U.S. patent application number 10/946873 was filed with the patent office on 2005-07-28 for modified irna agents.
Invention is credited to Manoharan, Muthiah, Rajeev, Kallanthottathil G..
Application Number | 20050164235 10/946873 |
Document ID | / |
Family ID | 35530789 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050164235 |
Kind Code |
A1 |
Manoharan, Muthiah ; et
al. |
July 28, 2005 |
Modified iRNA agents
Abstract
The invention relates to iRNA agents, which preferably include a
monomer in which the ribose moiety has been replaced by a moiety
other than ribose. The inclusion of such a monomer can allow for
modulation of a property of the iRNA agent into which it is
incorporated, e.g., by using the non-ribose moiety as a point to
which a ligand or other entity, e.g., a carbohydrate; or a steroid,
e.g., cholesterol, which is optionally substituted with at least
one carbohydrate. is directly, or indirectly, tethered. The
invention also relates to methods of making and using such modified
iRNA agents.
Inventors: |
Manoharan, Muthiah; (Weston,
MA) ; Rajeev, Kallanthottathil G.; (Cambridge,
MA) |
Correspondence
Address: |
FISH & RICHARDSON PC
225 FRANKLIN ST
BOSTON
MA
02110
US
|
Family ID: |
35530789 |
Appl. No.: |
10/946873 |
Filed: |
September 21, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10946873 |
Sep 21, 2004 |
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PCT/US04/11829 |
Apr 16, 2004 |
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60493986 |
Aug 8, 2003 |
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60494597 |
Aug 11, 2003 |
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60506341 |
Sep 26, 2003 |
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60518453 |
Nov 7, 2003 |
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60463772 |
Apr 17, 2003 |
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60465802 |
Apr 25, 2003 |
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60469612 |
May 9, 2003 |
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60510246 |
Oct 9, 2003 |
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60510318 |
Oct 10, 2003 |
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60503414 |
Sep 15, 2003 |
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60465665 |
Apr 25, 2003 |
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Current U.S.
Class: |
435/6.12 ;
536/25.32 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 47/554 20170801; C12N 2320/51 20130101; C12N 2310/344
20130101; C12N 2310/3515 20130101; C12N 2310/323 20130101; C12N
15/111 20130101; A61P 35/02 20180101; A61P 31/14 20180101; C12N
2310/14 20130101 |
Class at
Publication: |
435/006 ;
536/025.32 |
International
Class: |
C12Q 001/68; C07H
021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 8, 2004 |
WO |
PCT/US04/07070 |
Apr 5, 2004 |
WO |
PCT/US04/10586 |
Apr 9, 2004 |
WO |
PCT/US04/11255 |
Apr 16, 2004 |
WO |
PCT/US04/11822 |
Apr 16, 2004 |
WO |
PCT/US04/11829 |
Claims
What is claimed is:
1. An iRNA agent comprising a first strand and a second strand,
wherein at 2 least one subunit having a formula (I) is incorporated
into at least one of said strands: 111wherein: X is N(CO)R.sup.7,
NR.sup.7 or CH.sub.2; Y is NR.sup.8, O, S, CR.sup.9R.sup.10, or
absent; Z is CR.sup.11R.sup.12 or absent; Each of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.9, and R.sup.10 is, independently, H,
OR.sup.a, OR.sup.b, (CH.sub.2).sub.nOR.sup.a, or
(CH.sub.2).sub.nOR.sup.b, provided that at least one of R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.9, and R.sup.10 is OR.sup.a or
OR.sup.b and that at least one of R.sup.1, R.sup.2, R.sup.3,
R.sup.4, R.sup.9, and R.sup.10 is (CH.sub.2).sub.nOR.sup.a, or
(CH.sub.2).sub.nOR.sup.b; Each of R.sup.5, R.sup.6, R.sup.11, and
R.sup.12 is, independently, H, C.sub.1-C.sub.6 alkyl optionally
substituted with 1-3 R.sup.13, or C(O)NHR.sup.7; or R.sup.5 and
R.sup.11 together are C.sub.3-C.sub.8 cycloalkyl optionally
substituted with R.sup.14; R.sup.7 is Rd; or C.sub.1-C.sub.20 alkyl
substituted with NR.sup.cR.sup.d or NHC(O)R.sup.d; R.sup.8 is
C.sub.1-C.sub.6 alkyl; R.sup.13 is hydroxy, C.sub.1-C.sub.4 alkoxy,
or halo; R.sup.14 is NR.sup.cR.sup.7; R.sup.a is: 112R.sup.b is
113Each of A and C is, independently, O or S; B is OH, O.sup.-, or
114R.sup.c is H or C.sub.1-C.sub.6 alkyl; R.sup.d is a carbohydrate
radical; or a steroid radical, which is optionally tethered to at
least one carbohydrate radical; and n is 1-4.
2. The compound of claim 1, wherein X is N(CO)R.sup.7 or NR.sup.7,
Y is CR.sup.9R.sup.10, and Z is absent.
3. The compound of claim 2, wherein R.sup.1 is
(CH.sub.2).sub.nOR.sup.b and R.sup.3 is OR.sup.a.
4. The compound of claim 3, wherein R.sup.1 and R.sup.3 are
cis.
5. The compound of claim 3, wherein R.sup.1 and R.sup.3 are
trans.
6. The compound of claim 3, wherein n is 1.
7. The compound of claims 3, A is 0.
8. The compound of claim 3, wherein A is S.
9. The compound of claim 2, wherein R.sup.1 is
(CH.sub.2).sub.nOR.sup.b and R.sup.3 is OR.sup.b.
10. The compound of claim 2, wherein R.sup.1 is
(CH.sub.2).sub.nOR.sup.a and R.sup.3 is OR.sup.b.
11. The compound of claim 3, wherein R.sup.7 is
(CH.sub.2).sub.5NHR.sup.d or (CH.sub.2).sub.5NHC(O)R.sup.d.
12. The compound of claim 3, wherein Rd is chosen from the group of
a galactose radical, an N-acetylgalactose radical, or a mannose
radical.
13. The compound of claim 2, wherein R.sup.1 is OR.sup.b and
R.sup.3 is (CH.sub.2).sub.nOR.sup.b.
14. The compound of claim 2, wherein R.sup.1 is OR.sup.b and
R.sup.3 is (CH.sub.2).sub.nOR.sup.b.
15. The compound of claim 2, wherein R.sup.1 is OR.sup.a and
R.sup.3 is (CH.sub.2)OR.sup.b.
16. The compound of claim 15, wherein R.sup.1 and R.sup.3 are
cis.
17. The compound of claim 15, wherein R.sup.1 and R.sup.3 are
trans.
18. The compound of claim 15, wherein n is 1.
19. The compound of claim 2, wherein R.sup.1 is
(CH.sub.2).sub.nOR.sup.b and R.sup.9 is OR.sup.a.
20. The compound of claim 19, wherein R.sup.1 and R.sup.9 are
cis.
21. The compound of claim 19, wherein R.sup.1 and R.sup.9 are
trans.
22. The compound of claim 19, wherein n is 1.
23. The compound of claim 2, wherein R.sup.1 is OR.sup.a and
R.sup.9 is (CH.sub.2).sub.nOR.sup.b.
24. The compound of claim 23, wherein R.sup.1 and R.sup.9 are
cis.
25. The compound of claim 23, wherein R.sup.1 and R.sup.9 are
trans.
26. The compound of claim 23, wherein n is 1.
27. The compound of claim 2, wherein R.sup.1 is
(CH.sub.2).sub.nOR.sup.b and R.sup.9 is OR.sup.b.
28. The compound of claim 2, wherein R.sup.1 is
(CH.sub.2).sub.nOR.sup.a and R.sup.9 is OR.sup.b.
29. The compound of claim 2, wherein R.sup.1 is OR.sup.b and
R.sup.9 is (CH.sub.2).sub.nOR.sup.b.
30. The compound of claim 2, wherein R.sup.1 is OR.sup.b and
R.sup.9 is (CH.sub.2).sub.nOR.sup.a.
31. The compound of claim 2, wherein R.sup.3 is
(CH.sub.2).sub.nOR.sup.b and R.sup.9 is OR.sup.a.
32. The compound of claim 2, wherein R.sup.3 is
(CH.sub.2).sub.nOR.sup.b and R.sup.9 is OR.sup.b.
33. The compound of claim 2, wherein R.sup.3 is
(CH.sub.2).sub.nOR.sup.a and R.sup.9 is OR.sup.b.
34. The compound of claim 2, wherein R.sup.3 is OR.sup.a and
R.sup.9 is (CH.sub.2).sub.nOR.sup.b.
35. The compound of claim 2, wherein R.sup.3 is OR.sup.b and
R.sup.9 is (CH.sub.2).sub.nOR.sup.b.
36. The compound of claim 2, wherein R.sup.3 is OR.sup.b and
R.sup.9 is (CH.sub.2).sub.nOR.sup.a.
37. The compound of claim 1, wherein one of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.9, and R.sup.10 is OR.sup.a and one of
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.9, and R.sup.10 is
(CH.sub.2).sub.nOR.sup.b.
38. The compound of claim 1, wherein one of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.9, and R.sup.10 is (CH.sub.2).sub.nOR.sup.a
and one of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.9, and
R.sup.10 is OR.sup.b.
39. The compound of claim 1, wherein one of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.9, and R.sup.10 is OR.sup.b and one of
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.9, and R.sup.10 is
(CH.sub.2).sub.nOR.sup.b.
40. An iRNA agent comprising a first strand and a second strand,
wherein at least one subunit is derivatized with a moiety
comprising a carbohydrate radical or a steroid radical, the steroid
radical being optionally tethered to at least one carbohydrate
radical, which enhances entrance into a cell.
41. The compound of claim 40, wherein the subunit is derivatized
with a galactose radical, an N-acetylgalactose radical, or a
mannose radical.
42. The compound of claim 40, wherein the subunit is derivatized
with a steroid tethered to at least one carbohydrate radical.
43. The iRNA agent of claim 40, comprising at least one one subunit
having a formula (I) is incorporated into at least one of said
strands: 115wherein: X is N(CO)R.sup.7, NR.sup.7 or CH.sub.2; Y is
NR.sup.8, O, S, CR.sup.9R.sup.10, or absent; Z is CR.sup.11R.sup.12
or absent; Each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.9, and
R.sup.10 is, independently, H, OR.sup.a, OR.sup.b,
(CH.sub.2).sub.nOR.sup.a, or (CH.sub.2).sub.nOR.sup.b, provided
that at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.9,
and R.sup.10 is OR.sup.a or OR.sup.b and that at least one of
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.9, and R.sup.10 is
(CH.sub.2).sub.nOR.sup.a, or (CH.sub.2).sub.nOR.sup.b; Each of
R.sup.5, R.sup.6, R.sup.11, and R.sup.12 is, independently, H,
C.sub.1-C.sub.6 alkyl optionally substituted with 1-3 R.sup.13, or
C(O)NHR.sup.7; or R.sup.5 and R.sup.11 together are C.sub.3-C.sub.8
cycloalkyl optionally substituted with R.sup.14; R.sup.7 is Rd; or
C.sub.1-C.sub.20 alkyl substituted with NR.sup.cR.sup.d or
NHC(O)Rd; R.sup.8 is C.sub.1-C.sub.6 alkyl; R.sup.13 is hydroxy,
C.sub.1-C.sub.4 alkoxy, or halo; R.sup.14 is NR.sup.cR.sup.7;
R.sup.a is: 116R.sup.b is 117Each of A and C is, independently, O
or S; B is OH, O.sup.-, or 118R.sup.c is H or C.sub.1-C.sub.6
alkyl; R.sup.d is a carbohydrate radical; or a steroid radical,
which is optionally tethered to at least one carbohydrate radical;
and n is 1-4.
44. The compound of claim 43, wherein R.sup.d is chosen from a
galactose radical, an N-acetylgalactose radical, or a mannose
radical.
45. The compound of claim 42, wherein R.sup.d is steroid tethered
to at least one carbohydrate radical.
46. The compound of claim 43, wherein X is N(CO)R.sup.7 or
NR.sup.7, Y is CR.sup.9R.sup.10, and Z is absent.
47. The compound of claim 46, wherein R.sup.1 is
(CH.sub.2).sub.nOR.sup.b and R.sup.3 is OR.sup.a.
48. The iRNA agent of claim 40, wherein the iRNA is 21 nucleotides
in length and there is a duplex region of about 19 pairs.
49. The iRNA agent of claim 40, wherein the iRNA agent includes a
duplex region between 17 and 23 pairs in length.
50. The compound of claim 40, wherein the first or second strand is
a sense strand, and the moiety is attached to the sense strand.
51. The compound of claim 50, wherein the moiety is attached to a
3' terminal subunit.
52. The compound of claim 50, wherein the moiety is attached to a
5' terminal subunit.
53. The compound of claim 50, wherein the moiety is attached an
internal subunit.
54. The iRNA agent of claim 40, wherein the moiety enhances
entrance into a primary cell.
55. The iRNA agent of claim 40, wherein the moiety enhances
entrance into a hepatocyte cell.
56. The iRNA agent of claim 40, wherein the cell comprises an
exogenous gene, and the iRNA agent targets the exogenous gene.
57. The iRNA agent of claim 56, wherein the exogenous gene is a
hepatitis viral gene.
58. A composition comprising an iRNA agent to which a carbohydrate
radical or a steroid radical, which is optionally substiuted with
at least one carbohydrate radical is conjugated, which is free of,
has a reduced amount of, or is essentially free of, other reagents
that facilitate or enhance delivery through the cell membrane.
59. The composition of claim 58, wherein said composition is free
of, has a reduced amount of, or is essentially free of: an
additional lipophilic moiety; a transfection agent, e.g.,
concentrations of an ion or other substance which substantially
alters cell permeability to an iRNA agent.
60. A method of modulating expression of a target gene in a
subject, the method comprising: administering an iRNA agent of
claim 1 to a subject.
61. A pharmaceutical composition comprising an iRNA agent of claim
1 and a pharmaceutically acceptable carrier.
62. A kit comprising an iRNA agent of claim 1, a sterile container
in which the iRNA agent is disclosed, and instructions for use.
63. A method of treating a human having or at risk for developing a
disorder of the liver, the method comprising administering an iRNA
agent of claim 1 or 40.
64. The method of claim 63, wherein the disorder of the liver is
infection with a hepatitis virus.
65. The method of claim 64, wherein the virus is HCV.
66. The method of claim 64, wherein the virus is HBV.
67. The iRNA agent of claim 1, wherein the iRNA agent comprises a
first subunit in which R.sup.d is a carbohydrate radical, and a
second subunit in which R.sup.d is a steroid radical, which is
optionally tethered to at least one carbohydrate radical.
68. The iRNA agent of claim 67, wherein the first and second
subunits are incorporated into the same strand.
69. The iRNA agent of claim 67, wherein the first and second
subunits are incorporated into different strands.
70. The iRNA of claim 67, wherein R.sup.d in the first subunit is a
galactose radical.
71. The iRNA agent of claim 67, wherein R.sup.d in the first
subunit is an N-acetylgalactoseamine radical.
72. The iRNA agent of claim 67, wherein R.sup.d in the first
subunit is mannose radical.
73. The iRNA agent of claim 67, wherein R.sup.d in the second
subunit is a steroid radical.
74. The iRNA agent of claim 73, wherein R.sup.d in the second
subunit is a cholesterol radical.
75. The iRNA agent of claim 73, wherein R.sup.d in the second
subunit is a cholanic acid radical.
76. The iRNA agent of claim 1, wherein the iRNA agent comprises a
first subunit and a second subunit in which R.sup.d is a
carbohydrate radical in both the first and second subunits.
77. The iRNA agent of claim 76, wherein R.sup.d is the same
carbohydrate radical in the first and second subunit.
78. The iRNA agent of claim 76, wherein the carbohydrate radical in
the first subunit is different from the carbohydrate radical in the
second subunit.
79. An iRNA agent comprising a first strand and a second strand,
wherein at least one subunit is derivatized with a porphyrin, which
enhances entrance into a cell.
80. The iRNA agent of claim 79, wherein the porphyrin further
comprises a carbohydrate radical; or a steroid radical optionally
substituted with at least one carbohydrate radical.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application No. PCT/US2004/011829, filed on Apr. 16, 2004, which
claims the benefit of U.S. Provisional Application No. 60/493,986,
filed on Aug. 8, 2003; U.S. Provisional Application No. 60/494,597,
filed on Aug. 11, 2003; U.S. Provisional Application No.
60/506,341, filed on Sep. 26, 2003; U.S. Provisional Application
No. 60/518,453, filed on Nov. 7, 2003;U.S. Provisional Application
No. 60/463, 772, filed on Apr. 17, 2003; U.S. Provisional
Application No. 60/465,802, filed on Apr. 25, 2003; U.S.
Provisional Application No. 60/469,612, filed on May 9, 2003; U.S.
Provisional Application No. 60/510,246, filed on Oct. 9, 2003; U.S.
Provisional Application No. 60/510,318, filed on Oct. 10, 2003;
U.S. Provisional Application No. 60/503,414, filed on Sep. 15,
2003; U.S. Provisional Application No. 60/465,665, filed on Apr.
25, 2003; International Application No.: PCT/US04/07070, filed on
Mar. 8, 2004; International Application No.: PCT/US2004/10586,
filed on Apr. 5, 2004; International Application No.:
PCT/US2004/11255, filed on Apr. 9, 2004; and International
Application No.: PCT/US2004/011822, filed on Apr. 16, 2004. The
contents of all of these prior applications are hereby incorporated
by reference in their entireties.
TECHNICAL FIELD
[0002] The invention relates to iRNA agents, which preferably
include a monomer in which the ribose moiety has been replaced by a
moiety other than ribose. The inclusion of such a monomer can allow
for modulation of a property of the iRNA agent into which it is
incorporated, e.g., by using the non-ribose moiety as a point to
which a ligand or other entity, e.g., a carbohydrate; or a steroid,
e.g., cholesterol, which is optionally substituted with at least
one carbohydrate. is directly, or indirectly, tethered. The
invention also relates to methods of making and using such modified
iRNA agents.
BACKGROUND
[0003] Many diseases (e.g., cancers, hematopoietic disorders,
endocrine disorders, and immune disorders) arise from the abnormal
expression or activity of a particular gene or group of genes.
Similarly, disease can result through expression of a mutant form
of protein, as well as from expression of viral genes that have
been integrated into the genome of their host. The therapeutic
benefits of being able to selectively silence these abnormal or
foreign genes are obvious.
[0004] Double-stranded RNA molecules (dsRNA) can block gene
expression by virtue of a highly conserved regulatory mechanism
known as RNA interference (RNAi). Briefly, the RNA III Dicer enzyme
processes dsRNA into small interfering RNA (siRNA) of approximately
22 nucleotides. One strand of the siRNA (the "antisense strand")
then serves as a guide sequence to induce cleavage of messenger
RNAs (mRNAs) including a nucleotide sequence which is at least
partially complementary to the sequence of the antisense strand by
an RNA-induced silencing complex RISC (Hammond, S. M., et al.,
Nature (2000) 404:293-296). The antisense strand is not cleaved or
otherwise degraded in this process, and the RISC including the
antisense strand can subsequently effect the cleavage of further
mRNAs.
SUMMARY
[0005] The inventor has discovered, inter alia, that attachment of
a saccharide moiety to an iRNA agent can optimize one or more
properties of the iRNA agent. In many cases, the saccharide will be
attached to a modified subunit of the iRNA agent. E.g., the ribose
sugar of one or more ribonucleotide subunits of an iRNA agent can
be replaced with another moiety, e.g., a non-carbohydrate
(preferably cyclic) carrier to which is attached a saccharide. A
ribonucleotide subunit in which the ribose sugar of the subunit has
been so replaced is referred to herein as a ribose replacement
modification subunit (RRMS). A cyclic carrier may be a carbocyclic
ring system, i.e., all ring atoms are carbon atoms, or a
heterocyclic ring system, i.e., one or more ring atoms may be a
heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may
be a monocyclic ring system, or may contain two or more rings, e.g.
fused rings. The cyclic carrier may be a fully saturated ring
system, or it may contain one or more double bonds.
[0006] The carriers further include (i) at least two "backbone
attachment points" and (ii) at least one "tethering attachment
point." A "backbone attachment point" as used herein refers to a
functional group, e.g. a hydroxyl group, or generally, a bond
available for, and that is suitable for incorporation of the
carrier into the backbone, e.g., the phosphate, or modified
phosphate, e.g., sulfur containing, backbone, of a ribonucleic
acid. A "tethering attachment point" in some embodiments refers to
a constituent ring atom of the cyclic carrier, e.g., a carbon atom
or a heteroatom (distinct from an atom which provides a backbone
attachment point), that connects a selected moiety. The moiety can
be, e.g., a ligand, e.g., a targeting or delivery moiety, or a
moiety which alters a physical property. One of the most preferred
moieties is a moiety which promotes entry into a cell, e.g., a
lipophilic moiety, e.g., cholesterol. While not wishing to be bound
by theory it is believed the attachment of a lipohilic agent
increases the lipophilicity of an iRNA agent. Optionally, the
selected moiety is connected by an intervening tether to the cyclic
carrier. Thus, it will often include a functional group, e.g., an
amino group, or generally, provide a bond, that is suitable for
incorporation or tethering of another chemical entity, e.g., a
ligand to the constituent ring.
[0007] Incorporation of one or more RRMSs described herein into an
RNA agent, e.g., an iRNA agent, particularly when tethered to an
appropriate entity, can confer one or more new properties to the
RNA agent and/or alter, enhance or modulate one or more existing
properties in the RNA molecule. E.g., it can alter one or more of
lipophilicity or nuclease resistance. Incorporation of one or more
RRMSs described herein into an iRNA agent can, particularly when
the RRMS is tethered to an appropriate entity, modulate, e.g.,
increase, binding affinity of an iRNA agent to a target mRNA,
change the geometry of the duplex form of the iRNA agent, alter
distribution or target the iRNA agent to a particular part of the
body, or modify the interaction with nucleic acid binding proteins
(e.g., during RISC formation and strand separation).
[0008] Accordingly, in one aspect, the invention features, an iRNA
agent preferably comprising a first strand and a second strand,
wherein at least one subunit having a formula (I) is incorporated
into at least one of said strands: 1
[0009] wherein:
[0010] X is N(CO)R.sup.7, NR.sup.7 or CH.sub.2;
[0011] Y is NR.sup.8, O, S, CR.sup.9R.sup.10, or absent;
[0012] Z is CR.sup.11R.sup.12 or absent;
[0013] Each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.9, and
R.sup.10 is, independently, H, OR.sup.a, OR.sup.b,
(CH.sub.2).sub.nOR.sup.a, or (CH.sub.2).sub.nOR.sup.b, provided
that at least one of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.9,
and R.sup.10 is OR.sup.a or OR.sup.b and that at least one of
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.9, and R.sup.10 is
(CH.sub.2).sub.nOR.sup.a, or (CH.sub.2).sub.nOR.sup.b (when the
RRMS is terminal, one of R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.9, and R.sup.10 will include R.sup.a and one will include
R.sup.b; when the RRMSS is internal, two of R.sup.1, R.sup.2,
R.sup.3, R.sup.4, R.sup.9, and R.sup.10 will each include an
R.sup.b); further provided that preferably OR.sup.a may only be
present with (CH.sub.2).sub.nOR.sup.b and (CH.sub.2).sub.nOR.sup.a
may only be present with OR.sup.b;
[0014] Each of R.sup.5, R.sup.6, R.sup.11, and R.sup.12 is,
independently, H, C.sub.1-C.sub.6 alkyl optionally substituted with
1-3 R.sup.13, or C(O)NHR.sup.7; or R.sup.5 and R.sup.11 together
are C.sub.3-C.sub.8 cycloalkyl optionally substituted with
R.sup.14;
[0015] R.sup.7 can be a ligand, e.g., R.sup.7 can be R.sup.d, or
R.sup.7 can be a ligand tethered indirectly to the carrier, e.g.,
through a tethering moiety, e.g., C.sub.1-C.sub.20 alkyl
substituted with NR.sup.cR.sup.d; or C.sub.1-C.sub.20 alkyl
substituted with NHC(O)R.sup.d;
[0016] R.sup.8 is C.sub.1-C.sub.6 alkyl;
[0017] R.sup.13 is hydroxy, C.sub.1-C.sub.4 alkoxy, or halo;
[0018] R.sup.14 is NR.sup.cR.sup.7;
[0019] R.sup.a is: 2
[0020] R.sup.b is: 3
[0021] Each of A and C is, independently, O or S;
[0022] B is OH, O.sup.-, or 4
[0023] R.sup.c is H or C.sub.1-C.sub.6 alkyl;
[0024] R.sup.d is a carbohydrate radical (e.g., mannose, galactose,
or N-acetylgalactose); or a steroid radical, e.g., cholesterol,
which is optionally tethered to at least one carbohydrate radical
(e.g., mannose, galactose, or N-acetylgalactose); and
[0025] n is 1-4.
[0026] In another aspect, this invention relates to an iRNA agent
having a first subunit in which R.sup.d is a carbohydrate radical,
and a second subunit in which R.sup.d is a steroid radical, which
is optionally tethered to at least one carbohydrate radical. The
first and second subunits can be incorporated into the same strand
or different ones. Rd in the first subunit can be a galactose
radical. R.sup.d in the first subunit can be an
N-acetylgalactoseamine radical. R.sup.d in the first subunit can be
mannose radical. R.sup.d in the second subunit can be a steroid
radical. R.sup.d in the second subunit can be a cholesterol
radical. Rd in the second subunit is a cholanic acid radical.
[0027] In a further aspect, this invention relates to an iRNA agent
having a first subunit and a second subunit in which R.sup.d is a
carbohydrate radical in both the first and second subunits. R.sup.d
can be the same carbohydrate radical in the first and second
subunit. The carbohydrate radical in the first subunit can be
different from the carbohydrate radical in the second subunit.
[0028] In general, when more than one ligand is present on an iRNA
agent, the ligands can be distributed as desired, e.g., the ligands
can be attached to subunits on the same strand or different ones.
Some or all of the ligands can all be the same moiety.
Alternatively, all of the ligands can be different moieties.
[0029] In one aspect, this invention relates to an iRNA agent
having a first strand and a second strand, wherein at least one
subunit is derivatized with a porphyrin, which preferably enhances
entrance into a cell. The porphyrin further can have a carbohydrate
radical; or a steroid radical optionally substituted with at least
one carbohydrate radical.
[0030] Embodiments can include one or more of the following
features.
[0031] The iRNA agent can be 21 nucleotides in length and there can
be a duplex region of about 19 pairs.
[0032] The iRNA agent can include a duplex region between 17 and 23
pairs in length.
[0033] R.sup.1 can be CH.sub.2OR.sup.a and R.sup.3 can be OR.sup.b;
or R.sup.1 can be CH.sub.2OR.sup.a and R.sup.9 can be OR.sup.b; or
R.sup.1 can be CH.sub.2OR.sup.a and R.sup.2 can be OR.sup.b.
[0034] R.sup.1 can be CH.sub.2OR.sup.b and R.sup.3 can be OR.sup.b;
or R.sup.1 can be CH.sub.2OR.sup.b and R.sup.9 can be OR.sup.b; or
R.sup.1 can be CH.sub.2OR.sup.b and R.sup.2 can be OR.sup.b; or
R.sup.1 can be CH.sub.2OR.sup.b and R.sup.3 can be OR.sup.a; or
R.sup.1 can be CH.sub.2OR.sup.band R.sup.9 can be OR.sup.a; or
R.sup.1 can be CH.sub.2OR.sup.b and R.sup.2 can be OR.sup.a.
[0035] R.sup.1 can be OR.sup.a and R.sup.3 can be CH.sub.2OR.sup.b;
or R.sup.1 can be OR.sup.a and R.sup.9 can be CH.sub.2OR.sup.b; or
R.sup.1 can be OR.sup.a and R.sup.2 can be CH.sub.2OR.sup.b.
[0036] R.sup.1 can be OR.sup.b and R.sup.3 can be CH.sub.2OR.sup.b;
or R.sup.1 can be OR.sup.b and R.sup.9 can be CH.sub.2OR.sup.b; or
R.sup.1 can be OR.sup.b and R.sup.2 can be CH.sub.2OR.sup.b; or
R.sup.1 can be OR.sup.b and R.sup.3 can be CH.sub.2OR.sup.a; or
R.sup.1 can be OR.sup.b and R.sup.9 can be CH.sub.2OR.sup.a; or
R.sup.1 can be OR.sup.b and R.sup.2 can be CH.sub.2OR.sup.a.
[0037] R.sup.3 can be CH.sub.2OR.sup.a and R.sup.9 can be OR.sup.b;
or R.sup.3 can be CH.sub.2OR.sup.aand R.sup.4 can be OR.sup.b.
[0038] R.sup.3 can be CH.sub.2OR.sup.b and R.sup.9 can be OR.sup.b;
or R.sup.3 can be CH.sub.2OR.sup.b and R.sup.4 can be OR.sup.b; or
R.sup.3 can be CH.sub.2OR.sup.b and R.sup.9 can be OR.sup.a; or
R.sup.3 can be CH.sub.2OR.sup.b and R.sup.4 can be OR.sup.a.
[0039] R.sup.3 can be OR.sup.b and R.sup.9 can be CH.sub.2OR.sup.a;
or R.sup.3 can be OR.sup.b and R.sup.4 can be CH.sub.2OR.sup.a; or
R.sup.3 can be OR.sup.b and R.sup.9 can be CH.sub.2OR.sup.b; or
R.sup.3 can be OR.sup.b and R.sup.4 can be CH.sub.2OR.sup.b.
[0040] R.sup.3 can be OR.sup.a and R.sup.9 can be CH.sub.2OR.sup.b;
or R.sup.3 can be OR.sup.a and R.sup.4 can be CH.sub.2OR.sup.b.
[0041] R.sup.9 can be CH.sub.2OR.sup.a and R.sup.10 can be
OR.sup.b.
[0042] R.sup.9 can be CH.sub.2OR.sup.b and R.sup.10 can be
OR.sup.b; or R.sup.9 can be CH.sub.2OR.sup.b and R.sup.10 can be
OR.sup.a.
[0043] In a preferred embodiment the ribose is replaced with a
pyrroline scaffold or with a 4-hydroxyproline-derived scaffold, and
X is N(CO)R.sup.7 or NR.sup.7, Y is CR.sup.9R.sup.10, and Z is
absent.
[0044] R.sup.1 and R.sup.3 can be cis or R.sup.1 and R.sup.3 can be
trans.
[0045] n can be 1.
[0046] A can be O or S.
[0047] R.sup.1 can be (CH.sub.2).sub.nOR.sup.b and R.sup.3 can be
OR.sup.b; or R.sup.1 can be (CH.sub.2).sub.nOR.sup.a and R.sup.3
can be OR.sup.b.
[0048] R.sup.7 can be (CH.sub.2).sub.nNHR.sup.d or
(CH.sub.2).sub.5NHR.sup- .d.
[0049] R.sup.1 can be OR.sup.b and R.sup.3 can be
(CH.sub.2).sub.nOR.sup.b- ; or R.sup.1 can be OR.sup.b and R.sup.3
can be (CH.sub.2).sub.nOR.sup.a; or R.sup.1 can be OR.sup.a and
R.sup.3 can be (CH.sub.2).sub.nOR.sup.b; or R.sup.1 can be
(CH.sub.2).sub.nOR.sup.b and R.sup.9 can be OR.sup.a.
[0050] R.sup.1 and R.sup.9 can be cis or R.sup.1 and R.sup.9 can be
trans.
[0051] R.sup.1 can be OR.sup.a and R.sup.9 can be
(CH.sub.2).sub.nOR.sup.b- ; or R.sup.1 can be
(CH.sub.2).sub.nOR.sup.b and R.sup.9 can be OR.sup.b; or R.sup.1
can be (CH.sub.2).sub.nOR.sup.a and R.sup.9 can be OR.sup.b; or
R.sup.1 can be OR.sup.b and R.sup.9 can be
(CH.sub.2).sub.nOR.sup.b; or R.sup.1 can be OR.sup.b and R.sup.9
can be (CH.sub.2).sub.nOR.sup.a.
[0052] R.sup.3 can be (CH.sub.2).sub.nOR.sup.b and R.sup.9 can be
OR.sup.a; or R.sup.3 can be (CH.sub.2).sub.nOR.sup.b and R.sup.9
can be OR.sup.b; or R.sup.3 can be (CH.sub.2).sub.nOR.sup.a and
R.sup.9 can be OR.sup.b; or R.sup.3 can be OR.sup.a and R.sup.9 can
be (CH.sub.2).sub.nOR.sup.b; R.sup.3 can be OR.sup.b and R.sup.9
can be (CH.sub.2).sub.nOR.sup.b; or R.sup.3 can be OR.sup.b and
R.sup.9 can be (CH.sub.2).sub.nOR.sup.a.
[0053] R.sup.3 and R.sup.9 can be cis or R.sup.3 and R.sup.9 can be
trans.
[0054] In other preferred embodiments the ribose is replaced with a
piperidine scaffold, and X is N(CO)R.sup.7 or NR.sup.7, Y is
CR.sup.9R.sup.10, and Z is CR.sup.11R.sup.12.
[0055] R.sup.9 can be (CH.sub.2).sub.nOR.sup.b and R.sup.10 can be
OR.sup.a.
[0056] n can be 1 or 2.
[0057] R.sup.9 can be (CH.sub.2).sub.nOR.sup.b and R.sup.10 can be
OR.sup.b; or R.sup.9 can be (CH.sub.2).sub.nOR.sup.a and R.sup.10
can be OR.sup.b.
[0058] A can be O or S.
[0059] R.sup.7 can be (CH.sub.2).sub.5NHR.sup.d or
(CH.sub.2).sub.5NHR.sup- .d.
[0060] R.sup.3 can be (CH.sub.2).sub.nOR.sup.b and R.sup.4 can be
OR.sup.a; or R.sup.3 can be (CH.sub.2).sub.nOR.sup.b and R.sup.4
can be OR.sup.b; or
[0061] R.sup.3 can be (CH.sub.2).sub.nOR.sup.a and R.sup.4 can be
OR.sup.b.
[0062] R.sup.1 can be (CH.sub.2).sub.nOR.sup.b and R.sup.2 can be
OR.sup.a; or R.sup.1 can be (CH.sub.2).sub.nOR.sup.b and R.sup.2
can be OR.sup.b; or R.sup.1 can be (CH.sub.2).sub.nOR.sup.a and
R.sup.2 can be OR.sup.b.
[0063] R.sup.3 can be (CH.sub.2).sub.nOR.sup.b and R.sup.9 can be
OR.sup.a.
[0064] R.sup.3 and R.sup.9 can be cis, or R.sup.3 and R.sup.9 can
be trans.
[0065] R.sup.3 can be (CH.sub.2).sub.nOR.sup.b and R.sup.9 can be
OR.sup.b; or R.sup.3 can be (CH.sub.2).sub.nOR.sup.1 and R.sup.9
can be OR.sup.a; or R.sup.3 can be (CH.sub.2).sub.nOR.sup.a and
R.sup.9 can be OR.sup.b.
[0066] R.sup.1 can be (CH.sub.2).sub.nOR.sup.b and R.sup.3 can be
OR.sup.a.
[0067] R.sup.1 and R.sup.3 can be cis, or R.sup.1 and R.sup.3 can
be trans.
[0068] R.sup.3 can be OR.sup.a and R.sup.9 can be
(CH.sub.2).sub.nOR.sup.b- .
[0069] R.sup.1 can be OR.sup.a and R.sup.3 can be
(CH.sub.2).sub.nOR.sup.a- .
