the 2'-substituted
adenosine diphosphates. It is not clear whether the 3'-endo
preference of the conformation or the presence of the substituent
is responsible for the increased binding. However, greater overlap
of adjacent bases (stacking) can be achieved with the 3'-endo
conformation.
[0214] One synthetic 2'-modification that imparts increased
nuclease resistance and a very high binding affinity to nucleotides
is the 2-methoxyethoxy (2'-MOE, 2'-OCH.sub.2CH.sub.2OCH.sub.3) side
chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). One
of the immediate advantages of the 2'-MOE substitution is the
improvement in binding affinity, which is greater than many similar
2' modifications such as O-methyl, O-propyl, and O-aminopropyl.
Oligonucleotides having the 2'-O-methoxyethyl substituent also have
been shown to be antisense inhibitors of gene expression with
promising features for in vivo use (Martin, P., Helv. Chim. Acta,
1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176;
Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and
Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).
Relative to DNA, the oligonucleotides having the 2'-MOE
modification displayed improved RNA affinity and higher nuclease
resistance. Chimeric oligonucleotides having 2'-MOE substituents in
the wing nucleosides and an internal region of
deoxy-phosphorothioate nucleotides (also termed a gapped
oligonucleotide or gapmer) have shown effective reduction in the
growth of tumors in animal models at low doses. 2'-MOE substituted
oligonucleotides have also shown outstanding promise as antisense
agents in several disease states. One such MOE substituted
oligonucleotide is presently being investigated in clinical trials
for the treatment of CMV retinitis.
[0215] Chemistries Defined
[0216] Unless otherwise defined herein, alkyl means
C.sub.1-C.sub.12, C.sub.1-C.sub.8, or C.sub.1-C.sub.6, straight or
(where possible) branched chain aliphatic hydrocarbyl.
[0217] Unless otherwise defined herein, heteroalkyl means
C.sub.1-C.sub.12, C.sub.1-C.sub.8, or C.sub.1-C.sub.6, straight or
(where possible) branched chain aliphatic hydrocarbyl containing at
least one or about 1 to about 3 hetero atoms in the chain,
including the terminal portion of the chain. Suitable heteroatoms
include N, O and S.
[0218] Unless otherwise defined herein, cycloalkyl means
C.sub.3-C.sub.12, C.sub.3-C.sub.8, or C.sub.3-C.sub.6, aliphatic
hydrocarbyl ring.
[0219] Unless otherwise defined herein, alkenyl means
C.sub.2-C.sub.12, C.sub.2-C.sub.8, or C.sub.2-C.sub.6 alkenyl,
which may be straight or (where possible) branched hydrocarbyl
moiety, which contains at least one carbon-carbon double bond.
[0220] Unless otherwise defined herein, alkynyl means
C.sub.2-C.sub.12, C.sub.2-C.sub.8, or C.sub.2-C.sub.6 alkynyl,
which may be straight or (where possible) branched hydrocarbyl
moiety, which contains at least one carbon-carbon triple bond.
[0221] Unless otherwise defined herein, heterocycloalkyl means a
ring moiety containing at least three ring members, at least one of
which is carbon, and of which 1, 2 or three ring members are other
than carbon. The number of carbon atoms can vary from 1 to about
12, or 1 to about 6, and the total number of ring members can vary
from three to about 15, or from about 3 to about 8. Suitable ring
heteroatoms are N, O and S. Suitable heterocycloalkyl groups
include morpholino, thiomorpholino, piperidinyl, piperazinyl,
homopiperidinyl, homopiperazinyl, homomorpholino,
homothiomorpholino, pyrrolodinyl, tetrahydrooxazolyl,
tetrahydroimidazolyl, tetrahydrothiazolyl, tetrahydroisoxazolyl,
tetrahydropyrrazolyl, furanyl, pyranyl, and
tetrahydroisothiazolyl.
[0222] Unless otherwise defined herein, aryl means any hydrocarbon
ring structure containing at least one aryl ring. Suitable aryl
rings have about 6 to about 20 ring carbons. Suitable aryl rings
include phenyl, napthyl, anthracenyl, and phenanthrenyl.
[0223] Unless otherwise defined herein, hetaryl means a ring moiety
containing at least one fully unsaturated ring, the ring consisting
of carbon and non-carbon atoms. The ring system can contain about 1
to about 4 rings. The number of carbon atoms can vary from 1 to
about 12, or 1 to about 6, and the total number of ring members can
vary from three to about 15, or from about 3 to about 8. Suitable
ring heteroatoms are N, O and S. Suitable hetaryl moieties include,
but are not limited to, pyrazolyl, thiophenyl, pyridyl, imidazolyl,
tetrazolyl, pyridyl, pyrimidinyl, purinyl, quinazolinyl,
quinoxalinyl, benzimidazolyl, benzothiophenyl, and the like.
[0224] Unless otherwise defined herein, where a moiety is defined
as a compound moiety, such as hetarylalkyl (hetaryl and alkyl),
aralkyl (aryl and alkyl), etc., each of the sub-moieties is as
defined herein.
[0225] Unless otherwise defined herein, an electron withdrawing
group is a group, such as the cyano or isocyanato group that draws
electronic charge away from the carbon to which it is attached.
Other electron withdrawing groups of note include those whose
electronegativities exceed that of carbon, for example halogen,
nitro, or phenyl substituted in the ortho- or para-position with
one or more cyano, isothiocyanato, nitro or halo groups.
[0226] Unless otherwise defined herein, the terms halogen and halo
have their ordinary meanings. Suitable halo (halogen) substituents
are Cl, Br, and I.
[0227] The aforementioned optional substituents are, unless
otherwise herein defined, suitable substituents depending upon
desired properties. Included are halogens (Cl, Br, I), alkyl,
alkenyl, and alkynyl moieties, NO.sub.2, NH.sub.3 (substituted and
unsubstituted), acid moieties (e.g.--CO.sub.2H, --OSO.sub.3H.sub.2,
etc.), heterocycloalkyl moieties, hetaryl moieties, aryl moieties,
etc. In all the preceding formulae, the squiggle (.about.)
indicates a bond to an oxygen or sulfur of the 5'-phosphate.
[0228] Phosphate protecting groups include those described in U.S.
Pat. No. 5,760,209, U.S. Pat. No. 5,614,621, U.S. Pat. No.
6,051,699, U.S. Pat. No. 6,020,475, U.S. Pat. No. 6,326,478, U.S.
Pat. No. 6,169,177, U.S. Pat. No. 6,121,437, U.S. Pat. No.
6,465,628.
[0229] The present invention discloses novel nucleosides comprising
bicyclic sugar moieties and oligomeric compounds comprising at
least one such nucleoside. The bicylcic sugar moieties have a
locked 3'-endo sugar conformation which provides nucleosides having
A form, RNA like without having some of the undesirable properties
associated with native RNA nucleosides. One of the immediate
advantages is the nuclease stability gained by replacing RNA
nucleosides with locked e.g. bicyclic sugar nucleosides. The
bicyclic sugar modified nucleosides are also expected to have
enhanced binding affinity that has been previously reported for LNA
(3-8.degree. C. per modification).
[0230] The nucleosides of the present invention have a bridge from
the 2', to the 4'-position defined by -Q.sub.1-Q.sub.2-Q.sub.3- as
shown below in structure I. 21
[0231] wherein:
[0232] Bx is a heterocyclic base moiety;
[0233] each T.sub.a and T.sub.b is, independently, H, a hydroxyl
protecting group, an activated phosphorus moiety, a conjugate group
or a covalent attachment to a support medium;
[0234] -Q.sub.1-Q.sub.2-Q.sub.3- is
--CH.sub.2--N(R.sub.1)-CH.sub.2--,
--C(.dbd.O)--N(R.sub.1)-CH.sub.2--, --CH.sub.2--O--N(R.sub.1)- or
N(R.sub.1)--O--CH.sub.2--; and
[0235] R.sub.1 is C.sub.1-C.sub.12 alkyl or an amino protecting
group.
[0236] The present invention also provides for oligomeric compounds
having at least one nucleoside having a bicyclic sugar moiety of
structure I. The nucleosides of structure I can be used to modify
the properties an oligomeric compound. Such a nucleoside can be put
into an oligonucleotide or an oligonucleoside in a single position
or at multiple positions to create a hemimer, blockmer, gapmer or a
more complicated alternating or chimeric oligomeric compound. The
nucleosides having structure I can also be used to modify
properties of oligomeric comounds that comprise more complicated
chemistries to prepare oligomeric compounds that are essentially
oligonucleotide mimics such as, for example, peptide nucleic acids
(PNA), morpholino nucleic acids, cyclohexenyl nucleic acids (CeNA),
anhydrohexitol nucleic acids, locked nucleic acids (LNA and ENA),
bicyclic and tricyclic nucleic acids, phosphonomonoester nucleic
acids and cyclobutyl nucleic acids.
[0237] The compositions of the present invention illustrate the use
of activated phosphorus compositions (e.g. compounds having
activated phosphorus-containing substituent groups) in coupling
reactions. As used herein, the term "activated phosphorus
composition" includes monomers and oligomers that have an activated
phosphorus-containing substituent group that is reactive with a
hydroxyl group of another monomeric or oligomeric compound to form
a phosphorus-containing internucleotide linkage. Such activated
phosphorus groups contain activated phosphorus atoms in P.sup.III
valence state and are known in the art and include, but are not
limited to, phosphoramidite, H-phosphonate, phosphate triesters and
chiral auxiliaries. One snthetic solid phase synthesis utilizes
phosphoramidites as activated phosphates. The phosphoramidites
utilize P.sup.III chemistry. The intermediate phosphite compounds
are subsequently oxidized to the P.sup.V state using known methods
to yield, in some embodiments, phosphodiester or phosphorothioate
internucleotide linkages. Additional activated phosphates and
phosphites are disclosed in Tetrahedron Report Number 309 (Beaucage
and Iyer, Tetrahedron, 1992, 48, 2223-2311).
