U.S. patent application number 10/936115 was filed with the patent office on 2005-06-02 for nuclease resistant double-stranded ribonucleic acid.
Invention is credited to Manoharan, Muthiah, Rajeev, Kallanthottathil G..
Application Number | 20050119214 10/936115 |
Document ID | / |
Family ID | 35530789 |
Filed Date | 2005-06-02 |
United States Patent
Application |
20050119214 |
Kind Code |
A1 |
Manoharan, Muthiah ; et
al. |
June 2, 2005 |
Nuclease resistant double-stranded ribonucleic acid
Abstract
This invention features modified iRNA agents having improved
stability in cells and biological fluids, and methods of making and
identifying iRNA agents having improved stability, and of using the
iRNA agents to inhibit the expression or function of a target
gene.
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.: |
10/936115 |
Filed: |
September 7, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10936115 |
Sep 7, 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: |
514/44A ;
435/6.16; 536/23.1 |
Current CPC
Class: |
A61K 47/554 20170801;
C12N 2310/3515 20130101; C12N 15/111 20130101; A61P 35/00 20180101;
C12N 2310/344 20130101; A61P 35/02 20180101; A61P 31/14 20180101;
C12N 2310/14 20130101; C12N 2320/51 20130101; C12N 2310/323
20130101 |
Class at
Publication: |
514/044 ;
435/006; 536/023.1 |
International
Class: |
A61K 048/00; C12Q
001/68; C07H 021/02 |
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 |
Claims
What is claimed is:
1. An iRNA agent comprising at least one ribonucleotide
modification, wherein the modification confers improved exonuclease
stability to the iRNA agent when the iRNA agent is contacted with a
biological sample.
2. The iRNA agent of claim 1, wherein the iRNA agent comprises at
least two ribonucleotide modifications.
3. The iRNA agent of claim 1, wherein the iRNA agent comprises at
least three ribonucleotide modifications.
4. The iRNA agent of claim 1, wherein the iRNA agent comprises at
least four ribonucleotide modifications.
5. The iRNA agent of claim 1, wherein the iRNA agent comprises at
least five ribonucleotide modifications.
6. The iRNA agent of claim 1, wherein biological sample comprises
serum.
7. The iRNA agent of claim 1, wherein the modification comprises a
backbone modification between one or more of the terminal
nucleotides on an iRNA strand of the iRNA agent.
8. The iRNA agent of claim 7, wherein the backbone modification
comprises a phosphorothioate linkage between one or more of the
terminal nucleotides of an iRNA agent.
9. The iRNA agent of claim 1, wherein the modification comprises a
modification of a nucluotide sugar.
10. The iRNA agent of claim 9, wherein the nucleotide is a terminal
nucleotide.
11. The iRNA agent of claim 10, wherein the terminal nucleotide
comprises a 2' or 3' sugar modification.
12. The iRNA agent of claim 10, wherein the iRNA agent comprises a
2'-O-methylated nucleotide, a 2'-deoxy nucleotide, a 2'-deoxyfluoro
nucleotide, a 2'-O-methoxyethyl nucleotide, a 2'-O-NMA nucleotide,
a 2'-DMAEOE nucleotide, a 2'-AP nucleotide, 2'-hydroxy nucleotide,
a 2'-ara-fluoro nucleotide, a locked nucleic acid, an extended
nucleic acid, hexose nucleic acid, or cyclohexene nucleic acid.
13. The iRNA agent of claim 12, wherein the iRNA agent comprises a
2'-O-methylated nucleotide.
14. The iRNA agent of claim 12, wherein the iRNA agent comprises a
2'-deoxyfluoro nucleotide.
15. The iRNA agent of claim 12, wherein the iRNA agent comprises a
locked nucleic acid and the locked nucleic acid is on a 5' or 3'
end of a sense strand of the iRNA agent, and not on a 5' or 3' end
of an antisense strand of the iRNA agent.
16. The iRNA agent of claim 11, wherein the iRNA agent comprises a
3'-O-methylated nucleotide.
17. The iRNA agent of claim 11, wherein the 3'-O-methylated
nucleotide is on a 3' or 5' end of a sense strand of the iRNA
agent.