[0070] In other preferred embodiments the ribose is replaced with a
piperazine scaffold, and X is N(CO)R.sup.7 or NR.sup.7, Y is
NR.sup.8, and Z is CR.sup.11R.sup.12.
[0071] R.sup.1 can be (CH.sub.2).sub.nOR.sup.b and R.sup.3 can be
OR.sup.a.
[0072] R.sup.1 and R.sup.3 can be cis or R.sup.1 and R.sup.3 can be
trans.
[0073] n can be 1.
[0074] R.sup.1 can be (CH.sub.2).sub.nOR.sup.b and R.sup.3 can be
OR.sup.b; or R.sup.1 can be (CH.sub.2).sub.nOR.sup.a and R.sup.3
can be OR.sup.b.
[0075] A can be O or S, preferably S.
[0076] R.sup.7 can be (CH.sub.2).sub.5NHR.sup.d or
(CH.sub.2).sub.5NHR.sup- .d. R.sup.d can be chosen from the group
of a folic acid radical; a cholesterol radical; a carbohydrate
radical; a vitamin A radical; a vitamin E radical; a vitamin K
radical. Preferably, Rd is a cholesterol radical.
[0077] R.sup.8 can be CH.sub.3.
[0078] R.sup.1 can be OR.sup.a and R.sup.3 can be
(CH.sub.2).sub.nOR.sup.b- .
[0079] In other preferred embodiments the ribose is replaced with a
morpholino scaffold, and X is N(CO)R.sup.7 or NR.sup.7, Y is O, and
Z is CR.sup.11R.sup.12.
[0080] R.sup.1 can be (CH.sub.2).sub.nOR.sup.b and R.sup.3 can be
OR.sup.a.
[0081] R.sup.1 and R.sup.3 can be cis, or R.sup.1 and R.sup.3 can
be trans.
[0082] n can be 1.
[0083] R.sup.1 can be (CH.sub.2).sub.nOR.sup.b and R.sup.3 can be
OR.sup.b; of R.sup.1 can be (CH.sub.2).sub.nOR.sup.a and R.sup.3
can be OR.sup.b.
[0084] A can be O or S.
[0085] R.sup.7 can be (CH.sub.2).sub.5NHR.sup.d or
(CH.sub.2).sub.5NHR.sup- .d.
[0086] R.sup.8 can be CH.sub.3.
[0087] R.sup.1 can be OR.sup.a and R.sup.3 can be
(CH.sub.2).sub.nOR.sup.b- .
[0088] In other preferred embodiments the ribose is replaced with a
decalin scaffold, and X is CH.sub.2; Y is CR.sup.9R.sup.10; and Z
is CR.sup.11R.sup.12; and R.sup.5 and R.sup.11 together are C.sup.6
cycloalkyl.
[0089] R.sup.6 can be C(O)NHR.sup.7.
[0090] R.sup.12 can be hydrogen.
[0091] R.sup.6 and R.sup.2 can be trans.
[0092] R.sup.3 can be OR.sup.a and R.sup.9 can be
(CH.sub.2).sub.nOR.sup.b- .
[0093] R.sup.3 and R.sup.9 can be cis, or R.sup.3 and R.sup.9 can
be trans.
[0094] n can be 1 or 2.
[0095] R.sup.3 can be OR.sup.b and R.sup.9 can be
(CH.sub.2).sub.nOR.sup.b- ; or R.sup.3 can be OR.sup.b and R.sup.9
can be (CH.sub.2).sub.nOR.sup.a.
[0096] A can be Q or S.
[0097] R.sup.7 can be (CH.sub.2).sub.5NHR.sup.d or
(CH.sub.2).sub.5NHR.sup- .d.
[0098] In other preferred embodiments the ribose is replaced with a
decalin/indane scafold, e.g., X is CH.sub.2; Y is CR.sup.9R.sup.10;
and Z is CR.sup.11R.sup.12; and R.sup.5 and R.sup.11 together are
C.sup.5 cycloalkyl.
[0099] R.sup.6 can be CH.sub.3.
[0100] R.sup.12 can be hydrogen.
[0101] R.sup.11 and R.sup.12 can be trans.
[0102] R.sup.3 can be OR.sup.a and R.sup.9 can be
(CH.sub.2).sub.nOR.sup.b- .
[0103] R.sup.3 and R.sup.9 can be cis, or R.sup.3 and R.sup.9 can
be trans.
[0104] n can be 1 or 2.
[0105] R.sup.3 can be OR.sup.b and R.sup.9 can be
(CH.sub.2).sub.nOR.sup.a- ; or R.sup.3 can be OR.sup.b and R.sup.9
can be (CH.sub.2).sub.nOR.sup.a.
[0106] A can be O or S.
[0107] R.sup.14 can be N(CH3)R.sup.7. R.sup.7 can be
(CH.sub.2).sub.5NHR.sup.d or (CH.sub.2).sub.5NHR.sup.d.
[0108] In another aspect, this invention features an iRNA agent
comprising a first strand and a second strand, wherein at least one
one subunit having a formula (II) is incorporated into at least one
of said strands: 5
[0109] X is N(CO)R.sup.7 or NR.sup.7;
[0110] Each of R.sup.1 and R.sup.2 is, independently, OR.sup.a,
OR.sup.b, (CH.sub.2).sub.nOR.sup.a, or (CH.sub.2).sub.nOR.sup.b,
provided that one of R.sup.1 and R.sup.2 is OR.sup.a or OR.sup.b
and the other is (CH.sub.2).sub.nOR.sup.a or
(CH.sub.2).sub.nOR.sup.b (when the RRMS is terminal, one of R.sup.1
or R.sup.2 will include R.sup.a and one will include R.sup.b; when
the RRMSS is internal, both R.sup.1 and R.sup.2 will each include
an R.sup.b); further provided that preferably OR.sup.a may only be
present with (CH.sub.2).sub.nOR.sup.b and (CH.sub.2).sub.nOR.sup.a
may only be present with OR.sup.b;
[0111] R.sup.7 is C.sub.1-C.sub.20 alkyl substituted with
NR.sup.cR.sup.d;
[0112] R.sup.8 is C.sub.1-C.sub.6 alkyl;
[0113] R.sup.13 is hydroxy, C.sub.1-C.sub.4 alkoxy, or halo;
[0114] R.sup.14 is NR.sup.cR.sup.7;
[0115] R.sup.a is: 6
[0116] R.sup.b is 7
[0117] Each of A and C is, independently, O or S;
[0118] B is OH, O.sup.-, or 8
[0119] R.sup.c is H or C.sub.1-C.sub.6 alkyl;
[0120] R.sup.d is a carbohydrate radical (e.g., mannose, galactose,
or N-acetylgalactose); or a steroid radical, e.g., cholesterol,
which is optionally tethered to at least one carbohydrate radical
(e.g., mannose, galactose, or N-acetylgalactose); and
[0121] n is 1-4.
[0122] Embodiments can include one or more of the features
described above.
[0123] In a further aspect, this invention features an iRNA agent
having a first strand and a second strand, wherein at least one
subunit having a formula (I) or formula (II) is incorporated into
at least one of said strands.
[0124] In one aspect, this invention features an iRNA agent having
a first strand and a second strand, wherein at least two subunits
having a formula (I) and/or formula (II) are incorporated into at
least one of said strands.
[0125] In another aspect, this invention provides a method of
making an iRNA agent described herein having a first strand and a
second strand in which at least one subunit of formula (I) and/or
(II) is incorporated in the strands. The method includes contacting
the first strand with the second strand.
[0126] In a further aspect, this invention provides a method of
modulating expression of a target gene, the method includes
administering an iRNA agent described herein having a first strand
and a second strand in which at least one subunit of formula (I)
and/or (II) is incorporated in the strands. to a subject.
[0127] In one aspect, this invention features a pharmaceutical
composition having an iRNA agent described herein having a first
strand and a second strand in which at least one subunit of formula
(I) and/or (II) is incorporated in the strands and a
pharmaceutically acceptable carrier.
[0128] RRMSs described herein may be incorporated into any
double-stranded RNA-like molecule described herein, e.g., an iRNA
agent. An iRNA agent may include a duplex comprising a hybridized
sense and antisense strand, in which the antisense strand and/or
the sense strand may include one or more of the RRMSs described
herein. An RRMS can be introduced at one or more points in one or
both strands of a double-stranded iRNA agent. An RRMS can be placed
at or near (within 1, 2, or 3 positions) of the 3' or 5' end of the
sense strand or at near (within 2 or 3 positions of) the 3' end of
the antisense strand. In some embodiments it is preferred to not
have an RRMS at or near (within 1, 2, or 3 positions of) the 5' end
of the antisense strand. An RRMS can be internal, and will
preferably be positioned in regions not critical for antisense
binding to the target.
[0129] In an embodiment, an iRNA agent may have an RRMS at (or
within 1, 2, or 3 positions of) the 3' end of the antisense strand.
In an embodiment, an iRNA agent may have an RRMS at (or within 1,
2, or 3 positions of) the 3' end of the antisense strand and at (or
within 1, 2, or 3 positions of) the 3' end of the sense strand. In
an embodiment, an iRNA agent may have an RRMS at (or within 1, 2,
or 3 positions of) the 3' end of the antisense strand and an RRMS
at the 5' end of the sense strand, in which both ligands are
located at the same end of the iRNA agent.
[0130] In certain embodiments, two ligands are tethered,
preferably, one on each strand and are hydrophobic moieties. While
not wishing to be bound by theory, it is believed that pairing of
the hydrophobic ligands can stabilize the iRNA agent via
intermolecular van der Waals interactions.
[0131] In an embodiment, an iRNA agent may have an RRMS at (or
within 1, 2, or 3 positions of) the 3' end of the antisense strand
and an RRMS at the 5' end of the sense strand, in which both RRMSs
may share the same ligand (e.g., cholic acid) via connection of
their individual tethers to separate positions on the ligand. A
ligand shared between two proximal RRMSs is referred to herein as a
"hairpin ligand."
[0132] In other embodiments, an iRNA agent may have an RRMS at the
3' end of the sense strand and an RRMS at an internal position of
the sense strand. An iRNA agent may have an RRMS at an internal
position of the sense strand; or may have an RRMS at an internal
position of the antisense strand; or may have an RRMS at an
internal position of the sense strand and an RRMS at an internal
position of the antisense strand.
[0133] In preferred embodiments the iRNA agent includes a first and
second sequences, which are preferably two separate molecules as
opposed to two sequences located on the same strand, have
sufficient complementarity to each other to hybridize (and thereby
form a duplex region), e.g., under physiological conditions, e.g.,
under physiological conditions but not in contact with a helicase
or other unwinding enzyme.
[0134] It is preferred that the first and second sequences be
chosen such that the ds iRNA agent includes a single strand or
unpaired region at one or both ends of the molecule. Thus, a ds
iRNA agent contains first and second sequences, preferable paired
to contain an overhang, e.g., one or two 5' or 3' overhangs but
preferably a 3' overhang of 2-3 nucleotides. Most embodiments will
have a 3' overhang. Preferred sRNA agents will have single-stranded
overhangs, preferably 3' overhangs, of 1 or preferably 2 or 3
nucleotides in length at each end. The overhangs can be the result
of one strand being longer than the other, or the result of two
strands of the same length being staggered. 5' ends are preferably
phosphorylated.
[0135] Other modifications to sugars, bases, or backbones described
herein can be incorpoated into the iRNA agents.
[0136] The iRNA agents can take an architecture or structure
described herein. The iRNA agents can be palindromic, or double
targeting, as described herein.
[0137] The iRNA agents can have a sequence such that a
non-cannonical or other than cannonical Watson-Crick structure is
formed beween two monomers of the iRNA agent or between a strand of
the iRNA agent and another sequence, e.g., a target or off-target
sequence, as is described herein.
[0138] The iRNA agent can be selected to target any of a broad
spectrum of genes, including any of the genes described herein.
[0139] In a preferred embodiment the iRNA agent has an architecture
(architecture refers to one or more of overall length, length of a
duplex region, the presence, number, location, or length of
overhangs, single strand versus double strand form) described
herein. E.g., the iRNA agent can be less than 30 nucleotides in
length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21
nucleotides in length and there is a duplex region of about 19
pairs. In one embodiment, the iRNA is 21 nucleotides in length, and
the duplex region of the iRNA is 19 nucleotides. In another
embodiment, the iRNA is greater than 30 nucleotides in length.
[0140] In some embodiment the duplex region of the iRNA agent will
have, mismatches. Preferably it will have no more than 1, 2, 3, 4,
or 5 bases, which do not form canonical Watson-Crick pairs or which
do not hybridize. Overhangs are discussed in detail elsewhere
herein but are preferably about 2 nucleotides in length. The
overhangs can be complementary to the gene sequences being targeted
or can be other sequence. TT is a preferred overhang sequence. The
first and second iRNA agent sequences can also be joined, e.g., by
additional bases to form a hairpin, or by other non-base
linkers.
[0141] In addition ot the RRMS-containing bases the iRNA agents
described herein can include nuclease resistant monomers
(NRMs).
[0142] In another aspect, the invention features an iRNA agent to
which is conjugated to a carbohydrate radical (e.g., mannose,
galactose, or N-acetylgalactose); or a steroid radical, e.g.,
cholesterol, which is optionally tethered to at least one
carbohydrate radical (e.g., mannose, galactose, or
N-acetylgalactose); e.g., by conjugation to an RRMS of an iRNA
agent. In a preferred embodiment, the lipophilic moiety enhances
entry of the iRNA agent into a cell. In a preferred embodiment, the
cell is part of an organism, tissue, or cell line, e.g., a primary
cell line, immortalized cell line, or any type of cell line
disclosed herein. Thus, the conjugated iRNA agent an be used to
silence a target gene in an organism, e.g., a mammal, e.g., a
human, or to silence a target gene in a cell line or in cells which
are outside an organism.
[0143] The iRNA agent can have a first strand and a second strand,
wherein at least one subunit having formula (I) or formula (II) is
incorporated into at least one of the strands. The iRNA agent can
have one or more of any of the features described herein. For
example, when the subunit is of formula (I), Rd can be a
carbohydrate, a steroid, e.g., cholesterol, a steroid, e.g.,
cholesterol, tethered to a carbohydrate; X can be N(CO)R.sup.7 or
NR.sup.7, Y can be CR.sup.9R.sup.10, and Z can be absent, and
R.sup.1 can be (CH.sub.2).sub.nOR.sup.b and R.sup.3 can be
OR.sup.a; X can be N(CO)R.sup.7 or NR.sup.7, Y can be
CR.sup.9R.sup.10, and Z can be CR.sup.11R.sup.12, and R.sup.9 can
be (CH.sub.2).sub.nOR.sup.b and R.sup.10 can be OR.sup.a; X can be
N(CO)R.sup.7 or NR.sup.7, Y can be NR.sup.8, and Z can be
CR.sup.11R.sup.12, and R.sup.1 can be (CH.sub.2).sub.nOR.sup.b and
R.sup.3 can be OR.sup.a; X can be CH.sub.2; Y can be
CR.sup.9R.sup.10; and Z can be CR.sup.11R.sup.12, in which R.sup.6
can be C(O)NHR.sup.7; or X can be CH.sub.2; Y can be
CR.sup.9R.sup.10; and Z can be CR.sup.11R.sup.12, in which R.sup.11
or R.sup.12 can be C(O)NHR.sup.7 or R.sup.5 and R.sup.11 together
can be C.sub.5 or C.sub.6 cycloalkyl substituted with
N(CH3)R.sup.7.
[0144] In a preferred embodiment, the ligands described herein can
enhance entry of the iRNA agent into a hepatocyte cell.
[0145] In a preferred embodiment, the iRNA agent targets an
exogenous gene of a genetically modified cell. An exogenous gene
can be, for example, a viral or bacterial gene that derives from an
organism that has invaded or infected the cell, or the exogenous
gene can be any gene introduced into the cell by natural or
artificial means, such as by a genetic recombination event. An iRNA
agent can target a viral gene, for example, such as a hepatitis
viral gene (e.g., a gene of an HAV, HBV, or HCV). Alternatively, or
in addition, the iRNA agent can silence a reporter gene, such as
GFP or beta galatosidase and the like. These iRNA agents can be
used to silence exogenous genes in an adherent tumor cell line.
[0146] In another aspect, the invention provides, methods of
silencing a target gene by providing an iRNA agent to which a
carbohydrate, a steroid, or a steroid tethered to at least one
carbohydrate is conjugated. In a preferred embodiment the
conjugated iRNA agent an be used to silence a target gene in an
organism, e.g., a mammal, e.g., a human, or to silence a target
gene in a cell line or in cells which are outside an organism. In
the case of a whole organism, the method can be used to silence a
gene, e.g., a gene described herein, and treat a condition mediated
by the gene. In the case of use on a cell which is not part of an
organism, e.g., a primary cell line, secondary cell line, tumor
cell line, or transformed or immortalized cell line, the iRNA agent
to which a a carbohydrate, a steroid, or a steroid tethered to at
least one carbohydrate is conjugated can be used to silence a gene,
e.g., one described herein. Cells which are not part of a whole
organism can be used in an initial screen to determine if an iRNA
agent is effective in silencing a gene. A test in cells which are
not part of a whole organism can be followed by testing the iRNA
agent in a whole animal. In preferred embodiments, the iRNA agent
which is conjugated to a a carbohydrate, a steroid, or a steroid
tethered to at least one carbohydrate is conjugated is administered
to an organism, or contacted with a cell which is not part of an
organism, in the absence of (or in a reduced amount of) other
reagents that facilitate or enhance delivery, e.g., a compound
which enhances transit through the cell membrane. (A reduced amount
can be an amount of such reagent which is reduced in comparison to
what would be needed to get an equal amount of nonconjugated iRNA
agent into the target cell). E.g., the iRNA agent which is
conjugated to a lipophilic moiety is administered to an organism,
or contacted with a cell which is not part of an organism, in the
absence (or reduced amount) of: an additional lipophilic moiety; a
transfection agent, e.g., concentrations of an ion or other
substance which substantially alters cell permeability to an iRNA
agent; a transfecting agent such as Lipofectamine.TM. (Invitrogen,
Carlsbad, Calif.), Lipofectamine 2000.TM., TransIT-TKO.TM. (Mirus,
Madison, Wis.), FuGENE 6 (Roche, Indianapolis, Ind.),
polyethylenimine, X-tremeGENE Q2 (Roche, Indianapolis, Ind.),
DOTAP, DOSPER, Metafectene.TM. (Biontex, Munich, Germany), and the
like.
[0147] In a preferred embodiment the iRNA agent is suitable for
delivery to a cell in vivo, e.g., to a cell in an organism. In
another aspect, the iRNA agent is suitable for delivery to a cell
in vitro, e.g., to a cell in a cell line.
[0148] An iRNA agent to which a lipophilic moiety is attached can
target any gene described herein and can be delivered to any cell
type described herein, e.g., a cell type in an organism, tissue, or
cell line. Delivery of the iRNA agent can be in vivo, e.g., to a
cell in an organism, or in vitro, e.g., to a cell in a cell
line.
[0149] In another aspect, the invention provides compositions of
iRNA agents described herein, and in particular compositions of an
iRNA agent to which a lipophilic moiety is conjugated, e.g., a
lipophilic conjugated iRNA agent described herein. In a preferred
embodiment the composition is a pharmaceutically acceptable
composition.
[0150] In preferred embodiments, the composition, e.g.,
pharmaceutically acceptable composition, is free of, has a reduced
amount of, or is essentially free of other reagents that facilitate
or enhance delivery, e.g., compounds which enhance transit through
the cell membrane. (A reduced amount can be an amount of such
reagent which is reduced in comparison to what would be needed to
get an equal amount of nonconjugated iRNA agent into the target
cell). E.g., the composition is free of, has a reduced amount of,
or is essentially free of: an additional lipophilic moiety; a
transfection agent, e.g., concentrations of an ion or other
substance which substantially alters cell permeability to an iRNA
agent; a transfecting agent such as Lipofectamine.TM. (Invitrogen,
Carlsbad, Calif.), Lipofectamine 2000.TM., TransIT-TKO.TM. (Mirus,
Madison, Wis.), FuGENE 6 (Roche, Indianapolis, Ind.),
polyethylenimine, X-tremeGENE Q2 (Roche, Indianapolis, Ind.),
DOTAP, DOSPER, Metafectene.TM. (Biontex, Munich, Germany), and the
like.
[0151] In a preferred embodiment the composition is suitable for
delivery to a cell in vivo, e.g., to a cell in an organism. In
another aspect, the iRNA agent is suitable for delivery to a cell
in vitro, e.g., to a cell in a cell line.
[0152] The RRMS-containing iRNA agents can be used in any of the
methods described herein, e.g., to target any of the genes
described herein or to treat any of the disorders described herein.
They can be incorporated into any of the formulations, modes of
delivery, delivery modalities, kits or preparations, e.g.,
pharmaceutical preparations, described herein. E.g, a kit which
inlcudes one or more of the iRNA aents described herein, a sterile
container in which the iRNA agent is discolsed, and instructions
for use.
[0153] The methods and compositions of the invention, e.g., the
RRSM-containing iRNA agents described herein, can be used with any
of the iRNA agents described herein. In addition, the methods and
compositions of the invention can be used for the treatment of any
disease or disorder described herein, and for the treatment of any
subject, e.g., any animal, any mammal, such as any human.
[0154] The methods and compositions of the invention, e.g., the the
RRMS-containing iRNA agents described herein, can be used with any
dosage and/or formulation described herein, as well as with any
route of administration described herein.
[0155] The non-ribose scaffolds, as well as monomers and dimers of
the RRMSs described herein are within the invention In one aspect,
an iRNA agent includes a carbohydrate modification, e.g., galactose
and/or analogues thereof. These agents target, in particular, the
parenchymal cells of the liver (see Table 1). In one embodiment,
the iRNA agent includes more than one galactose moiety, preferably
two or three. In another embodiment, the iRNA agent includes at
least one (e.g., two or three or more) lactose molecules (lactose
is a glucose coupled to a galactose). In another embodiment, the
iRNA agent includes at least one (e.g., two or three or more)
N-Acetyl-Galactosamine, N-Ac-Glucosamine, or mannose (e.g.,
mannose-6-phosphate). In one embodiment, iRNA agents include
mannose conjugates, and the iRNA agents target macrophages.
[0156] In one aspect, an iRNA agent includes a carbohydrate
modification, e.g., galactose and/or analogues thereof. These
carbohydrate-conjugated iRNA agents target, in particular, the
parenchymal cells of the liver (see Table 1). In one embodiment,
the iRNA agent includes more than one galactose moiety, preferably
two or three. In another embodiment, the iRNA agent includes at
least one (e.g., two or three or more) lactose molecules (lactose
is a glucose coupled to a galactose). In another embodiment, the
iRNA agent includes at least one (e.g., two or three or more)
N-Acetyl-Galactosamine, N-Ac-Glucosamine, or mannose (e.g.,
mannose-6-phosphate). In one embodiment, iRNA agents include
mannose conjugates, and the iRNA agents target macrophages, e.g.,
macrophages in the liver.
[0157] In one aspect, the invention features an iRNA agent
including a carbohydrate modification, and the presence of the
carbohydrate modification can increase delivery of the iRNA agent
to the liver. Thus an iRNA agent including a carbohydrate
modification can be useful for targeting a gene for which
expression is undesired in the liver. For example, an iRNA agent
including a carbohydrate modification can target a nucleic acid
expresses by a hepatitis virus (e.g., hepatitis C, hepatitis B,
hepatitis A, hepatitis D, hepatitis E, hepatitis F, hepatitis G, or
hepatitis H).
[0158] In a preferred embodiment, the carbohydrate-conjugated iRNA
agent targets a gene of the hepatitis C virus. In another
embodiment, the iRNA agent that targets a gene of the hepatitis C
virus can be administered to a human having or at risk for
developing hepatitis, e.g., acute or chronic hepatitis, or
inflammation of the liver. A human who is a candidate for treatment
with a carbohydrate-conjugated iRNA agent, e.g., an iRNA agent that
targets a gene of HCV, can present symptoms indicative of HCV
infection, such as jaundice, abdominal pain, liver enlargement and
fatigue.
[0159] In one embodiment, a carbohydrate-conjugated iRNA agent
targets the 5' core region of HCV. This region lies just downstream
of the ribosomal toe-print straddling the initiator methionine. In
another embodiment, an iRNA agent targets any one of the
nonstructural proteins of HCV, such as NS3, NS4A, NS4B, NS5A, or
NS5B. In another embodiment, an iRNA agent targets the E1, E2, or C
gene of HCV.
[0160] In another embodiment, the carbohydrate-conjugated iRNA
agent targets a hepatitis B virus (HBV), and the iRNA agent has a
sequence that is substantially similar to a sequence of a gene of
HBV, e.g., the protein X (HBx) gene of HBV.
[0161] Carbohydrate-conjugated iRNA agents can also be used to
treat other liver disorders, including disorders characterized by
unwanted cell proliferation, hematological disorders, metabolic
disorders, and disorders characterized by inflammation. A
proliferation disorder of the liver can be, for example, a benign
or malignant disorder, e.g., a cancer, e.g, a hepatocellular
carcinoma (HCC), hepatic metastasis, or hepatoblastoma. A hepatic
hematology or inflammation disorder can be a disorder involving
clotting factors, a complement-mediated inflammation or a fibrosis,
for example. Metabolic diseases of the liver include dyslipidemias
and irregularities in glucose regulation. In one embodiment, a
liver disorder is treated by administering one or more iRNA agents
that have a sequence that is substantially identical to a sequence
in a gene involved in the liver disorder.
[0162] In one embodiment, a carbohydrate-conjugated iRNA agent
targets a nucleic acid expressed in the liver, such as an ApoB RNA,
c-jun RNA, beta-catenin RNA, or glucose-6-phosphatase mRNA.
[0163] An iRNA that targets glucose-6-phosphatase can be
administered to a subject to inhibit hepatic glucose production,
e.g., for the treatment of glucose-metabolism-related disorders,
such as diabetes, e.g., type-2-diabetes mellitus. The iRNA agent
can be administered to an individual at risk for the disorder to
delay onset of the disorder or a symptom of the disorder.
[0164] In other embodiments, a carbohydrate-conjugated iRNA agent
has sequence-similarity to the following genes, and the iRNA agent
is useful for inhibiting hepatic glucose production. These other
genes include "forkhead homologue in rhabdomyosarcoma" (FKHR);
glucagon; glucagon receptor; glycogen phosphorylase; PPAR-Gamma
Coactivator (PGC-1); Fructose-1,6-bisphosphatase;
glucose-6-phosphate locator; glucokinase inhibitory regulatory
protein; and phosphoenolpyruvate carboxykinase (PEPCK).
[0165] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF DRAWINGS
[0166] FIG. 1 a general synthetic scheme for incorporation of RRMS
monomers into an oligonucleotide.
[0167] FIG. 2A is a list of substituents that may be present on
silicon in OFG.sup.1.
[0168] FIG. 2B is a list of substituents that may be present on the
C2'-orthoester group.
[0169] FIG. 3 is list of representative RRMS cyclic carriers. Panel
1 shows pyrroline-based RRMSs; panel 2 shows 3-hydroxyproline-based
RRMSs; panel 3 shows piperidine-based RRMSs; panel 4 shows
morpholine and piperazine-based RRMSs; and panel 5 shows
decalin-based RRMSs. R.sup.1 is succinate or phosphoramidate and
R.sup.2 is H or a conjugate ligand.
[0170] FIG. 4 is a general reaction scheme for 3' conjugation of
peptide into iRNA.
[0171] FIG. 5 is a general reaction scheme for 5' conjugation of
peptide into iRNA.
[0172] FIG. 6 is a general reaction scheme for the synthesis of
aza-peptides.
[0173] FIG. 7 is a general reaction scheme for the synthesis of
N-methyl amino acids and peptides.
[0174] FIG. 8 is a general reaction scheme for the synthesis of
.beta.-methyl amino acids and Ant and Tat peptides.
[0175] FIG. 9 is a general reaction scheme for the synthesis of Ant
and Tat oligocarbamates.
[0176] FIG. 10 is a a general reaction scheme for the synthesis of
Ant and Tat oligoureas.
[0177] FIG. 11 is a schematic representation of peptide
carriers.
[0178] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0179] Double-stranded (dsRNA) directs the sequence-specific
silencing of mRNA through a process known as RNA interference
(RNAi). The process occurs in a wide variety of organisms,
including mammals and other vertebrates.
[0180] It has been demonstrated that 21-23 nt fragments of dsRNA
are sequence-specific mediators of RNA silencing, e.g., by causing
RNA degradation. While not wishing to be bound by theory, it may be
that a molecular signal, which may be merely the specific length of
the fragments, present in these 21-23 nt fragments recruits
cellular factors that mediate RNAi. Described herein are methods
for preparing and administering these 21-23 nt fragments, and other
iRNAs agents, and their use for specifically inactivating gene
function. The use of iRNAs agents (or recombinantly produced or
chemically synthesized oligonucleotides of the same or similar
nature) enables the targeting of specific mRNAs for silencing in
mammalian cells. In addition, longer dsRNA agent fragments can also
be used, e.g., as described below.
[0181] Although, in mammalian cells, long dsRNAs can induce the
interferon response, which is frequently deleterious, sRNAs do not
trigger the interferon response, at least not to an extent that is
deleterious to the cell and host. In particular, the length of the
iRNA agent strands in an sRNA agent can be less than 31, 30, 28,
25, or 23 nt, e.g., sufficiently short to avoid inducing a
deleterious interferon response. Thus, the administration of a
composition of sRNA agent (e.g., formulated as described herein) to
a mammalian cell can be used to silence expression of a target gene
while circumventing the interferon response. Further, use of a
discrete species of iRNA agent can be used to selectively target
one allele of a target gene, e.g., in a subject heterozygous for
the allele.
[0182] In a typical embodiment, the subject is a mammal such as a
cow, horse, mouse, rat, dog, pig, goat, or a primate. The subject
can be a dairy mammal (e.g., a cow, or goat) or other farmed animal
(e.g., a chicken, turkey, sheep, pig, fish, shrimp). In a much
preferred embodiment, the subject is a human, e.g., a normal
individual or an individual that has, is diagnosed with, or is
predicted to have a disease or disorder.
[0183] Targeting to the Liver
[0184] An iRNA agent containing a carbohydrate modification can be
targeted to a particular cell type in the liver. Exemplary
carbohydrate moieties and their associated receptors are presented
in Table 1.
1TABLE 1 Targeting agents (Ligands) and their associated receptors
Liver Cells Ligand Receptor 1) Parenchymal Galactose ASGP-R Cell
(PC) (Asiologlycoprotein (Hepatocytes) receptor) Gal NAc ASPG-R
(n-acetyl- Gal NAc Receptor galactosamine) Lactose Asialofetuin
ASPG-r 2) Sinusoidal Hyaluronan Hyaluronan receptor Endothelial
Procollagen Procollagen receptor Cell (SEC) Negatively Scavenger
receptors charged molecules Mannose Mannose receptors N-acetyl
Scavenger receptors Glucosamine Immunoglobulins Fc Receptor LPS
CD14 Receptor Insulin Receptor mediated transcytosis Transferrin
Receptor mediated transcytosis Albumins Non-specific Sugar-Albumin
conjugates Mannose-6- Mannose-6-phosphate receptor phosphate 3)
Kupffer Mannose Mannose receptors Cell (KC) Fucose Fucose receptors
Albumins Non-specific Mannose-albumin conjugates
[0185] Liver Diseases
[0186] Exemplary diseases and disorders that can be treated by the
carbohydrate-conjugated iRNA agents described herein.
[0187] Disorders involving the liver include, but are not limited
to, hepatic injury; jaundice and cholestasis, such as bilirubin and
bile formation; hepatic failure and cirrhosis, such as cirrhosis,
portal hypertension, including ascites, portosystemic shunts, and
splenomegaly; infectious disorders, such as viral hepatitis,
including hepatitis A-E infection and infection by other hepatitis
viruses, clinicopathologic syndromes, such as the carrier state,
asymptomatic infection, acute viral hepatitis, chronic viral
hepatitis, and fulminant hepatitis; autoimmune hepatitis; drug- and
toxin-induced liver disease, such as alcoholic liver disease;
inborn errors of metabolism and pediatric liver disease, such as
hemochromatosis, Wilson disease, a1-antitrypsin deficiency, and
neonatal hepatitis; intrahepatic biliary tract disease, such as
secondary biliary cirrhosis, primary biliary cirrhosis, primary
sclerosing cholangitis, and anomalies of the biliary tree;
circulatory disorders, such as impaired blood flow into the liver,
including hepatic artery compromise and portal vein obstruction and
thrombosis, impaired blood flow through the liver, including
passive congestion and centrilobular necrosis and peliosis hepatis,
hepatic vein outflow obstruction, including hepatic vein thrombosis
(Budd-Chiari syndrome) and veno-occlusive disease; hepatic disease
associated with pregnancy, such as preeclampsia and eclampsia,
acute fatty liver of pregnancy, and intrehepatic cholestasis of
pregnancy; hepatic complications of organ or bone marrow
transplantation, such as drug toxicity after bone marrow
transplantation, graft-versus-host disease and liver rejection, and
nonimmunologic damage to liver allografts; tumors and tumorous
conditions, such as nodular hyperplasias, adenomas, and malignant
tumors, including primary carcinoma of the liver and metastatic
tumors.
[0188] An iRNA agent can also be administered to inhibit Factor V
expression in the liver. Two to five percent of the United States
population is heterozygous for an allele of the Factor V gene that
encodes a single amino acid change at position 1961. These
heterozygous individuals have a 3-8 fold increased risk of venous
thrombosis, a risk that is associated with increased factor V
activity. The increased activity leads to increased thrombin
generation from the prothrombinase complex. An iRNA agent directed
against Factor V can treat or prevent venous thrombosis or treat a
human who has Factor V Leiden. The iRNA agent that targets Factor V
can be also be used as a prophylaxis in patients with Factor V
Leiden who undergo high-risk surgical procedures, and this
prophylaxis can be an adjunct to the therapeutic use of low
molecular weight (LMW) heparin prophylaxis.
[0189] An iRNA agent that targets Factor V can also be administered
to patients with Factor V Leiden to treat deep vein thrombosis
(DVT) or pulmonary embolism (PE), and this treatment can be an
adjunct to therapeutic uses of heparin or coumadin. Any other
disorder caused by elevated or otherwise unwanted levels of Factor
V protein can be treated by administering an iRNA agent against
Factor V.
[0190] iRNA agents of the invention can be targeted to any gene
whose overexpression is associated with the liver diseases.
[0191] Ligand-Conjugated Monomer Subunits and Monomers for
Oligonucleotide Synthesis
[0192] Definitions
[0193] The term "halo" refers to any radical of fluorine, chlorine,
bromine or iodine.
[0194] The term "alkyl" refers to a hydrocarbon chain that may be a
straight chain or branched chain, containing the indicated number
of carbon atoms. For example, C.sub.1-C.sub.12 alkyl indicates that
the group may have from 1 to 12 (inclusive) carbon atoms in it. The
term "haloalkyl" refers to an alkyl in which one or more hydrogen
atoms are replaced by halo, and includes alkyl moieties in which
all hydrogens have been replaced by halo (e.g., perfluoroalkyl).
Alkyl and haloalkyl groups may be optionally inserted with O, N, or
S. The terms "aralkyl" refers to an alkyl moiety in which an alkyl
hydrogen atom is replaced by an aryl group. Aralkyl includes groups
in which more than one hydrogen atom has been replaced by an aryl
group. Examples of "aralkyl" include benzyl, 9-fluorenyl,
benzhydryl, and trityl groups.