[0238] Activated phosphorus groups are useful in the preparation of
a wide range of oligomeric compounds including but not limited to
oligonucleosides and oligonucleotides as well as oligonucleotides
that have been modified or conjugated with other groups at the base
or sugar or both. Also included are oligonucleotide mimetics
including but not limited to peptide nucleic acids (PNA),
morpholino nucleic acids, cyclohexenyl nucleic acids (CeNA),
anhydrohexitol nucleic acids, locked nucleic acids (LNA and ENA),
bicyclic and tricyclic nucleic acids, phosphonomonoester nucleic
acids and cyclobutyl nucleic acids. A representative example of one
type of oligomer synthesis that utilizes the coupling of an
activated phosphorus group with a reactive hydroxyl group is the
widely used phosphoramidite approach. A phosphoramidite synthon is
reacted under appropriate conditions with a reactive hydroxyl group
to form a phosphite linkage that is further oxidized to a
phosphodiester or phosphorothioate linkage. This approach commonly
utilizes nucleoside phosphoramidites of the formula: 22
[0239] wherein:
[0240] Bx' is an optionally protected heterocyclic base moiety;
[0241] R.sub.1' is, independently, H or an optionally protected
sugar substituent group;
[0242] T.sub.3' is H, a hydroxyl protecting group, a nucleoside, a
nucleotide, an oligonucleoside or an oligonucleotide;
[0243] L.sub.1 is N(R.sub.1)R.sub.2;
[0244] R.sub.1 and R.sub.2 is, independently, C.sub.1-C.sub.12
straight or branched chain alkyl;
[0245] or R.sub.1 and R.sub.2 are joined together to form a 4- to
7-membered heterocyclic ring system including the nitrogen atom to
which R.sub.1 and R.sub.2 are attached, wherein the ring system
optionally includes at least one additional heteroatom selected
from O, N and S;
[0246] L.sub.2 is Pg-O--, Pg-S--, C.sub.1-C.sub.12 straight or
branched chain alkyl, CH.sub.3(CH.sub.2).sub.0-10--O-- or
--NR.sub.5R.sub.6;
[0247] Pg is a protecting/blocking group; and
[0248] R.sub.5 and R.sub.6 is, independently, hydrogen,
C.sub.1-C.sub.12 straight or branched chain alkyl, cycloalkyl or
aryl;
[0249] or optionally, R.sub.5 and R.sub.6, together with the
nitrogen atom to which they are attached form a cyclic moiety that
may include an additional heteroatom selected from O, S and N;
or
[0250] L.sub.1 and L.sub.2 together with the phosphorus atom to
which L.sub.1 and L.sub.2 are attached form a chiral auxiliary.
[0251] Groups that are attached to the phosphorus atom of
internucleotide linkages before and after oxidation (L.sub.1 and
L.sub.2) can include nitrogen containing cyclic moieties such as
morpholine. Such oxidized internucleoside linkages include a
phosphoromorpholidothioate linkage (Wilk et al., Nucleosides and
nucleotides, 1991, 10, 319-322). Further cyclic moieties amenable
to the present invention include mono-, bi- or tricyclic ring
moieties which may be substituted with groups such as oxo, acyl,
alkoxy, alkoxycarbonyl, alkyl, alkenyl, alkynyl, amino, amido,
azido, aryl, heteroaryl, carboxylic acid, cyano, guanidino, halo,
haloalkyl, haloalkoxy, hydrazino, ODMT, alkylsulfonyl, nitro,
sulfide, sulfone, sulfonamide, thiol and thioalkoxy. A suitable
bicyclic ring structure that includes nitrogen is phthalimido.
[0252] Hybridization
[0253] In the context of this invention, "hybridization" means the
pairing of complementary strands of oligomeric compounds. In the
present invention, one mechanism of pairing involves hydrogen
bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding, between complementary nucleoside or nucleotide
bases (nucleobases) of the strands of oligomeric compounds. For
example, adenine and thymine are complementary nucleobases which
pair through the formation of hydrogen bonds. Hybridization can
occur under varying circumstances.
[0254] An antisense oligomeric compound is specifically
hybridizable when binding of the compound to the target nucleic
acid interferes with the normal function of the target nucleic acid
to cause a loss of activity, and there is a sufficient degree of
complementarity to avoid non-specific binding of the antisense
oligomeric compound to non-target nucleic acid sequences under
conditions in which specific binding is desired, i.e., under
physiological conditions in the case of in vivo assays or
therapeutic treatment, and under conditions in which assays are
performed in the case of in vitro assays.
[0255] In the present invention the phrase "stringent hybridization
conditions" or "stringent conditions" refers to conditions under
which an oligomeric compound of the invention will hybridize to its
target sequence, but to a minimal number of other sequences.
Stringent conditions are sequence-dependent and will vary with
different circumstances and in the context of this invention,
"stringent conditions" under which oligomeric compounds hybridize
to a target sequence are determined by the nature and composition
of the oligomeric compounds and the assays in which they are being
investigated.
[0256] "Complementary," as used herein, refers to the capacity for
precise pairing of two nucleobases regardless of where the two are
located. For example, if a nucleobase at a certain position of an
oligomeric compound is capable of hydrogen bonding with a
nucleobase at a certain position of a target nucleic acid, the
target nucleic acid being a DNA, RNA, or oligonucleotide molecule,
then the position of hydrogen bonding between the oligonucleotide
and the target nucleic acid is considered to be a complementary
position. The oligomeric compound and the further DNA, RNA, or
oligonucleotide molecule are complementary to each other when a
sufficient number of complementary positions in each molecule are
occupied by nucleobases which can hydrogen bond with each other.
Thus, "specifically hybridizable" and "complementary" are terms
which are used to indicate a sufficient degree of precise pairing
or complementarity over a sufficient number of nucleobases such
that stable and specific binding occurs between the oligonucleotide
and a target nucleic acid.
[0257] It is understood in the art that the sequence of an
antisense oligomeric compound need not be 100% complementary to
that of its target nucleic acid to be specifically hybridizable.
Moreover, an oligonucleotide may hybridize over one or more
segments such that intervening or adjacent segments are not
involved in the hybridization event (e.g., a loop structure or
hairpin structure). The oligomeric compounds of the present
invention can comprise at least 70%, at least 80%, at least 90%, at
least 95%, or at least 99% sequence complementarity to a target
region within the target nucleic acid sequence to which they are
targeted. For example, an antisense oligomeric compound in which 18
of 20 nucleobases of the antisense oligomeric compound are
complementary to a target region, and would therefore specifically
hybridize, would represent 90 percent complementarity. In this
example, the remaining noncomplementary nucleobases may be
clustered or interspersed with complementary nucleobases and need
not be contiguous to each other or to complementary nucleobases. As
such, an antisense oligomeric compound which is 18 nucleobases in
length having 4 (four) noncomplementary nucleobases which are
flanked by two regions of complete complementarity with the target
nucleic acid would have 77.8% overall complementarity with the
target nucleic acid and would thus fall within the scope of the
present invention. Percent complementarity of an antisense
oligomeric compound with a region of a target nucleic acid can be
determined routinely using BLAST programs (basic local alignment
search tools) and PowerBLAST programs known in the art (Altschul et
al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome
Res., 1997, 7, 649-656).
[0258] Targets of the Invention
[0259] "Targeting" an antisense oligomeric compound to a particular
nucleic acid molecule, in the context of this invention, can be a
multistep process. The process usually begins with the
identification of a target nucleic acid whose function is to be
modulated. This target nucleic acid 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
nucleic acid molecule from an infectious agent.
[0260] The targeting process usually also includes determination of
at least one target region, segment, or site within the target
nucleic acid for the antisense interaction to occur such that the
desired effect, e.g., modulation of expression, will result. Within
the context of the present invention, the term "region" is defined
as a portion of the target nucleic acid having at least one
identifiable structure, function, or characteristic. Within regions
of target nucleic acids are segments. "Segments" are defined as
smaller or sub-portions of regions within a target nucleic acid.
"Sites," as used in the present invention, are defined as positions
within a target nucleic acid. The terms region, segment, and site
can also be used to describe an oligomeric compound of the
invention such as for example a gapped oligomeric compound having 3
separate segments.
[0261] Since, 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.
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 (in 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 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 transcribed from a gene encoding a nucleic
acid target, 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).
[0262] The terms "start codon region" and "translation initiation
codon 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 codon 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. Consequently, the "start codon region" (or
"translation initiation codon region") and the "stop codon region"
(or "translation termination codon region") are all regions which
may be targeted effectively with the oligomeric compounds of the
present invention.
[0263] The open reading frame (ORF) or "coding region," which is
known in the art to refer to the region between the translation
initiation codon and the translation termination codon, is also a
region which may be targeted effectively. Within the context of the
present invention, a suitable region is the intragenic region
encompassing the translation initiation or termination codon of the
open reading frame (ORF) of a gene.
[0264] Other target regions include the 5' untranslated region
(5'UTR), known in the art to refer to the portion of an mRNA in the
5' direction from the translation initiation codon, and thus
including nucleotides between the 5' cap site and the translation
initiation codon of an mRNA (or corresponding nucleotides on the
gene), and the 3' untranslated region (3'UTR), known in the art to
refer to the portion of an mRNA in the 3' direction from the
translation termination codon, and thus including nucleotides
between the translation termination codon and 3' end of an mRNA (or
corresponding nucleotides on the gene). The 5' cap site of an mRNA
comprises an N7-methylated guanosine residue joined to the 5'-most
residue of the mRNA via a 5'-5' triphosphate linkage. The 5' cap
region of an mRNA is considered to include the 5' cap structure
itself as well as the first 50 nucleotides adjacent to the cap
site. It is also suitable to target the 5' cap region.
[0265] Although some eukaryotic mRNA transcripts are directly
translated, many contain one or more regions, known as "introns,"
which are excised from a transcript before it is translated. The
remaining (and therefore translated) regions are known as "exons"
and are spliced together to form a continuous mRNA sequence.
Targeting splice sites, i.e., intron-exon junctions or exon-intron
junctions, may also be particularly useful in situations where
aberrant splicing is implicated in disease, or where an
overproduction of a particular splice product is implicated in
disease. Aberrant fusion junctions due to rearrangements or
deletions are also suitable target sites. mRNA transcripts produced
via the process of splicing of two (or more) mRNAs from different
gene sources are known as "fusion transcripts." It is also known
that introns can be effectively targeted using antisense oligomeric
compounds targeted to, for example, DNA or pre-mRNA.
[0266] It is also known in the art that alternative RNA transcripts
can be produced from the same genomic region of DNA. These
alternative transcripts are generally known as "variants." More
specifically, "pre-mRNA variants" are transcripts produced from the
same genomic DNA that differ from other transcripts produced from
the same genomic DNA in either their start or stop position and
contain both intronic and exonic sequences.
[0267] Upon excision of one or more exon or intron regions, or
portions thereof during splicing, pre-mRNA variants produce smaller
"mRNA variants." Consequently, mRNA variants are processed pre-mRNA
variants and each unique pre-mRNA variant must always produce a
unique mRNA variant as a result of splicing. These mRNA variants
are also known as "alternative splice variants." If no splicing of
the pre-mRNA variant occurs then the pre-mRNA variant is identical
to the mRNA variant.
[0268] It is also known in the art that variants can be produced
through the use of alternative signals to start or stop
transcription and that pre-mRNAs and mRNAs can possess more that
one start codon or stop codon. Variants that originate from a
pre-mRNA or mRNA that use alternative start codons are known as
"alternative start variants" of that pre-mRNA or mRNA. Those
transcripts that use an alternative stop codon are known as
"alternative stop variants" of that pre-mRNA or mRNA. One specific
type of alternative stop variant is the "polyA variant" in which
the multiple transcripts produced result from the alternative
selection of one of the "polyA stop signals" by the transcription
machinery, thereby producing transcripts that terminate at unique
polyA sites. Within the context of the invention, the types of
variants described herein are also suitable target nucleic
acids.
[0269] The locations on the target nucleic acid to which the
oligomeric compounds hybridize are hereinbelow referred to as
"suitable target segments." As used herein the term "suitable
target segment" is defined as at least an 8-nucleobase portion of a
target region to which an active oligomeric compound is targeted.