18. The iRNA agent of claim 1, wherein the modification comprises a
nucleobase modification.
19. The iRNA agent of claim 18, wherein the iRNA agent comprises a
5'-methyl-pyrimidine.
20. The iRNA agent of claim 19, wherein the iRNA agent comprises a
5'-methyl-uridine.
21. The iRNA agent of claim 19, wherein the iRNA agent comprises a
5'-methyl-cytodine.
22. The iRNA agent of claim 18, wherein the iRNA agent comprises at
least one terminal nucleotide having a nucleobase modification.
23. The iRNA agent of claim 22, wherein the iRNA agent comprises a
3'-abasic cationic modification.
24. The iRNA agent of claim 23, wherein the cationic modification
comprises an alkylamino-dT, an allylamino conjugate, a pyrrolidine
conjugate, a pthalamido or a hydroxyprolinol conjugate, on one or
more of the terminal nucleotides of the iRNA agent.
25. The iRNA agent of claim 1, wherein the iRNA agent comprises a
comprises a backbone modification between one or more of the
terminal nucleotides on an iRNA strand of the iRNA agent and a
terminal nucleotide having a 2' or 3' sugar modification.
26. The iRNA agent of claim 1, wherein the iRNA agent comprises a
terminal nucleotide having a nucleobase modification and a terminal
nucleotide having a 2' or 3' sugar modification.
27. The iRNA agent of claim 1, wherein the iRNA agent comprises at
least one conjugate.
28. The iRNA agent of claim 27, wherein the conjugate is attached
to one or more terminal nucleotides of the iRNA agent.
29. The iRNA agent of claim 27, wherein the conjugate is selected
from a lipophile, a terpene, a protein binding agent, a vitamin, a
carbohydrate, or a peptide.
30. The iRNA agent of claim 27, wherein the conjugate is on a 3'
end of an antisense strand of the iRNA agent, or on a 5' or 3' end
of a sense strand on the iRNA agent.
31. The iRNA agent of claim 30, the conjugate is not on the 3' end
of the antisense strand and on the 3' end of the sense strand.
32. The iRNA agent of claim 27, wherein conjugate is naproxen.
32. The iRNA agent of claim 27, wherein the conjugate is
cholesterol.
33. The iRNA agent of claim 32, wherein the cholesterol is
conjugated to the iRNA agent by a pyrrolidine linker, or serinol
linker, or hydroxyprolinol linker.
34. The iRNA agent of claim 27, wherein the conjugate is cholanic
acid.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of International
Application No. PCT/U.S.2004/011829, 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/U.S.04/07070, filed on
Mar. 8, 2004; International Application No.: PCT/U.S.2004/10586,
filed on Apr. 5, 2004; International Application No.:
PCT/U.S.2004/11255, filed on Apr. 9, 2004; and International
Application No.: PCT/U.S.2004/011822, filed on Apr. 16, 2004. The
contents of all of these prior applications are hereby incorporated
by reference in their entireties.
TECHNICAL FIELD
[0002] This invention features modified iRNA agents having improved
stability in cells and biological fluids, and methods of making and
identifying iRNA agents having improved stability, and of using the
iRNA agents to inhibit the expression or function of a target
gene.
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 invention features iRNA agents having improved stability
in cells and biological fluids, methods of making and identifying
iRNA agents having improved stability, as well as compositions and
methods for inhibiting the expression of a target gene in a cell
using the iRNA agent. The invention also features compositions and
methods for treating diseases caused by the expression or activity
of the target gene. The invention features iRNA agents that contain
modifications that increase resistance of the iRNA agents to
exonucleolytic degradation, including 5' exonucleolytice and 3'
exonucleolytic degradation. The modifications render the iRNA
agents more resistant to enzymatic or chemical degradation, and
thus more stable and bioavailable than an otherwise identical iRNA
agent that does not include the modification.