[0195] The term "alkenyl" refers to a straight or branched
hydrocarbon chain containing 2-8 carbon atoms and characterized in
having one or more double bonds. Examples of a 15 typical alkenyl
include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl
and 3-octenyl groups. The term "alkynyl" refers to a straight or
branched hydrocarbon chain containing 2-8 carbon atoms and
characterized in having one or more triple bonds. Some examples of
a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and
propargyl. The sp.sup.2 and sp.sup.3 carbons may optionally serve
as the point of attachment of the alkenyl and alkynyl groups,
respectively.
[0196] The terms "alkylamino" and "dialkylamino" refer to
--NH(alkyl) and --N(alkyl).sub.2 radicals respectively. The term
"aralkylamino" refers to a --NH(aralkyl) radical. The term "alkoxy"
refers to an --O-alkyl radical, and the terms "cycloalkoxy" and
"aralkoxy" refer to an --O-cycloalkyl and O-aralkyl radicals
respectively. The term "siloxy" refers to a R.sub.3SiO-- radical.
The term "mercapto" refers to an SH radical. The term "thioalkoxy"
refers to an --S-alkyl radical.
[0197] The term "alkylene" refers to a divalent alkyl (i.e.,
--R--), e.g., --CH.sub.2--, --CH.sub.2CH.sub.2--, and
--CH.sub.2CH.sub.2CH.sub.2--. The term "alkylenedioxo" refers to a
divalent species of the structure --O--R--O--, in which R
represents an alkylene.
[0198] The term "aryl" refers to an aromatic monocyclic, bicyclic,
or tricyclic hydrocarbon ring system, wherein any ring atom can be
substituted. Examples of aryl moieties include, but are not limited
to, phenyl, naphthyl, anthracenyl, and pyrenyl.
[0199] The term "cycloalkyl" as employed herein includes saturated
cyclic, bicyclic, tricyclic, or polycyclic hydrocarbon groups
having 3 to 12 carbons, wherein any ring atom can be substituted.
The cycloalkyl groups herein described may also contain fused
rings. Fused rings are rings that share a common carbon-carbon bond
or a common carbon atom (e.g., spiro-fused rings). Examples of
cycloalkyl moieties include, but are not limited to, cyclohexyl,
adamantyl, and norbornyl.
[0200] The term "heterocyclyl" refers to a nonaromatic 3-10
membered monocyclic, 8-12 membered bicyclic, or 11-14 membered
tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6
heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said
heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3,
1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or
tricyclic, respectively), wherein any ring atom can be substituted.
The heterocyclyl groups herein described may also contain fused
rings. Fused rings are rings that share a common carbon-carbon bond
or a common carbon atom (e.g., spiro-fused rings). Examples of
heterocyclyl include, but are not limited to tetrahydrofuranyl,
tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and
pyrrolidinyl.
[0201] The term "cycloalkenyl" as employed herein includes
partially unsaturated, nonaromatic, cyclic, bicyclic, tricyclic, or
polycyclic hydrocarbon groups having 5 to 12 carbons, preferably 5
to 8 carbons, wherein any ring atom can be substituted. The
cycloalkenyl groups herein described may also contain fused rings.
Fused rings are rings that share a common carbon-carbon bond or a
common carbon atom (e.g., spiro-fused rings). Examples of
cycloalkenyl moieties include, but are not limited to cyclohexenyl,
cyclohexadienyl, or norbornenyl.
[0202] The term "heterocycloalkenyl" refers to a partially
saturated, nonaromatic 5-10 membered monocyclic, 8-12 membered
bicyclic, or 11-14 membered tricyclic ring system having 1-3
heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9
heteroatoms if tricyclic, said heteroatoms selected from O, N, or S
(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S
if monocyclic, bicyclic, or tricyclic, respectively), wherein any
ring atom can be substituted. The heterocycloalkenyl groups herein
described may also contain fused rings. Fused rings are rings that
share a common carbon-carbon bond or a common carbon atom (e.g.,
spiro-fused rings). Examples of heterocycloalkenyl include but are
not limited to tetrahydropyridyl and dihydropyran.
[0203] The term "heteroaryl" refers to an aromatic 5-8 membered
monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic
ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms
if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms
selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9
heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic,
respectively), wherein any ring atom can be substituted. The
heteroaryl groups herein described may also contain fused rings
that share a common carbon-carbon bond.
[0204] The term "oxo" refers to an oxygen atom, which forms a
carbonyl when attached to carbon, an N-oxide when attached to
nitrogen, and a sulfoxide or sulfone when attached to sulfur.
[0205] The term "acyl" refers to an alkylcarbonyl,
cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or
heteroarylcarbonyl substituent, any of which may be further
substituted by substituents.
[0206] The term "substituents" refers to a group "substituted" on
an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl,
heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any
atom of that group. Suitable substituents include, without
limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano,
nitro, amino, SO.sub.3H, sulfate, phosphate, perfluoroalkyl,
perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo,
thioxo, imino (alkyl, aryl, aralkyl), S(O).sub.nalkyl (where n is
0-2), S(O).sub.n aryl (where n is 0-2), S(O).sub.n heteroaryl
(where n is 0-2), S(O).sub.n heterocyclyl (where n is 0-2), amine
(mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and
combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide
(mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations
thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl,
and combinations thereof), unsubstituted aryl, unsubstituted
heteroaryl, unsubstituted heterocyclyl, and unsubstituted
cycloalkyl. In one aspect, the substituents on a group are
independently any one single, or any subset of the aforementioned
substituents.
[0207] The terms "adeninyl, cytosinyl, guaninyl, thyminyl, and
uracilyl" and the like refer to radicals of adenine, cytosine,
guanine, thymine, and uracil.
[0208] A "protected" moiety refers to a reactive functional group,
e.g., a hydroxyl group or an amino group, or a class of molecules,
e.g., sugars, having one or more functional groups, in which the
reactivity of the functional group is temporarily blocked by the
presence of an attached protecting group. Protecting groups useful
for the monomers and methods described herein can be found, e.g.,
in Greene, T. W., Protective Groups in Organic Synthesis (John
Wiley and Sons: New York), 1981, which is hereby incorporated by
reference.
[0209] General
[0210] An RNA agent, e.g., an iRNA agent, containing a preferred,
but nonlimiting ligand-conjugated monomer subunit is presented as
formula (II) below and in the scheme in FIG. 1. The carrier (also
referred to in some embodiments as a "linker") can be a cyclic or
acyclic moiety and includes two "backbone attachment points" (e.g.,
hydroxyl groups) and a ligand. The ligand can be directly attached
(e.g., conjugated) to the carrier or indirectly attached (e.g.,
conjugated) to the carrier by an intervening tether (e.g., an
acyclic chain of one or more atoms; or a nucleobase, e.g., a
naturally occurring nucleobase optionally having one or more
chemical modifications, e.g., an unusual base; or a universal
base). The carrier therefore also includes a "ligand or tethering
attachment point" for the ligand and tether/tethered ligand,
respectively.
[0211] The ligand-conjugated monomer subunit may be the 5' or 3'
terminal subunit of the RNA molecule, i.e., one of the two "W"
groups may be a hydroxyl group, and the other "W" group may be a
chain of two or more unmodified or modified ribonucleotides.
Alternatively, the ligand-conjugated monomer subunit may occupy an
internal position, and both "W" groups may be one or more
unmodified or modified ribonucleotides. More than one
ligand-conjugated monomer subunit may be present in a RNA molecule,
e.g., an iRNA agent. Preferred positions for inclusion of a
tethered ligand-conjugated monomer subunits, e.g., one in which a
lipophilic moiety, e.g., cholesterol, is tethered to the carrier
are at the 3' terminus, the 5' terminus, or an internal position of
the sense strand. 9
[0212] The modified RNA molecule of formula (II) can be obtained
using oligonucleotide synthetic methods known in the art. In a
preferred embodiment, the modified RNA molecule of formula (II) can
be prepared by incorporating one or more of the corresponding
monomer compounds (see, e.g., A, B, and C below and in the scheme
in FIG. 1) into a growing sense or antisense strand, utilizing,
e.g., phosphoramidite or H-phosphonate coupling strategies.
[0213] The monomers, e.g., a ligand-conjugated monomer, generally
include two differently functionalized hydroxyl groups (OFG.sup.1
and OFG.sup.2), which are linked to the carrier molecule (see A
below and in FIG. 1), and a ligand/tethering attachment point. As
used herein, the term "functionalized hydroxyl group" means that
the hydroxyl proton has been replaced by another substituent. As
shown in representative structures B and C below and in FIG. 1, one
hydroxyl group (OFG.sup.1) on the carrier is functionalized with a
protecting group (PG). The other hydroxyl group (OFG.sup.2) can be
functionalized with either (1) a liquid or solid phase synthesis
support reagent (solid circle) directly or indirectly through a
linker, L, as in B, or (2) a phosphorus-containing moiety, e.g., a
phosphoramidite as in C. The tethering attachment point may be
connected to a hydrogen atom, a suitable protecting group, a
tether, or a tethered ligand at the time that the monomer is
incorporated into the growing sense or antisense strand (see
variable "R" in A below). Thus, the tethered ligand can be, but
need not be attached to the monomer at the time that the monomer is
incorporated into the growing strand. In certain embodiments, the
tether, the ligand or the tethered ligand may be linked to a
"precursor" ligand-conjugated monomer subunit after a "precursor"
ligand-conjugated monomer subunit has been incorporated into the
strand. The wavy line used below (and elsewhere herein) refers to a
connection, and can represent a direct bond between the moiety and
the attachment point or a tethering molecule which is interposed
between the moiety and the attachment point. Directly tethered
means the moiety is bound directly to the attachment point.
Indirectly tethered means that there is a tether molecule
interposed between the attachment point and the moiety. 10
[0214] The (OFG.sup.1) protecting group may be selected as desired,
e.g., from T. W. Greene and P. G. M. Wuts, Protective Groups in
Organic Synthesis, 2d. Ed., John Wiley and Sons (1991). The
protecting group is preferably stable under amidite synthesis
conditions, storage conditions, and oligonucleotide synthesis
conditions. Hydroxyl groups, --OH, are nucleophilic groups (i.e.,
Lewis bases), which react through the oxygen with electrophiles
(i.e., Lewis acids). Hydroxyl groups in which the hydrogen has been
replaced with a protecting group, e.g., a triarylmethyl group or a
trialkylsilyl group, are essentially unreactive as nucleophiles in
displacement reactions. Thus, the protected hydroxyl group is
useful in preventing e.g., homocoupling of compounds exemplified by
structure C during oligonucleotide synthesis. In some embodiments,
a preferred protecting group is the dimethoxytrityl group. In other
embodiments, a preferred protecting group is a silicon-based
protecting group having the formula below: 11
[0215] X5', X5", and X5'" can be selected from substituted or
unsubstituted alkyl, cycloalkyl, aryl, araklyl, heteroaryl, alkoxy,
cycloalkoxy, aralkoxy, aryloxy, heteroaryloxy, or siloxy (i.e.,
R.sub.3SiO--, the three "R" groups can be any combination of the
above listed groups). X.sup.5', X.sup.5", and X.sup.5'" may all be
the same or different; also contemplated is a combination in which
two of X.sup.5', X.sup.5", and X.sup.5'" are identical and the
third is different. In certain embodiments X.sup.5', X.sup.5", and
X.sup.5'" include at least one alkoxy or siloxy groups and may be
any one of the groups listed in FIG. 2A, a preferred combination
includes X.sup.5', X.sup.5"=trimethylsiloxy and
X.sup.5'"=1,3-(triphenylmethoxy)-2-propoxy or cyclododecyloxy.
[0216] Other preferred combinations of X.sup.5', X.sup.5", and
X.sup.5'" include those that result in OFG.sup.1 groups that meet
the deprotection and stability criteria delineated below. The group
is preferably stable under amidite synthesis conditions, storage
conditions, and oligonucleotide synthesis conditions. Rapid
removal, i.e., less than one minute, of the silyl group from e.g.,
a support-bound oligonucleotide is desirable because it can reduce
synthesis times and thereby reduce exposure time of the growing
oligonucleotide chain to the reagents. Oligonucleotide synthesis
can be improved if the silyl protecting group is visible during
deprotection, e.g., from the addition of a chromophore silyl
substituent.
[0217] Selection of silyl protecting groups can be complicated by
the competing demands of the essential characteristics of stability
and facile removal, and the need to balance these competitive
goals. Most substituents that increase stability can also increase
the reaction time required for removal of the silyl group,
potentially increasing the level of difficulty in removal of the
group.
[0218] The addition of alkoxy and siloxy substituents to OFG.sup.1
silicon-containing protecting groups increases the susceptibility
of the protecting groups to fluoride cleavage of the silylether
bonds. Increasing the steric bulk of the substituents preserves
stability while not decreasing fluoride lability to an equal
extent. An appropriate balance of substituents on the silyl group
makes a silyl ether a viable nucleoside protecting group.
[0219] Candidate OFG.sup.1 silicon-containing protecting groups may
be tested by exposing a tetrahydrofuran solution of a preferred
carrier bearing the candidate OFG.sup.1 group to five molar
equivalents of tetrahydrofuran at room temperature. The reaction
time may be determined by monitoring the disappearance of the
starting material by thin layer chromatography.
[0220] When the OFG.sup.2 in B includes a linker, e.g., a
relatively long organic linker, connected to a soluble or insoluble
support reagent, solution or solid phase synthesis techniques can
be employed to build up a chain of natural and/or modified
ribonucleotides once OFG.sup.1 is deprotected and free to react as
a nucleophile with another nucleoside or monomer containing an
electrophilic group (e.g., an amidite group). Alternatively, a
natural or modified ribonucleotide or oligoribonucleotide chain can
be coupled to monomer C via an amidite group or H-phosphonate group
at OFG.sup.2. Subsequent to this operation, OFG.sup.1 can be
deblocked, and the restored nucleophilic hydroxyl group can react
with another nucleoside or monomer containing an electrophilic
group. R' can be substituted or unsubstituted alkyl or alkenyl. In
preferred embodiments, R' is methyl, allyl or 2-cyanoethyl. R" may
a C.sub.1-C.sub.10 alkyl group, preferably it is a branched group
containing three or more carbons, e.g., isopropyl.
[0221] OFG.sup.2 in B can be hydroxyl functionalized with a linker,
which in turn contains a liquid or solid phase synthesis support
reagent at the other linker terminus. The support reagent can be
any support medium that can support the monomers described herein.
The monomer can be attached to an insoluble support via a linker,
L, which allows the monomer (and the growing chain) to be
solubilized in the solvent in which the support is placed. The
solubilized, yet immobilized, monomer can react with reagents in
the surrounding solvent; unreacted reagents and soluble by-products
can be readily washed away from the solid support to which the
monomer or monomer-derived products is attached. Alternatively, the
monomer can be attached to a soluble support moiety, e.g.,
polyethylene glycol (PEG) and liquid phase synthesis techniques can
be used to build up the chain. Linker and support medium selection
is within skill of the art. Generally the linker may be
--C(O)(CH.sub.2).sub.qC(O)--, or --C(O)(CH.sub.2).sub.qS--, in
which q can be 0, 1, 2, 3, or 4; preferably, it is oxalyl, succinyl
or thioglycolyl. Standard control pore glass solid phase synthesis
supports can not be used in conjunction with fluoride labile 5'
silyl protecting groups because the glass is degraded by fluoride
with a significant reduction in the amount of full-length product.
Fluoride-stable polystyrene based supports or PEG are
preferred.
[0222] The ligand/tethering attachment point can be any divalent,
trivalent, tetravalent, pentavalent or hexavalent atom. In some
embodiments, ligand/tethering attachment point can be a carbon,
oxygen, nitrogen or sulfur atom. For example, a ligand/tethering
attachment point precursor functional group can have a nucleophilic
heteroatom, e.g., --SH, --NH.sub.2, secondary amino, ONH.sub.2, or
NH.sub.2NH.sub.2. As another example, the ligand/tethering
attachment point precursor functional group can be an olefin, e.g.,
--CH.dbd.CH.sub.2, and the precursor functional group can be
attached to a ligand, a tether, or tethered ligand using, e.g.,
transition metal catalyzed carbon-carbon (for example olefin
metathesis) processes or cycloadditions (e.g., Diels-Alder). As a
further example, the ligand/tethering attachment point precursor
functional group can be an electrophilic moiety, e.g., an aldehyde.
When the carrier is a cyclic carrier, the ligand/tethering
attachment point can be an endocyclic atom (i.e., a constituent
atom in the cyclic moiety, e.g., a nitrogen atom) or an exocyclic
atom (i.e., an atom or group of atoms attached to a constituent
atom in the cyclic moiety).
[0223] The carrier can be any organic molecule containing
attachment points for OFG.sup.1, OFG.sup.2, and the ligand. In
certain embodiments, carrier is a cyclic molecule and may contain
heteroatoms (e.g., O, N or S). E.g., carrier molecules may include
aryl (e.g., benzene, biphenyl, etc.), cycloalkyl (e.g.,
cyclohexane, cis or trans decalin, etc.), cycloalkenyl (e.g.,
cyclohexenyl), or heterocyclyl (tetrahydropyran, piperazine,
pyrrolidine, etc.). In other embodiments, the carrier can be an
acyclic moiety, e.g., based on serinol. Any of the above cyclic
systems may include substituents in addition to OFG.sup.1,
OFG.sup.2, and the ligand.
[0224] Sugar-Based Monomers
[0225] In some embodiments, the carrier molecule is an oxygen
containing heterocycle. Preferably the carrier is a ribose sugar as
shown in structure LCM-I. In this embodiment, the monomer, e.g., a
ligand-conjugated monomer is a nucleoside. 12
[0226] "B" represents a nucleobase, e.g., a naturally occurring
nucleobase optionally having one or more chemical modifications,
e.g., and unusual base; or a universal base.
[0227] As used herein, an "unusual" nucleobase can include any one
of the following:
[0228] 2-methyladeninyl,
[0229] N6-methyladeninyl,
[0230] 2-methylthio-N-6-methyladeninyl,
[0231] N6-isopentenyladeninyl,
[0232] 2-methylthio-N-6-isopentenyladeninyl,
[0233] N6-(cis-hydroxyisopentenyl)adeninyl,
[0234] 2-methylthio-N-6-(cis-hydroxyisopentenyl) adeninyl,
[0235] N6-glycinylcarbamoyladeninyl,
[0236] N6-threonylcarbamoyladeninyl,
[0237] 2-methylthio-N-6-threonylcarbamoyladeninyl,
[0238] N6-methyl-N-6-threonylcarbamoyladeninyl,
[0239] N6-hydroxynorvalylcarbamoyladeninyl,
[0240] 2-methylthio-N-6-hydroxynorvalyl carbamoyladeninyl,
[0241] N6,N6-dimethyladeninyl,
[0242] 3-methylcytosinyl,
[0243] 5-methylcytosinyl,
[0244] 2-thiocytosinyl,
[0245] 5-formylcytosinyl, 13
[0246] N4-methylcytosinyl,
[0247] 5-hydroxymethylcytosinyl,
[0248] 1-methylguaninyl,
[0249] N2-methylguaninyl,
[0250] 7-methylguaninyl,
[0251] N2,N2-dimethylguaninyl, 141516
[0252] N2,7-dimethylguaninyl,
[0253] N2,N2,7-trimethylguaninyl,
[0254] 1-methylguaninyl,
[0255] 7-cyano-7-deazaguaninyl,
[0256] 7-aminomethyl-7-deazaguaninyl,
[0257] pseudouracilyl,
[0258] dihydrouracilyl,
[0259] 5-methyluracilyl,
[0260] 1-methylpseudouracilyl,
[0261] 2-thiouracilyl,
[0262] 4-thiouracilyl,
[0263] 2-thiothyminyl
[0264] 5-methyl-2-thiouracilyl,
[0265] 3-(3-amino-3-carboxypropyl)uracilyl,
[0266] 5-hydroxyuracilyl,
[0267] 5-methoxyuracilyl,
[0268] uracilyl 5-oxyacetic acid,
[0269] uracilyl 5-oxyacetic acid methyl ester,
[0270] 5-(carboxyhydroxymethyl)uracilyl,
[0271] 5-(carboxyhydroxymethyl)uracilyl methyl ester,
[0272] 5-methoxycarbonylmethyluracilyl,
[0273] 5-methoxycarbonylmethyl-2-thiouracilyl,
[0274] 5-aminomethyl-2-thiouracilyl,
[0275] 5-methylaminomethyluracilyl,
[0276] 5-methylaminomethyl-2-thiouracilyl,
[0277] 5-methylaminomethyl-2-selenouracilyl,
[0278] 5-carbamoylmethyluracilyl,
[0279] 5-carboxymethylaminomethyluracilyl,
[0280] 5-carboxymethylaminomethyl-2-thiouracilyl,
[0281] 3-methyluracilyl,
[0282] 1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl,
[0283] 5-carboxymethyluracilyl,
[0284] 5-methyldihydrouracilyl, or
[0285] 3-methylpseudouracilyl.
[0286] A universal base can form base pairs with each of the
natural DNA/RNA bases, exhibiting relatively little discrimination
between them. In general, the universal bases are non-hydrogen
bonding, hydrophobic, aromatic moieties which can stabilize e.g.,
duplex RNA or RNA-like molecules, via stacking interactions. A
universal base can also include hydrogen bonding substituents. As
used herein, a "universal base" can include anthracenes, pyrenes or
any one of the following: 1718
[0287] In some embodiments, B can form part of a tether that
connects a ligand to the carrier. For example, the tether can be
B--CH.dbd.CH--C(O)NH--(CH.sub.2).sub.5--NHC(O)-LIGAND. In a
preferred embodiment, the double bond is trans, and the ligand is a
substituted or unsubstituted cholesterolyl radical (e.g., attached
through the D-ring side chain or the C-3 hydroxyl); an aralkyl
moiety having at least one sterogenic center and at least one
substituent on the aryl portion of the aralkyl group; or a
nucleobase. In certain embodiments, B, in the tether described
above, is uracilyl or a universal base, e.g., an aryl moiety, e.g.,
phenyl, optionally having additional substituents, e.g., one or
more fluoro groups. B can be substituted at any atom with the
remainder of the tether.
[0288] X.sup.2 can include "oxy" or "deoxy" substituents in place
of the 2'-OH; or be a ligand or a tethered ligand.
[0289] Examples of "oxy"-substituents include alkoxy or aryloxy
(OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl,
sugar, or protecting group); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nC- H.sub.2CH.sub.2OR; "locked" nucleic
acids (LNA) in which the 2' hydroxyl is connected, e.g., by a
methylene bridge, to the 4' carbon of the same ribose sugar;
O-PROTECTED AMINE (AMINE=NH.sub.2; alkylamino, dialkylamino,
heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or
diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy,
O(CH.sub.2).sub.nPROTECTED AMINE, (e.g., AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, or diheteroaryl amino, ethylene diamine,
polyamino), and orthoester. Amine protecting groups can include
formyl, amido, benzyl, allyl, etc.
[0290] Preferred orthoesters have the general formula J. The groups
R.sup.31 and R.sup.32 may be the same or different and can be any
combination of the groups listed in FIG. 2B. A preferred orthoester
is the "ACE" group, shown below as structure K. 19
[0291] "Deoxy" substituents include hydrogen (i.e. deoxyribose
sugars); halo (e.g., fluoro); protected amino (e.g. NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid in which all
amino are protected); fully protected polyamino (e.g.,
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.s- ub.2-AMINE, wherein
AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino,
diaryl amino, heteroaryl amino, or diheteroaryl amino and all amino
groups are protected), --NHC(O)R (R=alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar), cyano; alkyl-thio-alkyl; thioalkoxy;
and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be
optionally substituted with e.g., a protected amino functionality.
Preferred substitutents are 2'-methoxyethyl, 2'-OCH3, 2'-O-allyl,
2'-C-- allyl, and 2'-fluoro.
[0292] X.sup.3 is as described for OFG.sup.2 above.
[0293] PG can be a triarylmethyl group (e.g., a dimethoxytrityl
group) or Si(X.sup.5')(X.sup.5")(X.sup.5'") in which (X.sup.5'),
(X.sup.5"), and (X.sup.5'") are as described elsewhere.
[0294] Sugar Replacement-Based Monomers, e.g., Ligand-Conjugated
Monomers (Cyclic)
[0295] Cyclic sugar replacement-based monomers, e.g., sugar
replacement-based ligand-conjugated monomers, are also referred to
herein as ribose replacement monomer subunit (RRMS) monomer
compounds. Preferred carriers have the general formula (LCM-2)
provided below (In that structure preferred backbone attachment
points can be chosen from R.sup.1 or R.sup.2; R.sup.3 or R.sup.1;
or R.sup.9 and R.sup.10 if Y is CR.sup.9R.sup.10 (two positions are
chosen to give two backbone attachment points, e.g., R.sup.1 and
R.sup.4, or R.sup.4 and R.sup.9)). Preferred tethering attachment
points include R.sup.7; R.sup.5 or R.sup.6 when X is CH.sub.2. The
carriers are described below as an entity, which can be
incorporated into a strand. Thus, it is understood that the
structures also encompass the situations wherein one (in the case
of a terminal position) or two (in the case of an internal
position) of the attachment points, e.g., R.sup.1 or R.sup.2;
R.sup.3 or R.sup.4; or R.sup.9 or R.sup.10 (when Y is
CR.sup.9R.sup.10), is connected to the phosphate, or modified
phosphate, e.g., sulfur containing, backbone. E.g., one of the
above-named R groups can be --CH.sub.2--, wherein one bond is
connected to the carrier and one to a backbone atom, e.g., a
linking oxygen or a central phosphorus atom.) 20
[0296] in which,
[0297] X is N(CO)R.sup.7, NR.sup.7 or CH.sub.2;
[0298] Y is NR.sup.8, O, S, CR.sup.9R.sup.10;
[0299] Z is CR.sup.11R.sup.12 or absent;
[0300] Each of R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.9, and
R.sup.10 is, independently, H, OR.sup.a, or
(CH.sub.2).sub.nOR.sup.b, provided that at least two of R.sup.1,
R.sup.2, R.sup.3, R.sup.4, R.sup.9, and R.sup.10 are OR.sup.a
and/or (CH.sub.2).sub.nOR.sup.b;
[0301] Each of R.sup.5, R.sup.6, R.sup.11, and R.sup.12 is,
independently, a ligand, H, C.sub.1-C.sub.6 alkyl optionally
substituted with 1-3 R.sup.13, or C(O)NHR.sup.7; or R.sup.5 and
R.sup.11 together are C.sub.3-C.sub.8 cycloalkyl optionally
substituted with R.sup.14;
[0302] R.sup.7 can be a ligand, e.g., R.sup.7 can be R.sup.d, or
R.sup.7 can be a ligand tethered indirectly to the carrier, e.g.,
through a tethering moiety, e.g., C.sub.1-C.sub.20 alkyl
substituted with NR.sup.cR.sup.d; or C.sub.1-C.sub.20 alkyl
substituted with NHC(O)Rd;
[0303] R.sup.8 is H or C.sub.1-C.sub.6 alkyl;
[0304] R.sup.13 is hydroxy, C.sub.1-C.sub.4 alkoxy, or halo;
[0305] R.sup.14 is NR.sup.cR.sup.7;
[0306] R.sup.15 is C.sub.1-C.sub.6 alkyl optionally substituted
with cyano, or C.sub.2-C.sub.6 alkenyl;
[0307] R.sup.16 is C.sub.1-C.sub.10 alkyl;
[0308] R.sup.17 is a liquid or solid phase support reagent;
[0309] L is --C(O)(CH.sub.2).sub.qC(O)--, or
--C(O)(CH.sub.2).sub.qS--;
[0310] R.sup.a is a protecting group, e.g., CAr.sub.3; (e.g., a
dimethoxytrityl group) or Si(X.sup.5')(X.sup.5")(X.sup.5'") in
which (X.sup.5'), (X.sup.5"), and (X.sup.5'") are as described
elsewhere.
[0311] R.sup.b is P(O)(O.sup.-)H, P(OR.sup.15)N(R.sup.16).sub.2 or
L-R.sup.17;
[0312] R.sup.c is H or C.sub.1-C.sub.6 alkyl;
[0313] R.sup.d is carbohydrate radical or a steroid optionally
tethered to at least one carbohydrate radical;
[0314] Each Ar is, independently, C.sub.6-C.sub.10 aryl optionally
substituted with C.sub.1-C.sub.4 alkoxy;
[0315] n is 1-4; and q is 0-4.
[0316] Exemplary carriers include those in which, e.g., X is
N(CO)R.sup.7 or NR.sup.7, Y is CR.sup.9R.sup.10, and Z is absent;
or X is N(CO)R.sup.7 or NR.sup.7, Y is CR.sup.9R.sup.10, and Z is
CR.sup.11R.sup.12; or X is N(CO)R.sup.7 or NR.sup.7, Y is NR.sup.8,
and Z is CR.sup.11R.sup.12; or X is N(CO)R.sup.7 or NR.sup.7, Y is
O, and Z is CR.sup.11R.sup.12; or X is CH.sub.2; Y is
CR.sup.9R.sup.10; Z is CR.sup.11R.sup.12, and R.sup.5 and R.sup.11
together form C.sub.6 cycloalkyl (H, z=2), or the indane ring
system, e.g., X is CH.sub.2; Y is CR.sup.9R.sup.10; Z is
CR.sup.11R.sup.12, and R.sup.5 and R.sup.11 together form C.sub.5
cycloalkyl (H, z=1).
[0317] In certain embodiments, the carrier may be based on the
pyrroline ring system or the 4-hydroxyproline ring system, e.g., X
is N(CO)R.sup.7 or NR.sup.7, Y is CR.sup.9R.sup.10, and Z is absent
(D). OFG.sup.1 is preferably attached to a primary carbon, e.g., an
exocyclic alkylene 21
[0318] group, e.g., a methylene group, connected to one of the
carbons in the five-membered ring (--CH.sub.2OFG.sup.1 in D).
OFG.sup.2 is preferably attached directly to one of the carbons in
the five-membered ring (--OFG.sup.2 in D). For the pyrroline-based
carriers, --CH.sub.2OFG.sup.1 may be attached to C-2 and OFG.sup.2
may be attached to C-3; or --CH.sub.2OFG.sup.1 may be attached to
C-3 and OFG.sup.2 may be attached to C-4. In certain embodiments,
CH.sub.2OFG.sup.1 and OFG.sup.2 may be geminally substituted to one
of the above-referenced carbons. For the 3-hydroxyproline-based
carriers, --CH.sub.2OFG.sup.1 may be attached to C-2 and OFG.sup.2
may be attached to C-4. The pyrroline- and 4-hydroxyproline-based
monomers may therefore contain linkages (e.g., carbon-carbon bonds)
wherein bond rotation is restricted about that particular linkage,
e.g. restriction resulting from the presence of a ring. Thus,
CH.sub.2OFG.sup.1 and OFG.sup.2 may be cis or trans with respect to
one another in any of the pairings delineated above Accordingly,
all cis/trans isomers are expressly included. The monomers may also
contain one or more asymmetric centers and thus occur as racemates
and racemic mixtures, single enantiomers, individual diastereomers
and diastereomeric mixtures. All such isomeric forms of the
monomers are expressly included (e.g., the centers bearing
CH.sub.2OFG.sup.1 and OFG.sup.2 can both have the R configuration;
or both have the S configuration; or one center can have the R
configuration and the other center can have the S configuration and
vice versa). The tethering attachment point is preferably nitrogen.
Preferred examples of carrier D include the following: 22
[0319] In certain embodiments, the carrier may be based on the
piperidine ring system (E), e.g., X is N(CO)R.sup.7 or NR.sup.7, Y
is CR.sup.9R.sup.10, and Z is CR.sup.11R.sup.12. OFG.sup.1 is
preferably 23
[0320] attached to a primary carbon, e.g., an exocyclic alkylene
group, e.g., a methylene group (n=1) or ethylene group (n=2),
connected to one of the carbons in the six-membered ring
[--(CH.sub.2).sub.nOFG.sup.1 in E]. OFG.sup.2 is preferably
attached directly to one of the carbons in the six-membered ring
(--OFG.sup.2 in E). --(CH.sub.2).sub.nOFG.sup.1 and OFG.sup.2 may
be disposed in a geminal manner on the ring, i.e., both groups may
be attached to the same carbon, e.g., at C-2, C-3, or C-4.
Alternatively, --(CH.sub.2).sub.nOFG.sup.1 and OFG.sup.2 may be
disposed in a vicinal manner on the ring, i.e., both groups may be
attached to adjacent ring carbon atoms, e.g.,
[0321] --(CH.sub.2).sub.nOFG.sup.1 may be attached to C-2 and
OFG.sup.2 may be attached to C-3; --(CH.sub.2).sub.nOFG.sup.1 may
be attached to C-3 and OFG.sup.2 may be attached to C-2;
--(CH.sub.2).sub.nOFG.sup.1 may be attached to C-3 and OFG.sup.2
may be attached to C-4; or --(CH.sub.2).sub.nOFG.sup.1 may be
attached to C-4 and OFG.sup.2 may be attached to C-3. The
piperidine-based monomers may therefore contain linkages (e.g.,
carbon-carbon bonds) wherein bond rotation is restricted about that
particular linkage, e.g. restriction resulting from the presence of
a ring. Thus, --(CH.sub.2).sub.nOFG.sup.1 and OFG.sup.2 may be cis
or trans with respect to one another in any of the pairings
delineated above. Accordingly, all cis/trans isomers are expressly
included. The monomers may also contain one or more asymmetric
centers and thus occur as racemates and racemic mixtures, single
enantiomers, individual diastereomers and diastereomeric mixtures.
All such isomeric forms of the monomers are expressly included
(e.g., the centers bearing CH.sub.2OFG.sup.1 and OFG.sup.2 can both
have the R configuration; or both have the S configuration; or one
center can have the R configuration and the other center can have
the S configuration and vice versa). The tethering attachment point
is preferably nitrogen.
[0322] In certain embodiments, the carrier may be based on the
piperazine ring system (F), e.g., X is N(CO)R.sup.7 or NR.sup.7, Y
is NR.sup.8, and Z is CR.sup.11R.sup.12, or the morpholine ring
system (G), e.g., X is N(CO)R.sup.7 or NR.sup.7, Y is O, and Z is
CR.sup.11R.sup.12. OFG.sup.1 is preferably 24
[0323] attached to a primary carbon, e.g., an exocyclic alkylene
group, e.g., a methylene group, connected to one of the carbons in
the six-membered ring (--CH.sub.2OFG.sup.1 in F or G). OFG.sup.2 is
preferably attached directly to one of the carbons in the
six-membered rings (--OFG.sup.2 in F or G). For both F and G,
--CH.sub.2OFG.sup.1 may be attached to C-2 and OFG.sup.2 may be
attached to C-3; or vice versa. In certain embodiments,
CH.sub.2OFG.sup.1 and OFG.sup.2 may be geminally substituted to one
of the above-referenced carbons. The piperazine- and
morpholine-based monomers may therefore contain linkages (e.g.,
carbon-carbon bonds) wherein bond rotation is restricted about that
particular linkage, e.g. restriction resulting from the presence of
a ring. Thus, CH.sub.2OFG.sup.1 and OFG.sup.2 may be cis or trans
with respect to one another in any of the pairings delineated
above. Accordingly, all cis/trans isomers are expressly included.
The monomers may also contain one or more asymmetric centers and
thus occur as racemates and racemic mixtures, single enantiomers,
individual diastereomers and diastereomeric mixtures. All such
isomeric forms of the monomers are expressly included (e.g., the
centers bearing CH.sub.2OFG.sup.1 and OFG.sup.2 can both have the R
configuration; or both have the S configuration; or one center can
have the R configuration and the other center can have the S
configuration and vice versa). R'" can be, e.g., C.sub.1-C.sub.6
alkyl, preferably CH.sub.3. The tethering attachment point is
preferably nitrogen in both F and G.