While not wishing to be bound by theory, it is presently believed
that these target segments represent portions of the target nucleic
acid which are accessible for hybridization.
[0270] Exemplary suitable oligomeric compounds include oligomeric
compounds that comprise at least the 8 consecutive nucleobases from
the 5'-terminus of a targeted nucleic acid e.g. a cellular gene or
mRNA transcribed from the gene (the remaining nucleobases being a
consecutive stretch of the same oligonucleotide beginning
immediately upstream of the 5'-terminus of the compound which is
specifically hybridizable to the target nucleic acid and continuing
until the oligonucleotide contains from about 8 to about 80
nucleobases). Additional suitable oligomeric compounds are
represented by oligonucleotide sequences that comprise at least the
8 consecutive nucleobases from the 3'-terminus of one of the
illustrative compounds (the remaining nucleobases being a
consecutive stretch of the same oligonucleotide beginning
immediately downstream of the 3'-terminus of the compound which is
specifically hybridizable to the target nucleic acid and continuing
until the oligonucleotide contains from about 8 to about 80
nucleobases). One having skill in the art armed with the suitable
compounds illustrated herein will be able, without undue
experimentation, to identify further oligomeric compounds.
[0271] Once one or more target regions, segments or sites have been
identified, oligomeric compounds are chosen which are sufficiently
complementary to the target, i.e., hybridize sufficiently well and
with sufficient specificity, to give the desired effect.
[0272] In accordance with one embodiment of the present invention,
a series of nucleic acid duplexes comprising the oligomeric
compounds of the present invention and their complements can be
designed for a specific target or targets. The ends of the strands
may be modified by the addition of one or more natural or modified
nucleobases to form an overhang. The sense strand of the duplex is
then designed and synthesized as the complement of the antisense
strand and may also contain modifications or additions to either
terminus. For example, in one embodiment, both strands of the
duplex would be complementary over the central nucleobases, each
having overhangs at one or both termini.
[0273] For example, a duplex comprising an antisense oligomeric
compound having the sequence CGAGAGGCGGACGGGACCG (SEQ ID NO:1) and
having a two-nucleobase overhang of deoxythymidine(dT) would have
the following structure:
2 cgagaggcggacgggaccgTT Antisense Strand (SEQ ID NO:1)
.vertline..vertline..vertline..vertline..vertline..vertline..vertline..-
vertline..vertline..vertline..vertline..vertline..vertline..vertline..vert-
line..vertline..vertline..vertline..vertline. TTgctctccgcctgccctggc
Complement Strand (SEQ ID NO:2)
[0274] RNA strands of the duplex can be synthesized by methods
disclosed herein or purchased from various RNA synthesis companies
such as for example Dharmacon Research Inc., (Lafayette, Colo.)
(see also the section on RNA synthesis below). Once synthesized,
the complementary strands are annealed. The single strands are
aliquoted and diluted to a concentration of 50 .mu.M. Once diluted,
30 .mu.L of each strand is combined with 15 .mu.L of a 5.times.
solution of annealing buffer. The final concentration of the buffer
is 100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, and 2 mM
magnesium acetate. The final volume is 75 .mu.L. This solution is
incubated for 1 minute at 90.degree. C. and then centrifuged for 15
seconds. The tube is allowed to sit for 1 hour at 37.degree. C. at
which time the dsRNA duplexes are used in experimentation. The
final concentration of the dsRNA compound is 20 uM. This solution
can be stored frozen (-20.degree. C.) and freeze-thawed up to 5
times.
[0275] Once prepared, the desired synthetic duplexs are evaluated
for their ability to modulate target expression. When cells reach
80% confluency, they are treated with synthetic duplexs comprising
at least one oligomeric compound of the invention. For cells grown
in 96-well plates, wells are washed once with 200 .mu.L OPTI-MEM-1
reduced-serum medium (Gibco BRL) and then treated with 130 .mu.L of
OPTI-MEM-1 containing 12 .mu.g/mL LIPOFECTIN (Gibco BRL) and the
desired dsRNA compound at a final concentration of 200 nM. After 5
hours of treatment, the medium is replaced with fresh medium. Cells
are harvested 16 hours after treatment, at which time RNA is
isolated and target reduction measured by RT-PCR.
[0276] In another embodiment, the "suitable target segments"
identified herein may be employed in a screen for additional
oligomeric compounds that modulate the expression of a target.
"Modulators" are those oligomeric compounds that decrease or
increase the expression of a nucleic acid molecule encoding a
target and which comprise at least an 8-nucleobase portion which is
complementary to a suitable target segment. The screening method
comprises the steps of contacting a suitable target segment of a
nucleic acid molecule encoding a target with one or more candidate
modulators, and selecting for one or more candidate modulators
which decrease or increase the expression of a nucleic acid
molecule encoding a target. Once it is shown that the candidate
modulator or modulators are capable of modulating (e.g. either
decreasing or increasing) the expression of a nucleic acid molecule
encoding a target, the modulator may then be employed in further
investigative studies of the function of a target, or for use as a
research, diagnostic, or therapeutic agent in accordance with the
present invention.
[0277] The suitable target segments of the present invention may
also be combined with their respective complementary antisense
oligomeric compounds of the present invention to form stabilized
double-stranded (duplexed) oligonucleotides.
[0278] Screening and Target Validation
[0279] In a further embodiment, "suitable target segments" may be
employed in a screen for additional oligomeric compounds that
modulate the expression of a selected protein. "Modulators" are
those oligomeric compounds that decrease or increase the expression
of a nucleic acid molecule encoding a protein and which comprise at
least an 8-nucleobase portion which is complementary to a suitable
target segment. The screening method comprises the steps of
contacting a suitable target segment of a nucleic acid molecule
encoding a protein with one or more candidate modulators, and
selecting for one or more candidate modulators which decrease or
increase the expression of a nucleic acid molecule encoding a
protein. Once it is shown that the candidate modulator or
modulators are capable of modulating (e.g. either decreasing or
increasing) the expression of a nucleic acid molecule encoding a
peptide, the modulator may then be employed in further
investigative studies of the function of the peptide, or for use as
a research, diagnostic, or therapeutic agent in accordance with the
present invention.
[0280] The oligomeric compounds of the present invention can also
be applied in the areas of drug discovery and target validation.
The present invention comprehends the use of the oligomeric
compounds and suitable targets identified herein in drug discovery
efforts to elucidate relationships that exist between proteins and
a disease state, phenotype, or condition. These methods include
detecting or modulating a target peptide comprising contacting a
sample, tissue, cell, or organism with the oligomeric compounds of
the present invention, measuring the nucleic acid or protein level
of the target and/or a related phenotypic or chemical endpoint at
some time after treatment, and optionally comparing the measured
value to a non-treated sample or sample treated with a further
oligomeric compound of the invention. These methods can also be
performed in parallel or in combination with other experiments to
determine the function of unknown genes for the process of target
validation or to determine the validity of a particular gene
product as a target for treatment or prevention of a particular
disease, condition, or phenotype.
[0281] Effect of nucleoside modifications on RNAi activity is
evaluated according to existing literature (Elbashir et al., Nature
(2001), 411, 494-498; Nishikura et al., Cell (2001), 107, 415-416;
and Bass et al., Cell (2000), 101, 235-238.)
[0282] Kits, Research Reagents, Diagnostics, and Therapeutics
[0283] The oligomeric compounds of the present invention can be
utilized for diagnostics, therapeutics, prophylaxis and as research
reagents and kits. Furthermore, 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 or to distinguish between functions of various
members of a biological pathway.
[0284] For use in kits and diagnostics, the oligomeric compounds of
the present invention, either alone or in combination with other
oligomeric compounds or therapeutics, can be used as tools in
differential and/or combinatorial analyses to elucidate expression
patterns of a portion or the entire complement of genes expressed
within cells and tissues.
[0285] As one nonlimiting example, expression patterns within cells
or tissues treated with one or more antisense oligomeric compounds
are compared to control cells or tissues not treated with antisense
oligomeric compounds and the patterns produced are analyzed for
differential levels of gene expression as they pertain, for
example, to disease association, signaling pathway, cellular
localization, expression level, size, structure or function of the
genes examined. These analyses can be performed on stimulated or
unstimulated cells and in the presence or absence of other
compounds and or oligomeric compounds which affect expression
patterns.
[0286] Examples of methods of gene expression analysis known in the
art include DNA arrays or microarrays (Brazma and Vilo, FEBS Lett.,
2000, 480, 17-24; Celis, et al., FEBS Lett., 2000, 480, 2-16), SAGE
(serial analysis of gene expression)(Madden, et al., Drug Discov.
Today, 2000, 5, 415-425), READS (restriction enzyme amplification
of digested cDNAs) (Prashar and Weissman, Methods Enzymol., 1999,
303, 258-72), TOGA (total gene expression analysis) (Sutcliffe, et
al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 1976-81), protein
arrays and proteomics (Celis, et al., FEBS Lett., 2000, 480, 2-16;
Jungblut, et al., Electrophoresis, 1999, 20, 2100-10), expressed
sequence tag (EST) sequencing (Celis, et al., FEBS Lett., 2000,
480, 2-16; Larsson, et al., J. Biotechnol., 2000, 80, 143-57),
subtractive RNA fingerprinting (SuRF) (Fuchs, et al., Anal.
Biochem., 2000, 286, 91-98; Larson, et al., Cytometry, 2000, 41,
203-208), subtractive cloning, differential display (DD) (Jurecic
and Belmont, Curr. Opin. Microbiol., 2000, 3, 316-21), comparative
genomic hybridization (Carulli, et al., J. Cell Biochem. Suppl.,
1998, 31, 286-96), FISH (fluorescent in situ hybridization)
techniques (Going and Gusterson, Eur. J. Cancer, 1999, 35,
1895-904) and mass spectrometry methods (To, Comb. Chem. High
Throughput Screen, 2000, 3, 235-41).
[0287] The oligomeric compounds of the invention are useful for
research and diagnostics, because these oligomeric compounds
hybridize to nucleic acids encoding proteins. For example,
oligonucleotides that are shown to hybridize with such efficiency
and under such conditions as disclosed herein as to be effective
protein inhibitors will also be effective primers or probes under
conditions favoring gene amplification or detection, respectively.
These primers and probes are useful in methods requiring the
specific detection of nucleic acid molecules encoding proteins and
in the amplification of the nucleic acid molecules for detection or
for use in further studies. Hybridization of the antisense
oligonucleotides, particularly the primers and probes, of the
invention with a nucleic acid 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 means. Kits using such detection means for
detecting the level of selected proteins in a sample may also be
prepared.
[0288] The specificity and sensitivity of antisense is also
harnessed by those of skill in the art for therapeutic uses.
Antisense oligomeric compounds have been employed as therapeutic
moieties in the treatment of disease states in animals, including
humans. Antisense oligonucleotide drugs, including ribozymes, have
been safely and effectively administered to humans and numerous
clinical trials are presently underway. It is thus established that
antisense oligomeric compounds can be useful therapeutic modalities
that can be configured to be useful in treatment regimes for the
treatment of cells, tissues and animals, especially humans.