[0006] In one aspect, the invention features iRNA agents having
improved stability when administered to a subject. These agents can
have, e.g., improved stability in cells and biological samples,
particularly serum. The iRNA agent includes one or more of various
modifications. For example, the iRNA agents can include a backbone
modification to one or more of the terminal nucleotides on an iRNA
strand. A terminal nucleotide can be the nucleotide at the last
position, and can include the nucleotide(s) at the second, third,
fourth, and fifth positions from the 5' or 3'-most ends of the
sense or antisense strand of an iRNA agent. In one embodiment, an
iRNA agent includes a phosphorothioate linkage or P-alkyl
modification in the linkages between one or more of the terminal
nucleotides of an iRNA agent. In another embodiment, one or more
terminal nucleotides of an iRNA agent include 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, a 2'-deoxyfluoro nucleotide, a 2'-O-methoxyethyl
nucleotide, a 2'-O-NMA, a 2'-DMAEOE, a 2'-AP, 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, more preferably,
2'fluoro. In one embodiment the modification is an LNA and the LNA
is on the 5' or 3' end of the sense strand, and not on the 5' or 3'
end of the antisense strand.
[0007] In one embodiment, the iRNA agent includes a 3' sugar
modification, e.g., a 3'OMe modification. Preferably a 3'OMe
modificationis on the 3' or 5' end of the sense strand of the iRNA
agent.
[0008] In some embodiments, an iRNA agent includes a
5'-methyl-pyrimidine (e.g., a 5'-methyl-uridine modification or a
5'-methyl-cytodine modification.
[0009] The modifications described herein can be combined onto a
single iRNA agent. For example, in one embodiment, at least one
terminal nucleotide of an iRNA agent has a phosphorothioate linkage
and a 2' sugar modification, e.g., a 2'OMe or 2.degree. F.
modification. In another embodiment, at least one terminal
nucleotide of an iRNA agent has a 5' Me-pyrimidine and a 2' sugar
modification, e.g., a 2'F or 2'OMe modification.
[0010] In one embodiment, an iRNA agent includes 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 or a hydroxyprolinol conjugate,
on one or more of the terminal nucleotides of the iRNA agent. An
alkylamino-dT conjugate is preferably attached to the 3' end of the
sense or antisense strand of an iRNA agent. A pyrrolidine linker is
preferably attached to the 3' or 5' end of the sense strand, or the
3' end of the antisense strand. An allyl amine uridine is
preferably on the 3' or 5' end of the sense strand, and not on the
5' end of the antisense strand.
[0011] In another embodiment, the iRNA agent includes an conjugate
on one or more of the terminal nucleotides of the iRNA agent. The
conjugate can be, for example, 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 C5 pyrimidine linker. In other embodiments, the
conjugates are glyceride lipid conjugates (e.g. a dialkyl glyceride
derivatives), vitamin E conjugates, or thio-cholesterols.
Preferably, conjugates are on the 3' end of the antisense strand,
or on the 5' or 3' end of the sense strand, and preferably the
conjugates are not on the 3' end of the antisense strand and on the
3' end of the sense strand.
[0012] In one embodiment, the conjugate is naproxen, and the
conjugate is preferably on the 5' or 3' end of the sense or
antisense strands. In one embodiment, the conjugate is cholesterol,
and the conjugate is preferably on the 5' or 3' end of the sense
strand and preferably not present on the antisense strand. In some
embodiments, the cholesterol is conjugated to the iRNA agent by a
pyrrolidine linker, or serinol linker, or hydroxyprolinol linker.
In other embodiments, the conjugate is a dU-cholesterol, or
cholesterol is conjugated to the iRNA agent by a disulfide linkage.
In a particularly prefered embodiment, the conjugate is cholanic
acid, and the cholanic acid is 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.
[0013] In another embodiment, one or more terminal nucleotides has
a 2'-5' linkage. Preferably, a 2'-5' linkage occurs on the sense
strand, e.g., the 5' end of the sense strand.
[0014] In one embodiment, the iRNA agent includes an L-sugar,
preferably at the 5' or 3' end of the sense strand.
[0015] In one embodiment, the iRNA agent includes a
methylphosphonate at one or more terminal nucleotides to enhance
exonuclease resistance, e.g., at the 3' end of the sense or
antisense strands of the iRNA agent.
[0016] In one embodiment, an iRNA agent has been 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.