[0324] In certain embodiments, the carrier may be based on the
decalin ring system, e.g., X is CH.sub.2; Y is CR.sup.9R.sup.10; Z
is CR.sup.11R.sup.12, and R.sup.5 and R.sup.11 together form
C.sub.6 cycloalkyl (H, z=2), or the indane ring system, e.g., X is
CH.sub.2; Y is CR.sup.9R.sup.10; Z is CR.sup.11R.sup.12, and
R.sup.5 and R.sup.11 together form C.sub.5 cycloalkyl (H, z=1).
OFG.sup.1 is preferably attached to a primary carbon, 25
[0325] e.g., an exocyclic methylene group (n=1) or ethylene group
(n=2) connected to one of C-2, C-3, C-4, or C-5
[--(CH.sub.2).sub.nOFG.sup.1 in H]. OFG.sup.2 is preferably
attached directly to one of C-2, C-3, C-4, or C-5 (--OFG.sup.2 in
H). --(CH.sub.2).sub.nOFG.sup.1 and OFG.sup.2 may be disposed in a
geminal manner on the ring, i.e., both groups may be attached to
the same carbon, e.g., at C-2, C-3, C-4, or C-5. Alternatively,
--(CH.sub.2).sub.nOFG.sup.1 and OFG.sup.2 may be disposed in a
vicinal manner on the ring, i.e., both groups may be attached to
adjacent ring carbon atoms, e.g., --(CH.sub.2).sub.nOFG.sup.1 may
be attached to C-2 and OFG.sup.2 may be attached to C-3;
--(CH.sub.2).sub.nOFG.sup.1 may be attached to C-3 and OFG.sup.2
may be attached to C-2; --(CH.sub.2).sub.nOFG.sup.1 may be attached
to C-3 and OFG.sup.2 may be attached to C-4; or
--(CH.sub.2).sub.nOFG.sup.1 may be attached to C-4 and OFG.sup.2
may be attached to C-3; --(CH.sub.2).sub.nOFG.sup.1 may be attached
to C-4 and OFG.sup.2 may be attached to C-5; or
--(CH.sub.2).sub.nOFG.sup.1 may be attached to C-5 and OFG.sup.2
may be attached to C-4. The decalin or indane-based monomers may
therefore contain linkages (e.g., carbon-carbon bonds) wherein bond
rotation is restricted about that particular linkage, e.g.
restriction resulting from the presence of a ring. Thus,
--(CH.sub.2).sub.nOFG.sup.1 and OFG.sup.2 may be cis or trans with
respect to one another in any of the pairings delineated above.
Accordingly, all cis/trans isomers are expressly included. The
monomers may also contain one or more asymmetric centers and thus
occur as racemates and racemic mixtures, single enantiomers,
individual diastereomers and diastereomeric mixtures. All such
isomeric forms of the monomers are expressly included (e.g., the
centers bearing CH.sub.2OFG.sup.1 and OFG.sup.2 can both have the R
configuration; or both have the S configuration; or one center can
have the R configuration and the other center can have the S
configuration and vice versa). In a preferred embodiment, the
substituents at C-1 and C-6 are trans with respect to one another.
The tethering attachment point is preferably C-6 or C-7.
[0326] Other carriers may include those based on 3-hydroxyproline
(J). Thus, --(CH.sub.2).sub.nOFG.sup.1 and OFG.sup.2 may be cis or
trans with respect to one another. Accordingly, all cis/trans
isomers are expressly included. The monomers may also contain one
or more asymmetric centers 26
[0327] and thus occur as racemates and racemic mixtures, single
enantiomers, individual diastereomers and diastereomeric mixtures.
All such isomeric forms of the monomers are expressly included
(e.g., the centers bearing CH.sub.2OFG.sup.1 and OFG.sup.2 can both
have the R configuration; or both have the S configuration; or one
center can have the R configuration and the other center can have
the S configuration and vice versa). The tethering attachment point
is preferably nitrogen.
[0328] Representative cyclic, sugar replacement-based carriers are
shown in FIG. 3.
[0329] Sugar Replacement-Based Monomers (Acyclic)
[0330] Acyclic sugar replacement-based monomers, e.g., sugar
replacement-based ligand-conjugated monomers, are also referred to
herein as ribose replacement monomer subunit (RRMS) monomer
compounds. Preferred acyclic carriers can have formula LCM-3 or
LCM-4 below. 27
[0331] In some embodiments, each of x, y, and z can be,
independently of one another, 0, 1, 2, or 3. In formula LCM-3, when
y and z are different, then the tertiary carbon can have either the
R or S configuration. In preferred embodiments, x is zero and y and
z are each 1 in formula LCM-3 (e.g., based on serinol), and y and z
are each 1 in formula LCM-3. Each of formula LCM-3 or LCM-4 below
can optionally be substituted, e.g., with hydroxy, alkoxy,
perhaloalkyl.
[0332] Tethers
[0333] In certain embodiments, a moiety, e.g., a ligand may be
connected indirectly to the carrier via the intermediacy of an
intervening tether. Tethers are connected to the carrier at a
tethering attachment point (TAP) and may include any
C.sub.1-C.sub.100 carbon-containing moiety, (e.g. C.sub.1-C.sub.75,
C.sub.1-C.sub.50, C.sub.1-C.sub.20, C.sub.1-C.sub.10; C.sub.1,
C.sub.2, C.sub.3, C.sub.4, C.sub.5, C.sub.6, C.sub.7, C.sub.8,
C.sub.9, or C.sub.10), preferably having at least one nitrogen
atom. In preferred embodiments, the nitrogen atom forms part of a
terminal amino or amido (NHC(O)--) group on the tether, which may
serve as a connection point for the ligand. Preferred tethers
(underlined) include TAP-(CH.sub.2).sub.nNH--;
TAP-C(O)(CH.sub.2).sub.nNH--; TAP-NR""(CH.sub.2).sub.nNH--,
TAP-C(O)--(CH.sub.2).sub.2--C(O)--;
TAP-C(O)--O--(CH.sub.2).sub.n--C(O)O--; TAP-C(O)--O--;
TAP-C(O)--(CH.sub.2).sub.n--NH--C(O)--;
TAP-C(O)--(CH.sub.2).sub.n--; TAP-C(O)--NH--; TAP-C(O)--;
TAP-(CH.sub.2).sub.n--C(O)--; TAP-(CH.sub.2).sub.n--C(O)O--;
TAP-(CH.sub.2).sub.n--; or TAP-(CH.sub.2).sub.n--NH--C(O)--; in
which n is 1-20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, or 20) and R"" is C.sub.1-C.sub.6 alkyl.
Preferably, n is 5, 6, or 11. In other embodiments, the nitrogen
may form part of a terminal oxyamino group, e.g., --ONH.sub.2, or
hydrazino group, --NHNH.sub.2. The tether may optionally be
substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or
optionally inserted with one or more additional heteroatoms, e.g.,
N, O, or S. Preferred tethered ligands may include, e.g.,
TAP-(CH.sub.2).sub.nNH(LIGAND); TAP-C(O)(CH.sub.2).sub.nNH(LIGAND);
TAP-NR""(CH.sub.2).sub.nNH(LIGAND);
TAP-(CH.sub.2).sub.nONH(LIGAND);
TAP-C(O)--(CH.sub.2).sub.nONH(LIGAND);
TAP-NR""(CH.sub.2).sub.nONH(LIGAND- );
TAP-(CH.sub.2).sub.nNHNH.sub.2(LIGAND),
TAP-C(O)(CH.sub.2).sub.nNHNH.su- b.2(LIGAND);
TAP-NR""(CH.sub.2).sub.nNHNH.sub.2(LIGAND); TAP-C(O)--(CH.sub.2
--C(O(LIGAND); TAP-C(O)--(CH).sub.n--C(O)O(LIGAND);
TAP-C(O)--O(LIGAND); TAP-C(O)--(CH.sub.2).sub.n--NH--C(O)(LIGAND);
TAP-C(O)--(CH.sub.2).sub.n(LIGAND); TAP-C(O)--NH(LIGAND);
TAP-C(O)(LIGAND); TAP-(CH.sub.2).sub.n--C(O)(LIGAND);
TAP-(CH.sub.2).sub.n--C(O)O(LIGAND); TAP-(CH.sub.2).sub.n(LIGAND);
or TAP-(CH.sub.2).sub.n--NH--C(O)(LIGAND). In some embodiments,
amino terminated tethers (e.g., NH.sub.2, ONH.sub.2,
NH.sub.2NH.sub.2) can form an imino bond (i.e., C.dbd.N) with the
ligand. In some embodiments, amino terminated tethers (e.g.,
NH.sub.2, ONH.sub.2, NH.sub.2NH.sub.2) can acylated, e.g., with
C(O)CF.sub.3.
[0334] In some embodiments, the tether can terminate with a
mercapto group (i.e., SH) or an olefin (e.g., CH.dbd.CH.sub.2). For
example, the tether can be TAP-(CH.sub.2).sub.2--SH,
TAP-C(O)(CH.sub.2).sub.nSH,
TAP-(CH.sub.2).sub.n--(CH.dbd.CH.sub.2), or
TAP-C(O)(CH.sub.2).sub.n(CH.d- bd.CH.sub.2), in which n can be as
described elsewhere. The tether may optionally be substituted,
e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally
inserted with one or more additional heteroatoms, e.g., N, O, or S.
The double bond can be cis or trans or E or Z.
[0335] In other embodiments the tether may include an electrophilic
moiety, preferably at the terminal position of the tether.
Preferred electrophilic moieties include, e.g., an aldehyde, alkyl
halide, mesylate, tosylate, nosylate, or brosylate, or an activated
carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl
ester. Preferred tethers (underlined) include
TAP-(CH.sub.2).sub.nCHO; TAP-C(O)(CH.sub.2).sub.nCHO; or
TAP-NR""(CH.sub.2).sub.nCHO, in which n is 1-6 and R"" is
C.sub.1-C.sub.6 alkyl; or TAP-(CH.sub.2).sub.nC(O)ONHS;
TAP-C(O)(CH.sub.2).sub.nC(O)ONHS; or
TAP-NR""(CH.sub.2).sub.nC(O)ONHS, in which n is 1-6 and R"" is
C.sub.1-C.sub.6 alkyl; TAP-(CH.sub.2).sub.nC(O)- OC.sub.6F.sub.5;
TAP-C(O)(CH.sub.2).sub.2C(O)OC.sub.6F.sub.5; or
TAP-NR""(CH.sub.2).sub.nC(O)OC.sub.6F.sub.5, in which n is 1-11 and
R"" is C.sub.1-C.sub.6 alkyl; or --(CH.sub.2).sub.nCH.sub.2LG;
TAP-C(O)(CH.sub.2).sub.nCH.sub.2LG; or
TAP-NR""(CH.sub.2).sub.nCH.sub.2LG- , in which n can be as
described elsewhere and R"" is C.sub.1-C.sub.6 alkyl (LG can be a
leaving group, e.g., halide, mesylate, tosylate, nosylate,
brosylate). Tethering can be carried out by coupling a nucleophilic
group of a ligand, e.g., a thiol or amino group with an
electrophilic group on the tether.
[0336] In other embodiments, it can be desirable for the monomer to
include a phthalimido group (K) at the terminal position of the
tether. 28
[0337] In other embodiments, other protected amino groups can be at
the terminal position of the tether, e.g., alloc, monomethoxy
trityl (MMT), trifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the
aryl portion can be ortho-nitrophenyl or ortho,
para-dinitrophenyl).
[0338] Any of the tethers described herein may further include one
or more additional linking groups, e.g., --O--(CH.sub.2).sub.n--,
--(CH.sub.2).sub.n--SS--, --(CH.sub.2).sub.n--, or
--(CH.dbd.CH)--.
[0339] Tethered Ligands
[0340] A wide variety of entities, e.g., ligands, can be tethered
to an iRNA agent, e.g., to the carrier of a ligand-conjugated
monomer subunit. Examples are described below in the context of a
ligand-conjugated monomer subunit but that is only preferred,
entities can be coupled at other points to an iRNA agent.
[0341] Preferred moieties are ligands, which are coupled,
preferably covalently, either directly or indirectly via an
intervening tether, to the carrier. In preferred embodiments, the
ligand is attached to the carrier via an intervening tether. As
discussed above, the ligand or tethered ligand may be present on
the ligand-conjugated monomer.backslash. when the ligand-conjugated
monomer is incorporated into the growing strand. In some
embodiments, the ligand may be incorporated into a "precursor"
ligand-conjugated monomer subunit after a "precursor"
ligand-conjugated monomer subunit has been incorporated into the
growing strand. For example, a monomer having, e.g., an
amino-terminated tether, e.g., TAP-(CH.sub.2).sub.nNH.sub.2 may be
incorporated into a growing sense or antisense strand. In a
subsequent operation, i.e., after incorporation of the precursor
monomer subunit into the strand, a ligand having an electrophilic
group, e.g., a pentafluorophenyl ester or aldehyde group, can
subsequently be attached to the precursor ligand-conjugated monomer
by coupling the electrophilic group of the ligand with the terminal
nucleophilic group of the precursor ligand-conjugated monomer
subunit tether.
[0342] In preferred embodiments, a ligand alters the distribution,
targeting or lifetime of an iRNA agent into which it is
incorporated. In preferred embodiments a ligand provides an
enhanced affinity for a selected target, e.g, molecule, cell or
cell type, compartment, e.g., a cellular or organ compartment,
tissue, organ or region of the body, as, e.g., compared to a
species absent such a ligand.
[0343] Preferred ligands can improve transport, hybridization, and
specificity properties and may also improve nuclease resistance of
the resultant natural or modified oligoribonucleotide, or a
polymeric molecule comprising any combination of monomers described
herein and/or natural or modified ribonucleotides.
[0344] Ligands in general can include therapeutic modifiers, e.g.,
for enhancing uptake; diagnostic compounds or reporter groups e.g.,
for monitoring distribution; cross-linking agents;
nuclease-resistance conferring moieties; and natural or unusual
nucleobases. General examples include lipophiles, lipids, steroids
(e.g., cholesterol, uvaol, hecigenin, diosgenin), terpenes (e.g.,
triterpenes, e.g., sarsasapogenin, Friedelin, epifriedelanol
derivatized lithocholic acid), vitamins (e.g., folic acid, vitamin
A, vitamin E, biotin, pyridoxal), carbohydrates, proteins, protein
binding agents, integrin targeting molecules, polycationics (e.g.,
porphyrins), peptides, polyamines, and peptide mimics.
[0345] In some embodiments, the ligand can be one of the following
triterpenes: 29
[0346] In some embodiments, the ligand can be a steroid, e.g., a
substituted or unsubstituted cholesterol or cholanic acid, or
stereoisomer thereof or one of the following steroids: 30
[0347] In some embodiments, the ligand can be a carbohydrate,
(e.g., a monosaccharide, e.g., an aldose or a ketose, (triose,
tetrose, pentose, hexose, etc.); a disaccharide; or
polysaccharide). In preferred embodiments the ligand can be, e.g.,
galactose, N-acetylgalactosamine, or mannose.
[0348] In some embodiments, the ligand can include a steroid that
is tethered to at least one carbohydrate (e.g., a monosaccharide).
Such a ligand can be attached to the tether or the tethering
attachment point through a atom or group of atoms that is
associated with either the steroid or the carbohydrate (e.g., an
amino group or a hydroxy group). Tethers can include any of those
described herein. In certain embodiments, the tether can further
include a multivalent moiety (e.g., a trihydroxybenzoate or
trihydroxybenzyl group) for tethering one or more carbohydrates.
Examples of such ligands include compounds 7, 8, 38, 39, 55, and
61.
[0349] In some embodiments, the ligand can be a porphyrin. In
certain embodiments, the porphyrin can be further substituted with
a tether or a tethered ligand (e.g., a steroid, a carbohydrate, or
a steroid tethered to at least one carbohydrate). In these
embodiments, the porphyrin can be or form part of a tether that
attaches a ligand to an iRNA agent.
[0350] Methods for Making iRNA Agents
[0351] A listing of ribonucleosides containing the unusual bases
described herein are described in "The RNA Modification Database"
maintained by Pamela F. Crain, Jef Rozenski and James A. McCloskey;
Departments of Medicinal Chemistry and Biochemistry, University of
Utah, Salt Lake City, Utah 84112, USA
(RNAmods@lib.med.utah.edu).
[0352] The 5' silyl protecting group can be used in conjunction
with acid labile orthoesters at the 2' position of ribonucleosides
to synthesize oligonucleotides via phosphoramidite chemistry. Final
deprotection conditions are known not to significantly degrade RNA
products. Functional groups on the unusual and universal bases are
blocked during oligonucleotide synthesis with protecting groups
that are compatible with the operations being performed that are
described herein. All syntheses can be can be conducted in any
automated or manual synthesizer on large, medium, or small scale.
The syntheses may also be carried out in multiple well plates or
glass slides.
[0353] The 5'-O-silyl group can be removed via exposure to fluoride
ions, which can include any source of fluoride ion, e.g., those
salts containing fluoride ion paired with inorganic counterions
e.g., cesium fluoride and potassium fluoride or those salts
containing fluoride ion paired with an organic counterion, e.g., a
tetraalkylammonium fluoride. A crown ether catalyst can be utilized
in combination with the inorganic fluoride in the deprotection
reaction. Preferred fluoride ion source are tetrabutylammonium
fluoride or aminehydrofluorides (e.g., combining aqueous HF with
triethylamine in a dipolar aprotic solvent, e.g.,
dimethylformamide).
[0354] The choice of protecting groups for use on the phosphite
triesters and phosphotriesters can alter the stability of the
triesters towards fluoride. Methyl protection of the
phosphotriester or phosphitetriester can stabilize the linkage
against fluoride ions and improve process yields.
[0355] Since ribonucleosides have a reactive 2' hydroxyl
substituent, it can be desirable to protect the reactive 2'
position in RNA with a protecting group that is compatible with a
5'-O-silyl protecting group, e.g. one stable to fluoride.
Orthoesters meet this criterion and can be readily removed in a
final acid deprotection step that can result in minimal RNA
degradation.
[0356] Tetrazole catalysts can be used in the standard
phosphoramidite coupling reaction. Preferred catalysts include e.g.
tetrazole, S-ethyl-tetrazole, p-nitrophenyltetrazole.
[0357] The general process is as follows. Nucleosides are suitably
protected and functionalized for use in solid-phase or
solution-phase synthesis of RNA oligonucleotides. The 2'-hydroxyl
group in a ribonucleotide can be modified using a tris orthoester
reagent. The 2'-hydroxyl can be modified to yield a 2'-O-orthoester
nucleoside by reacting the ribonucleoside with the tris orthoester
reagent in the presence of an acidic catalyst, e.g., pyridinium
p-toluene sulfonate. This reaction is known to those skilled in the
art. The product can then be subjected to further protecting group
reactions (e.g., 5'-O-silylation) and functionalizations (e.g.,
3'-O-phosphitylation) to produce a desired reagent (e.g.,
nucleoside phosphoramidite) for incorporation within an
oligonucleotide or polymer by reactions known to those skilled in
the art.
[0358] Preferred orthoesters include those comprising ethylene
glycol ligands which are protected with acyl or ester protecting
groups. Specifically, the preferred acyl group is acetyl. The
nucleoside reagents may then be used by those skilled in the art to
synthesize RNA oligonucleotides on commercially available
synthesizer instruments, e.g. Gene Assembler Plus (Pharmacia), 380B
(Applied Biosystems). Following synthesis (either solution-phase or
solid-phase) of an oligonucleotide or polymer, the product can be
subjected to one or more reactions using non-acidic reagents. One
of these reactions may be strong basic conditions, for example, 40%
methylamine in water for 10 minutes at 55.degree. C., which will
remove the acyl protecting groups from the ethylene glycol ligands
but leave the orthoester moiety attached. The resultant orthoester
may be left attached when the polymer or oligonucleotide is used in
subsequent applications, or it may be removed in a final
mildly-acidic reaction, for example, 10 minutes at 55.degree. C. in
50 mM acetic acid, pH 3.0, followed by addition of equal volume of
150 mM TRIS buffer for 10 minutes at 55.degree. C.
[0359] Universal bases are described in "Survey and Summary: The
Applications of Universal DNA base analogues" Loakes, D., Nucleic
Acid Research 2001, 29, 2437, which is incorporated by reference in
its entirety. Specific examples are described in the following:
Liu, D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926;
Morales, J. C.; Kool, E. T. Biochemistry, 2000, 39, 2626-2632;
Matray, T, J.; Kool, E. T. J. Am. Chem. Soc., 1998, 120, 6191-6192;
Moran, S. Ren, R. X. -F.; Rumney I V, S.; Kool, E. T. J. Am. Chem.
Soc., 1997, 119, 2056-2057; Guckian, K. M.; Morales, J. C.; Kool,
E. T. J. Org. Chem., 1998, 63, 9652-9656; Berger, M.; Wu. Y.;
Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E.
Nucleic Acids Res., 2000, 28, 2911-2914; Ogawa, A. K.; Wu, Y.;
McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am.
Chem. Soc., 2000, 122, 3274-3287; Ogawa, A. K.; Wu. Y.; Berger, M.;
Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122,
8803-8804; Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg,
F. E. J. Am. Chem. Soc., 2001, 123, 7439-7440; Wu, Y.; Ogawa, A.
K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J.
Am. Chem. Soc., 2000, 122, 7621-7632; McMinn, D. L.; Ogawa. A. K.;
Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem.
Soc., 1999, 121, 11585-11586; Brotschi, C.; Haberli, A.; Leumann,
C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014; Weizman, H.; Tor,
Y. J. Am. Chem. Soc., 2001, 123, 3375-3376; Lan, T.; McLaughlin, L.
W. J. Am. Chem. Soc., 2000, 122, 6512-13.
[0360] As discussed above, the monomers and methods described
herein can be used in the preparation of modified RNA molecules, or
polymeric molecules comprising any combination of monomer compounds
described herein and/or natural or modified ribonucleotides in
which one or more subunits contain an unusual or universal base.
Modified RNA molecules include e.g. those molecules containing a
chemically or stereochemically modified nucleoside (e.g., having
one or more backbone modifications, e.g., phosphorothioate or
P-alkyl; having one or more sugar modifications, e.g., 2'-OCH.sub.3
or 2'-F; and/or having one or more base modifications, e.g.,
5-alkylamino or 5-allylamino) or a nucleoside surrogate.
[0361] Coupling of 5'-hydroxyl groups with phosphoramidites forms
phosphite ester intermediates, which in turn are oxidized e.g.,
with iodine, to the phosphate diester. Alternatively, the
phosphites may be treated with e.g., sulfur, selenium, amino, and
boron reagents to form modified phosphate backbones. Linkages
between the monomers described herein and a nucleoside or
oligonucleotide chain can also be treated with iodine, sulfur,
selenium, amino, and boron reagents to form unmodified and modified
phosphate backbones respectively. Similarly, the monomers described
herein may be coupled with nucleosides or oligonucleotides
containing any of the modifications or nucleoside surrogates
described herein.
[0362] The synthesis and purification of oligonucleotide peptide
conjugates can be performed by established methods. See, for
example, Trufert et al., Tetrahedron, 52:3005, 1996; and Manoharan,
"Oligonucleotide Conjugates in Antisense Technology," in Antisense
Drug Technology, ed. S. T. Crooke, Marcel Dekker, Inc., 2001.
Exemplary methods are shown in FIGS. 4 and 5.
[0363] In one embodiment of the invention, a peptidomimetic can be
modified to create a constrained peptide that adopts a distinct and
specific preferred conformation, which can increase the potency and
selectivity of the peptide. For example, the constrained peptide
can be an azapeptide (Gante, Synthesis, 405-413, 1989). An
azapeptide is synthesized by replacing the .alpha.-carbon of an
amino acid with a nitrogen atom without changing the structure of
the amino acid side chain. For example, the azapeptide can be
synthesized by using hydrazine in traditional peptide synthesis
coupling methods, such as by reacting hydrazine with a "carbonyl
donor," e.g., phenylchloroformate. A general azapeptide synthesis
is shown in FIG. 6.
[0364] In one embodiment of the invention, a peptide or
peptidomimetic (e.g., a peptide or peptidomimetic tethered to an
ligand-conjugated monomer) can be an N-methyl peptide. N-methyl
peptides are composed of N-methyl amino acids, which provide an
additional methyl group in the peptide backbone, thereby
potentially providing additional means of resistance to proteolytic
cleavage. N-methyl peptides can by synthesized by methods known in
the art (see, for example, Lindgren et al., Trends Pharmacol. Sci.
21:99, 2000; Cell Penetrating Peptides: Processes and Applications,
Langel, ed., CRC Press, Boca Raton, Fla., 2002; Fische et al.,
Bioconjugate. Chem. 12: 825, 2001; Wander et al., J. Am. Chem.
Soc., 124:13382, 2002). For example, an Ant or Tat peptide can be
an N-methyl peptide. An exemplary synthesis is shown in FIG. 7.
[0365] In one embodiment of the invention, a peptide or
peptidomimetic (e.g., a peptide or peptidomimetic tethered to a
ligand-conjugated monomer) can be a .beta.-peptide. .beta.-peptides
form stable secondary structures such as helices, pleated sheets,
turns and hairpins in solutions. Their cyclic derivatives can fold
into nanotubes in the solid state. .beta.-peptides are resistant to
degradation by proteolytic enzymes. .beta.-peptides can be
synthesized by methods known in the art. For example, an Ant or Tat
peptide can be a .beta.-peptide. An exemplary synthesis is shown in
FIG. 8.
[0366] In one embodiment of the invention, a peptide or
peptidomimetic (e.g., a peptide or peptidomimetic tethered to a
ligand-conjugated monomer) can be a oligocarbamate. Oligocarbamate
peptides are internalized into a cell by a transport pathway
facilitated by carbamate transporters. For example, an Ant or Tat
peptide can be an oligocarbamate. An exemplary synthesis is shown
in FIG. 9.
[0367] In one embodiment of the invention, a peptide or
peptidomimetic (e.g., a peptide or peptidomimetic tethered to a
ligand-conjugated monomer) can be an oligourea conjugate (or an
oligothiourea conjugate), in which the amide bond of a
peptidomimetic is replaced with a urea moiety. Replacement of the
amide bond provides increased resistance to degradation by
proteolytic enzymes, e.g., proteolytic enzymes in the
gastrointestinal tract. In one embodiment, an oligourea conjugate
is tethered to an iRNA agent for use in oral delivery. The backbone
in each repeating unit of an oligourea peptidomimetic can be
extended by one carbon atom in comparison with the natural amino
acid. The single carbon atom extension can increase peptide
stability and lipophilicity, for example. An oligourea peptide can
therefore be advantageous when an iRNA agent is directed for
passage through a bacterial cell wall, or when an iRNA agent must
traverse the blood-brain barrier, such as for the treatment of a
neurological disorder. In one embodiment, a hydrogen bonding unit
is conjugated to the oligourea peptide, such as to create an
increased affinity with a receptor. For example, an Ant or Tat
peptide can be an oligourea conjugate (or an oligothiourea
conjugate). An exemplary synthesis is shown in FIG. 10.
[0368] The siRNA peptide conjugates of the invention can be
affiliated with, e.g., tethered to, ligand-conjugated monomers
occurring at various positions on an iRNA agent. For example, a
peptide can be terminally conjugated, on either the sense or the
antisense strand, or a peptide can be bisconjugated (one peptide
tethered to each end, one conjugated to the sense strand, and one
conjugated to the antisense strand). In another option, the peptide
can be internally conjugated, such as in the loop of a short
hairpin iRNA agent. In yet another option, the peptide can be
affiliated with a complex, such as a peptide-carrier complex.
[0369] A peptide-carrier complex consists of at least a carrier
molecule, which can encapsulate one or more iRNA agents (such as
for delivery to a biological system and/or a cell), and a peptide
moiety tethered to the outside of the carrier molecule, such as for
targeting the carrier complex to a particular tissue or cell type.
A carrier complex can carry additional targeting molecules on the
exterior of the complex, or fusogenic agents to aid in cell
delivery. The one or more iRNA agents encapsulated within the
carrier can be conjugated to lipophilic molecules, which can aid in
the delivery of the agents to the interior of the carrier.
[0370] A carrier molecule or structure can be, for example, a
micelle, a liposome (e.g., a cationic liposome), a nanoparticle, a
microsphere, or a biodegradable polymer. A peptide moiety can be
tethered to the carrier molecule by a variety of linkages, such as
a disulfide linkage, an acid labile linkage, a peptide-based
linkage, an oxyamino linkage or a hydrazine linkage. For example, a
peptide-based linkage can be a GFLG peptide. Certain linkages will
have particular advantages, and the advantages (or disadvantages)
can be considered depending on the tissue target or intended use.
For example, peptide based linkages are stable in the blood stream
but are susceptible to enzymatic cleavage in the lysosomes. A
schematic of preferred carriers is shown in FIG. 11.
[0371] The monomer compounds can be separated from a reaction
mixture and further purified by a method such as column
chromatography, high pressure liquid chromatography, or
recrystallization. As can be appreciated by the skilled artisan,
further methods of synthesizing the compounds of the formulae
herein will be evident to those of ordinary skill in the art.
Additionally, the various synthetic steps may be performed in an
alternate sequence or order to give the desired compounds. Other
synthetic chemistry transformations, protecting groups (e.g., for
hydroxyl, amino, etc. present on the bases) and protecting group
methodologies (protection and deprotection) useful in synthesizing
the compounds described herein are known in the art and include,
for example, those such as described in R. Larock, Comprehensive
Organic Transformations, VCH Publishers (1989); T. W. Greene and P.
G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John
Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's
Reagents for Organic Synthesis, John Wiley and Sons (1994); and L.
Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John
Wiley and Sons (1995), and subsequent editions thereof.
[0372] The monomer compounds may contain one or more asymmetric
centers and thus occur as racemates and racemic mixtures, single
enantiomers, individual diastereomers and diastereomeric mixtures.
All such isomeric forms of these compounds are expressly included
in the present invention. The compounds described herein can also
contain linkages (e.g., carbon-carbon bonds, carbon-nitrogen bonds,
e.g., amides) or substituents that can restrict bond rotation, e.g.
restriction resulting from the presence of a ring or double bond.
Accordingly, all cis/trans, E/Z isomers, and rotational isomers
(rotamers) are expressly included herein. The compounds of this
invention may also be represented in multiple tautomeric forms, in
such instances, the invention expressly includes all tautomeric
forms of the compounds described herein (e.g., alkylation of a ring
system may result in alkylation at multiple sites, the invention
expressly includes all such reaction products). All such isomeric
forms of such compounds are expressly included in the present
invention. All crystal forms of the compounds described herein are
expressly included in the present invention.
[0373] Representative ligand-conjugated monomers and typical
syntheses for preparing ligand-conjugated monomers and related
compounds described herein are provided below. As discussed
elsewhere, protecting groups for ligand-conjugated monomer hydroxyl
groups, e.g., OFG.sup.1, include but are not limited to the
dimethoxytrityl group (DMT). For example, it can be desirable in
some embodiments to use silicon-based protecting groups as a
protecting group for OFG.sup.1. Silicon-based protecting groups can
therefore be used in conjunction with or in place of the DMT group
as necessary or desired. Thus, the ligand-conjugated monomers and
syntheses delineated below, which feature the DMT protecting group
as a protecting group for OFG.sup.1, is not to be construed as
limiting in any way to the invention.
[0374] Carbohydrate Conjugated Oligonucleotides
[0375] Galactose, N-Acetylgalactosamine and Mannose conjugate
building blocks for oligonucleotide conjugation.
2 31 32 33 34 2R,4R; 2R,4S; 2S,4S; 2S,4R and Racemic 2R,4R; 2R,4S;
2S,4S; 2S,4R and Racemic 35 36 3R,4R; 3R,4S; 3S,4S; 3S,4R and
Racemic 3R,4R; 3R,4S; 3S,4S; 3S,4R and Racemic 37 38 3R,5R; 3R,5S;
3S,5S; 3S,5R and Racemic 3R,5R; 3R,5S; 3S,5S; 3S,5R and Racemic 39
40 2R,4R; 2R,4S; 2S,4S; 2S,4R and Racemic 2R,4R; 2R,4S; 2S,4S;
2S,4R and Racemic 41
[0376] Bis(Galactose), bis(N-Acetylgalactosamine) and bis(mannose)
conjugate building blocks for oligonucleotide conjugation.
3 42 43 44 45 2R,4R; 2R,4S; 2S,4S; 2S,4R and Racemic 2R,4R; 2R,4S;
2S,4S; 2S,4R and Racemic 46 47 3R,4R; 3R,4S; 3S,4S; 3S,4R and
Racemic 3R,4R; 3R,4S; 3S,4S; 3S,4R and Racemic 48 49 3R,5R; 3R,5S;
3S,5S; 3S,5R and Racemic 3R,5R; 3R,5S; 3S,5S; 3S,5R and Racemic 50
51 2R,4R; 2R,4S; 2S,4S; 2S,4R and Racemic 2R,4R; 2R,4S; 2S,4S;
2S,4R and Racemic 52
[0377]
4TABLE 3 Galactose, N-Acetylgalactosamine and Mannose conjugate
building blocks for postsynthetic oligonucleotide conjugation. 53
54 55 56 57 58 59 60 61
[0378] 62 63 64 65 6667 6869
[0379] iRNA Agent Structure
[0380] The monomers described herein can be used to make
oligonucleotides which are useful as iRNA agents, e.g., RNA
molecules, (double-stranded; single-stranded) that mediate RNAi,
e.g., with respect to an endogenous gene of a subject or to a gene
of a pathogen. In most cases the iRNA agent will incorporate
momomers described herein together with naturally occuring
nucleosides or nucleotides or with other modified nucleosides or
nucleotides. The modified monomers can be present at any position
in the iRNA agent, e.g., at the terminii or in the middle region of
an iRNA agent or in a duplex region or in an unpaired region. In a
preferred embodiment iRNA agent can have any architecture, e.g.,
architecture described herein. E.g., it can be incorporated into an
iRNA agent having an overhang structure, a hairpin or other single
strand structure or a two-strand structure, as described
herein.
[0381] An "RNA agent" as used herein, is an unmodified RNA,
modified RNA, or nucleoside surrogate, all of which are defined
herein (see, e.g., the section below entitled RNA Agents). While
numerous modified RNAs and nucleoside surrogates are described,
preferred examples include those which have greater resistance to
nuclease degradation than do unmodified RNAs. Preferred examples
include those which have a 2' sugar modification, a modification in
a single strand overhang, preferably a 3' single strand overhang,
or, particularly if single stranded, a 5' modification which
includes one or more phosphate groups or one or more analogs of a
phosphate group.
[0382] An "iRNA agent" as used herein, is an RNA agent which can,
or which can be cleaved into an RNA agent which can, down regulate
the expression of a target gene, preferably an endogenous or
pathogen target RNA. While not wishing to be bound by theory, an
iRNA agent may act by one or more of a number of mechanisms,
including post-transcriptional cleavage of a target mRNA sometimes
referred to in the art as RNAi, or pre-transcriptional or
pre-translational mechanisms. An iRNA agent can include a single
strand or can include more than one strands, e.g., it can be a
double stranded iRNA agent. If the iRNA agent is a single strand it
is particularly preferred that it include a 5' modification which
includes one or more phosphate groups or one or more analogs of a
phosphate group.