[0289] For therapeutics, an animal, such as a human, suspected of
having a disease or disorder which can be treated by modulating the
expression of a selected protein is treated by administering
antisense oligomeric compounds in accordance with this invention.
For example, in one non-limiting embodiment, the methods comprise
the step of administering to the animal in need of treatment, a
therapeutically effective amount of a protein inhibitor. The
protein inhibitors of the present invention effectively inhibit the
activity of the protein or inhibit the expression of the protein.
In some embodiments, the activity or expression of a protein in an
animal or in vitro is inhibited by at least 10%, by at least 20%,
by at least 30%, by at least 40%, by at least 50%, by at least 60%,
by at least 70%, by at least 80%, by at least 90%, by at least 95%,
by at least 99%, or by 100%.
[0290] For example, the reduction of the expression of a protein
may be measured in serum, adipose tissue, liver or any other body
fluid, tissue or organ of the animal. The cells contained within
the fluids, tissues or organs being analyzed can contain a nucleic
acid molecule encoding a protein and/or the protein itself.
[0291] The oligomeric compounds of the invention can be utilized in
pharmaceutical compositions by adding an effective amount of a
oligomeric compound to a suitable pharmaceutically acceptable
diluent or carrier. Use of the oligomeric compounds and methods of
the invention may also be useful prophylactically.
[0292] Formulations
[0293] The oligomeric compounds of the invention may also be
admixed, encapsulated, conjugated or otherwise associated with
other molecules, molecule structures or mixtures of compounds, as
for example, liposomes, receptor-targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption. Representative United States
patents that teach the preparation of such uptake, distribution
and/or absorption-assisting formulations include, but are not
limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756.
[0294] The antisense oligomeric compounds of the invention
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters, or any other compound which, upon administration to
an animal, including a human, is capable of providing (directly or
indirectly) the biologically active metabolite or residue thereof.
Accordingly, for example, the disclosure is also drawn to prodrugs
and pharmaceutically acceptable salts of the oligomeric compounds
of the invention, pharmaceutically acceptable salts of such
prodrugs, and other bioequivalents. 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 or in WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et
al.
[0295] The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
oligomeric compounds of the invention: i.e., salts that retain the
desired biological activity of the parent compound and do not
impart undesired toxicological effects thereto. For
oligonucleotides, examples of pharmaceutically acceptable salts and
their uses are further described in U.S. Pat. No. 6,287,860.
[0296] The present invention also includes pharmaceutical
compositions and formulations which include the antisense
oligomeric compounds of the invention. 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. Administration may be
topical (including ophthalmic and to mucous membranes including
vaginal and rectal delivery), pulmonary, e.g., by inhalation or
insufflation of powders or aerosols, including by nebulizer;
intratracheal, intranasal, epidermal and transdermal), oral or
parenteral. Parenteral administration includes intravenous,
intraarterial, subcutaneous, intraperitoneal or intramuscular
injection or infusion; or intracranial, e.g., intrathecal or
intraventricular, administration. Oligonucleotides with at least
one 2'-O-methoxyethyl modification are believed to be particularly
useful for oral administration. Pharmaceutical compositions and
formulations for topical administration may include transdermal
patches, ointments, lotions, creams, gels, drops, suppositories,
sprays, liquids and powders. Conventional pharmaceutical carriers,
aqueous, powder or oily bases, thickeners and the like may be
necessary or desirable. Coated condoms, gloves and the like may
also be useful.
[0297] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0298] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, gel capsules, liquid syrups, soft gels,
suppositories, and enemas. The compositions of the present
invention may also be formulated as suspensions in aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain
substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
[0299] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, foams and
liposome-containing formulations. The pharmaceutical compositions
and formulations of the present invention may comprise one or more
penetration enhancers, carriers, excipients or other active or
inactive ingredients.
[0300] Emulsions are typically heterogenous systems of one liquid
dispersed in another in the form of droplets usually exceeding 0.1
.mu.m in diameter. Emulsions may contain additional components in
addition to the dispersed phases, and the active drug which may be
present as a solution in either the aqueous phase, oily phase or
itself as a separate phase. Microemulsions are included as an
embodiment of the present invention. Emulsions and their uses are
well known in the art and are further described in U.S. Pat. No.
6,287,860.
[0301] Formulations of the present invention include liposomal
formulations. As used in the present invention, the term "liposome"
means a vesicle composed of amphiphilic lipids arranged in a
spherical bilayer or bilayers. Liposomes are unilamellar or
multilamellar vesicles which have a membrane formed from a
lipophilic material and an aqueous interior that contains the
composition to be delivered. Cationic liposomes are positively
charged liposomes which are believed to interact with negatively
charged DNA molecules to form a stable complex. Liposomes that are
pH-sensitive or negatively-charged are believed to entrap DNA
rather than complex with it. Both cationic and noncationic
liposomes have been used to deliver DNA to cells.
[0302] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome comprises one or more glycolipids or is
derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. Liposomes and their uses are
further described in U.S. Pat. No. 6,287,860.
[0303] The pharmaceutical formulations and compositions of the
present invention may also include surfactants. The use of
surfactants in drug products, formulations and in emulsions is well
known in the art. Surfactants and their uses are further described
in U.S. Pat. No. 6,287,860.
[0304] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly oligonucleotides. In addition to aiding the
diffision of non-lipophilic drugs across cell membranes,
penetration enhancers also enhance the permeability of lipophilic
drugs. Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants.
Penetration enhancers and their uses are further described in U.S.
Pat. No. 6,287,860.
[0305] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
[0306] Suitable formulations for topical administration include
those in which the oligonucleotides of the invention are in
admixture with a topical delivery agent such as lipids, liposomes,
fatty acids, fatty acid esters, steroids, chelating agents and
surfactants. Suitable lipids and liposomes include neutral (e.g.
dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl
choline DMPC, distearolyphosphatidyl choline) negative (e.g.
dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.
dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl
ethanolamine DOTMA).
[0307] For topical or other administration, oligonucleotides of the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, oligonucleotides may be complexed to lipids, in
particular to cationic lipids. Suitable fatty acids and esters,
pharmaceutically acceptable salts thereof, and their uses are
further described in U.S. Pat. No. 6,287,860. Topical formulations
are described in detail in U.S. patent application Ser. No.
09/315,298 filed on May 20, 1999.
[0308] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Suitable oral formulations are those in which
oligonucleotides of the invention are administered in conjunction
with one or more penetration enhancers surfactants and chelators.
Suitable surfactants include fatty acids and/or esters or salts
thereof, bile acids and/or salts thereof. Suitable bile acids/salts
and fatty acids and their uses are further described in U.S. Pat.
No. 6,287,860. Also suitable are combinations of penetration
enhancers, for example, fatty acids/salts in combination with bile
acids/salts. A suitable combination is the sodium salt of lauric
acid, capric acid and UDCA. Further penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
Oligonucleotides of the invention may be delivered orally, in
granular form including sprayed dried particles, or complexed to
form micro or nanoparticles. Oligonucleotide complexing agents and
their uses are further described in U.S. Pat. No. 6,287,860. Oral
formulations for oligonucleotides and their preparation are
described in detail in U.S. application Ser. Nos. 09/108,673 (filed
Jul. 1, 1998), 09/315,298 (filed May 20, 1999) and 10/071,822,
filed Feb. 8, 2002.
[0309] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0310] Certain embodiments of the invention provide pharmaceutical
compositions containing one or more oligomeric compounds and one or
more other chemotherapeutic agents which function by a
non-antisense mechanism. Examples of such chemotherapeutic agents
include but are not limited to cancer chemotherapeutic drugs such
as 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, 5-azacytidine, hydroxyurea,
deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil
(5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX),
colchicine, taxol, vincristine, vinblastine, etoposide (VP-16),
trimetrexate, irinotecan, topotecan, gemcitabine, teniposide,
cisplatin and diethylstilbestrol (DES). When used with the
oligomeric 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).
Anti-inflammatory drugs, including but not limited to nonsteroidal
anti-inflammatory drugs and corticosteroids, and antiviral drugs,
including but not limited to ribivirin, vidarabine, acyclovir and
ganciclovir, may also be combined in compositions of the invention.
Combinations of antisense oligomeric compounds and other
non-antisense drugs are also within the scope of this invention.
Two or more combined compounds such as two oligomeric compounds or
one oligomeric compound combined further compounds may be used
together or sequentially.
[0311] In another related embodiment, compositions of the invention
may contain one or more antisense oligomeric compounds,
particularly oligonucleotides, targeted to a first nucleic acid and
one or more additional antisense oligomeric compounds targeted to a
second nucleic acid target. Alternatively, compositions of the
invention may contain two or more antisense oligomeric compounds
targeted to different regions of the same nucleic acid target.
[0312] Numerous examples of antisense oligomeric compounds are
known in the art. Two or more combined compounds may be used
together or sequentially
[0313] Dosing
[0314] The formulation of therapeutic compositions and their
subsequent administration (dosing) is believed to be within the
skill of those in the art. 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, from 0.1 .mu.g
to 10 g per kg of body weight, from 1 .mu.g to 1 g per kg of body
weight, from 10 .mu.g to 100 mg per kg of body weight, from 100
.mu.g to 10 mg per kg of body weight, or from 100 .mu.g to 1 mg 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. Persons of
ordinary skill in the art can easily estimate repetition rates for
dosing based on measured residence times and concentrations of the
drug in bodily fluids or tissues. 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.
[0315] While the present invention has been described with
specificity in accordance with certain of its embodiments, the
following examples serve only to illustrate the invention and are
not intended to limit the same.
EXAMPLES
Example 1
LNA Gapmers Targeted to Mouse TRADD: In Vivo Study
[0316] Six-week old male Balb/c mice (Jackson Laboratory, Bar
Harbor, Me.) were injected with compounds targeted to TRADD. Each
treatment group was comprised of four animals. Animals were dosed
via intraperitoneal injection twice per week for three weeks. The
LNA gapmers ISIS 335385, ISIS 353878, and ISIS 353879 were
evaluated at doses of 40, 12, or 4 mg/kg (4.5, 1.5, or 0.5
.mu.mol/kg, respectively). The 2'-MOE gapmers ISIS 325585,
ISIS325587, and ISIS 325588 were similarly administered.
Saline-injected animals and animals injected with ISIS 141923, a
control 2'-MOE gapmer, served as control groups. Animals were
sacrificed after the last dose of oligonucleotide was administered,
and liver, kidney, and fat tissues were harvested.
[0317] Target reduction in liver was measured at the conclusion of
the study using real-time PCR. Results are presented in Table 2 for
animals treated with 40 mg/kg (4.5 .mu.mol/kg) of the compounds as
described. The data were normalized to saline-treated controls and
are expressed as percent of control, wherein a percentage less than
100 is indicative of target mRNA reduction.