[0017] In some embodiments, an iRNA agent having increased
stability in cells and biological samples includes a difluorotoluyl
(DFT) modification, e.g., 2,4-difluorotoluyl uracil, or a guanidine
to inosine substitution.
[0018] In one embodiment the iRNA agent includes a terminal
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'-AP, 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 the modifications described
herein are particularly stabilized against exonuclease
activity.
[0019] In one particularly preferred embodiment, an iRNA agent
includes a cholesterol molecule on the 3' end of an iRNA agent, and
conjugated to the iRNA agent by a pyrrolidine linker, and at least
one additional modification, e.g., a modification described herein,
such as a phosphorothioate linkage, for inhibiting exonucleolytic
degradation. In another particularly preferred embodiment, an iRNA
agent includes a cholanic acid modification on one or both of the
3' ends of the sense or antisense strands, and at least one
additional modification, e.g., a modification described herein,
such as a phosphorothioate linkage, for inhibiting exonucleolytic
degradation.
[0020] In one embodiment, the iRNA agent has 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. In another embodiment,
the iRNA agent has a double overhang, e.g., both ends of the iRNA
agent have a 3' or 5' overhang, such as a dinuclotide overhang. In
another embodiment, both ends of the iRNA agent have blunt
ends.
[0021] 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. In one embodiment, the doublestrand region of the iRNA
agent includes phosphorothioate dinucleotide linkages on one or
both of the sense and antisense strands. In one embodiment, the
antisense RNA strand and the sense RNA strand are connected with a
linker. The chemical linker may be a hexaethylene glycol linker, a
poly-(oxyphosphinico-oxy-1,- 3-propandiol) linker, an allyl linker,
or a polyethylene glycol linker.
[0022] In another aspect, the invention relates to a method of
making an iRNA agent. The method includes preparing an antisense
RNA strand and a sense RNA strand, wherein at least one of the RNA
strands includes a modification described herein, e.g., a
modification that confers an enhanced resistance to exonucleolytic
cleavage. The RNA strands may then be mixed to form a double
stranded iRNA agent. The RNA strands may be prepared using
solid-phase synthesis in a 3' to 5' direction.
[0023] In yet another aspect, the invention relates to a method for
inhibiting the expression of a target gene in a cell. The method
includes introducing an iRNA agent, as described above, into the
cell, and maintaining the cell for a time sufficient to obtain iRNA
agent-mediated downregulation, e.g., by degradation, of an mRNA
transcript of the target gene. The cell may be a mammalian cell,
such as a human cell.
[0024] In still another aspect, the invention relates to a
pharmaceutical composition for inhibiting the expression of a
target gene in an organism. The compositions include an iRNA agent,
as described above, and a pharmaceutically acceptable carrier. The
pharmaceutically acceptable carrier may be an aqueous solution,
such as phosphate buffered saline, or it may include a micellar
structure, such as a liposome, capsid, capsoid, polymeric
nanocapsule, or polymeric microcapsule.
[0025] In yet a further aspect, the invention relates to a method
for treating a disease caused by the expression of a target gene in
a subject. The method includes administering a pharmaceutical
composition containing an iRNA agent, as described above, and a
pharmaceutically acceptable carrier. The subject may be a mammal,
such as a human.
[0026] In another aspect, the invention relates to methods of
identifying an iRNA agent having increased stability in a
biological sample. The method may include preparing a mixture of
iRNA agent, incubating the mixture of iRNA agent in the biological
sample, and identifying an iRNA agent exhibiting an increased
stability as compared to other iRNA agent in the biological sample.
The mixture of iRNA agent may be prepared by chemical synthesis or
by extraction from a biological sample. Alternatively, the method
may include introducing an iRNA agent into a cell, maintaining the
cell under conditions suitable for expressing a protein encoded by
a target gene, measuring an amount of the protein produced in the
cell, comparing the amount of the protein produced in the cell to
that in a control cell, and identifying an iRNA agent that causes a
reduction in the amount of protein in the cell as compared to the
control cell. The cell may be a mammalian cell.
[0027] In still another aspect, the invention relates to a method
of increasing the nuclease resistance of an iRNA agent. The method
includes forming an iRNA agent, as described above, wherein the
nuclease resistance of the iRNA agent is increased compared to a
control composition. Such method includes the steps of
incorporating into the iRNA agent, a modification described herein.