[0383] The RRMS-containing iRNA agent should include a region of
sufficient homology to the target gene, and be of sufficient length
in terms of nucleotides, such that the iRNA agent, or a fragment
thereof, can mediate down regulation of the target gene. (For ease
of exposition the term nucleotide or ribonucleotide is sometimes
used herein in reference to one or more monomeric subunits of an
RNA agent. It will be understood herein that the usage of the term
"ribonucleotide" or "nucleotide", herein can, in the case of a
modified RNA or nucleotide surrogate, also refer to a modified
nucleotide, or surrogate replacement moiety at one or more
positions.) Thus, the iRNA agent is or includes a region which is
at least partially, and in some embodiments fully, complementary to
the target RNA. It is not necessary that there be perfect
complementarity between the iRNA agent and the target, but the
correspondence must be sufficient to enable the iRNA agent, or a
cleavage product thereof, to direct sequence specific silencing,
e.g., by RNAi cleavage of the target RNA, e.g., mRNA.
[0384] As discussed elsewhere herein, an iRNA agent will often be
modified or include nucleoside surrogates in addition to the ribose
replacement modification subunit (RRMS). Single stranded regions of
an iRNA agent will often be modified or include nucleoside
surrogates, e.g., the unpaired region or regions of a hairpin
structure, e.g., a region which links two complementary regions,
can have modifications or nucleoside surrogates. Modification to
stabilize one or more 3'- or 5'-terminus of an iRNA agent, e.g.,
against exonucleases, or to favor the antisense sRNA agent to enter
into RISC are also favored. Modifications can include C3 (or C6,
C7, C12) amino linkers, thiol linkers, carboxyl linkers,
non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene
glycol, hexaethylene glycol), special biotin or fluorescein
reagents that come as phosphoramidites and that have another
DMT-protected hydroxyl group, allowing multiple couplings during
RNA synthesis.
[0385] In addition to homology to target RNA and the ability to
down regulate a target gene, an iRNA agent will preferably have one
or more of the following properties:
[0386] (1) it will be of the Formula 1, 2, 3, or 4 set out in the
RNA Agent section below;
[0387] (2) if single stranded it will have a 5' modification which
includes one or more phosphate groups or one or more analogs of a
phosphate group;
[0388] (3) it will, despite modifications, even to a very large
number, or all of the nucleosides, have an antisense strand that
can present bases (or modified bases) in the proper three
dimensional framework so as to be able to form correct base pairing
and form a duplex structure with a homologous target RNA which is
sufficient to allow down regulation of the target, e.g., by
cleavage of the target RNA;
[0389] (4) it will, despite modifications, even to a very large
number, or all of the nucleosides, still have "RNA-like"
properties, i.e., it will possess the overall structural, chemical
and physical properties of an RNA molecule, even though not
exclusively, or even partly, of ribonucleotide-based content. For
example, an iRNA agent can contain, e.g., a sense and/or an
antisense strand in which all of the nucleotide sugars contain
e.g., 2' fluoro in place of 2' hydroxyl. This
deoxyribonucleotide-containing agent can still be expected to
exhibit RNA-like properties. While not wishing to be bound by
theory, the electronegative fluorine prefers an axial orientation
when attached to the C2' position of ribose. This spatial
preference of fluorine can, in turn, force the sugars to adopt a
C.sub.3'-endo pucker. This is the same puckering mode as observed
in RNA molecules and gives rise to the RNA-characteristic
A-family-type helix. Further, since fluorine is a good hydrogen
bond acceptor, it can participate in the same hydrogen bonding
interactions with water molecules that are known to stabilize RNA
structures. (Generally, it is preferred that a modified moiety at
the 2' sugar position will be able to enter into H-bonding which is
more characteristic of the OH moiety of a ribonucleotide than the H
moiety of a deoxyribonucleotide. A preferred iRNA agent will:
exhibit a C.sub.3'-endo pucker in all, or at least 50, 75, 80, 85,
90, or 95% of its sugars; exhibit a C.sub.3'-endo pucker in a
sufficient amount of its sugars that it can give rise to a the
RNA-characteristic A-family-type helix; will have no more than 20,
10, 5, 4, 3, 2, or 1 sugar which is not a C.sub.3'-endo pucker
structure. These limitations are particularly preferably in the
antisense strand;
[0390] (4) regardless of the nature of the modification, and even
though the RNA agent can contain deoxynucleotides or modified
deoxynucleotides, particularly in overhang or other single strand
regions, it is preferred that DNA molecules, or any molecule in
which more than 50, 60, or 70% of the nucleotides in the molecule,
or more than 50, 60, or 70% of the nucleotides in a duplexed region
are deoxyribonucleotides, or modified deoxyribonucleotides which
are deoxy at the 2' position, are excluded from the definition of
RNA agent.
[0391] A "single strand iRNA agent" as used herein, is an iRNA
agent which is made up of a single molecule. It may include a
duplexed region, formed by intra-strand pairing, e.g., it may be,
or include, a hairpin or pan-handle structure. Single strand iRNA
agents are preferably antisense with regard to the target molecule.
In preferred embodiments single strand iRNA agents are 5'
phosphorylated or include a phosphoryl analog at the 5' prime
terminus. 5'-phosphate modifications include those which are
compatible with RISC mediated gene silencing. Suitable
modifications include: 5'-monophosphate ((HO)2(O)P--O-5');
5'-diphosphate ((HO)2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate
((HO)2(O)P--O--(HO)(O)P--- O--P(HO)(O)--O-5'); 5'-guanosine cap
(7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine
cap (Appp), and any modified or unmodified nucleotide cap structure
(N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5');
5'-monothiophosphate (phosphorothioate; (HO)2(S)P--O-5');
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO)2(O)P--S-5'); any additional combination
of oxygen/sulfur replaced monophosphate, diphosphate and
triphosphates (e.g. 5'-alpha-thiotriphosphate,
5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates
((HO)2(O)P--NH-5', (HO)(NH2)(O)P--O-5'), 5'-alkylphosphonates
(R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g.
RP(OH)(O)--O-5'--, (OH).sub.2(O)P-5'-CH2--),
5'-alkyletherphosphonat- es (R=alkylether=methoxymethyl (MeOCH2--),
ethoxymethyl, etc., e.g. RP(OH)(O)--O-5'--). (These modifications
can also be used with the antisense strand of a double stranded
iRNA.)
[0392] It may be desirable to modify one or both of the antisense
and sense strands of a double strand iRNA agent. In some cases they
will have the same modification or the same class of modification
but in other cases the sense and antisense strand will have
different modifications, e.g., in some cases it is desirable to
modify only the sense strand. It may be desirable to modify only
the sense strand, e.g., to inactivate it, e.g., the sense strand
can be modified in order to inactivate the sense strand and prevent
formation of an active sRNA/protein or RISC. This can be
accomplished by a modification which prevents 5'-phosphorylation of
the sense strand, e.g., by modification with a 5'-O-methyl
ribonucleotide (see Nyknen et al., (2001) ATP requirements and
small interfering RNA structure in the RNA interference pathway.
Cell 107, 309-321.) Other modifications which prevent
phosphorylation can also be used, e.g., simply substituting the
5'-OH by H rather than O--Me. Alternatively, a large bulky group
may be added to the 5'-phosphate turning it into a phosphodiester
linkage, though this may be less desirable as phosphodiesterases
can cleave such a linkage and release a functional sRNA 5'-end.
Antisense strand modifications include 5' phosphorylation as well
as any of the other 5' modifications discussed herein, particularly
the 5' modifications discussed above in the section on single
stranded iRNA molecules.
[0393] In some cases the sense and the antisense strands will
include different modifications. Multiple different modifications
can be included on the sense and antisense strands. The
modifications on each strand may differ from each other, and may
also differ from the various modifications on the other strand. For
example, the sense strand may have a modification, e.g., a
modification described herein, and the antisense strand may have a
different modification, e.g., a different modification described
herein. In other cases, one strand, such as the sense strand may
have two different modifications, and the antisense strand may
include a modification that differs from the at least two
modifications on the sense strand.
[0394] It is preferred that the sense and antisense strands be
chosen such that the ds iRNA agent includes a single strand or
unpaired region at one or both ends of the molecule. Thus, a ds
iRNA agent contains sense and antisense strands, preferable paired
to contain an overhang, e.g., one or two 5' or 3' overhangs but
preferably a 3' overhang of 2-3 nucleotides. Most embodiments will
have a 3' overhang. Preferred sRNA agents will have single-stranded
overhangs, preferably 3' overhangs, of 1 or preferably 2 or 3
nucleotides in length at each end. The overhangs can be the result
of one strand being longer than the other, or the result of two
strands of the same length being staggered. 5' ends are preferably
phosphorylated.
[0395] Preferred lengths for the duplexed region is between 15 and
30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in
length, e.g., in the sRNA agent range discussed above. sRNA agents
can resemble in length and structure the natural Dicer processed
products from long dsRNAs. Embodiments in which the two strands of
the sRNA agent are linked, e.g., covalently linked are also
included. Hairpin, or other single strand structures which provide
the required double stranded region, and preferably a 3' overhang
are also within the invention.
[0396] As nucleic acids are polymers of subunits or monomers, many
of the modifications described below occur at a position which is
repeated within a nucleic acid, e.g., a modification of a base, or
a phosphate moiety, or the a non-linking O of a phosphate moiety.
In some cases the modification will occur at all of the subject
positions in the nucleic acid but in many, and infact in most cases
it will not. By way of example, a modification may only occur at a
3' or 5' terminal position, may only occur in a terminal regions,
e.g. at a position on a terminal nucleotide or in the last 2, 3, 4,
5, or 10 nucleotides of a strand. A modification may occur in a
double strand region, a single strand region, or in both. A
modification may occur only in the double strand region of an RNA
or may only occur in a single strand region of an RNA. E.g., a
phosphorothioate modification at a non-linking O position may only
occur at one or both termini, may only occur in a terminal regions,
e.g., at a position on a terminal nucleotide or in the last 2, 3,
4, 5, or 10 nucleotides of a strand, or may occur in double strand
and single strand regions, particularly at termini. The 5' end or
ends can be phosphorylated.
[0397] In some embodiments it is particularly preferred, e.g., to
enhance stability, to include particular bases in overhangs, or to
include modified nucleotides or nucleotide surrogates, in single
strand overhangs, e.g., in a 5' or 3' overhang, or in both. E.g.,
it can be desirable to include purine nucleotides in overhangs. In
some embodiments all or some of the bases in a 3' or 5' overhang
will be modified, e.g., with a modification described herein.
Modifications can include, e.g., the use of modifications at the 2'
OH group of the ribose sugar, e.g., the use of
deoxyribonucleotides, e.g., deoxythymidine, instead of
ribonucleotides, and modifications in the phosphate group, e.g.,
phosphothioate modifications. Overhangs need not be homologous with
the target sequence.
[0398] Modifications and nucleotide surrogates are discussed below.
70
[0399] The scaffold presented above in Formula 1 represents a
portion of a ribonucleic acid. The basic components are the ribose
sugar, the base, the terminal phosphates, and phosphate
internucleotide linkers. Where the bases are naturally occurring
bases, e.g., adenine, uracil, guanine or cytosine, the sugars are
the unmodified 2' hydroxyl ribose sugar (as depicted) and W, X, Y,
and Z are all O, Formula 1 represents a naturally occurring
unmodified oligoribonucleotide.
[0400] Unmodified oligoribonucleotides may be less than optimal in
some applications, e.g., unmodified oligoribonucleotides can be
prone to degradation by e.g., cellular nucleases. Nucleases can
hydrolyze nucleic acid phosphodiester bonds. However, chemical
modifications to one or more of the above RNA components can confer
improved properties, and, e.g., can render oligoribonucleotides
more stable to nucleases. Umodified oligoribonucleotides may also
be less than optimal in terms of offering tethering points for
attaching ligands or other moieties to an iRNA agent.
[0401] Modified nucleic acids and nucleotide surrogates can include
one or more of:
[0402] (i) alteration, e.g., replacement, of one or both of the
non-linking (X and Y) phosphate oxygens and/or of one or more of
the linking (W and Z) phosphate oxygens (When the phosphate is in
the terminal position, one of the positions W or Z will not link
the phosphate to an additional element in a naturally occurring
ribonucleic acid. However, for simplicity of terminology, except
where otherwise noted, the W position at the 5'end of a nucleic
acid and the terminal Z position at the 3'end of a nucleic acid,
are within the term "linking phosphate oxygens" as used
herein.);
[0403] (ii) alteration, e.g., replacement, of a constituent of the
ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar, or
wholesale replacement of the ribose sugar with a structure other
than ribose, e.g., as described herein;
[0404] (iii) wholesale replacement of the phosphate moiety (bracket
I) with "dephospho" linkers;
[0405] (iv) modification or replacement of a naturally occurring
base;
[0406] (v) replacement or modification of the ribose-phosphate
backbone (bracket II);
[0407] (vi) modification of the 3' end or 5' end of the RNA, e.g.,
removal, modification or replacement of a terminal phosphate group
or conjugation of a moiety, e.g. a fluorescently labeled moiety, to
either the 3' or 5' end of RNA.
[0408] The terms replacement, modification, alteration, and the
like, as used in this context, do not imply any process limitation,
e.g., modification does not mean that one must start with a
reference or naturally occurring ribonucleic acid and modify it to
produce a modified ribonucleic acid bur rather modified simply
indicates a difference from a naturally occurring molecule.
[0409] It is understood that the actual electronic structure of
some chemical entities cannot be adequately represented by only one
canonical form (i.e. Lewis structure). While not wishing to be
bound by theory, the actual structure can instead be some hybrid or
weighted average of two or more canonical forms, known collectively
as resonance forms or structures. Resonance structures are not
discrete chemical entities and exist only on paper. They differ
from one another only in the placement or "localization" of the
bonding and nonbonding electrons for a particular chemical entity.
It can be possible for one resonance structure to contribute to a
greater extent to the hybrid than the others. Thus, the written and
graphical descriptions of the embodiments of the present invention
are made in terms of what the art recognizes as the predominant
resonance form for a particular species. For example, any
phosphoroamidate (replacement of a nonlinking oxygen with nitrogen)
would be represented by X.dbd.O and Y.dbd.N in the above
figure.
[0410] Specific modifications are discussed in more detail
below.
[0411] The Phosphate Group
[0412] The phosphate group is a negatively charged species. The
charge is distributed equally over the two non-linking oxygen atoms
(i.e., X and Y in Formula 1 above). However, the phosphate group
can be modified by replacing one of the oxygens with a different
substituent. One result of this modification to RNA phosphate
backbones can be increased resistance of the oligoribonucleotide to
nucleolytic breakdown. Thus while not wishing to be bound by
theory, it can be desirable in some embodiments to introduce
alterations which result in either an uncharged linker or a charged
linker with unsymmetrical charge distribution.
[0413] Examples of modified phosphate groups include
phosphorothioate, phosphoroselenates, borano phosphates, borano
phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl
or aryl phosphonates and phosphotriesters. Phosphorodithioates have
both non-linking oxygens replaced by sulfur. Unlike the situation
where only one of X or Y is altered, the phosphorus center in the
phosphorodithioates is achiral which precludes the formation of
oligoribonucleotides diastereomers. Diastereomer formation can
result in a preparation in which the individual diastereomers
exhibit varying resistance to nucleases. Further, the hybridization
affinity of RNA containing chiral phosphate groups can be lower
relative to the corresponding unmodified RNA species. Thus, while
not wishing to be bound by theory, modifications to both X and Y
which eliminate the chiral center, e.g. phosphorodithioate
formation, may be desirable in that they cannot produce
diastereomer mixtures. Thus, X can be any one of S, Se, B, C, H, N,
or OR (R is alkyl or aryl). Thus Y can be any one of S, Se, B, C,
H, N, or OR (R is alkyl or aryl). Replacement of X and/or Y with
sulfur is preferred.
[0414] The phosphate linker can also be modified by replacement of
a linking oxygen (i.e., W or Z in Formula 1) with nitrogen (bridged
phosphoroamidates), sulfur (bridged phosphorothioates) and carbon
(bridged methylenephosphonates). The replacement can occur at a
terminal oxygen (position W (3') or position Z (5'). Replacement of
W with carbon or Z with nitrogen is preferred.
[0415] Candidate agents can be evaluated for suitability as
described below.
[0416] The Sugar Group
[0417] A modified RNA can include modification of all or some of
the sugar groups of the ribonucleic acid. E.g., the 2' hydroxyl
group (OH) can be modified or replaced with a number of different
"oxy" or "deoxy" substituents. While not being bound by theory,
enhanced stability is expected since the hydroxyl can no longer be
deprotonated to form a 2' alkoxide ion. The 2' alkoxide can
catalyze degradation by intramolecular nucleophilic attack on the
linker phosphorus atom. Again, while not wishing to be bound by
theory, it can be desirable to some embodiments to introduce
alterations in which alkoxide formation at the 2' position is not
possible.
[0418] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2C- H.sub.2OR; "locked" nucleic
acids (LNA) in which the 2' hydroxyl is connected, e.g., by a
methylene bridge, to the 4' carbon of the same ribose sugar;
O-AMINE (AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino) and aminoalkoxy,
O(CH.sub.2).sub.nAMINE, (e.g., AMINE=NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, or diheteroaryl amino, ethylene diamine, polyamino). It is
noteworthy that oligonucleotides containing only the methoxyethyl
group (MOE), (OCH.sub.2CH.sub.2OCH.sub.3, a PEG derivative),
exhibit nuclease stabilities comparable to those modified with the
robust phosphorothioate modification.
[0419] "Deoxy" modifications include hydrogen (i.e. deoxyribose
sugars, which are of particular relevance to the overhang portions
of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2C- H.sub.2-AMINE
(AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino,
diaryl amino, heteroaryl amino, or diheteroaryl amino), --NHC(O)R
(R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano;
mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl,
aryl, alkenyl and alkynyl, which may be optionally substituted with
e.g., an amino functionality. Preferred substitutents are
2'-methoxyethyl, 2'-OCH3, 2'-O-allyl, 2'-C-allyl, and
2'-fluoro.
[0420] The sugar group can also contain one or more carbons that
possess the opposite stereochemical configuration than that of the
corresponding carbon in ribose. Thus, a modified RNA can include
nucleotides containing e.g., arabinose, as the sugar.
[0421] Modified RNAs can also include "abasic" sugars, which lack a
nucleobase at C-1'. These abasic sugars can also be further contain
modifications at one or more of the constituent sugar atoms.
[0422] To maximize nuclease resistance, the 2' modifications can be
used in combination with one or more phosphate linker modifications
(e.g., phosphorothioate). The so-called "chimeric" oligonucleotides
are those that contain two or more different modifications.
[0423] The modificaton can also entail the wholesale replacement of
a ribose structure with another entity at one or more sites in the
iRNA agent.
[0424] Candidate modifications can be evaluated as described
below.
[0425] Replacement of the Phosphate Group
[0426] The phosphate group can be replaced by non-phosphorus
containing connectors (cf. Bracket I in Formula 1 above). While not
wishing to be bound by theory, it is believed that since the
charged phosphodiester group is the reaction center in nucleolytic
degradation, its replacement with neutral structural mimics should
impart enhanced nuclease stability. Again, while not wishing to be
bound by theory, it can be desirable, in some embodiment, to
introduce alterations in which the charged phosphate group is
replaced by a neutral moiety.
[0427] Examples of moieties which can replace the phosphate group
include siloxane, carbonate, carboxymethyl, carbamate, amide,
thioether, ethylene oxide linker, sulfonate, sulfonamide,
thioformacetal, formacetal, oxime, methyleneimino,
methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo
and methyleneoxymethylimino. Preferred replacements include the
methylenecarbonylamino and methylenemethylimino groups.
[0428] Candidate modifications can be evaluated as described
below.
[0429] Replacement of Ribophosphate Backbone
[0430] Oligonucleotide-mimicking scaffolds can also be constructed
wherein the phosphate linker and ribose sugar are replaced by
nuclease resistant nucleoside or nucleotide surrogates (see Bracket
II of Formula 1 above). While not wishing to be bound by theory, it
is believed that the absence of a repetitively charged backbone
diminishes binding to proteins that recognize polyanions (e.g.
nucleases). Again, while not wishing to be bound by theory, it can
be desirable in some embodiment, to introduce alterations in which
the bases are tethered by a neutral surrogate backbone.
[0431] Examples include the mophilino, cyclobutyl, pyrrolidine and
peptide nucleic acid (PNA) nucleoside surrogates. A preferred
surrogate is a PNA surrogate.
[0432] Candidate modifications can be evaluated as described
below.
[0433] Terminal Modifications
[0434] The 3' and 5' ends of an oligonucleotide can be modified.
Such modifications can be at the 3' end, 5' end or both ends of the
molecule. They can include modification or replacement of an entire
terminal phosphate or of one or more of the atoms of the phosphate
group. E.g., the 3' and 5' ends of an oligonucleotide can be
conjugated to other functional molecular entities such as labeling
moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3
or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon,
boron or ester). The functional molecular entities can be attached
to the sugar through a phosphate group and/or a spacer. The
terminal atom of the spacer can connect to or replace the linking
atom of the phosphate group or the C-3' or C-5' O, N, S or C group
of the sugar. Alternatively, the spacer can connect to or replace
the terminal atom of a nucleotide surrogate (e.g., PNAs). These
spacers or linkers can include e.g., --(CH.sub.2).sub.n--,
--(CH.sub.2).sub.nN--, --(CH.sub.2).sub.nO--,
--(CH.sub.2).sub.nS--, O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OH
(e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine,
oxyimine, thioether, disulfide, thiourea, sulfonamide, or
morpholino, or biotin and fluorescein reagents. When a
spacer/phosphate-functional molecular entity-spacer/phosphate array
is interposed between two strands of iRNA agents, this array can
substitute for a hairpin RNA loop in a hairpin-type RNA agent. The
3' end can be an --OH group. While not wishing to be bound by
theory, it is believed that conjugation of certain moieties can
improve transport, hybridization, and specificity properties.
Again, while not wishing to be bound by theory, it may be desirable
to introduce terminal alterations that improve nuclease resistance.
Other examples of terminal modifications include dyes,
intercalating agents (e.g. acridines), cross-linkers (e.g.
psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin),
polycyclic aromatic hydrocarbons (e.g., phenazine,
dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic
carriers (e.g., cholesterol, cholic acid, adamantane acetic acid,
1-pyrene butyric acid, dihydrotestosterone,
1,3-Bis-O(hexadecyl)gly- cerol, geranyloxyhexyl group,
hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl
group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid,
O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and
peptide conjugates (e.g., antennapedia peptide, Tat peptide),
alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K),
MPEG, [MPEG].sub.2, polyamino, alkyl, substituted alkyl,
radiolabeled markers, enzymes, haptens (e.g. biotin),
transport/absorption facilitators (e.g., aspirin, vitamin E, folic
acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,
histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+
complexes of tetraazamacrocycles).
[0435] Terminal modifications can be added for a number of reasons,
including as discussed elsewhere herein to modulate activity or to
modulate resistance to degradation. Terminal modifications useful
for modulating activity include modification of the 5' end with
phosphate or phosphate analogs. E.g., in preferred embodiments iRNA
agents, especially antisense strands, are 5' phosphorylated or
include a phosphoryl analog at the 5' prime terminus. 5'-phosphate
modifications include those which are compatible with RISC mediated
gene silencing. Suitable modifications include: 5'-monophosphate
((HO)2(O)P--O-5'); 5'-diphosphate ((HO)2(O)P--O--P(HO)(O)--O-5');
5'-triphosphate ((HO)2(O)P--O--(HO)(O)P--- O--P(HO)(O)--O-5');
5'-guanosine cap (7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine
cap (Appp), and any modified or unmodified nucleotide cap structure
(N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5');
5'-monothiophosphate (phosphorothioate; (HO)2(S)P--O-5');
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO)2(O)P--S-5'); any additional combination
of oxgen/sulfur replaced monophosphate, diphosphate and
triphosphates (e.g. 5'-alpha-thiotriphosphate,
5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates
((HO)2(O)P--NH-5', (HO)(NH2)(O)P--O-5'), 5'-alkylphosphonates
(R-alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g.
RP(OH)(O)--O-5'-, (OH)2(O)P-5'-CH2-), 5'-alkyletherphosphonates
(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.
RP(OH)(O)--O-5'-).
[0436] Terminal modifications can also be useful for monitoring
distribution, and in such cases the preferred groups to be added
include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa
488. Terminal modifications can also be useful for enhancing
uptake, useful modifications for this include cholesterol. Terminal
modifications can also be useful for cross-linking an RNA agent to
another moiety; modifications useful for this include mitomycin
C.
[0437] Candidate modifications can be evaluated as described
below.
[0438] The Bases
[0439] Adenine, guanine, cytosine and uracil are the most common
bases found in RNA. These bases can be modified or replaced to
provide RNA's having improved properties. E.g., nuclease resistant
oligoribonucleotides can be prepared with these bases or with
synthetic and natural nucleobases (e.g., inosine, thymine,
xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine)
and any one of the above modifications. Alternatively, substituted
or modified analogs of any of the above bases, e.g., "unusual
bases" and "universal bases" described herein, can be employed.
Examples include without limitation 2-aminoadenine, 6-methyl and
other alkyl derivatives of adenine and guanine, 2-propyl and other
alkyl derivatives of adenine and guanine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine
and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,
5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino,
thiol, thioalkyl, hydroxyl and other 8-substituted adenines and
guanines, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine, 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine,
2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,
7-deazaadenine, N6,N6-dimethyladenine, 2,6-diaminopurine,
5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles,
2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil,
uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,
5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,
5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,
3-methylcytosine, 5-methylcytosine, N.sup.4-acetyl cytosine,
2-thiocytosine, N6-methyladenine, N6-isopentyladenine,
2-methylthio-N6-isopentenyladenine, N-methylguanines, or
O-alkylated bases. Further purines and pyrimidines 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, and
those disclosed by Englisch et al., Angewandte Chemie,
International Edition, 1991, 30, 613.
[0440] Generally, base changes are less preferred for promoting
stability, but they can be useful for other reasons, e.g., some,
e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent.
Modified bases can reduce target specificity. This should be taken
into consideration in the design of iRNA agents.
[0441] Candidate modifications can be evaluated as described
below.
[0442] Exemplary Modifications and Placement Within an iRNA
Agent
[0443] Some modifications may preferably be included on an iRNA
agent at a particular location, e.g., on the sense strand or
antisense strand, or on the 5' or 3' end of the sense or antisense
strand of an iRNA agent. A preferred location of a modification on
an iRNA agent, may confer preferred properties on the agent. For
example, preferred locations of particular modifications may confer
optimum gene silencing properties, or increased resistance to
endonuclease or exonuclease activity. A modification described
herein and below may be the sole modification, or the sole type of
modification included on multiple ribonucleotides, or a
modification can be combined with one or more other modifications
described herein and below. For example, a modification on a sense
strand of a dsRNA agent can be different than a modification on the
antisense strand of an iRNA agent. Similary, two different
modifications on the sense strand can differ from a modification on
the antisense strand. Other additional unique modifications,
without limitation, can be incorporates into the sense and
antisense strands.
[0444] An iRNA agent may include a backbone modification to any
nucleotide on an iRNA strand. For example, an iRNA agent may
include a phosphorothioate linkage or P-alkyl modification in the
linkages between one or more nucleotides of an iRNA agent. The
nucleotides can be terminal nucleotides, e.g., nucleotides at the
last position of a sense or antisense strand, or internal
nucleotides.
[0445] An iRNA agent can include a a sugar modification, e.g., a 2'
or 3' sugar modification. Exemplary sugar modifications include,
for example, a 2'-O-methylated nucleotide, a 2'-deoxy nucleotide,
(e.g., a 2'-deoxyfluoro nucleotide), a 2'-O-methoxyethyl
nucleotide, a 2'-O-NMA, a 2'-DMAEOE, a 2'-aminopropyl, 2'-hydroxy,
or a 2'-ara-fluoro or a locked nucleic acid (LNA), extended nucleic
acid (ENA), hexose nucleic acid (HNA), or cyclohexene nucleic acid
(CeNA). A 2' modification is preferably 2'OMe, and more preferably,
2'-deoxyfluoro. When the modification is 2'OMe, the modification is
preferably on the sense strand. When the modification is a 2'
fluoro, and the modification may be on the sense or antisense
strand, or on both strands. A 2'-ara-fluoro modification will
preferably be on the sense strand of the iRNA agent. An LNA
modification will preferably be on the sense strand of the iRNA
agent or on the
[0446] An iRNA agent may include a 3' sugar modification, e.g., a
3'OMe modification. Preferably a 3'OMe modification is on the sense
strand of the iRNA agent.
[0447] An iRNA agent may includes a 5'-methyl-pyrimidine (e.g., a
5'-methyl-uridine modification or a 5'-methyl-cytodine)
modification.
[0448] The modifications described herein can be combined onto a
single iRNA agent. For example, an iRNA agent may have a
phosphorothioate linkage and a 2' sugar modification, e.g., a 2'OMe
or 2.degree. F. modification. In another example, an iRNA agent may
include at least one 5' Me-pyrimidine and a 2' sugar modification,
e.g., a 2.degree. F. or 2'OMe modification.
[0449] An iRNA agent may include a nucleobase modification, such as
a cationic modification, such as a 3'-abasic cationic modification.
The cationic modification can be e.g., an alkylamino-dT (e.g., a C6
amino-dT), an allylamino conjugate, a pyrrolidine conjugate, a
pthalamido, a porphyrin, or a hydroxyprolinol conjugate, on one or
more of the terminal nucleotides of the iRNA agent. When an
alkylamino-dT conjugate is attached to the terminal nucleotide of
an iRNA agent, the conjugate is preferably attached to the 3' end
of the sense or antisense strand of an iRNA agent. When a
pyrrolidine linker is attached to the terminal nucleotide of an
iRNA agent, the linker is preferably attached to the 3' or 5' end
of the sense strand, or the 3' end of the antisense strand. When a
pyrrolidine linker is attached to the terminal nucleotide of an
iRNA agent, the linker is preferably on the 3' or 5' end of the
sense strand, and not on the 5' end of the antisense strand.
[0450] An iRNA agent may include at least one conjugate, such as a
lipophile, a terpene, a protein binding agent, a vitamin, a
carbohydrate, or a peptide. For example, the conjugate can be
naproxen, nitroindole (or another conjugate that contributes to
stacking interactions), folate, ibuprofen, or a Cs pyrimidine
linker. The conjugate can also be a glyceride lipid conjugate
(e.g., a dialkyl glyceride derivatives), vitamin E conjugate, or a
thio-cholesterol. In generally, and except where noted to the
contrary below, when a conjugate is on the terminal nucleotide of a
sense or antisense strand, the conjugate is preferably on the 5' or
3' end of the sense strand or on the 5' end of the antisense
strand, and preferably the conjugate is not on the 3' end of the
antisense strand.
[0451] When the conjugate is naproxen, and the conjugate is on the
terminal nucleotide of a sense or antisense strand, the conjugate
is preferably on the 5' or 3' end of the sense or antisense
strands. When the conjugate is cholesterol, and the conjugate is on
the terminal nucleotide of a sense or antisense strand, the
cholesterol conjugate is preferably on the 5' or 3' end of the
sense strand and preferably not present on the antisense strand.
Cholesterol may be conjugated to the iRNA agent by a pyrrolidine
linker, serinol linker, hydroxyprolinol linker, or disulfide
linkage. A dU-cholesterol conjugate may also be conjugated to the
iRNA agent by a disulfide linkage. When the conjugate is cholanic
acid, and the conjugate is on the terminal nucleotide of a sense or
antisense strand, the cholanic acid is preferably attached to the
5' or 3' end of the sense strand, or the 3' end of the antisense
strand. In one embodiment, the cholanic acid is attached to the 3'
end of the sense strand and the 3' end of the antisense strand.
[0452] One or more nucleotides of an iRNA agent may have a 2'-5'
linkage. Preferably, the 2'-5' linkage is on the sense strand. When
the 2'-5' linkage is on the terminal nucleotide of an iRNA agent,
the 2'-5' linkage occurs on the 5' end of the sense strand.
[0453] The iRNA agent may include an L-sugar, preferably on the
sense strand, and not on the antisense strand.
[0454] The iRNA agent may include a methylphosphonate modification.
When the methylphosphonate is on the terminal nucleotide of an iRNA
agent, the methylphosphonate is at the 3' end of the sense or
antisense strands of the iRNA agent.
[0455] An iRNA agent may be modified by replacing one or more
ribonucleotides with deoxyribonucleotides. Preferably, adjacent
deoxyribonucleotides are joined by phosphorothioate linkages, and
the iRNA agent does not include more than four consecutive
deoxyribonucleotides on the sense or the antisense strands.
[0456] An iRNA agent may include a difluorotoluyl (DFT)
modification, e.g., 2,4-difluorotoluyl uracil, or a guanidine to
inosine substitution.
[0457] The iRNA agent may include at least one
5'-uridine-adenine-3' (5'-UA-3') dinucleotide wherein the uridine
is a 2'-modified nucleotide, or a terminal 5'-uridine-guanine-3'
(5'-UG-3') dinucleotide, wherein the 5'-uridine is a 2'-modified
nucleotide, or a terminal 5'-cytidine-adenine-3' (5'-CA-3')
dinucleotide, wherein the 5'-cytidine is a 2'-modified nucleotide,
or a terminal 5'-uridine-uridine-3' (5'-UU-3') dinucleotide,
wherein the 5'-uridine is a 2'-modified nucleotide, or a terminal
5'-cytidine-cytidine-3' (5'-CC-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-cytidine-uridine-3' (5'-CU-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-uridine-cytidine-3' (5'-UC-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide. The chemically modified
nucleotide in the iRNA agent may be a 2'-O-methylated nucleotide.
In some embodiments, the modified nucleotide can be a 2'-deoxy
nucleotide, a 2'-deoxyfluoro nucleotide, a 2'-O-methoxyethyl
nucleotide, a 2'-O-NMA, a 2'-DMAEOE, a 2'-aminopropyl, 2'-hydroxy,
or a 2'-ara-fluoro, or a locked nucleic acid (LNA), extended
nucleic acid (ENA), hexose nucleic acid (HNA), or cyclohexene
nucleic acid (CeNA). The iRNA agents including these modifications
are particularly stabilized against exonuclease activity, when the
modified dinucleotide occurs on a terminal end of the sense or
antisense strand of an iRNA agent, and are otherwise particularly
stabilized against endonuclease activity.
[0458] An iRNA agent may have a single overhang, e.g., one end of
the iRNA agent has a 3' or 5' overhang and the other end of the
iRNA agent is a blunt end, or the iRNA agent may have a double
overhang, e.g., both ends of the iRNA agent have a 3' or 5'
overhang, such as a dinuclotide overhang. In another altervative,
both ends of the iRNA agent may have blunt ends.
[0459] The iRNA agent may further include a sense RNA strand and an
antisense RNA strand, wherein the antisense RNA strand is 25 or
fewer nucleotides in length, and includes an antisense nucleotide
sequence having 18-25 nucleotides in length. The iRNA agent may
further include a nucleotide overhang having 1 to 4 unpaired
nucleotides, which may be at the 3'-end of the antisense RNA
strand, and the nucleotide overhang may have the nucleotide
sequence 5'-GC-3' or 5'-CGC-3'. The unpaired nucleotides may have
at least one phosphorothioate dinucleotide linkage, and at least
one of the unpaired nucleotides may be chemically modified in the
2'-position. The doublestrand region of the iRNA agent may include
phosphorothioate dinucleotide linkages on one or both of the sense
and antisense strands. The antisense RNA strand and the sense RNA
strand may be connected with a linker, e.g., a chemical linker such
as hexaethylene glycol linker, a
poly-(oxyphosphinico-oxy-1,3-propandiol) linker, an allyl linker,
or a polyethylene glycol linker.