3TABLE 2 Target reduction by LNA compounds targeted to TRADD in
mouse liver SEQ ID % ISIS # SEQUENCE CHEMISTRY NO Control 141923
CCTTCCCTGAAGGTTCCTCC 5-10-5 MOE 3 94 335385 CTCATACTCGTAGGCC 3-10-3
LNA 4 20 353878 GCTCATACTCGTAGGCCA 3-12-3 LNA 5 17 353879
GCTCATACTCGTAGGCCA 2-14-2 LNA 6 30 325585 CTCATACTCGTAGGCC 3-10-3
MOE 7 65 325588 GCTCATACTCGTAGGCCA 3-12-3 MOE 8 19 325587
GCTCATACTCGTAGGCCA 2-14-2 MOE 9 17
[0318] As shown in Table 2, all of the compounds targeted to TRADD
were effective at reducing mRNA levels in mouse liver, whereas ISIS
141923, a control oligonucleotide, did not cause substantial target
reduction. ISIS 335385, an oligomeric compound comprised of a ten
2'-deoxynucleotide gap flanked by three LNAs on both the 3' and 5'
ends, substantially reduced TRADD mRNA levels as compared to ISIS
325585, an oligomeric compound comprised of a ten
2'-deoxynucleotide gap flanked by three 2'-MOE nucleotides on both
the 3' and 5' ends. ISIS 353878, an oligomeric compound comprised
of a twelve 2'-deoxynucleotide gap flanked by three LNAs on both
the 3' and 5' ends, caused reduction in TRADD mRNA levels
comparable to that produced by ISIS 325588, an oligomeric compound
comprised of a twelve 2'-deoxynucleotide gap flanked by three
2'-MOE nucleotides on both the 3' and 5' ends.
Example 2
Synthesis of Nucleoside Phosphoramidites
[0319] The following compounds, including amidites and their
intermediates were prepared as described in U.S. Pat. No. 6,426,220
and published PCT WO 02/36743; 5'-O-Dimethoxytrityl-thymidine
intermediate for 5-methyl dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-5-methylcytidine intermediate for
5-methyl-dC amidite,
5'-O-Dimethoxytrityl-2'-deoxy-N.sup.4-benzoyl-5-meth- ylcytidine
penultimate intermediate for 5-methyl dC amidite,
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-deoxy-N-4-benzoyl-5-methylcytidi-
n-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (5-methyl dC
amidite), 2'-Fluorodeoxy-adenosine, 2'-Fluorodeoxyguanosine,
2'-Fluorouridine, 2'-Fluorodeoxycytidine, 2'-O-(2-Methoxyethyl)
modified amidites, 2'-O-(2-methoxyethyl)-5-methyluridine
intermediate, 5'-O-DMT-2'-O-(2-methoxyethyl)-5-methyluridine
penultimate intermediate,
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-5-methyluridi-
n-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite (MOE T
amidite),
5'-O-Dimethoxytrityl-2'-O-(2-methoxy-ethyl)-5-methylcytidine
intermediate,
5'-O-dimethoxytrityl-2'-O-(2-methoxyethyl)-N.sup.4-benzoyl--
5-methyl-cytidine penultimate intermediate,
(5'-O-(4,4'-Dimethoxytriphenyl-
methyl)-2'-O-(2-methoxyethyl)-N.sup.4-benzoyl-5-methylcytidin-3'-O-yl)-2-c-
yanoethyl-N,N-diisopropylphosphoramidite (MOE 5-Me-C amidite),
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.sup.6-benzo-
yladenosin-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidite
(MOE A amdite),
(5'-O-(4,4'-Dimethoxytriphenylmethyl)-2'-O-(2-methoxyethyl)-N.su-
b.4-isobutyrylguanosin-3'-O-yl)-2-cyanoethyl-N,N-diisopropylphosphoramidit-
e (MOE G amidite), 2'-O-(Aminooxyethyl) nucleoside amidites and
2'-O-(dimethylaminooxyethyl) nucleoside amidites,
2'-(Dimethylaminooxyeth- oxy) nucleoside amidites,
5'-O-tert-Butyldiphenylsilyl-O.sup.2-2'-anhydro-- 5-methyluridine,
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-meth-
yluridine,
2'-O-((2-phthalimidoxy)ethyl)-5'-t-butyldiphenylsilyl-5-methylu-
ridine,
5'-O-tert-butyldiphenylsilyl-2'-O-((2-formadoximinooxy)ethyl)-5-me-
thyluridine, 5'-O-tert-Butyldiphenylsilyl-2'-O--(N,N
dimethylaminooxyethyl)-5-methyluridine,
2'-O-(dimethyl-aminooxyethyl)-5-m- ethyluridine,
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine,
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-((2-cyanoe-
thyl)-N,N-diisopropylphosphoramidite), 2'-(Aminooxyethoxy)
nucleoside amidites,
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(-
4,4'-dimethoxytrityl)guanosine-3'-((2-cyanoethyl)-N,N-diisopropylphosphora-
midite), 2'-dimethylaminoethoxyethoxy (2'-DMAEOE) nucleoside
amidites, 2'-O-(2(2-N,N-dimethylaminoethoxy)ethyl)-5-methyl
uridine,
5'-O-dimethoxytrityl-2'-O-(2(2-N,N-dimethylaminoethoxy)-ethyl))-5-methyl
uridine and
5'-O-Dimethoxytrityl-2'-O-(2(2-N,N-dimethylaminoethoxy)-ethyl-
))-5-methyl
uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.
Example 3
Oligonucleotide and Oligonucleoside Synthesis
[0320] The antisense oligomeric compounds 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 well known to use similar techniques to prepare
oligonucleotides such as the phosphorothioates and alkylated
derivatives.
[0321] Oligonucleotides: Unsubstituted and substituted
phosphodiester (P.dbd.O) oligonucleotides are synthesized on an
automated DNA synthesizer (Applied Biosystems model 394) using
standard phosphoramidite chemistry with oxidation by iodine.
[0322] Phosphorothioates (P.dbd.S) are synthesized similar to
phosphodiester oligonucleotides with the following exceptions:
thiation was effected by utilizing a 10% w/v solution of
3,H-1,2-benzodithiole-3-o- ne 1,1-dioxide in acetonitrile for the
oxidation of the phosphite linkages. The thiation reaction step
time was increased to 180 sec and preceded by the normal capping
step. After cleavage from the CPG column and deblocking in
concentrated ammonium hydroxide at 55.degree. C. (12-16 hr), the
oligonucleotides were recovered by precipitating with >3 volumes
of ethanol from a 1 M NH.sub.4OAc solution. Phosphinate
oligonucleotides are prepared as described in U.S. Pat. No.
5,508,270.
[0323] Alkyl phosphonate oligonucleotides are prepared as described
in U.S. Pat. No. 4,469,863.
[0324] 3'-Deoxy-3'-methylene phosphonate oligonucleotides are
prepared as described in U.S. Pat. No. 5,610,289 or 5,625,050.
[0325] Phosphoramidite oligonucleotides are prepared as described
in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.
[0326] Alkylphosphonothioate oligonucleotides are prepared as
described in published PCT applications PCT/US94/00902 and
PCT/US93/06976 (published as WO 94/17093 and WO 94/02499,
respectively).
[0327] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are
prepared as described in U.S. Pat. No. 5,476,925.
[0328] Phosphotriester oligonucleotides are prepared as described
in U.S. Pat. No. 5,023,243.
[0329] Borano phosphate oligonucleotides are prepared as described
in U.S. Pat. Nos. 5,130,302 and 5,177,198.
[0330] Oligonucleosides: Methylenemethylimino linked
oligonucleosides, also identified as MMI linked oligonucleosides,
methylenedimethylhydrazo linked oligonucleosides, also identified
as MDH linked oligonucleosides, and methylenecarbonylamino linked
oligonucleosides, also identified as amide-3 linked
oligonucleosides, and methyleneaminocarbonyl linked
oligonucleosides, also identified as amide-4 linked
oligo-nucleosides, as well as mixed backbone oligomeric compounds
having, for instance, alternating MMI and P.dbd.O or P.dbd.S
linkages are prepared as described in U.S. Pat. Nos. 5,378,825,
5,386,023, 5,489,677, 5,602,240 and 5,610,289.
[0331] Formacetal and thioformacetal linked oligonucleosides are
prepared as described in U.S. Pat. Nos. 5,264,562 and
5,264,564.
[0332] Ethylene oxide linked oligonucleosides are prepared as
described in U.S. Pat. No. 5,223,618.
Example 4
RNA Synthesis
[0333] In general, RNA synthesis chemistry is based on the
selective incorporation of various protecting groups at strategic
intermediary reactions. Although one of ordinary skill in the art
will understand the use of protecting groups in organic synthesis,
a useful class of protecting groups includes silyl ethers. In
particular bulky silyl ethers are used to protect the 5'-hydroxyl
in combination with an acid-labile orthoester protecting group on
the 2'-hydroxyl. This set of protecting groups is then used with
standard solid-phase synthesis technology. It is important to
lastly remove the acid labile orthoester protecting group after all
other synthetic steps. Moreover, the early use of the silyl
protecting groups during synthesis ensures facile removal when
desired, without undesired deprotection of 2' hydroxyl.
[0334] Following this procedure for the sequential protection of
the 5'-hydroxyl in combination with protection of the 2'-hydroxyl
by protecting groups that are differentially removed and are
differentially chemically labile, RNA oligonucleotides were
synthesized.
[0335] RNA oligonucleotides are synthesized in a stepwise fashion.
Each nucleotide is added sequentially (3'- to 5'-direction) to a
solid support-bound oligonucleotide. The first nucleoside at the
3'-end of the chain is covalently attached to a solid support. The
nucleotide precursor, a ribonucleoside phosphoramidite, and
activator are added, coupling the second base onto the 5'-end of
the first nucleoside. The support is washed and any unreacted
5'-hydroxyl groups are capped with acetic anhydride to yield
5'-acetyl moieties. The linkage is then oxidized to the more stable
and ultimately desired P(V) linkage. At the end of the nucleotide
addition cycle, the 5'-silyl group is cleaved with fluoride. The
cycle is repeated for each subsequent nucleotide.
[0336] Following synthesis, the methyl protecting groups on the
phosphates are cleaved in 30 minutes utilizing 1 M
disodium-2-carbamoyl-2-cyanoethyl- ene-1,1-dithiolate trihydrate
(S.sub.2Na.sub.2) in DMF. The deprotection solution is washed from
the solid support-bound oligonucleotide using water. The support is
then treated with 40% methylamine in water for 10 minutes at
55.degree. C. This releases the RNA oligonucleotides into solution,
deprotects the exocyclic amines, and modifies the 2'-groups. The
oligonucleotides can be analyzed by anion exchange HPLC at this
stage.
[0337] The 2'-orthoester groups are the last protecting groups to
be removed. The ethylene glycol monoacetate orthoester protecting
group developed by Dharmacon Research, Inc. (Lafayette, Colo.), is
one example of a useful orthoester protecting group which, has the
following important properties. It is stable to the conditions of
nucleoside phosphoramidite synthesis and oligonucleotide synthesis.