The modification can be added to one or more of the 5' or 3'
terminal nucleotides of the sense and antisense strands of the iRNA
agent.
[0028] While RNA interference using iRNA agent has been shown to be
an effective means for selective gene silencing, RNA can have less
than desired stability in some bodily fluids, particularly in
serum. Thus, RNA, including iRNA agent, can be degraded between the
time it is administered to a subject and the time it enters a
target cell. Even within the cell, RNA can undergo rapid
degradation by nucleases. Methods of the invention can provide more
stable or nuclease resistant iRNA agents that can offer better
bioavailability and hence improved effectiveness. This is an
improvement over the current insufficient methods for stabilizing
iRNA agent against degradation.
[0029] 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
[0030] FIG. 1 a general synthetic scheme for incorporation of RRMS
monomers into an oligonucleotide.
[0031] FIG. 2A is a list of substituents that may be present on
silicon in OFG.sup.1.
[0032] FIG. 2B is a list of substituents that may be present on the
C2'-orthoester group.
[0033] 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. R1 is succinate or phosphoramidate and R2 is H
or a conjugate ligand.
[0034] FIG. 4 is a general reaction scheme for 3' conjugation of
peptide into iRNA.
[0035] FIG. 5 is a general reaction scheme for 5' conjugation of
peptide into iRNA.
[0036] FIG. 6 is a general reaction scheme for the synthesis of
aza-peptides.
[0037] FIG. 7 is a general reaction scheme for the synthesis of
N-methyl amino acids and peptides.
[0038] FIG. 8 is a general reaction scheme for the synthesis of
.beta.-methyl amino acids and Ant and Tat peptides.
[0039] FIG. 9 is a general reaction scheme for the synthesis of Ant
and Tat oligocarbamates.
[0040] FIG. 10 is a a general reaction scheme for the synthesis of
Ant and Tat oligoureas.
[0041] FIG. 11 is a schematic representation of peptide
carriers.
[0042] FIG. 12 shows a gel electrophoretic separation of 3'
phosphorothiate modified dsRNAs following incubation in human
serum.
[0043] FIG. 13 shows a gel electrophoretic separation of 3'
modified dsRNAs following incubation in human serum.
[0044] FIG. 14 shows a gel electrophoretic separation of 3' abasic
pyrrolidine conjugated dsRNAs following incubation in human
serum.
[0045] FIG. 15 shows a gel electrophoretic separation of 3'
cationic C6 amino dT conjugated dsRNAs following incubation in
human serum.
[0046] FIG. 16 is a graph showing luciferase gene expression
activity in cells following transfection of 3'Naproxen modified
dsRNAs.
[0047] FIG. 17 is a graph showing luciferase gene expression
activity in cells following transfection of 3'abasic pyrrolidine
modified dsRNAs.
[0048] FIG. 18 is a graph showing luciferase gene expression
activity in cells following transfection of 3' cationic C6 amino dT
modified dsRNAs.
[0049] FIG. 19 shows a gel electrophoretic separation of 3'
naproxen conjugated dsRNAs following incubation in mouse serum.
[0050] FIG. 20 shows a gel electrophoretic separation of 3'
naproxen conjugated dsRNAs following incubation in liver
extract.
[0051] FIG. 21 is a denaturing gel analysis of the human serum
stability assay for AL-DUP-1000. C is the 4 hour time point for
siRNA duplex incubated in PBS buffer alone, OH-- is the partial
alkaline hydrolysis marker, *s/as represents siRNA duplex
containing 5' end-labeled sense RNA and s/*as represents duplex
containing 5' end-labeled antisense RNA. Samples were incubated in
90% human serum and time points were assayed at 10 seconds, 5 min,
15 min, 30 min, 1 hour, 2 hours and 4 hours. Black lines to the
right of bands indicate exonucleolytic degradation fragments and
the red lines highlight a few of the endonucleolytic degradation
fragment.