[0460] Evaluation of Candidate RNA's
[0461] One can evaluate a candidate RNA agent, e.g., a modified
RNA, for a selected property by exposing the agent or modified
molecule and a control molecule to the appropriate conditions and
evaluating for the presence of the selected property. For example,
resistance to a degradent can be evaluated as follows. A candidate
modified RNA (and preferably a control molecule, usually the
unmodified form) can be exposed to degradative conditions, e.g.,
exposed to a milieu, which includes a degradative agent, e.g., a
nuclease. E.g., one can use a biological sample, e.g., one that is
similar to a milieu, which might be encountered, in therapeutic
use, e.g., blood or a cellular fraction, e.g., a cell-free
homogenate or disrupted cells. The candidate and control could then
be evaluated for resistance to degradation by any of a number of
approaches. For example, the candidate and control could be
labeled, preferably prior to exposure, with, e.g., a radioactive or
enzymatic label, or a fluorescent label, such as Cy3 or Cy5.
Control and modified RNA's can be incubated with the degradative
agent, and optionally a control, e.g., an inactivated, e.g., heat
inactivated, degradative agent. A physical parameter, e.g., size,
of the modified and control molecules are then determined. They can
be determined by a physical method, e.g., by polyacrylamide gel
electrophoresis or a sizing column, to assess whether the molecule
has maintained its original length, or assessed functionally.
Alternatively, Northern blot analysis can be used to assay the
length of an unlabeled modified molecule.
[0462] A functional assay can also be used to evaluate the
candidate agent. A functional assay can be applied initially or
after an earlier non-functional assay, (e.g., assay for resistance
to degradation) to determine if the modification alters the ability
of the molecule to silence gene expression. For example, a cell,
e.g., a mammalian cell, such as a mouse or human cell, can be
co-transfected with a plasmid expressing a fluorescent protein,
e.g., GFP, and a candidate RNA agent homologous to the transcript
encoding the fluorescent protein (see, e.g., WO 00/44914). For
example, a modified dsRNA homologous to the GFP mRNA can be assayed
for the ability to inhibit GFP expression by monitoring for a
decrease in cell fluorescence, as compared to a control cell, in
which the transfection did not include the candidate dsRNA, e.g.,
controls with no agent added and/or controls with a non-modified
RNA added. Efficacy of the candidate agent on gene expression can
be assessed by comparing cell fluorescence in the presence of the
modified and unmodified dsRNA agents.
[0463] In an alternative functional assay, a candidate dsRNA agent
homologous to an endogenous mouse gene, preferably a maternally
expressed gene, such as c-mos, can be injected into an immature
mouse oocyte to assess the ability of the agent to inhibit gene
expression in vivo (see, e.g., WO 01/36646). A phenotype of the
oocyte, e.g., the ability to maintain arrest in metaphase II, can
be monitored as an indicator that the agent is inhibiting
expression. For example, cleavage of c-mos mRNA by a dsRNA agent
would cause the oocyte to exit metaphase arrest and initiate
parthenogenetic development (Colledge et al. Nature 370: 65-68,
1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of the
modified agent on target RNA levels can be verified by Northern
blot to assay for a decrease in the level of target mRNA, or by
Western blot to assay for a decrease in the level of target
protein, as compared to a negative control. Controls can include
cells in which with no agent is added and/or cells in which a
non-modified RNA is added.
[0464] References
[0465] General References
[0466] The oligoribonucleotides and oligoribonucleosides used in
accordance with this invention may be with solid phase synthesis,
see for example "Oligonucleotide synthesis, a practical approach",
Ed. M. J. Gait, IRL Press, 1984; "Oligonucleotides and Analogues, A
Practical Approach", Ed. F. Eckstein, IRL Press, 1991 (especially
Chapter 1, Modern machine-aided methods of oligodeoxyribonucleotide
synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter
3,2'-O-Methyloligoribonucleotide-s- : synthesis and applications,
Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis
of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of
oligo-2'-deoxyribonucleoside methylphosphonates, and. Chapter 7,
Oligodeoxynucleotides containing modified bases. Other particularly
useful synthetic procedures, reagents, blocking groups and reaction
conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78,
486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,
2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993,
49, 6123-6194, or references referred to therein.
[0467] Modification described in WO 00/44895, WO01/75164, or
WO02/44321 can be used herein.
[0468] The disclosure of all publications, patents, and published
patent applications listed herein are hereby incorporated by
reference.
[0469] Phosphate Group References
[0470] The preparation of phosphinate oligoribonucleotides is
described in U.S. Pat. No. 5,508,270. The preparation of alkyl
phosphonate oligoribonucleotides is described in U.S. Pat. No.
4,469,863. The preparation of phosphoramidite oligoribonucleotides
is described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.
The preparation of phosphotriester oligoribonucleotides is
described in U.S. Pat. No. 5,023,243. The preparation of borano
phosphate oligoribonucleotide is described in U.S. Pat. Nos.
5,130,302 and 5,177,198. The preparation of 3'-Deoxy-3'-amino
phosphoramidate oligoribonucleotides is described in U.S. Pat. No.
5,476,925. 3'-Deoxy-3'-methylenephosphonate oligoribonucleotides is
described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801.
Preparation of sulfur bridged nucleotides is described in Sproat et
al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.
Tetrahedron Lett. 1989, 30, 4693.
[0471] Sugar Group References
[0472] Modifications to the 2' modifications can be found in Verma,
S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references
therein. Specific modifications to the ribose can be found in the
following references: 2'-fluoro (Kawasaki et. al., J. Med. Chem.,
1993, 36, 831-841), 2'-MOE (Martin, P. Helv. Chim. Acta 1996, 79,
1930-1938), "LNA" (Wengel, J. Acc. Chem. Res. 1999, 32,
301-310).
[0473] Replacement of the Phosphate Group References
[0474] Methylenemethylimino linked oligoribonucleosides, also
identified herein as MMI linked oligoribonucleosides,
methylenedimethylhydrazo linked oligoribonucleosides, also
identified herein as MDH linked oligoribonucleosides, and
methylenecarbonylamino linked oligonucleosides, also identified
herein as amide-3 linked oligoribonucleosides, and
methyleneaminocarbonyl linked oligonucleosides, also identified
herein as amide-4 linked oligoribonucleosides as well as mixed
backbone compounds having, as for instance, alternating MMI and PO
or PS linkages can be prepared as is described in U.S. Pat. Nos.
5,378,825, 5,386,023, 5,489,677 and in published PCT applications
PCT/US92/04294 and PCT/US92/04305 (published as WO 92/20822 WO and
92/20823, respectively). Formacetal and thioformacetal linked
oligoribonucleosides can be prepared as is described in U.S. Pat.
Nos. 5,264,562 and 5,264,564. Ethylene oxide linked
oligoribonucleosides can be prepared as is described in U.S. Pat.
No. 5,223,618. Siloxane replacements are described in Cormier, J.
F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements
are described in Tittensor, J. R. J. Chem. Soc. C 1971, 1933.
Carboxymethyl replacements are described in Edge, M. D. et al. J.
Chem. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are
described in Stirchak, E. P. Nucleic Acids Res. 1989, 17, 6129.
[0475] Replacement of the Phosphate-Ribose Backbone References
[0476] Cyclobutyl sugar surrogate compounds can be prepared as is
described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate
can be prepared as is described in U.S. Pat. No. 5,519,134.
Morpholino sugar surrogates can be prepared as is described in U.S.
Pat. Nos. 5,142,047 and 5,235,033, and other related patent
disclosures. Peptide Nucleic Acids (PNAs) are known per se and can
be prepared in accordance with any of the various procedures
referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties
and Potential Applications, Bioorganic & Medicinal Chemistry,
1996, 4, 5-23. They may also be prepared in accordance with U.S.
Pat. No. 5,539,083.
[0477] Terminal Modification References
[0478] Terminal modifications are described in Manoharan, M. et al.
Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and
references therein.
[0479] Bases References
[0480] N-2 substitued purine nucleoside amidites can be prepared as
is described in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside
amidites can be prepared as is described in U.S. Pat. No.
5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can be
prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl
pyrimidine nucleoside amidites can be prepared as is described in
U.S. Pat. No. 5,484,908. Additional references can be disclosed in
the above section on base modifications.
Preferred iRNA Agents
[0481] Preferred RNA agents have the following structure (see
Formula 2 below): 71
[0482] Referring to Formula 2 above, R.sup.1, R.sup.2, and R.sup.3
are each, independently, H, (i.e. abasic nucleotides), adenine,
guanine, cytosine and uracil, inosine, thymine, xanthine,
hypoxanthine, nubularine, tubercidine, isoguanisine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,
6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl
uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other
8-substituted adenines and guanines, 5-trifluoromethyl and other
5-substituted uracils and cytosines, 7-methylguanine, 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted
purines, including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine,
2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,
7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine,
2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil,
substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole,
3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,
5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,
5-methoxycarbonylmethyl-2-thiouracil,
5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,
3-methylcytosine, 5-methylcytosine, N4-acetyl cytosine,
2-thiocytosine, N6-methyladenine, N6-isopentyladenine,
2-methylthio-N-6-isopentenyladenine, N-methylguanines, or
O-alkylated bases.
[0483] R.sup.4, R.sup.5, and R.sup.6 are each, independently,
OR.sup.8, O(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2OR.sup.8;
O(CH.sub.2).sub.nR.sup.9; O(CH.sub.2).sub.nOR.sup.9, H; halo;
NH.sub.2; NHR.sup.8; N(R.sup.8).sub.2;
NH(CH.sub.2CH.sub.2NH).sub.mCH.sub.2CH.sub.2- NHR.sup.9;
NHC(O)R.sup.8; cyano; mercapto, SR.sup.8; alkyl-thio-alkyl; alkyl,
aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of
which may be optionally substituted with halo, hydroxy, oxo, nitro,
haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino,
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,
arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,
alkanesulfonyl, alkanesulfonamido, arenesulfonamido,
aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido; or
R.sup.4, R.sup.5, or R.sup.6 together combine with R.sup.7 to form
an [--O--CH.sub.2--] covalently bound bridge between the sugar 2'
and 4' carbons.
[0484] A.sup.1 is: 72
[0485] H; OH; OCH.sub.3; W.sup.1; an abasic nucleotide; or
absent;
[0486] (a preferred A1, especially with regard to anti-sense
strands, is chosen from 5'-monophosphate ((HO).sub.2(O)P--O-5'),
5'-diphosphate ((HO).sub.2(O)P--O--P(HO)(O)--O-5'), 5'-triphosphate
((HO).sub.2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'), 5'-guanosine cap
(7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(H- O)(O)--O-5'),
5'-adenosine cap (Appp), and any modified or unmodified nucleotide
cap structure (N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5- '),
5'-monothiophosphate (phosphorothioate; (HO).sub.2(S)P--O-5'),
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO).sub.2(O)P--S-5'); any additional
combination of oxgen/sulfur replaced monophosphate, diphosphate and
triphosphates (e.g. 5'-alpha-thiotriphosphate,
5'-gamma-thiotriphosphate, etc.), 5'-phosphoramidates
((HO).sub.2(O)P--NH-5', (HO)(NH.sub.2)(O)P-0-5'),
5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl,
etc., e.g. RP(OH)(O)--O-5'-, (OH).sub.2(O)P-5'-CH.sub.2--),
5'-alkyletherphosphonates (R=alkylether=methoxymethyl
(MeOCH.sub.2--), ethoxymethyl, etc., e.g. RP(OH)(O)--O-5'-)).
[0487] A.sup.2 is: 73
[0488] A.sup.3 is: 74
[0489] and
[0490] A.sup.4 is: 75
[0491] H; Z.sup.4; an inverted nucleotide; an abasic nucleotide; or
absent.
[0492] W.sup.1 is OH, (CH.sub.2).sub.nR.sup.10,
(CH.sub.2).sub.nNHR.sup.10- , (CH.sub.2).sub.nOR.sup.10,
(CH.sub.2).sub.nSR.sup.10; O(CH.sub.2).sub.nR.sup.10;
O(CH.sub.2).sub.nOR.sup.10, O(CH.sub.2).sub.nNR.sup.10,
O(CH.sub.2).sub.nSR.sup.10;
O(CH.sub.2).sub.nSS(CH.sub.2).sub.nOR.sup.10,
O(CH.sub.2).sub.nC(O)OR.sup- .10, NH(CH.sub.2).sub.nR.sup.10;
NH(CH.sub.2).sub.nNR.sup.10; NH(CH.sub.2).sub.nOR.sup.10,
NH(CH.sub.2).sub.nSR.sup.10; S(CH.sub.2).sub.nR.sup.10,
S(CH.sub.2).sub.nNR.sup.10, S(CH.sub.2).sub.nOR.sup.10,
S(CH.sub.2).sub.nSR.sup.10O(CH.sub.2CH.sub.2O-
).sub.nCH.sub.2CH.sub.2OR.sup.10;
O(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub- .2NHR.sup.10,
NH(CH.sub.2CH.sub.2NH).sub.mCH.sub.2CH.sub.2NHR.sup.10; Q-R.sup.10,
O-Q-R.sup.10 N-Q-R.sup.10, S-Q-R.sup.10 or --O--. W.sup.4 is O,
CH.sub.2, NH, or S.
[0493] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are each,
independently, O or S.
[0494] Y.sup.1, Y.sup.2, Y.sup.3, and Y.sup.4 are each,
independently, OH, O--, OR.sup.8, S, Se, BH.sub.3.sup.-, H,
NHR.sup.9, N(R.sup.9).sub.2 alkyl, cycloalkyl, aralkyl, aryl, or
heteroaryl, each of which may be optionally substituted.
[0495] Z.sup.1, Z.sup.2, and Z.sup.3 are each independently O,
CH.sub.2, NH, or S. Z.sup.4 is OH, (CH.sub.2).sub.nR.sup.10,
(CH.sub.2).sub.nNHR.sup.10, (CH.sub.2). OR.sup.10,
(CH.sub.2).sub.nSR.sup.10; O(CH.sub.2).sub.nR.sup.10;
O(CH.sub.2).sub.nOR.sup.10, O(CH.sub.2).sub.nNR.sup.10,
O(CH.sub.2).sub.nSR.sup.10,
O(CH.sub.2).sub.nSS(CH.sub.2).sub.nOR.sup.10,
O(CH.sub.2).sub.nC(O)OR.sup.10; NH(CH.sub.2).sub.nR.sup.10;
NH(CH.sub.2).sub.nNR.sup.10; NH(CH.sub.2).sub.nOR.sup.10,
NH(CH.sub.2).sub.nSR.sup.10; S(CH.sub.2).sub.nR.sup.10,
S(CH.sub.2).sub.nNR.sup.10, S(CH.sub.2).sub.nOR.sup.10,
S(CH.sub.2).sub.nSR.sup.10O(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2OR.su-
p.10, O(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2NHR.sup.10,
NH(CH.sub.2CH.sub.2NH).sub.mCH.sub.2CH.sub.2NHR.sup.10; Q-R.sup.10,
O-Q-R.sup.10 N-Q-R.sup.10, S-Q-R.sup.10.
[0496] x is 5-100, chosen to comply with a length for an RNA agent
described herein.
[0497] R.sup.7 is H; or is together combined with R.sup.4, R.sup.5,
or R.sup.6 to form an [--O--CH.sub.2--] covalently bound bridge
between the sugar 2' and 4' carbons.
[0498] R.sup.8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,
heteroaryl, amino acid, or sugar; R.sup.9 is NH.sub.2, alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, diheteroaryl amino, or amino acid; and R.sup.10 is H;
fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur,
silicon, boron or ester protecting group; intercalating agents
(e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C),
porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic
hydrocarbons (e.g., phenazine, dihydrophenazine), artificial
endonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic
acid, adamantane acetic acid, 1-pyrene butyric acid,
dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl
group, hexadecylglycerol, bomeol, menthol, 1,3-propanediol,
heptadecyl group, palmitic acid, myristic acid,
O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,
dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g.,
antennapedia peptide, Tat peptide), alkylating agents, phosphate,
amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG].sub.2,
polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl;
radiolabelled markers, enzymes, haptens (e.g. biotin),
transport/absorption facilitators (e.g., aspirin, vitamin E, folic
acid), synthetic ribonucleases (e.g., imidazole, bisimidazole,
histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+
complexes of tetraazamacrocycles); or an RNA agent. m is
0-1,000,000, and n is 0-20. Q is a spacer selected from the group
consisting of abasic sugar, amide, carboxy, oxyamine, oxyimine,
thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin
or fluorescein reagents.
[0499] Preferred RNA agents in which the entire phosphate group has
been replaced have the following structure (see Formula 3 below):
76
[0500] Referring to Formula 3, A.sup.10-A.sup.40 is L-G-L; A.sup.10
and/or A.sup.40 may be absent, in which L is a linker, wherein one
or both L may be present or absent and is selected from the group
consisting of CH.sub.2(CH.sub.2).sub.g; N(CH.sub.2).sub.g;
O(CH.sub.2).sub.g; S(CH.sub.2).sub.g. G is a functional group
selected from the group consisting of siloxane, carbonate,
carboxymethyl, carbamate, amide, thioether, ethylene oxide linker,
sulfonate, sulfonamide, thioformacetal, formacetal, oxime,
methyleneimino, methylenemethylimino, methylenehydrazo,
methylenedimethylhydrazo and methyleneoxymethylimino.
[0501] R.sup.10, R.sup.20, and R.sup.30 are each, independently, H,
(i.e. abasic nucleotides), adenine, guanine, cytosine and uracil,
inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine,
isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil
and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil
(pseudouracil), 4-thiouracil, 5-halouracil,
5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino,
thiol, thioalkyl, hydroxyl and other 8-substituted adenines and
guanines, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine, 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine,
2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,
7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine,
2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil
substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole,
3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,
5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,
5-methoxycarbonylmethyl-2-thiouracil,
5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,
3-methylcytosine, 5-methylcytosine, N.sup.4-acetyl cytosine,
2-thiocytosine, N6-methyladenine, N6-isopentyladenine,
2-methylthio-N-6-isopentenyladenine, N-methylguanines, or
O-alkylated bases.
[0502] R.sup.40, R.sup.50, and R.sup.60 are each, independently,
OR.sup.8, O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR.sup.8;
O(CH.sub.2).sub.nR.sup.9; O(CH.sub.2).sub.nOR.sup.9, H; halo;
NH.sub.2; NHR.sup.8; N(R.sup.8).sub.2;
NH(CH.sub.2CH.sub.2NH).sub.mCH.sub.2CH.sub.2- R.sup.9;
NHC(O)R.sup.8; cyano; mercapto, SR.sup.7; alkyl-thio-alkyl; alkyl,
aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of
which may be optionally substituted with halo, hydroxy, oxo, nitro,
haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino,
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl,
arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,
alkanesulfonyl, alkanesulfonamido, arenesulfonamido,
aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido
groups; or R.sup.40, R.sup.50, or R.sup.60 together combine with
R.sup.70 to form an [--O--CH.sub.2--] covalently bound bridge
between the sugar 2' and 4' carbons.
[0503] x is 5-100 or chosen to comply with a length for an RNA
agent described herein.
[0504] R.sup.70 is H; or is together combined with R.sup.40,
R.sup.50, or R.sup.60 to form an [--O--CH.sub.2--] covalently bound
bridge between the sugar 2' and 4' carbons.
[0505] R.sup.8 is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,
heteroaryl, amino acid, or sugar; and R.sup.9 is NH.sub.2,
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid. m is
0-1,000,000, n is 0-20, and g is 0-2.
[0506] Preferred nucleoside surrogates have the following structure
(see Formula 4 below):
SLR.sup.100-(M-SLR.sup.200).sub.x-M-SLR.sup.300 FORMULA 4
[0507] S is a nucleoside surrogate selected from the group
consisting of mophilino, cyclobutyl, pyrrolidine and peptide
nucleic acid. L is a linker and is selected from the group
consisting of CH.sub.2(CH.sub.2).sub.g; N(CH.sub.2).sub.g;
O(CH.sub.2).sub.g; S(CH.sub.2).sub.g; --C(O)(CH.sub.2).sub.n-or may
be absent. M is an amide bond; sulfonamide; sulfinate; phosphate
group; modified phosphate group as described herein; or may be
absent.
[0508] R.sup.100, R.sup.200, and R.sup.300 are each, independently,
H (i.e., abasic nucleotides), adenine, guanine, cytosine and
uracil, inosine, thymine, xanthine, hypoxanthine, nubularine,
tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl
derivatives of adenine and guanine, 2-propyl and other alkyl
derivatives of adenine and guanine, 5halouracil and cytosine,
5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine,
5-uracil (pseudouracil), 4-thiouracil, 5-halouracil,
5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino,
thiol, thioalkyl, hydroxyl and other 8-substituted adenines and
guanines, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine, 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine,
2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,
7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine,
2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil
substituted 1, 2,4,-triazoles, 2-pyridinones, 5-nitroindole,
3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid,
5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil,
5-methoxycarbonylmethyl-2-thiouracil,
5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil,
3-methylcytosine, 5-methylcytosine, N.sup.4-acetyl cytosine,
2-thiocytosine, N6-methyladenine, N6-isopentyladenine,
2-methylthio-N-6-isopentenyladenine, N-methylguanines, or
O-alkylated bases.
[0509] x is 5-100, or chosen to comply with a length for an RNA
agent described herein; and g is 0-2.
[0510] Nuclease Resistant Monomers
[0511] The monomers and methods described herein can be used to
prepare an RNA, e.g., an iRNA agent, that incorporates a nuclease
resistant monomer (NRM), such as those described herein and those
described in copending, co-owned U.S. Provisional Application Ser.
No. 60/469,612, filed on May 9, 2003, and International Application
No. PCT/US04/07070, both of which are hereby incorporated by
reference.
[0512] An iRNA agent can include monomers which have been modifed
so as to inhibit degradation, e.g., by nucleases, e.g.,
endonucleases or exonucleases, found in the body of a subject.
These monomers are referred to herein as NRMs, or nuclease
resistance promoting monomers or modifications. In many cases these
modifications will modulate other properties of the iRNA agent as
well, e.g., the ability to interact with a protein, e.g., a
transport protein, e.g., serum albumin, or a member of the
RISC(RNA-induced Silencing Complex), or the ability of the first
and second sequences to form a duplex with one another or to form a
duplex with another sequence, e.g., a target molecule.
[0513] While not wishing to be bound by theory, it is believed that
modifications of the sugar, base, and/or phosphate backbone in an
iRNA agent can enhance endonuclease and exonuclease resistance, and
can enhance interactions with transporter proteins and one or more
of the functional components of the RISC complex. Preferred
modifications are those that increase exonuclease and endonuclease
resistance and thus prolong the half-life of the iRNA agent prior
to interaction with the RISC complex, but at the same time do not
render the iRNA agent resistant to endonuclease activity in the
RISC complex. Again, while not wishing to be bound by any theory,
it is believed that placement of the modifications at or near the
3' and/or 5' end of antisense strands can result in iRNA agents
that meet the preferred nuclease resistance criteria delineated
above. Again, still while not wishing to be bound by any theory, it
is believed that placement of the modifications at e.g., the middle
of a sense strand can result in iRNA agents that are relatively
less likely to undergo off-targeting.
[0514] Modifications described herein can be incorporated into any
double-stranded RNA and RNA-like molecule described herein, e.g.,
an iRNA agent. An iRNA agent may include a duplex comprising a
hybridized sense and antisense strand, in which the antisense
strand and/or the sense strand may include one or more of the
modifications described herein. The anti sense strand may include
modifications at the 3' end and/or the 5' end and/or at one or more
positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides
from either end of the strand. The sense strand may include
modifications at the 3' end and/or the 5' end and/or at any one of
the intervening positions between the two ends of the strand. The
iRNA agent may also include a duplex comprising two hybridized
antisense strands. The first and/or the second antisense strand may
include one or more of the modifications described herein. Thus,
one and/or both antisense strands may include modifications at the
3' end and/or the 5' end and/or at one or more positions that occur
1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the
strand. Particular configurations are discussed below.
[0515] Modifications that can be useful for producing iRNA agents
that meet the preferred nuclease resistance criteria delineated
above can include one or more of the following chemical and/or
stereochemical modifications of the sugar, base, and/or phosphate
backbone:
[0516] (i) chiral (S.sub.P) thioates. Thus, preferred NRMs include
nucleotide dimers with an enriched or pure for a particular chiral
form of a modified phosphate group containing a heteroatom at the
nonbridging position, e.g., Sp or Rp, at the position X, where this
is the position normally occupied by the oxygen. The atom at X can
also be S, Se, Nr.sub.2, or Br.sub.3. When X is S, enriched or
chirally pure Sp linkage is preferred. Enriched means at least 70,
80, 90, 95, or 99% of the preferred form. Such NRMs are discussed
in more detail below;
[0517] (ii) attachment of one or more cationic groups to the sugar,
base, and/or the phosphorus atom of a phosphate or modified
phosphate backbone moiety. Thus, preferred NRMs include monomers at
the terminal position derivatized at a cationic group. As the 5'
end of an antisense sequence should have a terminal --OH or
phosphate group this NRM is preferably not used at the 5' end of an
anti-sense sequence. The group should be attached at a position on
the base which minimizes interference with H bond formation and
hybridization, e.g., away form the face which interacts with the
complementary base on the other strand, e.g, at the 5' position of
a pyrimidine or a 7-position of a purine. These are discussed in
more detail below;
[0518] (iii) nonphosphate linkages at the termini. Thus, preferred
NRMs include Non-phosphate linkages, e.g., a linkage of 4 atoms
which confers greater resistance to cleavage than does a phosphate
bond. Examples include 3'CH2--NCH.sub.3--O--CH2-5' and 3'
CH2--NH--(O.dbd.)--CH2-5';
[0519] (iv) 3'-bridging thiophosphates and 5'-bridging
thiophosphates. Thus, preferred NRM's can included these
structures;
[0520] (v) L-RNA, 2'-5' linkages, inverted linkages, a-nucleosides.
Thus, other preferred NRM's include: L nucleosides and dimeric
nucleotides derived from L-nucleosides; 2'-5' phosphate,
non-phosphate and modified phosphate linkages (e.g.,
thiophosphates, phosphoramidates and boronophosphates); dimers
having inverted linkages, e.g., 3'-3' or 5'-5' linkages; monomers
having an alpha linkage at the 1' site on the sugar, e.g., the
structures described herein having an alpha linkage;
[0521] (vi) conjugate groups. Thus, preferred NRM's can include
e.g., a targeting moiety or a conjugated ligand described herein
conjugated with the monomer, e.g., through the sugar, base, or
backbone;
[0522] (vi) abasic linkages. Thus, preferred NRM's can include an
abasic monomer, e.g., an abasic monomer as described herein (e.g.,
a nucleobaseless monomer); an aromatic or heterocyclic or
polyheterocyclic aromatic monomer as described herein; and
[0523] (vii) 5'-phosphonates and 5'-phosphate prodrugs. Thus,
preferred NRM's include monomers, preferably at the terminal
position, e.g., the 5' position, in which one or more atoms of the
phosphate group is derivatized with a protecting group, which
protecting group or groups, are removed as a result of the action
of a component in the subject's body, e.g, a carboxyesterase or an
enzyme present in the subject's body. E.g., a phosphate prodrug in
which a carboxy esterase cleaves the protected molecule resulting
in the production of a thioate anion which attacks a carbon
adjacent to the 0 of a phosphate and resulting in the production of
an unprotected phosphate.
[0524] One or more different NRM modifications can be introduced
into an iRNA agent or into a sequence of an iRNA agent. An NRM
modification can be used more than once in a sequence or in an iRNA
agent. As some NRM's interfere with hybridization the total number
incorporated, should be such that acceptable levels of iRNA agent
duplex formation are maintained.
[0525] In some embodiments NRM modifications are introduced into
the terminal the cleavage site or in the cleavage region of a
sequence (a sense strand or sequence) which does not target a
desired sequence or gene in the subject. This can reduce off-target
silencing.
[0526] Chiral S.sub.P Thioates
[0527] A modification can include the alteration, e.g.,
replacement, of one or both of the non-linking (X and Y) phosphate
oxygens and/or of one or more of the linking (W and Z) phosphate
oxygens. Formula X below depicts a phosphate moiety linking two
sugar/sugar surrogate-base moieties, SB.sub.1 and SB.sub.2. 77
[0528] In certain embodiments, one of the non-linking phosphate
oxygens in the phosphate backbone moiety (X and Y) can be replaced
by any one of the following: S, Se, BR.sub.3 (R is hydrogen, alkyl,
aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H,
NR.sub.2 (R is hydrogen, alkyl, aryl, etc.), or OR (R is alkyl or
aryl). The phosphorus atom in an unmodified phosphate group is
achiral. However, replacement of one of the non-linking oxygens
with one of the above atoms or groups of atoms renders the
phosphorus atom chiral; in other words a phosphorus atom in a
phosphate group modified in this way is a stereogenic center. The
stereogenic phosphorus atom can possess either the "R"
configuration (herein R.sub.P) or the "S" configuration (herein
S.sub.P). Thus if 60% of a population of stereogenic phosphorus
atoms have the R.sub.P configuration, then the remaining 40% of the
population of stereogenic phosphorus atoms have the S.sub.P
configuration.
[0529] In some embodiments, iRNA agents, having phosphate groups in
which a phosphate non-linking oxygen has been replaced by another
atom or group of atoms, may contain a population of stereogenic
phosphorus atoms in which at least about 50% of these atoms (e.g.,
at least about 60% of these atoms, at least about 70% of these
atoms, at least about 80% of these atoms, at least about 90% of
these atoms, at least about 95% of these atoms, at least about 98%
of these atoms, at least about 99% of these atoms) have the S.sub.P
configuration. Alternatively, iRNA agents having phosphate groups
in which a phosphate non-linking oxygen has been replaced by
another atom or group of atoms may contain a population of
stereogenic phosphorus atoms in which at least about 50% of these
atoms (e.g., at least about 60% of these atoms, at least about 70%
of these atoms, at least about 80% of these atoms, at least about
90% of these atoms, at least about 95% of these atoms, at least
about 98% of these atoms, at least about 99% of these atoms) have
the R.sub.P configuration. In other embodiments, the population of
stereogenic phosphorus atoms may have the S.sub.P configuration and
may be substantially free of stereogenic phosphorus atoms having
the R.sub.P configuration. In still other embodiments, the
population of stereogenic phosphorus atoms may have the R.sub.P
configuration and may be substantially free of stereogenic
phosphorus atoms having the S.sub.P configuration. As used herein,
the phrase "substantially free of stereogenic phosphorus atoms
having the R.sub.P configuration" means that moieties containing
stereogenic phosphorus atoms having the R.sub.P configuration
cannot be detected by conventional methods known in the art (chiral
HPLC, .sup.1H NMR analysis using chiral shift reagents, etc.). As
used herein, the phrase "substantially free of stereogenic
phosphorus atoms having the S.sub.P configuration" means that
moieties containing stereogenic phosphorus atoms having the S.sub.P
configuration cannot be detected by conventional methods known in
the art (chiral HPLC, .sup.1H NMR analysis using chiral shift
reagents, etc.).
[0530] In a preferred embodiment, modified iRNA agents contain a
phosphorothioate group, i.e., a phosphate groups in which a
phosphate non-linking oxygen has been replaced by a sulfur atom. In
an especially preferred embodiment, the population of
phosphorothioate stereogenic phosphorus atoms may have the S.sub.P
configuration and be substantially free of stereogenic phosphorus
atoms having the R.sub.P configuration.
[0531] Phosphorothioates may be incorporated into iRNA agents using
dimers e.g., formulas X-1 and X-2. The former can be used to
introduce phosphorothioate 78
[0532] at the 3' end of a strand, while the latter can be used to
introduce this modification at the 5' end or at a position that
occurs e.g., 1, 2, 3, 4, 5, or 6 nucleotides from either end of the
strand. In the above formulas, Y can be 2-cyanoethoxy, W and Z can
be O, R.sub.2' can be, e.g., a substituent that can impart the C-3
endo configuration to the sugar (e.g., OH, F, OCH.sub.3), DMT is
dimethoxytrityl, and "BASE" can be a natural, unusual, or a
universal base.
[0533] X-1 and X-2 can be prepared using chiral reagents or
directing groups that can result in phosphorothioate-containing
dimers having a population of stereogenic phosphorus atoms having
essentially only the R.sub.P configuration (i.e., being
substantially free of the S.sub.P configuration) or only the
S.sub.P configuration (i.e., being substantially free of the
R.sub.P configuration). Alternatively, dimers can be prepared
having a population of stereogenic phosphorus atoms in which about
50% of the atoms have the R.sub.P configuration and about 50% of
the atoms have the S.sub.P configuration. Dimers having stereogenic
phosphorus atoms with the R.sub.P configuration can be identified
and separated from dimers having stereogenic phosphorus atoms with
the S.sub.P configuration using e.g., enzymatic degradation and/or
conventional chromatography techniques.
[0534] Cationic Groups
[0535] Modifications can also include attachment of one or more
cationic groups to the sugar, base, and/or the phosphorus atom of a
phosphate or modified phosphate backbone moiety. A cationic group
can be attached to any atom capable of substitution on a natural,
unusual or universal base. A preferred position is one that does
not interfere with hybridization, i.e., does not interfere with the
hydrogen bonding interactions needed for base pairing. A cationic
group can be attached e.g., through the C2' position of a sugar or
analogous position in a cyclic or acyclic sugar surrogate. Cationic
groups can include e.g., protonated amino groups, derived from
e.g., O-AMINE (AMINE=NH.sub.2; alkylamino, dialkylamino,
heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or
diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy,
e.g., O(CH.sub.2).sub.nAMINE, (e.g., AMINE=NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, or diheteroaryl amino, ethylene diamine, polyamino); amino
(e.g. NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino,
diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid);
or NH(CH.sub.2CH.sub.2NH).sub.nCH.sub- .2CH.sub.2-AMINE
(AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino,
diaryl amino, heteroaryl amino, or diheteroaryl amino).
[0536] Nonphosphate Linkages
[0537] Modifications can also include the incorporation of
nonphosphate linkages at the 5' and/or 3' end of a strand. Examples
of nonphosphate linkages which can replace the phosphate group
include methyl phosphonate, hydroxylamino, siloxane, carbonate,
carboxymethyl, carbamate, amide, thioether, ethylene oxide linker,
sulfonate, sulfonamide, thioformacetal, formacetal, oxime,
methyleneimino, methylenemethylimino, methylenehydrazo,
methylenedimethylhydrazo and methyleneoxymethylimino. Preferred
replacements include the methyl phosphonate and hydroxylamino
groups.
[0538] 3'-bridging Thiophosphates and 5'-bridging Thiophosphates,
Locked-RNA, 2'-5' Likages, Inverted Linkages, .alpha.-nucleosides:
Conjugate Groups; Abasic Linkages; and 5'-phosphonates and
5'-phosphate Prodrugs
[0539] Referring to formula X above, modifications can include
replacement of one of the bridging or linking phosphate oxygens in
the phosphate backbone moiety (W and Z). Unlike the situation where
only one of X or Y is altered, the phosphorus center in the
phosphorodithioates is achiral which precludes the formation of
iRNA agents containing a stereogenic phosphorus atom.
[0540] Modifications can also include linking two sugars via a
phosphate or modified phosphate group through the 2' position of a
first sugar and the 5' position of a second sugar. Also
contemplated are inverted linkages in which both a first and second
sugar are eached linked through the respective 3' positions.
Modified RNA's can also include "abasic" sugars, which lack a
nucleobase at C-1'. The sugar group can also contain one or more
carbons that possess the opposite stereochemical configuration than
that of the corresponding carbon in ribose. Thus, a modified iRNA
agent can include nucleotides containing e.g., arabinose, as the
sugar. In another subset of this modification, the natural,
unusual, or universal base may have the .alpha.-configuration.