However, after oligonucleotide synthesis the oligonucleotide is
treated with methylamine which not only cleaves the oligonucleotide
from the solid support but also removes the acetyl groups from the
orthoesters. The resulting 2-ethyl-hydroxyl substituents on the
orthoester are less electron withdrawing than the acetylated
precursor. As a result, the modified orthoester becomes more labile
to acid-catalyzed hydrolysis. Specifically, the rate of cleavage is
approximately 10 times faster after the acetyl groups are removed.
Therefore, this orthoester possesses sufficient stability in order
to be compatible with oligonucleotide synthesis and yet, when
subsequently modified, permits deprotection to be carried out under
relatively mild aqueous conditions compatible with the final RNA
oligonucleotide product.
[0338] Additionally, methods of RNA synthesis are well known in the
art (Scaringe, S. A. Ph.D. Thesis, University of Colorado, 1996;
Scaringe, S. A., et al., J. Am. Chem. Soc., 1998, 120, 11820-11821;
Matteucci, M. D. and Caruthers, M. H. J. Am. Chem. Soc., 1981, 103,
3185-3191; Beaucage, S. L. and Caruthers, M. H. Tetrahedron Lett.,
1981, 22, 1859-1862; Dahl, B. J., et al., Acta Chem. Scand, 1990,
44, 639-641; Reddy, M. P., et al., Tetrahedrom Lett., 1994, 25,
4311-4314; Wincott, F. et al., Nucleic Acids Res., 1995, 23,
2677-2684; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2301-2313; Griffin, B. E., et al., Tetrahedron, 1967, 23,
2315-2331).
[0339] RNA antisense oligomeric compounds (RNA oligonucleotides) of
the present invention can be synthesized by the methods herein or
purchased from Dharmacon Research, Inc (Lafayette, Colo.). Once
synthesized, complementary RNA antisense oligomeric compounds can
then be annealed by methods known in the art to form double
stranded (duplexed) antisense oligomeric compounds. For example,
duplexes can be formed by combining 30 .mu.l of each of the
complementary strands of RNA oligonucleotides (50 uM RNA
oligonucleotide solution) and 15 .mu.l of 5.times. annealing buffer
(100 mM potassium acetate, 30 mM HEPES-KOH pH 7.4, 2 mM magnesium
acetate) followed by heating for 1 minute at 90.degree. C., then 1
hour at 37.degree. C. The resulting duplexed antisense oligomeric
compounds can be used in kits, assays, screens, or other methods to
investigate the role of a target nucleic acid.
Example 5
Synthesis of Chimeric Oligonucleotides
[0340] Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers."
[0341] (2'-O-Me)--(2'-deoxy)--(2'-O-Me) Chimeric Phosphorothioate
Oligonucleotides
[0342] Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate
and 2'-deoxy phosphorothioate oligonucleotide segments are
synthesized using an Applied Biosystems automated DNA synthesizer
Model 394, as above. Oligonucleotides are synthesized using the
automated synthesizer and
2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite for the DNA
portion and 5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for
5' and 3' wings. The standard synthesis cycle is modified by
incorporating coupling steps with increased reaction times for the
5'-dimethoxytrityl-2'-O-methyl-3'-O- -phosphoramidite. The fully
protected oligonucleotide is cleaved from the support and
deprotected in concentrated ammonia (NH.sub.4OH) for 12-16 hr at
55.degree. C. The deprotected oligo is then recovered by an
appropriate method (precipitation, column chromatography, volume
reduced in vacuo and analyzed spetrophotometrically for yield and
for purity by capillary electrophoresis and by mass
spectrometry.
[0343] (2'-O-(2-Methoxyethyl))--(2'-deoxy)--(2'-O-(Methoxyethyl))
Chimeric Phosphorothioate Oligonucleotides
[0344] (2'-O-(2-methoxyethyl))--(2'-deoxy)--(-2'-O-(methoxyethyl))
chimeric phosphorothioate oligonucleotides were prepared as per the
procedure above for the 2'-O-methyl chimeric oligonucleotide, with
the substitution of 2'-O-(methoxyethyl) amidites for the
2'-O-methyl amidites.
[0345] (2'-O-(2-Methoxyethyl)Phosphodiester)--(2'-deoxy
Phosphorothioate)--(2'-O-(2-Methoxyethyl) Phosphodiester) Chimeric
Oligonucleotides
[0346] (2'-O-(2-methoxyethyl phosphodiester)--(2'-deoxy
phosphorothioate)--(2'-O-(methoxyethyl) phosphodiester) chimeric
oligonucleotides are prepared as per the above procedure for the
2'-O-methyl chimeric oligonucleotide with the substitution of
2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites,
oxidation with iodine to generate the phosphodiester
internucleotide linkages within the wing portions of the chimeric
structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate
internucleotide linkages for the center gap.
[0347] Other chimeric oligonucleotides, chimeric oligonucleosides
and mixed chimeric oligonucleotides/oligonucleosides are
synthesized according to U.S. Pat. No. 5,623,065.
Example 6
Oligonucleotide Isolation
[0348] After cleavage from the controlled pore glass solid support
and deblocking in concentrated ammonium hydroxide at 55.degree. C.
for 12-16 hours, the oligonucleotides or oligonucleosides are
recovered by precipitation out of 1 M NH.sub.4OAc with >3
volumes of ethanol. Synthesized oligonucleotides were analyzed by
electrospray mass spectroscopy (molecular weight determination) and
by capillary gel electrophoresis and judged to be at least 70% full
length material. The relative amounts of phosphorothioate and
phosphodiester linkages obtained in the synthesis was determined by
the ratio of correct molecular weight relative to the -16 amu
product (+/-32+/-48). For some studies oligonucleotides were
purified by HPLC, as described by Chiang et al., J. Biol. Chem.
1991, 266, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified
material.
Example 7
Oligonucleotide Synthesis --96 Well Plate Format
[0349] Oligonucleotides were synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a 96-well format.
Phosphodiester internucleotide linkages were afforded by oxidation
with aqueous iodine. Phosphorothioate internucleotide linkages were
generated by sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) in anhydrous acetonitrile. Standard
base-protected beta-cyanoethyl-diiso-propyl phosphoramidites were
purchased from commercial vendors (e.g. PE-Applied Biosystems,
Foster City, Calif., or Pharmacia, Piscataway, N.J.). Non-standard
nucleosides are synthesized as per standard or patented methods.
They are utilized as base protected beta-cyanoethyldiisopropyl
phosphoramidites.
[0350] Oligonucleotides were cleaved from support and deprotected
with concentrated NH.sub.4OH at elevated temperature (55-60.degree.
C.) for 12-16 hours and the released product then dried in vacuo.
The dried product was then re-suspended in sterile water to afford
a master plate from which all analytical and test plate samples are
then diluted utilizing robotic pipettors.
Example 8
Oligonucleotide Analysis 96 Well Plate Format
[0351] The concentration of oligonucleotide in each well was
assessed by dilution of samples and UV absorption spectroscopy. The
full-length integrity of the individual products was evaluated by
capillary electrophoresis (CE) in either the 96-well format
(Beckman P/ACE.TM. MDQ) or, for individually prepared samples, on a
commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000, ABI 270).
Base and backbone composition was confirmed by mass analysis of the
oligomeric compounds utilizing electrospray-mass spectroscopy. All
assay test plates were diluted from the master plate using single
and multi-channel robotic pipettors. Plates were judged to be
acceptable if at least 85% of the oligomeric compounds on the plate
were at least 85% full length.
Example 9
Cell Culture and Oligonucleotide Treatment
[0352] The effect of oligomeric compounds on target nucleic acid
expression can be tested in any of a variety of cell types provided
that the target nucleic acid is present at measurable levels. This
can be routinely determined using, for example, PCR or Northern
blot analysis. The following cell types are provided for
illustrative purposes, but other cell types can be routinely used,
provided that the target is expressed in the cell type chosen. This
can be readily determined by methods routine in the art, for
example Northern blot analysis, ribonuclease protection assays, or
RT-PCR.
[0353] T-24 Cells:
[0354] The human transitional cell bladder carcinoma cell line T-24
was obtained from the American Type Culture Collection (ATCC)
(Manassas, Va.). T-24 cells were routinely cultured in complete
McCoy's 5A basal media (Invitrogen Corporation, Carlsbad, Calif.)
supplemented with 10% fetal calf serum (Invitrogen Corporation,
Carlsbad, Calif.), penicillin 100 units per mL, and streptomycin
100 micrograms per mL (Invitrogen Corporation, Carlsbad, Calif.).
Cells were routinely passaged by trypsinization and dilution when
they reached 90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #353872) at a density of 7000 cells/well for use
in RT-PCR analysis.
[0355] For Northern blotting or other analysis, cells may be seeded
onto 100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
[0356] A549 Cells:
[0357] The human lung carcinoma cell line A549 was obtained from
the American Type Culture Collection (ATCC) (Manassas, Va.). A549
cells were routinely cultured in DMEM basal media (Invitrogen
Corporation, Carlsbad, Calif.) supplemented with 10% fetal calf
serum (Invitrogen Corporation, Carlsbad, Calif.), penicillin 100
units per mL, and streptomycin 100 micrograms per mL (Invitrogen
Corporation, Carlsbad, Calif.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence.
[0358] NHDF Cells:
[0359] Human neonatal dermal fibroblast (NHDF) were obtained from
the Clonetics Corporation (Walkersville, Md.). NHDFs were routinely
maintained in Fibroblast Growth Medium (Clonetics Corporation,
Walkersville, Md.) supplemented as recommended by the supplier.
Cells were maintained for up to 10 passages as recommended by the
supplier.
[0360] HEK Cells:
[0361] Human embryonic keratinocytes (HEK) were obtained from the
Clonetics Corporation (Walkersville, Md.). HEKs were routinely
maintained in Keratinocyte Growth Medium (Clonetics Corporation,
Walkersville, Md.) formulated as recommended by the supplier. Cells
were routinely maintained for up to 10 passages as recommended by
the supplier.
[0362] MCF-7 Cells
[0363] The human breast carcinoma cell line MCF-7 is obtained from
the American Type Culture Collection (Manassas, Va.). These cells
contain a wild-type p53 gene. MCF-7 cells are routinely cultured in
DMEM low glucose (Gibco/Life Technologies, Gaithersburg, Md.)
supplemented with 10% fetal calf serum (Gibco/Life Technologies,
Gaithersburg, Md.). Cells are routinely passaged by trypsinization
and dilution when they reach 90% confluence. Cells are seeded into
96-well plates (Falcon-Primaria #3872) at a density of 7000
cells/well for treatment with the oligomeric compounds of the
invention.
[0364] HepB3 Cells
[0365] The human hepatoma cell line HepB3 (Hep3B2.1-7) is obtained
from the American Type Culture Collection (ATCC-ATCC Catalog #
HB-8064) (Manassas, Va.). This cell line was initially derived from
a hepatocellular carcinoma of an 8-yr-old black male. The cells are
epithelial in morphology and are tumorigenic in nude mice. HepB3
cells are routinely cultured in Minimum Essential Medium (MEM) with
Earle's Balanced Salt Solution, 2 mM L-glutamine, 1.5 g/L sodium
bicarbonate, 0.1 mM nonessential amino acids, 1.0 mM sodium
pyruvate (ATCC #20-2003, Manassas, Va.) and with 10%
heat-inactivated fetal bovine serum (Gibco/Life Technologies,
Gaithersburg, Md.). Cells are routinely passaged by trypsinization
and dilution when they reach 90% confluence.