[0052] FIG. 22A is a denaturing gel analysis of the human serum
stability assay for AL-DUP-1393. C is the 4 hour time point for
each siRNA duplex incubated in PBS buffer alone, *s/as represents
siRNA duplex containing 5' end-labeled sense RNA and s/*as
represents duplex containing 5' end-labeled antisense RNA. Samples
were assayed at 10 seconds, 15 min, 30 min, 1 hour, 2 hours and 4
hours.
[0053] FIG. 22B is a denaturing gel analysis of the human serum
stability assay for AL-DUP-1329. The lanes are labeled and the
experiment was performed as described for FIG. 22A.
[0054] FIG. 23 is a denaturing gel analysis of AL-DUP-1036,
AL-DUP-13ff, and AL-DUP-1363 (see Table 2 for sequences). Black
vertical lines highlight regions where exonuclease cleavage is
suppressed, stars indicate sites of strong endonucleolytic cleavage
in the antisense strand and weaker endonucleolytic cleavage in the
sense strand. C is the 4 hour time point for each siRNA duplex
incubated in PBS buffer alone, *s/as represents siRNA duplex
containing 5' end-labeled sense RNA and s/*as represents duplex
containing 5' end-labeled antisense RNA. Samples were assayed at 10
seconds, 15 min, 30 min, 1 hour, 2 hours and 4 hours.
[0055] FIG. 24. Human serum stability profile of siRNA duplexes
containing cationic modifications. Denaturing gel analysis of
AL-DUP-10aa (alkylamino-dT), AL-DUP-1ccc (abasic pyrrolidine
cationic), and AL-DUP-1403 (see Table 3 for sequences). Black line
highlights region where exonuclease cleavage is suppressed and red
star indicates site of strong endonucleolytic cleavage in the
antisense strand. C is the 4 hour time point for each siRNA duplex
incubated in PBS buffer alone, *s/as represents siRNA duplex
containing 5' end-labeled sense RNA and s/*as represents duplex
containing 5' end-labeled antisense RNA. Samples were assayed at 10
seconds, 15 min, 30 min, 1 hour, 2 hours and 4 hours.
[0056] FIG. 25 is a denaturing gel analysis of the human serum
stability assay for AL-DUP-1069. The black vertical line highlights
the region where exonuclease cleavage is suppressed. C is the 4
hour time point for each siRNA duplex incubated in PBS buffer
alone, *s/as represents siRNA duplex containing 5' end-labeled
sense RNA and s/*as represents duplex containing 5' end-labeled
antisense RNA. Samples were assayed at 10 seconds, 15 min, 30 min,
1 hour, 2 hours and 4 hours.
[0057] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0058] The invention is based, at least in part, on the discovery
that particular modifications to an iRNA agent are effective at
increasing resistance of the iRNA agent to exonucleolytic
degradation. Thus, the invention features iRNA agents that contain
modifications that increase resistance of the iRNA agents to
exonucleolytic degradation, including 5' exonucleolytic and 3'
exonucleolytic degradation. These iRNA agents may have improved
stability in cells and biological fluids.
[0059] The iRNA agents having improved stability can include one or
more of various modifications. For example, the iRNA agent can
include a backbone modification to one or more of the terminal
nucleotides on an iRNA strand. Exemplary backbone modifications
include phosphorothioate linkages or P-alkyl linkages between one
or more of the terminal nucleotides of an iRNA agent. One or more
terminal nucleotides of an iRNA agent can also include a sugar
modification, e.g., a 2' or 3' sugar modification. Exemplary sugar
modifications are described below. An iRNA agent can include
multiple modifications that confer an enhanced resistance to
exonucleolytic cleavage. For example, one or more terminal
nucleotides of an iRNA agent can have a phosphorothioate linkage
and a 2' sugar modification.
[0060] An iRNA agent having increased resistance to exonucleolytice
degradation can include a nucleobase modification, a cationic
modification on one or more of the terminal nucleotides of the iRNA
agent.