Modifcations can also include L-RNA.
[0541] Modifications can also include 5'-phosphonates, e.g.,
P(O)(O.sup.-).sub.2--X--C.sup.5'-sugar (X.dbd.CH2, CF2, CHF and
5'-phosphate prodrugs, e.g.,
P(O)[OCH2CH2SC(O)R].sub.2CH.sub.2C.sup.5'-su- gar. In the latter
case, the prodrug groups may be decomposed via reaction first with
carboxy esterases. The remaining ethyl thiolate group via
intramolecular S.sub.N2 displacement can depart as episulfide to
afford the underivatized phosphate group.
[0542] Modification can also include the addition of conjugating
groups described elseqhere herein, which are prefereably attached
to an iRNA agent through any amino group available for
conjugation.
[0543] Nuclease resistant modifications include some which can be
placed only at the terminus and others which can go at any
position. Generally the modifications that can inhibit
hybridization so it is preferably to use them only in terminal
regions, and preferrable to not use them at the cleavage site or in
the cleavage region of an sequence which targets a subject sequence
or gene. The can be used anywhere in a sense sequence, provided
that sufficient hybridization between the two sequences of the iRNA
agent is maintained. In some embodiments it is desirabable to put
the NRM at the cleavage site or in the cleavage region of a
sequence which does not target a subject sequence or gene, as it
can minimize off-target silencing.
[0544] In addition, an iRNA agent described herein can have an
overhang which does not form a duplex structure with the other
sequence of the iRNA agent--it is an overhang, but it does
hybridize, either with itself, or with another nucleic acid, other
than the other sequence of the iRNA agent.
[0545] In most cases, the nuclease-resistance promoting
modifications will be distributed differently depending on whether
the sequence will target a sequence in the subject (often referred
to as an anti-sense sequence) or will not target a sequence in the
subject (often referred to as a sense sequence). If a sequence is
to target a sequence in the subject, modifications which interfer
with or inhibit endonuclease cleavage should not be inserted in the
region which is subject to RISC mediated cleavage, e.g., the
cleavage site or the cleavage region (As described in Elbashir et
al., 2001, Genes and Dev. 15: 188, hereby incorporated by
reference, cleavage of the target occurs about in the middle of a
20 or 21 nt guide RNA, or about 10 or 11 nucleotides upstream of
the first nucleotide which is complementary to the guide sequence.
As used herein cleavage site refers to the nucleotide on either
side of the cleavage site, on the target or on the iRNA agent
strand which hybridizes to it. Cleavage region means an nucleotide
with 1, 2, or 3 nucletides of the cleave site, in either
direction.)
[0546] Such modifications can be introduced into the terminal
regions, e.g., at the terminal position or with 2, 3, 4, or 5
positions of the terminus, of a sequence which targets or a
sequence which does not target a sequence in the subject.
[0547] An iRNA agent can have a first and a second strand chosen
from the following:
[0548] a first strand which does not target a sequence and which
has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions
from the 3' end;
[0549] a first strand which does not target a sequence and which
has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions
from the 5' end;
[0550] a first strand which does not target a sequence and which
has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions
from the 3' end and which has a NRM modification at or within 1, 2,
3, 4, 5, or 6 positions from the 5' end;
[0551] a first strand which does not target a sequence and which
has an NRM modification at the cleavage site or in the cleavage
region;
[0552] a first strand which does not target a sequence and which
has an NRM modification at the cleavage site or in the cleavage
region and one or more of an NRM modification at or within 1, 2, 3,
4, 5, or 6 positions from the 3' end, a NRM modification at or
within 1, 2, 3, 4, 5, or 6 positions from the 5' end, or NRM
modifications at or within 1, 2, 3, 4, 5, or 6 positions from both
the 3' and the 5' end; and
[0553] a second strand which targets a sequence and which has an
NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from
the 3' end;
[0554] a second strand which targets a sequence and which has an
NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from
the 5' end (5' end NRM modifications are preferentially not at the
terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the
5' terminus of an antisense strand);
[0555] a second strand which targets a sequence and which has an
NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from
the 3' end and which has a NRM modification at or within 1, 2, 3,
4, 5, or 6 positions from the 5' end;
[0556] a second strand which targets a sequence and which
preferably does not have an an NRM modification at the cleavage
site or in the cleavage region;
[0557] a second strand which targets a sequence and which does not
have an NRM modification at the cleavage site or in the cleavage
region and one or more of an NRM modification at or within 1, 2, 3,
4, 5, or 6 positions from the 3' end, a NRM modification at or
within 1, 2, 3, 4, 5, or 6 positions from the 5' end, or NRM
modifications at or within 1, 2, 3, 4, 5, or 6 positions from both
the 3' and the 5' end (5' end NRM modifications are preferentially
not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6
away from the 5' terminus of an antisense strand).
[0558] An iRNA agent can also target two sequences and can have a
first and second strand chosen from:
[0559] a first strand which targets a sequence and which has an NRM
modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3'
end;
[0560] a first strand which targets a sequence and which has an NRM
modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5'
end (5' end NRM modifications are preferentially not at the
terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the
5' terminus of an antisense strand);
[0561] a first strand which targets a sequence and which has an NRM
modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3'
end and which has a NRM modification at or within 1, 2, 3, 4, 5, or
6 positions from the 5' end;
[0562] a first strand which targets a sequence and which preferably
does not have an an NRM modification at the cleavage site or in the
cleavage region;
[0563] a first strand which targets a sequence and which dose not
have an NRM modification at the cleavage site or in the cleavage
region and one or more of an NRM modification at or within 1, 2, 3,
4, 5, or 6 positions from the 3' end, a NRM modification at or
within 1, 2, 3, 4, 5, or 6 positions from the 5' end, or NRM
modifications at or within 1, 2, 3, 4, 5, or 6 positions from both
the 3' and the 5' end (5' end NRM modifications are preferentially
not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6
away from the 5' terminus of an antisense strand) and a second
strand which targets a sequence and which has an NRM modification
at or within 1, 2, 3, 4, 5, or 6 positions from the 3' end;
[0564] a second strand which targets a sequence and which has an
NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from
the 5' end (5' end NRM modifications are preferentially not at the
terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the
5' terminus of an antisense strand);
[0565] a second strand which targets a sequence and which has an
NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from
the 3' end and which has a NRM modification at or within 1, 2, 3,
4, 5, or 6 positions from the 5' end;
[0566] a second strand which targets a sequence and which
preferably does not have an an NRM modification at the cleavage
site or in the cleavage region;
[0567] a second strand which targets a sequence and which dose not
have an NRM modification at the cleavage site or in the cleavage
region and one or more of an NRM modification at or within 1, 2, 3,
4, 5, or 6 positions from the 3' end, a NRM modification at or
within 1, 2, 3, 4, 5, or 6 positions from the 5' end, or NRM
modifications at or within 1, 2, 3, 4, 5, or 6 positions from both
the 3' and the 5' end (5' end NRM modifications are preferentially
not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6
away from the 5' terminus of an antisense strand).
Ribose Mimics
[0568] The monomers and methods described herein can be used to
prepare an RNA, e.g., an iRNA agent, that incorporates a ribose
mimic, such as those described herein and those described in
copending co-owned U.S. Provisional Application Ser. No.
60/454,962, filed on Mar. 13, 2003, and International Application
No. PCT/US04/07070, both of which are hereby incorporated by
reference.
[0569] Thus, an aspect of the invention features an iRNA agent that
includes a secondary hydroxyl group, which can increase efficacy
and/or confer nuclease resistance to the agent. Nucleases, e.g.,
cellular nucleases, can hydrolyze nucleic acid phosphodiester
bonds, resulting in partial or complete degradation of the nucleic
acid. The secondary hydroxy group confers nuclease resistance to an
iRNA agent by rendering the iRNA agent less prone to nuclease
degradation relative to an iRNA which lacks the modification. While
not wishing to be bound by theory, it is believed that the presence
of a secondary hydroxyl group on the iRNA agent can act as a
structural mimic of a 3' ribose hydroxyl group, thereby causing it
to be less susceptible to degradation.
[0570] The secondary hydroxyl group refers to an "OH" radical that
is attached to a carbon atom substituted by two other carbons and a
hydrogen. The secondary hydroxyl group that confers nuclease
resistance as described above can be part of any acyclic
carbon-containing group. The hydroxyl may also be part of any
cyclic carbon-containing group, and preferably one or more of the
following conditions is met (1) there is no ribose moiety between
the hydroxyl group and the terminal phosphate group or (2) the
hydroxyl group is not on a sugar moiety which is coupled to a base.
The hydroxyl group is located at least two bonds (e.g., at least
three bonds away, at least four bonds away, at least five bonds
away, at least six bonds away, at least seven bonds away, at least
eight bonds away, at least nine bonds away, at least ten bonds
away, etc.) from the terminal phosphate group phosphorus of the
iRNA agent. In preferred embodiments, there are five intervening
bonds between the terminal phosphate group phosphorus and the
secondary hydroxyl group.
[0571] Preferred iRNA agent delivery modules with five intervening
bonds between the terminal phosphate group phosphorus and the
secondary hydroxyl group have the following structure (see formula
Y below): 79
[0572] Referring to formula Y, A is an iRNA agent, including any
iRNA agent described herein. The iRNA agent may be connected
directly or indirectly (e.g., through a spacer or linker) to "W" of
the phosphate group. These spacers or linkers can include e.g.,
--(CH.sub.2).sub.n--, --(CH.sub.2).sub.nN--, --(CH.sub.2).sub.nO--,
--(CH.sub.2).sub.nS--, O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OH
(e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine,
oxyimine, thioether, disulfide, thiourea, sulfonamide, or
morpholino, or biotin and fluorescein reagents.
[0573] The iRNA agents can have a terminal phosphate group that is
unmodified (e.g., W, X, Y, and Z are O) or modified. In a modified
phosphate group, W and Z can be independently NH, O, or S; and X
and Y can be independently S, Se, BH.sub.3.sup.-, C.sub.1-C.sub.6
alkyl, C.sub.6-C.sub.10 aryl, H, O, O.sup.-, alkoxy or amino
(including alkylamino, arylamino, etc.). Preferably, W, X and Z are
O and Y is S.
[0574] R.sub.1 and R.sub.3 are each, independently, hydrogen; or
C.sub.1-C.sub.100 alkyl, optionally substituted with hydroxyl,
amino, halo, phosphate or sulfate and/or may be optionally inserted
with N, O, S, alkenyl or alkynyl.
[0575] R.sub.2 is hydrogen; C.sub.1-C.sub.100 alkyl, optionally
substituted with hydroxyl, amino, halo, phosphate or sulfate and/or
may be optionally inserted with N, O, S, alkenyl or alkynyl; or,
when n is 1, R.sub.2 may be taken together with with R.sup.4 or
R.sup.6 to form a ring of 5-12 atoms.
[0576] R.sup.4 is hydrogen; C.sub.1-C.sub.100 alkyl, optionally
substituted with hydroxyl, amino, halo, phosphate or sulfate and/or
may be optionally inserted with N, O, S, alkenyl or alkynyl; or,
when n is 1, R.sub.4 may be taken together with with R.sub.2 or
R.sub.5 to form a ring of 5-12 atoms.
[0577] R.sub.5 is hydrogen, C.sub.1-C.sub.100 alkyl optionally
substituted with hydroxyl, amino, halo, phosphate or sulfate and/or
may be optionally inserted with N, O, S, alkenyl or alkynyl; or,
when n is 1, R.sub.5 may be taken together with with R.sub.4 to
form a ring of 5-12 atoms.
[0578] R.sub.6 is hydrogen, C.sub.1-C.sub.100 alkyl, optionally
substituted with hydroxyl, amino, halo, phosphate or sulfate and/or
may be optionally inserted with N, O, S, alkenyl or alkynyl, or,
when n is 1, R.sub.6 may be taken together with with R.sub.2 to
form a ring of 6-10 atoms;
[0579] R.sub.7 is hydrogen, C.sub.1-C.sub.100 alkyl, or
C(O)(CH.sub.2).sub.qC(O)NHR.sub.9; T is hydrogen or a functional
group; n and q are each independently 1-100; R.sub.8 is
C.sub.1-C.sub.10 alkyl or C.sub.6-C.sub.10 aryl; and R.sub.9 is
hydrogen, C1-C10 alkyl, C6-C10 aryl or a solid support agent.
[0580] Preferred embodiments may include one of more of the
following subsets of iRNA agent delivery modules.
[0581] In one subset of RNAi agent delivery modules, A can be
connected directly or indirectly through a terminal 3' or 5' ribose
sugar carbon of the RNA agent.
[0582] In another subset of RNAi agent delivery modules, X, W, and
Z are O and Y is S.
[0583] In still yet another subset of RNAi agent delivery modules,
n is 1, and R.sub.2 and R.sub.6 are taken together to form a ring
containing six atoms and R.sub.4 and R.sub.5 are taken together to
form a ring containing six atoms. Preferably, the ring system is a
trans-decalin. For example, the RNAi agent delivery module of this
subset can include a compound of Formula (Y-1): 80
[0584] The functional group can be, for example, a targeting group
(e.g., a steroid or a carbohydrate), a reporter group (e.g., a
fluorophore), or a label (an isotopically labelled moiety). The
targeting group can further include protein binding agents,
endothelial cell targeting groups (e.g., RGD peptides and
mimetics), cancer cell targeting groups (e.g., folate Vitamin B12,
Biotin), bone cell targeting groups (e.g., bisphosphonates,
polyglutamates, polyaspartates), multivalent mannose (for e.g.,
macrophage testing), lactose, galactose, N-acetyl-galactosamine,
monoclonal antibodies, glycoproteins, lectins, melanotropin, or
thyrotropin.
[0585] As can be appreciated by the skilled artisan, methods of
synthesizing the compounds of the formulae herein will be evident
to those of ordinary skill in the art. The synthesized compounds
can be separated from a reaction mixture and further purified by a
method such as column chromatography, high pressure liquid
chromatography, or recrystallization. Additionally, the various
synthetic steps may be performed in an alternate sequence or order
to give the desired compounds. Synthetic chemistry transformations
and protecting group methodologies (protection and deprotection)
useful in synthesizing the compounds described herein are known in
the art and include, for example, those such as described in R.
Larock, Comprehensive Organic Transformations, VCH Publishers
(1989); T. W. Greene and P. G. M. Wuts, Protective Groups in
Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser
and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis,
John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of
Reagents for Organic Synthesis, John Wiley and Sons (1995), and
subsequent editions thereof.
[0586] Pharmaceutical Compositions
[0587] In one embodiment, the invention relates to a pharmaceutical
composition containing a modified iRNA agent, as described in the
preceding sections, and a pharmaceutically acceptable carrier, as
described below. A pharmaceutical composition including the
modified iRNA agent is useful for treating a disease caused by
expression of a target gene. In this aspect of the invention, the
iRNA agent of the invention is formulated as described below. The
pharmaceutical composition is administered in a dosage sufficient
to inhibit expression of the target gene.
[0588] The pharmaceutical compositions of the present invention are
administered in dosages sufficient to inhibit the expression or
activity of the target gene. Compositions containing the iRNA agent
of the invention can be administered at surprisingly low dosages. A
maximum dosage of 5 mg iRNA agent per kilogram body weight per day
may be sufficient to inhibit or completely suppress the expression
or activity of the target gene.
[0589] In general, a suitable dose of modified iRNA agent will be
in the range of 0.001 to 500 milligrams per kilogram body weight of
the recipient per day (e.g., about 1 microgram per kilogram to
about 500 milligrams per kilogram, about 100 micrograms per
kilogram to about 100 milligrams per kilogram, about 1 milligrams
per kilogram to about 75 milligrams per kilogram, about 10
micrograms per kilogram to about 50 milligrams per kilogram, or
about 1 microgram per kilogram to about 50 micrograms per
kilogram). The pharmaceutical composition may be administered once
per day, or the iRNA agent may be administered as two, three, four,
five, six or more sub-doses at appropriate intervals throughout the
day. In that case, the iRNA agent contained in each sub-dose must
be correspondingly smaller in order to achieve the total daily
dosage. The dosage unit can also be compounded for delivery over
several days, e.g., using a conventional sustained release
formulation which provides sustained release of the iRNA agent over
a several day period. Sustained release formulations are well known
in the art. In this embodiment, the dosage unit contains a
corresponding multiple of the daily dose.
[0590] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to the severity of the infection
or disease, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a composition
can include a single treatment or a series of treatments. Estimates
of effective dosages and in vivo half-lives for the individual iRNA
agent encompassed by the invention can be made using conventional
methodologies or on the basis of in vivo testing using an
appropriate animal model, as described elsewhere herein.
[0591] Advances in mouse genetics have generated a number of mouse
models for the study of various human diseases. For example, mouse
repositories can be found at The Jackson Laboratory, Charles River
Laboratories, Taconic, Harlan, Mutant Mouse Regional Resource
Centers (MMRRC) National Network and at the European Mouse Mutant
Archive. Such models may be used for in vivo testing of iRNA agent,
as well as for determining a therapeutically effective dose.
[0592] The pharmaceutical compositions encompassed by the invention
may be administered by any means known in the art including, but
not limited to oral or parenteral routes, including intravenous,
intramuscular, intraperitoneal, subcutaneous, transdermal, airway
(aerosol), ocular, rectal, vaginal and topical (including buccal
and sublingual) administration. In preferred embodiments, the
pharmaceutical compositions are administered by intravenous or
intraparenteral infusion or injection. The pharmaceutical
compositions can also be administered intraparenchymally,
intrathecally, and/or by stereotactic injection.
[0593] For oral administration, the iRNA agent useful in the
invention will generally be provided in the form of tablets or
capsules, as a powder or granules, or as an aqueous solution or
suspension.
[0594] Tablets for oral use may include the active ingredients
mixed with pharmaceutically acceptable excipients such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavoring agents, coloring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn
starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent,
if present, will generally be magnesium stearate, stearic acid or
talc. If desired, the tablets may be coated with a material such as
glyceryl monostearate or glyceryl distearate, to delay absorption
in the gastrointestinal tract.
[0595] Capsules for oral use include hard gelatin capsules in which
the active ingredient is mixed with a solid diluent, and soft
gelatin capsules wherein the active ingredient is mixed with water
or an oil such as peanut oil, liquid paraffin or olive oil.
[0596] For intramuscular, intraperitoneal, subcutaneous and
intravenous use, the pharmaceutical compositions of the invention
will generally be provided in sterile aqueous solutions or
suspensions, buffered to an appropriate pH and isotonicity.
Suitable aqueous vehicles include Ringer's solution and isotonic
sodium chloride. In a preferred embodiment, the carrier consists
exclusively of an aqueous buffer. In this context, "exclusively"
means no auxiliary agents or encapsulating substances are present
which might affect or mediate uptake of iRNA agent in the cells
that harbor the target gene or virus. Such substances include, for
example, micellar structures, such as liposomes or capsids, as
described below. Although microinjection, lipofection, viruses,
viroids, capsids, capsoids, or other auxiliary agents are required
to introduce iRNA agent into cell cultures, surprisingly these
methods and agents are not necessary for uptake of iRNA agent in
vivo. The iRNA agent of the present invention are particularly
advantageous in that they do not require the use of an auxiliary
agent to mediate uptake of the iRNA agent into the cell, many of
which agents are toxic or associated with deleterious side effects.
Aqueous suspensions according to the invention may include
suspending agents such as cellulose derivatives, sodium alginate,
polyvinyl-pyrrolidone and gum tragacanth, and a wetting agent such
as lecithin. Suitable preservatives for aqueous suspensions include
ethyl and n-propyl p-hydroxybenzoate.
[0597] The pharmaceutical compositions can also include
encapsulated formulations to protect the iRNA agent against rapid
elimination from the body, such as a controlled release
formulation, including implants and microencapsulated delivery
systems. Biodegradable, biocompatible polymers can be used, such as
ethylene vinyl acetate, polyanhydrides, polyglycolic acid,
collagen, polyorthoesters, and polylactic acid. Methods for
preparation of such formulations will be apparent to those skilled
in the art. The materials can also be obtained commercially from
Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal
suspensions (including liposomes targeted to infected cells with
monoclonal antibodies to viral antigens) can also be used as
pharmaceutically acceptable carriers. These can be prepared
according to methods known to those skilled in the art, for
example, as described in U.S. Pat. No. 4,522,811; PCT publication
WO 91/06309; and European patent publication EP-A-43075, which are
incorporated by reference herein.
[0598] Toxicity and therapeutic efficacy of iRNA agent can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. iRNA agents that
exhibit high therapeutic indices are preferred.
[0599] The data obtained from cell culture assays and animal
studies can be used in formulating a range of dosage for use in
humans. The dosages of compositions of the invention are preferably
within a range of circulating concentrations that include the ED50
with little or no toxicity. The dosage may vary within this range
depending upon the dosage form employed and the route of
administration utilized. For any iRNA agent used in the method of
the invention, the therapeutically effective dose can be estimated
initially from cell culture assays. A dose may be formulated in
animal models to achieve a circulating plasma concentration range
of the iRNA agent or, when appropriate, of the polypeptide product
of a target sequence (e.g., achieving a decreased concentration of
the polypeptide) that includes the IC50 (i.e., the concentration of
the test iRNA agent which achieves a half-maximal inhibition of
symptoms) as determined in cell culture. Such information can be
used to more accurately determine useful doses in humans. Levels in
plasma may be measured, for example, by high performance liquid
chromatography.
[0600] In addition to their administration individually or as a
plurality, as discussed above, iRNA agents relating to the
invention can be administered in combination with other known
agents effective in treating viral infections and diseases. In any
event, the administering physician can adjust the amount and timing
of iRNA agent administration on the basis of results observed using
standard measures of efficacy known in the art or described
herein.
[0601] For oral administration, the iRNA agent useful in the
invention will generally be provided in the form of tablets or
capsules, as a powder or granules, or as an aqueous solution or
suspension.
[0602] Methods for Treating Diseases Caused by Expression of a
Target Gene.
[0603] In one embodiment, the invention relates to a method for
treating a subject having a disease or at risk of developing a
disease caused by the expression of a target gene. In this
embodiment, iRNA agents can act as novel therapeutic agents for
controlling one or more of cellular proliferative and/or
differentiative disorders, disorders associated with bone
metabolism, immune disorders, hematopoietic disorders,
cardiovascular disorders, liver disorders, viral diseases, or
metabolic disorders. The method includes administering a
pharmaceutical composition of the invention to the patient (e.g., a
human), such that expression of the target gene is silenced.
Because of their high efficiency and specificity, the iRNA agent of
the present invention specifically target mRNA of target genes of
diseased cells and tissues, as described below, and at surprisingly
low dosages. The pharmaceutical compositions are formulated as
described in the preceding section, which is hereby incorporated by
reference herein.
[0604] Examples of genes which can be targeted for treatment
include, without limitation, an oncogene (Hanahan, D. and R. A.
Weinberg, Cell (2000) 100:57; and Yokota, J., Carcinogenesis (2000)
21(3):497-503); a cytokine gene (Rubinstein, M., et al., Cytokine
Growth Factor Rev. (1998) 9(2):175-81); a idiotype (Id) protein
gene (Benezra, R., et al., Oncogene (2001) 20(58):8334-41; Norton,
J. D., J. Cell Sci. (2000) 113(22):3897-905); a prion gene
(Prusiner, S. B., et al., Cell (1998) 93(3):337-48; Safar, J., and
S. B. Prusiner, Prog. Brain Res. (1998) 117:421-34); a gene that
expresses molecules that induce angiogenesis (Gould, V. E. and B.
M. Wagner, Hum. Pathol. (2002) 33(11):1061-3); adhesion molecules
(Chothia, C. and E. Y. Jones, Annu. Rev. Biochem. (1997) 66:823-62;
Parise, L. V., et al., Semin. Cancer Biol. (2000) 10(6):407-14);
cell surface receptors (Deller, M. C., and Y. E. Jones, Curr. Opin.
Struct. Biol. (2000) 10(2):213-9); genes of proteins that are
involved in metastasizing and/or invasive processes (Boyd, D.,
Cancer Metastasis Rev. (1996) 15(1):77-89; Yokota, J.,
Carcinogenesis (2000) 21(3):497-503); genes of proteases as well as
of molecules that regulate apoptosis and the cell cycle (Matrisian,
L. M., Curr. Biol. (1999) 9(20):R776-8; Krepela, E., Neoplasma
(2001) 48(5):332-49; Basbaum and Werb, Curr. Opin. Cell Biol.
(1996) 8:731-738; Birkedal-Hansen, et al., Crit. Rev. Oral Biol.
Med. (1993) 4:197-250; Mignatti and Rifkin, Physiol. Rev. (1993)
73:161-195; Stetler-Stevenson, et al., Annu. Rev. Cell Biol. (1993)
9:541-573; Brinkerhoff, E., and L. M. Matrisan, Nature Reviews
(2002) 3:207-214; Strasser, A., et al., Annu. Rev. Biochem. (2000)
69:217-45; Chao, D. T. and S. J. Korsmeyer, Annu. Rev. Immunol.
(1998) 16:395-419; Mullauer, L., et al., Mutat. Res. (2001)
488(3):211-31; Fotedar, R., et al., Prog. Cell Cycle Res. (1996)
2:147-63; Reed, J. C., Am. J. Pathol. (2000) 157(5):1415-30; D'Ari,
R., Bioassays (2001) 23(7):563-5); genes that express the EGF
receptor; Mendelsohn, J. and J. Baselga, Oncogene (2000)
19(56):6550-65; Normanno, N., et al., Front. Biosci. (2001)
6:D685-707); and the multi-drug resistance 1 gene, NMDR1 gene
(Childs, S., and V. Ling, Imp. Adv. Oncol. (1994) 21-36).
[0605] In the prevention of disease, the target gene may be one
which is required for initiation or maintenance of the disease, or
which has been identified as being associated with a higher risk of
contracting the disease. In the treatment of disease, the iRNA
agent can be brought into contact with the cells or tissue
exhibiting the disease. For example, iRNA agent substantially
identical to all or part of a mutated gene associated with cancer,
or one expressed at high levels in tumor cells, may be brought into
contact with or introduced into a cancerous cell or tumor gene.
[0606] Examples of cellular proliferative and/or differentiative
disorders include cancer, e.g., a carcinoma, sarcoma, metastatic
disorder or hematopoietic neoplastic disorder, such as a leukemia.
A metastatic tumor can arise from a multitude of primary tumor
types, including but not limited to those of prostate, colon, lung,
breast and liver origin. As used herein, the terms "cancer,"
"hyperproliferative," and "neoplastic" refer to cells having the
capacity for autonomous growth, i.e., an abnormal state or
condition characterized by rapidly proliferating cell growth. These
terms are meant to include all types of cancerous growths or
oncogenic processes, metastatic tissues or malignantly transformed
cells, tissues, or organs, irrespective of histopathologic type or
stage of invasiveness. Proliferative disorders also include
hematopoietic neoplastic disorders, including diseases involving
hyperplastic/neoplastic cells of hematopoietic origin, e.g.,
arising from myeloid, lymphoid or erythroid lineages, or precursor
cells thereof.
[0607] The pharmaceutical compositions of the present invention can
also be used to treat a variety of immune disorders, in particular
those associated with overexpression or aberrant expression of a
gene or expression of a mutant gene. Examples of hematopoietic
disorders or diseases include, without limitation, autoimmune
diseases (including, for example, diabetes mellitus, arthritis
(including rheumatoid arthritis, juvenile rheumatoid arthritis,
osteoarthritis, psoriatic arthritis), multiple sclerosis,
encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,
automimmune thyroiditis, dermatitis (including atopic dermatitis
and eczematous dermatitis), psoriasis, Sjogren's Syndrome, Crohn's
disease, aphthous ulcer, iritis, conjunctivitis,
keratoconjunctivitis, ulcerative colitis, asthma, allergic asthma,
cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis,
drug eruptions, leprosy reversal reactions, erythema nodosum
leprosum, autoimmune uveitis, allergic encephalomyelitis, acute
necrotizing hemorrhagic encephalopathy, idiopathic bilateral
progressive sensorineural hearing, loss, aplastic anemia, pure red
cell anemia, idiopathic thrombocytopenia, polychondritis, Wegener's
granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome,
idiopathic sprue, lichen planus, Graves' disease, sarcoidosis,
primary biliary cirrhosis, uveitis posterior, and interstitial lung
fibrosis), graft-versus-host disease, cases of transplantation, and
allergy.
[0608] In another embodiment, the invention relates to methods for
treating viral diseases, including but not limited to hepatitis C,
hepatitis B, herpes simplex virus (HSV), HIV-AIDS, poliovirus, and
smallpox virus. iRNA agent of the invention are prepared as
described herein to target expressed sequences of a virus, thus
ameliorating viral activity and replication. The iRNA agents can be
used in the treatment and/or diagnosis of viral infected tissue,
both animal and plant. Also, such iRNA agent can be used in the
treatment of virus-associated carcinoma, such as hepatocellular
cancer.
[0609] For example, the iRNA agent of the present invention are
useful for treating a subject having an infection or a disease
associated with the replication or activity of a (+) strand RNA
virus having a 3'-UTR, such as HCV. In this embodiment, the iRNA
agent can act as novel therapeutic agents for inhibiting
replication of the virus. The method includes administering a
pharmaceutical composition of the invention to the patient (e.g., a
human), such that viral replication is inhibited. Examples of (+)
strand RNA viruses which can be targeted for inhibition include,
without limitation, picomaviruses, caliciviruses, nodaviruses,
coronaviruses, arteriviruses, flaviviruses, and togaviruses.
Examples of picomaviruses include enterovirus (poliovirus 1),
rhinovirus (human rhinovirus 1A), hepatovirus (hepatitis A virus),
cardiovirus (encephalomyocarditis virus), aphthovirus
(foot-and-mouth disease virus O), and parechovirus (human echovirus
22). Examples of caliciviruses include vesiculovirus (swine
vesicular exanthema virus), lagovirus (rabbit hemorrhagic disease
virus), "Norwalk-like viruses" (Norwalk virus), "Sapporo-like
viruses" (Sapporo virus), and "hepatitis E-like viruses" (hepatitis
E virus). Betanodavirus (striped jack nervous necrosis virus) is
the representative nodavirus. Coronaviruses include coronavirus
(avian infections bronchitis virus) and torovirus (Berne virus).
Arterivirus (equine arteritis virus) is the representative
arteriviridus. Togavirises include alphavirus (Sindbis virus) and
rubivirus (Rubella virus). Finally, the flaviviruses include
flavivirus (Yellow fever virus), pestivirus (bovine diarrhea
virus), and hepacivirus (hepatitis C virus). In a preferred
embodiment, the virus is hepacivirus, the hepatitis C virus.
Although the foregoing list exemplifies vertebrate viruses, the
present invention encompasses the compositions and methods for
treating infections and diseases caused by any (+) strand RNA virus
having a 3'-UTR, regardless of the host. For example, the invention
encompasses the treatment of plant diseases caused by sequiviruses,
comoviruses, potyviruses, sobemovirus, luteoviruses, tombusviruses,
tobavirus, tobravirus, bromoviruses, and closteroviruses.
[0610] The pharmaceutical compositions encompassed by the invention
may be administered by any means known in the art including, but
not limited to, oral or parenteral routes, including intravenous,
intramuscular, intraperitoneal, subcutaneous, transdermal, airway
(aerosol), ocular, rectal, vaginal, and topical (including buccal
and sublingual) administration. In preferred embodiments, the
pharmaceutical compositions are administered by intravenous or
intraparenteral infusion or injection. The pharmaceutical
compositions can also be administered intraparenchymally,
intrathecally, and/or by stereotactic injection.
[0611] Methods for Inhibiting Expression of a Target Gene.
[0612] In yet another aspect, the invention relates to a method for
inhibiting the expression of a target gene in a cell or organism.
In one embodiment, the method includes administering the inventive
iRNA agent or a pharmaceutical composition containing the iRNA
agent to a cell or an organism, such as a mammal, such that
expression of the target gene is silenced. Because of their
surprisingly improved stability and bioavailability, the iRNA agent
of the present invention effectively inhibit expression or activity
of target genes at surprisingly low dosages. Compositions and
methods for inhibiting the expression of a target gene using iRNA
agent can be performed as described in the preceding sections,
particularly Sections 4 and 5.
[0613] In this embodiment, a pharmaceutical composition containing
the iRNA agent may be administered by any means known in the art
including, but not limited to oral or parenteral routes, including
intravenous, intramuscular, intraperitoneal, subcutaneous,
transdermal, airway (aerosol), ocular, rectal, vaginal, and topical
(including buccal and sublingual) administration. In preferred
embodiments, the pharmaceutical compositions are administered by
intravenous or intraparenteral infusion or injection. The
pharmaceutical compositions can also be administered
intraparenchymally, intrathecally, and/or by stereotactic
injection.
[0614] The methods for inhibiting the expression of a target gene
can be applied to any gene one wishes to silence, thereby
specifically inhibiting its expression, provided the cell or
organism in which the target gene is expressed includes the
cellular machinery which effects RNA interference. Examples of
genes which can be targeted for silencing include, without
limitation, developmental genes including but not limited to
adhesion molecules, cyclin kinase inhibitors, Wnt family members,
Pax family members, Winged helix family members, Hox family
members, cytokines/lymphokines and their receptors,
growth/differentiation factors and their receptors, and
neurotransmitters and their receptors; (2) oncogenes including but
not limited to ABL1, BCL1, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA,
ERBB, EBRB2, ETS1, ETS1, ETV6, FGR, FOS, FYN, HCR, HRAS, JUN, KRAS,
LCK, LYN, MDM2, MLL, MYB, MYC, MYCL1, MYCN, NRAS, PIM1, PML, RET,
SRC, TAL1, TCL3 and YES; (3) tumor suppresser genes including but
not limited to APC, BRCA1, BRCA2, MADH4, MCC, NF1, NF2, RB1, TP53
and WT1; and (4) enzymes including but not limited to ACP
desaturases and hydroxylases, ADP-glucose pyrophorylases, ATPases,
alcohol dehydrogenases, amylases, amyloglucosidases, catalases,
cellulases, cyclooxygenases, decarboxylases, dextrinases, DNA and
RNA polymerases, galactosidases, glucanases, glucose oxidases,
GTPases, helicases, hemicellulases, integrases, invertases,
isomerases, kinases, lactases, lipases, lipoxygenases, lysozymes,
pectinesterases, peroxidases, phosphatases, phospholipases,
phosphorylases, polygalacturonases, proteinases and peptideases,
pullanases, recombinases, reverse transcriptases, topoisomerases,
and xylanases.
[0615] In addition to in vivo gene inhibition, the skilled artisan
will appreciate that the iRNA agent of the present invention are
useful in a wide variety of in vitro applications. Such in vitro
applications, include, for example, scientific and commercial
research (e.g., elucidation of physiological pathways, drug
discovery and development), and medical and veterinary diagnostics.
In general, the method involves the introduction of the iRNA agent
into a cell using known techniques (e.g., absorption through
cellular processes, or by auxiliary agents or devices, such as
electroporation and lipofection), then maintaining the cell for a
time sufficient to obtain degradation of an mRNA transcript of the
target gene.
[0616] Methods for Identifying iRNA Agent Having Increased
Stability.