[0366] Primary Mouse Hepatocytes
[0367] Primary mouse hepatocytes are prepared from CD-1 mice
purchased from Charles River Labs. Primary mouse hepatocytes are
routinely cultured in Hepatocyte Attachment Media (Invitrogen Life
Technologies, Carlsbad, Calif.) supplemented with 10% Fetal Bovine
Serum (Invitrogen Life Technologies, Carlsbad, Calif.), 250 nM
dexamethasone (Sigma-Aldrich Corporation, St. Louis, Mo.), 10 nM
bovine insulin (Sigma-Aldrich Corporation, St. Louis, Mo.). Cells
are seeded into 96-well plates (Falcon-Primaria #353872, BD
Biosciences, Bedford, Mass.) at a density of 4000-6000 cells/well
for treatment with the oligomeric compounds of the invention.
[0368] Treatment with Oligomeric Compounds:
[0369] When cells reached 65-75% confluency, they were treated with
oligonucleotide. For cells grown in 96-well plates, wells were
washed once with 100 .mu.L OPTI-MEM.TM.-1 reduced-serum medium
(Invitrogen Corporation, Carlsbad, Calif.) and then treated with
130 .mu.L of OPTI-MEM.TM.-1 containing 3.75 .mu.g/mL LIPOFECTIN.TM.
(Invitrogen Corporation, Carlsbad, Calif.) and the desired
concentration of oligonucleotide. Cells are treated and data are
obtained in triplicate. After 4-7 hours of treatment at 37.degree.
C., the medium was replaced with fresh medium. Cells were harvested
16-24 hours after oligonucleotide treatment.
[0370] The concentration of oligonucleotide used varies from cell
line to cell line. To determine the optimal oligonucleotide
concentration for a particular cell line, the cells are treated
with a positive control oligonucleotide at a range of
concentrations. For human cells the positive control
oligonucleotide is selected from either ISIS 13920
(TCCGTCATCGCTCCTCAGGG, SEQ ID NO:10) which is targeted to human
H-ras, or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO:11) which is
targeted to human Jun-N-terminal kinase-2 (JNK2). Both controls are
2'-O-methoxyethyl gapmers (2'-O-methoxyethyls shown in bold) with a
phosphorothioate backbone. For mouse or rat cells the positive
control oligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID
NO:12, a 2'-O-methoxyethyl gapmer (2'-O-methoxyethyls shown in
bold) with a phosphorothioate backbone which is targeted to both
mouse and rat c-raf. The concentration of positive control
oligonucleotide that results in 80% inhibition of c-H-ras (for ISIS
13920), JNK2 (for ISIS 18078) or c-raf (for ISIS 15770) mRNA is
then utilized as the screening concentration for new
oligonucleotides in subsequent experiments for that cell line. If
80% inhibition is not achieved, the lowest concentration of
positive control oligonucleotide that results in 60% inhibition of
c-H-ras, JNK2 or c-raf mRNA is then utilized as the oligonucleotide
screening concentration in subsequent experiments for that cell
line. If 60% inhibition is not achieved, that particular cell line
is deemed as unsuitable for oligonucleotide transfection
experiments. The concentrations of antisense oligonucleotides used
herein are from 50 nM to 300 mM.
Example 10
Analysis of Oligonucleotide Inhibition of a Target Expression
[0371] Antisense modulation of a target expression can be assayed
in a variety of ways known in the art. For example, a target mRNA
levels can be quantitated by, e.g., Northern blot analysis,
competitive polymerase chain reaction (PCR), or real-time PCR
(RT-PCR). Real-time quantitative PCR is presently suitable. RNA
analysis can be performed on total cellular RNA or poly(A)+ mRNA.
One method of RNA analysis of the present invention is the use of
total cellular RNA as described in other examples herein. Methods
of RNA isolation are well known in the art. Northern blot analysis
is also routine in the art. Real-time quantitative (PCR) can be
conveniently accomplished using the commercially available ABI
PRISM.TM. 7600, 7700, or 7900 Sequence Detection System, available
from PE-Applied Biosystems, Foster City, Calif. and used according
to manufacturer's instructions.
[0372] Protein levels of a target can be quantitated in a variety
of ways well known in the art, such as immunoprecipitation, Western
blot analysis (immunoblotting), enzyme-linked immunosorbent assay
(ELISA) or fluorescence-activated cell sorting (FACS). Antibodies
directed to a target can be identified and obtained from a variety
of sources, such as the MSRS catalog of antibodies (Aerie
Corporation, Birmingham, Mich.), or can be prepared via
conventional monoclonal or polyclonal antibody generation methods
well known in the art.
Example 11
Design of Phenotypic Assays and In Vivo Studies for the Use of a
Target Inhibitors
[0373] Phenotypic Assays
[0374] Once a target inhibitors have been identified by the methods
disclosed herein, the oligomeric compounds are further investigated
in one or more phenotypic assays, each having measurable endpoints
predictive of efficacy in the treatment of a particular disease
state or condition.
[0375] Phenotypic assays, kits and reagents for their use are well
known to those skilled in the art and are herein used to
investigate the role and/or association of a target in health and
disease. Representative phenotypic assays, which can be purchased
from any one of several commercial vendors, include those for
determining cell viability, cytotoxicity, proliferation or cell
survival (Molecular Probes, Eugene, Oreg.; PerkinElmer, Boston,
Mass.), protein-based assays including enzymatic assays (Panvera,
LLC, Madison, Wis.; BD Biosciences, Franklin Lakes, N.J.; Oncogene
Research Products, San Diego, Calif.), cell regulation, signal
transduction, inflammation, oxidative processes and apoptosis
(Assay Designs Inc., Ann Arbor, Mich.), triglyceride accumulation
(Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tube
formation assays, cytokine and hormone assays and metabolic assays
(Chemicon International Inc., Temecula, Calif.; Amersham
Biosciences, Piscataway, N.J.).
[0376] In one non-limiting example, cells determined to be
appropriate for a particular phenotypic assay (i.e., MCF-7 cells
selected for breast cancer studies; adipocytes for obesity studies)
are treated with a target inhibitors identified from the in vitro
studies as well as control compounds at optimal concentrations
which are determined by the methods described above. At the end of
the treatment period, treated and untreated cells are analyzed by
one or more methods specific for the assay to determine phenotypic
outcomes and endpoints. Phenotypic endpoints include changes in
cell morphology over time or treatment dose as well as changes in
levels of cellular components such as proteins, lipids, nucleic
acids, hormones, saccharides or metals. Measurements of cellular
status which include pH, stage of the cell cycle, intake or
excretion of biological indicators by the cell, are also endpoints
of interest.
[0377] Analysis of the geneotype of the cell (measurement of the
expression of one or more of the genes of the cell) after treatment
is also used as an indicator of the efficacy or potency of the a
target inhibitors. Hallmark genes, or those genes suspected to be
associated with a specific disease state, condition, or phenotype,
are measured in both treated and untreated cells.
[0378] In Vivo Studies
[0379] The individual subjects of the in vivo studies described
herein are warm-blooded vertebrate animals, which includes
humans.
[0380] The clinical trial is subjected to rigorous controls to
ensure that individuals are not unnecessarily put at risk and that
they are fully informed about their role in the study.
[0381] To account for the psychological effects of receiving
treatments, volunteers are randomly given placebo or a target
inhibitor. Furthermore, to prevent the doctors from being biased in
treatments, they are not informed as to whether the medication they
are administering is a a target inhibitor or a placebo. Using this
randomization approach, each volunteer has the same chance of being
given either the new treatment or the placebo.
[0382] Volunteers receive either the a target inhibitor or placebo
for eight week period with biological parameters associated with
the indicated disease state or condition being measured at the
beginning (baseline measurements before any treatment), end (after
the final treatment), and at regular intervals during the study
period. Such measurements include the levels of nucleic acid
molecules encoding a target or a target protein levels in body
fluids, tissues or organs compared to pre-treatment levels. Other
measurements include, but are not limited to, indices of the
disease state or condition being treated, body weight, blood
pressure, serum titers of pharmacologic indicators of disease or
toxicity as well as ADME (absorption, distribution, metabolism and
excretion) measurements. Information recorded for each patient
includes age (years), gender, height (cm), family history of
disease state or condition (yes/no), motivation rating
(some/moderate/great) and number and type of previous treatment
regimens for the indicated disease or condition.
[0383] Volunteers taking part in this study are healthy adults (age
18 to 65 years) and roughly an equal number of males and females
participate in the study. Volunteers with certain characteristics
are equally distributed for placebo and a target inhibitor
treatment. In general, the volunteers treated with placebo have
little or no response to treatment, whereas the volunteers treated
with the a target inhibitor show positive trends in their disease
state or condition index at the conclusion of the study.
Example 12
RNA Isolation
[0384] Poly(A)+ mRNA Isolation
[0385] Poly(A)+ mRNA was isolated according to Miura et al., (Clin.
Chem., 1996, 42, 1758-1764). Other methods for poly(A)+ mRNA
isolation are routine in the art. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10
mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl, 0.5% NP-40, 20 mM
vanadyl-ribonucleoside complex) was added to each well, the plate
was gently agitated and then incubated at room temperature for five
minutes. 55 .mu.L of lysate was transferred to Oligo d(T) coated
96-well plates (AGCT Inc., Irvine Calif.). Plates were incubated
for 60 minutes at room temperature, washed 3 times with 200 .mu.L
of wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl).
After the final wash, the plate was blotted on paper towels to
remove excess wash buffer and then air-dried for 5 minutes. 60
.mu.L of elution buffer (5 mM Tris-HCl pH 7.6), preheated to
70.degree. C., was added to each well, the plate was incubated on a
90.degree. C. hot plate for 5 minutes, and the eluate was then
transferred to a fresh 96-well plate.
[0386] Cells grown on 100 mm or other standard plates may be
treated similarly, using appropriate volumes of all solutions.
[0387] Total RNA Isolation
[0388] Total RNA was isolated using an RNEASY 96.TM. kit and
buffers purchased from Qiagen Inc. (Valencia, Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 150 .mu.L Buffer RLT was
added to each well and the plate vigorously agitated for 20
seconds. 150 .mu.L of 70% ethanol was then added to each well and
the contents mixed by pipetting three times up and down. The
samples were then transferred to the RNEASY 96.TM. well plate
attached to a QIAVAC.TM. manifold fitted with a waste collection
tray and attached to a vacuum source. Vacuum was applied for 1
minute. 500 .mu.L of Buffer RW1 was added to each well of the
RNEASY 96.TM. plate and incubated for 15 minutes and the vacuum was
again applied for 1 minute. An additional 500 .mu.L of Buffer RW1
was added to each well of the RNEASY 96.TM. plate and the vacuum
was applied for 2 minutes. 1 mL of Buffer RPE was then added to
each well of the RNEASY 96.TM. plate and the vacuum applied for a
period of 90 seconds. The Buffer RPE wash was then repeated and the
vacuum was applied for an additional 3 minutes. The plate was then
removed from the QIAVAC.TM. manifold and blotted dry on paper
towels. The plate was then re-attached to the QIAVAC.TM. manifold
fitted with a collection tube rack containing 1.2 mL collection
tubes. RNA was then eluted by pipetting 140 .mu.L of RNAse free
water into each well, incubating 1 minute, and then applying the
vacuum for 3 minutes.