[0617] In yet another aspect, the invention relates to methods for
identifying iRNA agent having increased stability in biological
tissues and fluids such as serum. iRNA agent having increased
stability have enhanced resistance to degradation, e.g., by
chemicals or nucleases (particularly endonucleases) which normally
degrade RNA molecules. Methods for detecting increases in nucleic
acid stability are well known in the art. Any assay capable of
measuring or detecting differences between a test iRNA agent and a
control iRNA agent in any measurable physical parameter may be
suitable for use in the methods of the present invention. In
general, because the inhibitory effect of an iRNA agent on a target
gene activity or expression requires that the molecule remain
intact, the stability of a particular iRNA agent can be evaluated
indirectly by observing or measuring a property associated with the
expression of the gene. Thus, the relative stability of an iRNA
agent can be determined by observing or detecting (1) an absence or
observable decrease in the level of the protein encoded by the
target gene, (2) an absence or observable decrease in the level of
mRNA product from the target gene, and (3) a change or loss in
phenotype associated with expression of the target gene. In the
context of a medical treatment, the stability of an iRNA agent may
be evaluated based on the degree of the inhibition of expression or
function of the target gene, which in turn may be assessed based on
a change in the disease condition of the patient, such as reduction
in symptoms, remission, or a change in disease state.
[0618] In one embodiment, the method includes preparing an iRNA
agent as described above (e.g., through chemical synthesis),
incubating the iRNA agent with a biological sample, then analyzing
and identifying those iRNA agent that exhibit an increased
stability as compared to a control iRNA agent.
[0619] In an exemplified embodiment, iRNA agent is produced in
vitro by mixing/annealing complementary single-stranded RNA
strands, preferably in a molar ratio of at least about 3:7, more
preferably in a molar ratio of about 4:6, and most preferably in
essentially equal molar amounts (e.g., a molar ratio of about 5:5).
Preferably, the single-stranded RNA strands are denatured prior to
mixing/annealing, and the buffer in which the mixing/annealing
reaction takes place contains a salt, preferably potassium
chloride. Single-stranded RNA strands may be synthesized by solid
phase synthesis using, for example, an Expedite 8909 synthesizer
(Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany),
as described above.
[0620] iRNA agent are incubated with a biological sample under the
conditions sufficient or optimal for enzymatic function. After
incubating with a biological sample, the stability of the iRNA
agent is analyzed by means conventional in the art, for example
using RNA gel electrophoresis as exemplified herein. For example,
when the sample is serum, the iRNA agent may be incubated at a
concentration of 1-10 .mu.M, preferably 2-8 .mu.M, more preferably
3-6 .mu.M, and most preferably 4-5 .mu.M. The incubation
temperature is preferably between 25.degree. C. and 45.degree. C.,
more preferably between 35.degree. C. and 40.degree. C., and most
preferably about 37.degree. C.
[0621] The biological sample used in the incubation step may be
derived from tissues, cells, biological fluids or isolates thereof.
For example, the biological sample may be isolated from a subject,
such as a whole organism or a subset of its tissues or cells. The
biological sample may also be a component part of the subject, such
as a body fluid, including but not limited to blood, serum, plasma,
mucus, lymphatic fluid, synovial fluid, cerebrospinal fluid,
saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid
and semen. Preferably, the biological sample is a serum derived
from a blood sample of a subject. The subject is preferably a
mammal, more preferably a human or a mouse.
[0622] In another embodiment, the method includes selecting an iRNA
agent having increased stability by measuring the mRNA and/or
protein expression levels of a target gene in a cell following
introduction of the iRNA agent. In this embodiment, an iRNA agent
of the invention inhibits expression of a target gene in a cell,
and thus the method includes selecting an iRNA agent that induces a
measurable reduction in expression of a target gene as compared to
a control iRNA agent. Assays that measure gene expression by
monitoring RNA and/or protein levels can be performed within about
24 hours following uptake of the iRNA agent by the cell. For
example, RNA levels can be measured by Northern blot techniques,
RNAse Protection Assays, or Quality Control-PCR (QC-PCR) (including
quantitative reverse transcription coupled PCR (RT-PCR)) and
analogous methods known in the art. Protein levels can be assayed,
for example, by Western blot techniques, flow cytometry, or
reporter gene expression (e.g., expression of a fluorescent
reporter protein, such as green fluorescent protein (GFP)). RNA
and/or protein levels resulting from target gene expression can be
measured at regular time intervals following introduction of the
test iRNA agent, and the levels are compared to those following
introduction of a control iRNA agent into cells. A control iRNA
agent can be a nonsensical iRNA agent (i.e., an iRNA agent having a
scrambled sequence that does not target any nucleotide sequence in
the subject), an iRNA agent that can target a gene not present in
the subject (e.g., a luciferase gene, when the iRNA agent is tested
in human cells), or an iRNA agent otherwise previously shown to be
ineffective at silencing the target gene. The mRNA and protein
levels of the test sample and the control sample can be compared.
The test iRNA agent is selected as having increased stability when
there is a measurable reduction in expression levels following
absorption of the test iRNA agent as compared to the control iRNA
agent. mRNA and protein measurements can be made using any
art-recognized technique (see, e.g., Chiang, M. Y., et al., J. Biol
Chem. (1991) 266:18162-71; Fisher, T, et al., Nucl. Acids Res.
(1993) 21:3857; and Chen et al., J. Biol. Chem. (1996)
271:28259).
[0623] The ability of an iRNA agent composition of the invention to
inhibit gene expression can be measured using a variety of
techniques known in the art. For example, Northern blot analysis
can be used to measure the presence of RNA encoding a target
protein. The level of the specific mRNA produced by the target gene
can be measured, e.g., using RT-PCR. Because iRNA agent directs the
sequence-specific degradation of endogenous mRNA through RNAi, the
selection methods of the invention encompass any technique that is
capable of detecting a measurable reduction in the target RNA. In
yet another example, Western blots can be used to measure the
amount of target protein present. In still another embodiment, a
phenotype influenced by the amount of the protein can be detected.
Techniques for performing Western blots are well known in the art
(see, e.g., Chen, et al., J. Biol. Chem. (1996) 271:28259).
[0624] When the target gene is to be silenced by an iRNA agent that
targets a promoter sequence of the target gene, the target gene can
be fused to a reporter gene, and reporter gene expression (e.g.,
transcription and/or translation) can be monitored. Similarly, when
the target gene is to be silenced by an iRNA agent that targets a
sequence other than a promoter, a portion of the target gene (e.g.,
a portion including the target sequence) can be fused with a
reporter gene so that the reporter gene is transcribed. By
monitoring a change in the expression of the reporter gene in the
presence of the iRNA agent, it is possible to determine the
effectiveness of the iRNA agent in inhibiting the expression of the
reporter gene. The expression levels of the reporter gene in the
presence of the test iRNA agent versus a control iRNA agent are
then compared. The test iRNA agent is selected as having increased
stability when there is a measurable reduction in expression levels
of the reporter gene as compared to the control iRNA agent.
Examples of reporter genes useful for use in the present invention
include, without limitation, those coding for luciferase, GFP,
chloramphenicol acetyl transferase (CAT), .beta.-galactosidase, and
alkaline phosphatase. Suitable reporter genes are described, for
example, in Current Protocols in Molecular Biology, John Wiley
& Sons, New York (Ausubel, F. A., et al., eds., 1989); Gould,
S. J., and S. Subramani, Anal. Biochem. (1988) 7:404-408; Gorman,
C. M., et al., Mol. Cell. Biol. (1982) 2:1044-1051; and Selden, R.,
et al., Mol. Cell. Biol. (1986) 6:3173-3179; each of which is
hereby incorporated by reference.
[0625] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. All
publications, patent applications, patents, and other references
mentioned herein are incorporated by reference in their entirety.
In case of conflict, the present specification, including
definitions, will control. In addition, the materials, methods, and
examples are illustrative only and not intended to be limiting.
EXAMPLES
[0626] 1. Introduction and Design Criteria
[0627] siRNA-based drugs can be targeted to specific cells.
Targeting moieties can be conjugated to siRNAs through covalent or
noncovalent approaches. Several factors are considered when
designing targeting moieties. For example, a targeting moiety can
be a cationic carrier molecule, such as a PEI polymers or PAMAM
dendrimer, both of which have been demonstrated to be effective
gene delivery agents. Inclusion of a water-soluble unit on a
targeting moiety has also been shown to increase the
bioavailability, especially for use in oral delivery. Incorporation
of a lipophilic group on a targeting moiety may also increase
membrane permeability. Ideally, siRNA-based drugs, including the
targeting moiety, are nontoxic and nonimmunogenic. A targeting unit
may be necessary to increase the specificity in delivery. Further,
if the targeting moiety (or moieties) has multiple recognition (or
binding) sites, the moiety should have conformational flexibility,
yet be stable enough to maintain its orientation. The number of
synthetic steps and the cost of synthesis are also preferably
minimized.
[0628] 2. Results and Discussion
[0629] 2.1. Cationic Porphyrins
[0630] (1) Cationic porphyrins have good water-solubility while
being lipophilic with the central porphyrin moiety, which serves as
a chromophore in itself. The cationic porphyrins are known to be
low in toxicity, and can be prepared in a few synthetic steps from
a wide variety of commercial porphyrin derivatives. Although there
are some exceptional cases, earlier reports proposed that there are
three types of binding modes in the porphyrin-DNA duplex: (i)
intercalation, (ii) outside binding without self-stacking, and
(iii) outside binding with self-stacking (Fiel, R. J. J. Biomol.
Struct. Dyn. 1989, 6(6), 1259-1274). In general, the binding mode
of a specific porphyrin depends on its effective thickness,
typically resulting from the peripheral substitution pattern and
the type of the central metal ion. Commercial compounds 1 and 2 are
the well-studied cationic porphyrins which are proven to deliver an
oligodeoxynucleotide the gene into the nucleus (Benimetskaya, et
al., Nucleic Acid Res. 1998, 26, 5310-531). 81
[0631] (2) The second generation cationic porphyrin derivatives 3
and 4 were designed and tested for oligodeoxynucleotide delivery
specifically into the primary leukemic cells showing good
activities (Kralova et al., J. Med. Chem. 2003, 46, 2049-2056).
Authors claimed that a very delicate balance between the number and
position of the positive charges and the lipophilicity of the
molecule is a key factor as shown by the improved cellular uptake
of 3 over 2. 82
[0632] Applying the method developed by Frechet and a coworker to
prepare the multivalent sugar-terminated dendrons using a DNA
synthesizer, the structure 5 is proposed as a potential siRNA
delivery vehicle. This model compound combines several major
factors (i.e., cationic porphyrin, polyethylene glycol for
water-solubility, conformational flexibility, biocompatibility, and
carbohydrate dendrons for targeting) for siRNA delivery purposes.
83
[0633] Other designs include combining the cationic lipids (i.e.,
amphiphiles) and cationic porphyrins to increase lipophilicity and
thus improve cellular uptake. These can be categorized with the
second generation cationic porphyrins (e.g., 3 and 4), where again
the subtle play between the number and location of the positive
charges and the lipophilicity may compensate for the deficit in the
first generation cationic porphyrins, 1 and 2. As a starting point,
substituting the periphery of a commercial cationic porphyrin with
one or two units of long aliphatic chains (e.g., C.sub.18H.sub.37)
is proposed.
[0634] 2.2. Multivalent Carbohydrate siRNA Conjugates
[0635] Target Design. Following the model compound 6 synthesized
and studied by Biessen and coworkers, two target molecules, 7 and
8, were designed by modifying the central branching unit and adding
a primary and secondary alcohol to the steroid for the covalent
attachment of the oligonucleotide and a solid support. 8485
[0636] The main factors to our carrier design were the multivalent
sugar unit (e.g., GalNAc) for targeting and the steroid unit, where
these two groups were connected through a flexible linker.
Commercially available chenodeoxycholic acid (CDCA) has two
secondary alcohols in its scaffold. One of the secondary alcohols
can be further modified to a primary alcohol protected with a
dimethoxytrityl (DMT) group for the automated oligonucleotide
synthesis on a solid support. Structures 7 and 8 only differ in the
type of branching unit (trihydroxybenzyl and GalNAc, respectively)
for the multivalent sugar attachment.
[0637] Further goals in this project include building the same
system with mannose as a targeting sugar instead of GalNAc. In
general, GalNAc has been used for liver cell targeting and mannose
has been used for cancer cell targeting. However, these sugars are
not limited to these uses.
[0638] Computer Modeling. When energy-minimized structures of 7 and
8 were obtained through HyperChem 7.0 (CompuChem, Germany), the
terminal carbohydrate groups in compound 8 were distributed in a
more globular way compared to those in compound 7, which has a
rather flat aromatic group (i.e., benzyl) as a central branching
unit.
[0639] Synthetic Plan. A synthetic scheme to prepare the first
target 7 is outlined in this section. Our strategy is first to
build the middle linker moiety (Scheme 1) followed by the one-pot
attachment of peripheral sugars using a commercially available
galactal (Senn Chemicals, Scheme 2), and 8687
[0640] the final conjugation of the derivatized CDCA unit.sup.14
with the linker carrying the trivalent sugar (Scheme 3 and Scheme
4). 8889 9091 9293
[0641] Synthesis of the Trivalent Linker 14. Synthesis of compound
7 started from the preparation of the middle spacer unit (Scheme
1). Protection of diol 9 using stoichiometric amount of
t-butyldiphenylsilyl chloride (TBDPSCl) afforded the desired
mono-silylated product 10 in a relatively low yield (43%) along
with the di-silylated compound and the unreacted starting material.
The alcohol 10 was then tosylated and iodinated to produce 12. Next
step was the triple etherification of methyl
3,4,5-trihydroxybenzoate 13 with a slightly more than three
equivalents of 12 under a basic condition using cesium carbonate
(Cs.sub.2CO.sub.3) in DMF at 50.degree. C. 94
[0642] Generally, iodides are used instead of bromides or chlorides
to avoid heating at higher temperatures (i.e., >100.degree. C.).
In addition, Cs.sub.2CO.sub.3 is more ionizable than potassium
carbonate (K.sub.2CO.sub.3), but has the drawback of being more
hygroscopic. .sup.1H NMR analysis indicated that the isolated
product was the dialkylated species 33 substituted at 3,4-positions
instead of trialkylated 14. This wa evidenced by the two small
doublets around 6.5 ppm resulting from two different ortho-protons.
Formation of the 3,4-dialkylated, but not the symmetrical
3,5-dialkylated product, indicated that the first alkylation
occurred at the para-position and not the meta-position under this
condition (similar results reported in References 15 and 16).
Approximately 23% of the iodide 12 used, and which did not react,
was recovered.
[0643] In another attempt, potassium carbonate (K.sub.2CO.sub.3)
was used instead of Cs.sub.2CO.sub.3, where the reaction was heated
at 110.degree. C. for 15 h. Here, ca. 6 equiv of iodide 12 was
added to the triol 13 with excess of K.sub.2CO.sub.3 (58 equiv to
13). Again this reaction only afforded the dialkylated compound 33
as a minor product with no formation of the desired molecule 14.
When a series of compounds detected on TLC was isolated and checked
by .sup.1H NMR spectroscopy, one of the major products was the
carbonate derivative of 12 (i.e., 34) as evidenced by the 95
[0644] appearance of a methylene peak from the glycol unit with
unusual downfield chemical shift at ca. 4.3 ppm. This also
coincides with the mass spectra result assuming the possible
fragmentation at one side of the carbonate linkage.
[0645] The failure to synthesize the desired compound 14 using
either condition mentioned above may be both attributed to the high
reactivity of the iodide 12 which reacted with the carbonate added
as a base before deprotonated phenoxide derivative of 13 may attack
12 to make the ether linkage. Most of the references found so far
to make these bonds have used either chloride or bromide with the
only exception of the addition of potassium iodide (KI) in
catalytic amount when bromide is used to generate the corresponding
iodide in situ.
[0646] In another example, the Mitsunobu condition was attempted to
prepare the trivalent linker 14 (Scheme 5), where again only the
spot having a similar R.sub.f to the dialkylated species 33 was
observed on TLC as the most hydrophobic compound. 96
[0647] Etherification reaction of methyl 3,4,5-trihydroxybenzoate
13 with alkyl halides to get the trivalent linker was problematic,
and additional methods were attempted. The investigations carried
out so far did not provide any efficient way to prepare this
linker, however several more methods could be tested before
changing to different types of linkers. Table 1 summarizes the
conditions to prepare trivalent linkers. Through the results
obtained so far, the alkylation seems to be occurring first at the
para-position, and the third alkylation at the meta-position
required harsh conditions. In addition, usage of iodide derivative
(# 3) was unfavorable due to its high reactivity, which resulted in
the formation of carbonate with excess amounts of K.sub.2CO.sub.3
before reacting with the phenolate anions or 13. Unlike the
reported result, the reaction condition using t-butoxide as a base
at lower temperature was not sufficient to complete the reaction
where the unreacted starting material 35 was mostly recovered.
5TABLE 1 Attempts to prepare trivalent linker 14. 97 Entry Reaction
Type Reactant Condition Result .sup..dagger. Mitsunobu BDPS H 10
PPh.sub.3, DIAD, THF, 0.degree. C. .fwdarw. rt, 2 d no desired
product Etherification BDPS 12 Cs.sub.2CO.sub.3, DMF, 50.degree. C.
.fwdarw. 80.degree. C., 7h .fwdarw. 3,4-dialkylated, sm rt. 16 h
Etherification BDPS 12 K.sub.2CO.sub.3, DMF, 110.degree. C., 4.5 h
.fwdarw. rt, 17 h 3,4-dialkylated, carbonate Etherification BDPS r
35 t-BuOK, CH.sub.3CN, 60.degree. C., 17 h mostly sm Etherification
BDPS l 36 K.sub.2CO.sub.3, 18-crown-6, DMF, 80.degree. C., 7 h
mostly sm Etherification l 37 K.sub.2CO.sub.3, DMF, 80.degree. C.,
7 h Not characterized sm = starting material "Reactant" recovered;
carbonate = carbonate derivative of the "Reactant";
.sup..dagger.product will be the triol analog of 14 (without
TBDPS).
[0648] This could be due to the presence of bulky TBDPS protecting
group, whereas the reported reactions used unprotected alcohol. The
last entry (#6) may be a possible alternative for avoiding the
usage of the TBDPS-protected alkyl halides. However, in this case,
using more than 6 equiv of alcohol 37 to 13 is recommended.
[0649] Modified Targets and Synthetic Plans. During the earlier
stages of the project, the target designs were modified. First, an
unstable carbamate linkage in the central branching unit was
replaced with an amide linkage. Second, in order to prevent
potential difficulties in derivatizing the secondary alcohol at C-7
of CDCA, usage of lithocholic acid (LCA) was suggested instead of
CDCA as shown in structure 38. In an alternative approach, the
primary alcohol could be appended to the C-7 instead of C-3 as
shown in structure 39. Synthetic plans to prepare new two target
molecules, 38 and 39, starting from 14 are shown in Schemes 6-10.
Here the mono-Boc protected amine 42 was prepared by using excess
amount of diamine 41. 9899 100101 102 103104105 106107 108109
[0650] Chemistry on the Bile Acids. Earlier attempts to esterify
CDCA 24 (or LCA 44) did not produce the correct structure 25 by
.sup.1H NMR. In these experiments, the integration ratio of the
methyl group from the ester to the protons at C3 and C7 in the 3-4
ppm region was used to measure the reaction progress. The protons
at C3 and C7 in the 3-4 ppm region were expected to manifest the
largest change in the chemical shift upon acylation.
[0651] Several minor peaks at 5.0-5.6 ppm in the .sup.1H NMR after
reaction originated from the alkene protons. This major product,
which was isolated by column chromatography, seemed to be the
mixture of all possible regioisomers containing one double bond
(e.g., 62) resulting from the dehydration under the acidic 110
[0652] conditions at high temperatures when the reaction was
continued for extended hours. By then, the verification of the
structure 25 was further puzzled by the existence of the MS peak
with the MW of 407, which matched the MW of 25 and was the current
major contaminant in the mass spectra. Furthermore, the peak
corresponding to the mass of the dehydrated compound 62 was found
in the form of a sodium adduct which was in the similar region
(i.e., MW 409) as the contaminant. The dehydrated species was again
found as a major compound by mass spectra, but in lesser amounts
when lithocholic acid 44 was used for the esterification reaction
under the same condition.
[0653] When trace amounts of concentrated HCl was added for shorter
periods of time for the esterification of CDCA 24 following the
procedure of the Jung group (Jung and Johnson, Tetrahedron 2001,
57, 1449-1481), the minor spot on TLC (lower R.sub.f than the
isolated major 62 and its regioisomers) detected in the previous
assays became the major spot; and was later confirmed to be the
correct structure 25. Selective protection of C-3 alcohol with the
TBDPS group proceeded smoothly (Scheme 9), however the remaining
TBDPSCl which coincided with the R.sub.f of the product 56 was
troublesome when carried through for the next step, as this
actually helped to protect the second alcohol at C-7 when sodium
hydride was used as a base, thus complicating the isolation of
desired product 57. Usage of less than one equivalent of TBDPSCl is
recommended to avoid this problem when preparing 56.
[0654] 3. Experimental Section
[0655] General. Glassware was dried in an oven and cooled to room
temperature in nitrogen (N.sub.2) or argon (Ar) atmosphere before
use. All reactions were carried out under dry N.sub.2 or Ar
atmosphere unless otherwise mentioned. Solvents were purchased from
Acros as anhydrous grades (<50 ppm of water) and used as
received. Reagents were of commercial grades and were used without
further purification.
[0656] Analytical thin layer chromatography (TLC) was performed on
0.2 mm silica glass coated sheets (E. Merck) with F-254 indicator.
Visualization of the products on TLC plate was performed by UV
light, iodine (12), p-anisaldehye, potassium permanganate
(KMnO.sub.4), and ninhydrin. Flash column chromatography was
performed on Merck 40-63 .mu.m silica gel. Nuclear magnetic
resonance (NMR) spectra were recorded on a Varian Unity 300 or a
Varian Unity 400 spectrometer unless otherwise mentioned. .sup.1H
NMR chemical shifts were measured relative to the residual solvent
peak at 7.26 ppm in CDCl.sub.3 and at 2.50 ppm in DMSO-d.sub.6. The
electrospray ionization mass spectrometry (ESI MS) experiments were
performed by Dr. Gary Levine using in-house facilities.
[0657]
2-(2-{2-[2-(t-Butyldiphenylsilyloxy)ethoxy]ethoxy}ethoxy)ethanol
(10). A mixture of tetra(ethylene glycol) 9 (5.00 mL, 28.7 mmol)
and imidazole (2.91 g, 42.6 mmol) was dissolved in 10 mL of
N,N-dimethylformamide (DMF). TBDPSCl (7.80 mL, 29.4 mmol) was added
to the solution dropwise over a 30 min-period using a syringe with
an additional amount of DMF (total 20 mL) being added to avoid the
reaction mixture becoming cloudy. The reaction was stirred at room
temperature for 16 h. DMF was removed under reduced pressure and
the crude product was chromatographed (SiO.sub.2, gradient,
hexane/EtOAc 1:1 to 2:3) to give 6.05 g (14.0 mmol, 49%) of 10 as a
light colorless oil. R.sub.f 0.21 (hexane/EtOAc 1:1); .sup.1H NMR
(400 MHz, CDCl.sub.3) .delta. 7.68 (m, 4H, H-2 of phenyl),
7.44-7.35 (m, 6H, H-3 and H-4 of phenyl), 3.81 (t, 2H, J=5.5 Hz,
CH.sub.2OSi), 3.71 (m, 2H, HOCH.sub.2CH.sub.2), 3.68-3.58 (m, 12H,
HOCH.sub.2CH.sub.2(OCH.sub.2CH.sub.2).sub.2OCH.sub.2CH.sub.2), 2.46
(br s, 1H, OH), 1.05 (s, 9H, C(CH.sub.3).sub.3); MS (ESI) Calcd for
C.sub.24H.sub.36O.sub.5SiNa (M+Na.sup.+): 455.2, Found: 455.2.
[0658]
2-(2-{2-[2-(4-Toluenesulfonyloxy)ethoxy]ethoxy}ethoxy)ethoxy-t-buty-
ldiphenylsilane (11). To a solution of 10 (1.06 g, 2.46 mmol) in 30
mL of CH.sub.2Cl.sub.2 was added triethylamine (0.62 mL, 4.45
mmol). 4-Toluenesulfonyl chloride (TsCl, 567 mg, 2.97 mmol) was
added to this solution in one portion at 0.degree. C., and the
mixture was stirred at under Ar for 24 h allowing it to warm to
room temperature slowly. The reaction was quenched by pouring into
a mixture of water (100 mL) and chloroform (70 mL), separated
organic layer, and the aqueous layer was further extracted with
chloroform (100 mL). The combined organic layers were dried over
sodium sulfate (Na.sub.2SO.sub.4) and concentrated. The crude
product was chromatographed (SiO.sub.2, gradient, hexane/EtOAc 5:1
to 2:1) to give 1.08 g (1.85 mmol, 75%) of 11 as a clear colorless
oil. R.sub.f 0.65 (hexane/EtOAc 1:1); .sup.1H NMR (400 MHz,
CDCl.sub.3) 7.79 (d, 2H, J=8.3 Hz, H-2 of tosyl), 7.68 (m, 4H, H-2
of phenyl), 7.44-7.35 (m, 6H, H-3 and H-4 of phenyl), 7.32 (d, 2H,
J=8.7 Hz, H-3 of tosyl), 4.14 (t, 2H, J=4.9 Hz,
TsOCH.sub.2CH.sub.2), 3.80 (t, 2H, J=5.3 Hz, CH.sub.2OSi), 3.66 (t,
2H, J=4.9 Hz, TsOCH.sub.2CH.sub.2), 3.64-3.54 (m, 10H,
(OCH.sub.2CH.sub.2).sub.2OCH.sub.2CH.sub.2OSi), 2.43 (s, 3H,
CH.sub.3 of tosyl), 1.04 (s, 9H, C(CH.sub.3).sub.3); MS (ESI) Calcd
for C.sub.31H.sub.42O.sub.7SSiNa (M+Na.sup.+): 609.2, Found:
609.2.
[0659]
2-{2-[2-(2-Iodoethoxy)ethoxy]ethoxy}ethoxy-t-butyldiphenylsilane
(12). Compound 11 (1.07 g, 1.83 mmol) and potassium iodide (970 mg,
5.84 mmol) were suspended in acetone (35 mL) and heated at
65.degree. C. for 6 h and stirred at room temperature for 16 h. The
solvent was removed under reduced pressure, water (100 mL) was
added to the mixture, and extracted with CH.sub.2Cl.sub.2 (100
mL.times.2). The combined organic layers were dried over
Na.sub.2SO.sub.4, concentrated, and the crude product was
chromatographed (SiO.sub.2, gradient, hexane/EtOAc 7:1 to 5:1) to
give 839 mg (1.55 mmol, 85%) of 12 as a clear colorless oil.
R.sub.f 0.34 (hexane/EtOAc 5:1); .sup.1H NMR (400 MHz, CDCl.sub.3)
7.68 (m, 4H, H-2 of phenyl), 7.45-7.35 (m, 6H, H-3 and H-4 of
phenyl), 3.81 (t, 2H, J=5.4 Hz, CH.sub.2CH.sub.2OSi), 3.74 (t, 2H,
J=6.9 Hz, CH.sub.2CH.sub.2I), 3.65 (m, 8H,
(OCH.sub.2CH.sub.2).sub.2OCH.sub.2CH.sub.2OSi), 3.61 (t, 2H, J=5.4
Hz, CH.sub.2CH.sub.2OSi), 3.24 (t, 2H, J=7.1 Hz,
CH.sub.2CH.sub.2I), 1.05 (s, 9H, C(CH.sub.3).sub.3); MS (ESI) Calcd
for C.sub.24H.sub.351O.sub.4Si- Na (M+Na.sup.+): 565.5, Found:
565.1.
[0660] Methyl 3.alpha.,7.alpha.-dihydroxycholan-24-oate (25). CDCA
(1.00 g, 2.50 mmol) was dissolved in methanol (43 mL), refluxed at
80.degree. C., and then 12 drops of conc. hydrochloric acid (HCl)
was added to this solution. The reaction mixture was heated at
reflux for 2.5 h, cooled to room temperature, neutralized with a
saturated solution of sodium bicarbonate, and the solvent was
removed. 15 mL of water was added to the mixture, and the crude
product was extracted with EtOAc (20 mL.times.3). The combined
organic layers were dried over Na.sub.2SO.sub.4, concentrated, and
the crude product was chromatographed (SiO.sub.2, gradient,
hexane/EtOAc 1:2 to EtOAc only) to give 1.08 g (2.64 mmol,
>100%, less dry) of 25 as a white solid. R.sub.f 0.40 (EtOAc);
.sup.1H NMR (400 MHz, CDCl.sub.3) 3.80 (m, 1H, H-3/H-7), 3.63 (s,
3H, CO.sub.2CH.sub.3), 3.40 (m, 1H, H-3/H-7), 2.35-0.93 (m, 28H),
0.89 (d, 3H, J=6.5 Hz, CH.sub.3), 0.87 (s, 3H, CH.sub.3), 0.62 (s,
3H, CH.sub.3); MS (ESI) Calcd for C.sub.25H.sub.42O.sub.4Na
(M+Na.sup.+): 429.3, Found: 429.3.
[0661]
2-{2-[2-(2-Bromoethoxy)ethoxy]ethoxy}ethoxy-t-butyldiphenylsilane
(35). Compound 10 (651 mg, 1.50 mmol) and carbon tetrabromide (628
mg, 1.88 mmol) were dissolved in tetrahydrofuran (THF, 0.5 mL).
Triphenylphosphine (498 mg, 1.88 mmol) was added to this solution
portionwise over 5 min with stirring and additional amount of THF
(0.5 mL) was added to the mixture. Reaction was monitored by TLC,
which indicated that it was over in 10 min. Solvent was removed at
room temperature and the crude product was chromatographed
(SiO.sub.2, hexane/EtOAc 2:1) to give 767 mg (1.55 mmol, >100%,
less dry) of 35 as a clear colorless oil. R.sub.f 0.74
(hexane/EtOAc 1:1); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.68 (m, 4H,
H-2 of phenyl), 7.45-7.35 (m, 6H, H-3 and H-4 of phenyl), 3.81 (t,
2H, J=5.5 Hz, CH.sub.2CH.sub.2OSi), 3.79 (t, 2H, J=7.1 Hz,
CH.sub.2CH.sub.2Br), 3.65 (m, 8H, (OCH.sub.2CH.sub.2).sub.2OCH.-
sub.2CH.sub.2OSi), 3.61 (t, 2H, J=5.3 Hz, CH.sub.2CH.sub.2OSi),
3.45 (t, 2H, J=6.4 Hz, CH.sub.2CH.sub.2Br), 1.05 (s, 9H,
C(CH.sub.3).sub.3); MS (ESI) Calcd for
C.sub.24H.sub.35BrO.sub.4SiNa (M+Na.sup.+): 517.1, Found:
517.1.
[0662]
2-{2-[2-(2-Chloroethoxy)ethoxy]ethoxy}ethoxy-t-butyldiphenylsilane
(36). Compound 10 (813 mg, 1.88 mmol) and triphenylphosphine (995
mg, 3.76 mmol) were dissolved in CH.sub.2Cl.sub.2, and then at
-78.degree. C., hexachloroacetone (0.57 mL, 3.76 mmol) was added
dropwise to the solution. The reaction temperature was raised to
0.degree. C., and the progress of the reaction was monitored by TLC
which indicated that it was over in 40 min. Solvent was removed at
room temperature and the crude product was chromatographed
(SiO.sub.2, gradient, hexane/EtOAc 10:1 to 8:1) to give 834 mg
(1.85 mmol, 98%) of 36 as a clear colorless oil. R.sub.f 0.89
(hexane/EtOAc 1:1); .sup.1H NMR (400 MHz, CDCl.sub.3) 7.68 (m, 4H,
H-2 of phenyl), 7.45-7.35 (m, 6H, H-3 and H-4 of phenyl), 3.81 (t,
2H, J=5.4 Hz, CH.sub.2CH.sub.2OSi), 3.74 (t, 2H, J=6.0 Hz,
CH.sub.2CH.sub.2Cl), 3.67-3.59 (m, 12H,
ClCH.sub.2CH.sub.2(OCH.sub.2CH.su- b.2).sub.2OCH.sub.2CH.sub.2OSi),
1.05 (s, 9H, C(CH.sub.3).sub.3); MS (ESI) Calcd for
C.sub.24H.sub.35ClO.sub.4SiNa (M+Na.sup.+): 473.2, Found:
473.2.
[0663] 2-{2-[2-(N-tert-Boc-amino)ethoxy]ethoxy}ethylamine (42). To
a solution of diamine 10 (1.00 mL, 6.71 mmol) in tert-butanol (10
mL) was added a 2 N aqueous solution of NaOH (3.5 mL, 7.0 mmol),
and then at 0.degree. C. di-tert-butyl-dicarbonate (1.44 g, 6.60
mmol) was added to this mixture in one portion. The mixture was
sonicated, and stirred at room temperature for 16 h. White
precipitate was observed. The reaction was quenched by carefully
neutralizing to pH 7 using a pH paper, evaporated the solvent to
dryness, added DMF (3 mL), sonicated, and filtered through a
pipet-size size exclusion chromatography (BIO-RAD, Bio Beads S-X1,
DMF). Solvent was removed from the filtrate, and chromatographed
(SiO.sub.2, gradient, CH.sub.2Cl.sub.2/MeOH 5:1 to 3:2), filtered
again through a pipet-size size exclusion chromatography (BIO-RAD,
Bio Beads S-X1, DMF) to remove any dissolved silica gel, and
concentrated to give 596 mg (2.40 mmol, 36%) of 42 as a yellowish
oil. R.sub.f 0.10 (CH.sub.2Cl.sub.2/MeOH 3:1); .sup.1H NMR (400
MHz, DMSO-d.sub.6) 6.78 (t, 1H, NHBoc), 3.49 (m, 4H,
CH.sub.2CH.sub.2OCH.sub.2- CH.sub.2NHBoc), 3.35 (m, 4H,
CH.sub.2OCH.sub.2CH.sub.2OCH.sub.2CH.sub.2NHB- oc), 3.05 (q, 2H,
J=5.9 Hz, CH.sub.2NHBoc), 1.37 (s, 9H, CH.sub.3 of Boc) (some peaks
were not identified and missing).
[0664] Methyl
3.alpha.-(t-butyldiphenylsilyloxy)-7.alpha.-hydroxycholan-24- -oate
(56). A mixture of 25 (950 mg, 2.34 mmol) and imidazole (396 mg,
5.81 mmol) was dissolved in 5 mL of DMF. TBDPSCl (0.74 mL, 2.79
mmol) was added to the solution dropwise over a 30 min-period using
a syringe. The reaction was stirred at room temperature for 16 h.
The crude product was chromatographed (SiO.sub.2, gradient,
hexane/EtOAc 10:1 to 5:1) to give 1.37 g (2.12 mmol, 91%) of 56
which was slightly contaminated with TBDPSCl. R.sub.f 0.46
(hexane/EtOAc 5:1); .sup.1H NMR (400 MHz, CDCl.sub.3) .delta. 7.68
(m, 4H, H-2 of phenyl), 7.43-7.32 (m, 6H, H-3 and H-4 of phenyl),
3.82 (m, 1H, H-3/H-7), 3.67 (s, 3H, CO.sub.2CH.sub.3), 3.46 (m, 1H,
H-3/H-7), 2.40-0.69 (m, 28H), 1.04 (s, 9H, C(CH.sub.3).sub.3), 0.93
(d, 3H, J=6.4 Hz, CH.sub.3), 0.79 (s, 3H, CH.sub.3), 0.64 (s, 3H,
CH.sub.3); MS (ESI) Calcd for C.sub.41H.sub.60O.sub.4SiNa
(M+Na.sup.+): 667.4, Found: 667.4.
[0665] A number of embodiments of the invention have been
described. Nevertheless, it will be understood that various
modifications may be made without departing from the spirit and
scope of the invention. Other embodiments are in the claims.
* * * * *