[0389] The repetitive pipetting and elution steps may be automated
using a QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.).
Essentially, after lysing of the cells on the culture plate, the
plate is transferred to the robot deck where the pipetting, DNase
treatment and elution steps are carried out.
Example 13
Real-Time Quantitative PCR Analysis of a Target mRNA Levels
[0390] Quantitation of a target mRNA levels was accomplished by
real-time quantitative PCR using the ABI PRISM.TM. 7600, 7700, or
7900 Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. This is a
closed-tube, non-gel-based, fluorescence detection system which
allows high-throughput quantitation of polymerase chain reaction
(PCR) products in real-time. As opposed to standard PCR in which
amplification products are quantitated after the PCR is completed,
products in real-time quantitative PCR are quantitated as they
accumulate. This is accomplished by including in the PCR reaction
an oligonucleotide probe that anneals specifically between the
forward and reverse PCR primers, and contains two fluorescent dyes.
A reporter dye (e.g., FAM or JOE, obtained from either PE-Applied
Biosystems, Foster City, Calif., Operon Technologies Inc., Alameda,
Calif. or Integrated DNA Technologies Inc., Coralville, Iowa) is
attached to the 5' end of the probe and a quencher dye (e.g.,
TAMRA, obtained from either PE-Applied Biosystems, Foster City,
Calif., Operon Technologies Inc., Alameda, Calif. or Integrated DNA
Technologies Inc., Coralville, Iowa) is attached to the 3' end of
the probe. When the probe and dyes are intact, reporter dye
emission is quenched by the proximity of the 3' quencher dye.
During amplification, annealing of the probe to the target sequence
creates a substrate that can be cleaved by the 5'-exonuclease
activity of Taq polymerase. During the extension phase of the PCR
amplification cycle, cleavage of the probe by Taq polymerase
releases the reporter dye from the remainder of the probe (and
hence from the quencher moiety) and a sequence-specific fluorescent
signal is generated. With each cycle, additional reporter dye
molecules are cleaved from their respective probes, and the
fluorescence intensity is monitored at regular intervals by laser
optics built into the ABI PRISM.TM. Sequence Detection System. In
each assay, a series of parallel reactions containing serial
dilutions of mRNA from untreated control samples generates a
standard curve that is used to quantitate the percent inhibition
after antisense oligonucleotide treatment of test samples.
[0391] Prior to quantitative PCR analysis, primer-probe sets
specific to the target gene being measured are evaluated for their
ability to be "multiplexed" with a GAPDH amplification reaction. In
multiplexing, both the target gene and the internal standard gene
GAPDH are amplified concurrently in a single sample. In this
analysis, mRNA isolated from untreated cells is serially diluted.
Each dilution is amplified in the presence of primer-probe sets
specific for GAPDH only, target gene only ("single-plexing"), or
both (multiplexing). Following PCR amplification, standard curves
of GAPDH and target mRNA signal as a function of dilution are
generated from both the single-plexed and multiplexed samples. If
both the slope and correlation coefficient of the GAPDH and target
signals generated from the multiplexed samples fall within 10% of
their corresponding values generated from the single-plexed
samples, the primer-probe set specific for that target is deemed
multiplexable. Other methods of PCR are also known in the art.
[0392] PCR reagents were obtained from Invitrogen Corporation,
(Carlsbad, Calif.). RT-PCR reactions were carried out by adding 20
.mu.L PCR cocktail (2.5.times.PCR buffer minus MgCl.sub.2, 6.6 mM
MgCl.sub.2, 375 .mu.M each of dATP, dCTP, dCTP and dGTP, 375 nM
each of forward primer and reverse primer, 125 nM of probe, 4 Units
RNAse inhibitor, 1.25 Units PLATINUM.RTM. Taq, 5 Units MULV reverse
transcriptase, and 2.5.times.ROX dye) to 96-well plates containing
30 .mu.L total RNA solution (20-200 ng). The RT reaction was
carried out by incubation for 30 minutes at 48.degree. C. Following
a 10 minute incubation at 95.degree. C. to activate the
PLATINUM.RTM. Taq, 40 cycles of a two-step PCR protocol were
carried out: 95.degree. C. for 15 seconds (denaturation) followed
by 60.degree. C. for 1.5 minutes (annealing/extension).
[0393] Gene target quantities obtained by real time RT-PCR are
normalized using either the expression level of GAPDH, a gene whose
expression is constant, or by quantifying total RNA using
RiboGreen.TM. (Molecular Probes, Inc. Eugene, Oreg.). GAPDH
expression is quantified by real time RT-PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RiboGreen.TM. RNA quantification reagent
(Molecular Probes, Inc. Eugene, Oreg.). Methods of RNA
quantification by RiboGreen.TM. are taught in Jones, L. J., et al,
(Analytical Biochemistry, 1998, 265, 368-374).
[0394] In this assay, 170 .mu.L of RiboGreen.TM. working reagent
(RiboGreen.TM. reagent diluted 1:350 in 10 mM Tris-HCl, 1 mM EDTA,
pH 7.5) is pipetted into a 96-well plate containing 30 .mu.L
purified, cellular RNA. The plate is read in a CytoFluor 4000 (PE
Applied Biosystems) with excitation at 485 nm and emission at 530
nm.
[0395] Probes and are designed to hybridize to a human a target
sequence, using published sequence information.
Example 14
Northern Blot Analysis of a Target mRNA Levels
[0396] Eighteen hours after antisense treatment, cell monolayers
were washed twice with cold PBS and lysed in 1 mL RNAZOL.TM.
(TEL-TEST "B" Inc., Friendswood, Tex.). Total RNA was prepared
following manufacturer's recommended protocols. Twenty micrograms
of total RNA was fractionated by electrophoresis through 1.2%
agarose gels containing 1.1% formaldehyde using a MOPS buffer
system (AMRESCO, Inc. Solon, Ohio). RNA was transferred from the
gel to HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) by overnight capillary transfer using a
Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,
Friendswood, Tex.). RNA transfer was confirmed by UV visualization.
Membranes were fixed by UV cross-linking using a STRATALINKER.TM.
UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.) and then
probed using QUICKHYB.TM. hybridization solution (Stratagene, La
Jolla, Calif.) using manufacturer's recommendations for stringent
conditions.
[0397] To detect human a target, a human a target specific primer
probe set is prepared by PCR To normalize for variations in loading
and transfer efficiency membranes are stripped and probed for human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,
Palo Alto, Calif.).
[0398] Hybridized membranes were visualized and quantitated using a
PHOSPHORIMAGER.TM. and IMAGEQUANT.TM. Software V3.3 (Molecular
Dynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels
in untreated controls.
Example 15
Antisense Inhibition of Human a Target Expression by
Oligonucleotides
[0399] In accordance with the present invention, a series of
oligomeric compounds are designed to target different regions of
the human target RNA. The oligomeric compounds are analyzed for
their effect on human target mRNA levels by quantitative real-time
PCR as described in other examples herein. Data are averages from
three experiments. The target regions to which these suitable
sequences are complementary are herein referred to as "suitable
target segments" and are therefore suitable for targeting by
oligomeric compounds of the present invention. The sequences
represent the reverse complement of the suitable antisense
oligomeric compounds.
[0400] As these "suitable target segments" have been found by
experimentation to be open to, and accessible for, hybridization
with the antisense oligomeric compounds of the present invention,
one of skill in the art will recognize or be able to ascertain,
using no more than routine experimentation, further embodiments of
the invention that encompass other oligomeric compounds that
specifically hybridize to these suitable target segments and
consequently inhibit the expression of a target.
[0401] According to the present invention, antisense oligomeric
compounds include antisense oligomeric compounds, antisense
oligonucleotides, ribozymes, external guide sequence (EGS)
oligonucleotides, alternate splicers, primers, probes, and other
short oligomeric compounds which hybridize to at least a portion of
the target nucleic acid.
Example 16
Western Blot Analysis of a Target Protein Levels
[0402] Western blot analysis (immunoblot analysis) is carried out
using standard methods. Cells are harvested 16-20 h after
oligonucleotide treatment, washed once with PBS, suspended in
Laemmli buffer (100 .mu.l/well), boiled for 5 minutes and loaded on
a 16% SDS-PAGE gel. Gels are run for 1.5 hours at 150 V, and
transferred to membrane for western blotting. Appropriate primary
antibody directed to a target is used, with a radiolabeled or
fluorescently labeled secondary antibody directed against the
primary antibody species. Bands are visualized using a
PHOSPHORIMAGER.TM. (Molecular Dynamics, Sunnyvale Calif.).
[0403] Various modifications of the invention, in addition to those
described herein, will be apparent to those skilled in the art from
the foregoing description. Such modifications are also intended to
fall within the scope of the appended claims. Each reference
(including, but not limited to, journal articles, U.S. and non-U.S.
patents, patent application publications, international patent
application publications, gene bank accession numbers, and the
like) cited in the present application is incorporated herein by
reference in its entirety.
Sequence CWU 1
1
12 1 19 DNA Artificial Sequence Description of Artificial Sequence
Oligomer 1 cgagaggcgg acgggaccg 19 2 21 DNA Artificial Sequence
Description of Artificial Sequence Oligomer 2 cggtcccgtc cgcctctcgt
t 21 3 20 DNA Artificial Sequence Description of Artificial
Sequence Oligomer 3 ccttccctga aggttcctcc 20 4 16 DNA Artificial
Sequence Description of Artificial Sequence Oligomer 4 ctcatactcg
taggcc 16 5 18 DNA Artificial Sequence Description of Artificial
Sequence Oligomer 5 gctcatactc gtaggcca 18 6 18 DNA Artificial
Sequence Description of Artificial Sequence Oligomer 6 gctcatactc
gtaggcca 18 7 16 DNA Artificial Sequence Description of Artificial
Sequence Oligomer 7 ctcatactcg taggcc 16 8 18 DNA Artificial
Sequence Description of Artificial Sequence Oligomer 8 gctcatactc
gtaggcca 18 9 18 DNA Artificial Sequence Description of Artificial
Sequence Oligomer 9 gctcatactc gtaggcca 18 10 20 DNA Artificial
Sequence Description of Artificial Sequence Oligomer 10 tccgtcatcg
ctcctcaggg 20 11 20 DNA Artificial Sequence Description of
Artificial Sequence Oligomer 11 gtgcgcgcga gcccgaaatc 20 12 20 DNA
Artificial Sequence Description of Artificial Sequence Oligomer 12
atgcattctg cccccaagga 20
* * * * *