[0061] Exemplary modifications for enhanced resistance to
endonucleolytic cleavage are described below. 1 2 3
[0062] Most Preferred Sugar with 2'-Substituents and Backbones
45
[0063] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where
R.sub.5R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0064] X=OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0065] Y=O, S or Se
[0066] Chiral Rp and Sp isomers, P=S preferably Sp 6
[0067] R.sub.1, R.sub.2=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0068] X=OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X-O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0069] Y=O, S or Se
[0070] Chiral Rp and Sp isomers, P=S preferably Sp 7
[0071] R.sub.1, R.sub.2=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0072] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0073] Y=O, S or Se
[0074] Chiral Rp and Sp isomers, P=S preferably Sp 8
[0075] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.6=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0076] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0077] Y=O, S or Se
[0078] Chiral Rp and Sp isomers, P=S preferably Sp 9
[0079] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.6=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0080] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0081] Y=O, S or Se
[0082] Z=O, S, CH.sub.2, CHF, CF.sub.2
[0083] Chiral Rp and Sp isomers, P=S preferably Sp 10
[0084] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0085] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8.dbd.H, Me, Et, COMe and
n=1-12
[0086] Y=O, S or Se
[0087] Chiral Rp and Sp isomers, P=S preferably Sp 11
[0088] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0089] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0090] Y=O, S or Se
[0091] Z.sub.1, Z.sub.2=H, OH, NR'R", SR'", OR""
[0092] Chiral Rp and Sp isomers, P=S preferably Sp 12
[0093] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.6=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0094] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0095] Y=O, S or Se
[0096] Chiral Rp and Sp isomers, P=S preferably Sp 13
[0097] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0098] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0099] Y=O, S or Se
[0100] Z.sub.1, Z.sub.2=O, S, NR', CH.sub.2, CHF, CF
[0101] Chiral Rp and Sp isomers, P=S preferably Sp 14
[0102] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0103] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8.dbd.H, Me, Et, COMe and
n=1-12
[0104] Y=O, S or Se
[0105] Z=O, S, NR', CH.sub.2, CHF, CF
[0106] Chiral Rp and Sp isomers, P=S preferably Sp 15
[0107] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0108] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8.dbd.H, Me, Et, COMe and
n=1-12
[0109] Y=O, S or Se
[0110] Z.sub.1=O, S, NR', CH.sub.2, CHF, CF
[0111] Z.sub.2.dbd.H, OR', SR', NR'R"
[0112] Chiral Rp and Sp isomers, P=S preferably Sp 16
[0113] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0114] Y=O, S, CH.sub.2, CHF, CF.sub.2 17 18
[0115] R=H, OH, OMe, F, O(CH.sub.2).sub.2O(CH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0116] X.ident.OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0117] Y=O, S or Se
[0118] Z=O, S, NR', CH.sub.2, CHF, CF.sub.2
[0119] Chiral Rp and Sp isomers, P=S preferably Sp 19
[0120] R=H, OH, OMe, F, O(CH.sub.2)OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.6=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0121] X=OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0122] Y=O, S or Se
[0123] Z=O, S, NR', CH.sub.2, CHF, CF.sub.2
[0124] R'=H, Me, Et, (CH.sub.2).sub.nOH; n=0-6
[0125] Chiral Rp and Sp isomers, P=S preferably Sp
[0126] L-Nucleoside Derived from 1-15
[0127] 2'-5' Linkage from 1-15 20
[0128] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0129] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0130] Y=O, S or Se 21
[0131] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0132] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0133] Y=O, S or Se 22
[0134] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0135] X.ident.OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0136] Y=O, S or Se 23
[0137] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0138] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0139] Y=O, S or Se 24
[0140] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0141] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl, --N
H-Alkyl, --N(dialkyl), --N(alkyl)(Aryl), --(CH.sub.2).sub.nXR.sub.7
or --NH(CH.sub.2)XR.sub.7, where X=O, S, NR.sub.8, R.sub.7=H, Me,
Et, and R.sub.8=H, Me, Et, COMe and n=1-12
[0142] Y=O, S or Se 25
[0143] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0144] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0145] Y=O, S or Se 26
[0146] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.6=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0147] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0148] Y=O, S or Se
[0149] Chiral Rp and Sp isomers, P=S preferably Sp 27
[0150] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0151] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0152] Y=O, S or Se
[0153] Z=OH, OMe, SH, SMe, NH.sub.2, NHMe, NMe.sub.2, Aryl
[0154] Chiral Rp and Sp isomers, P=S preferably Sp 28
[0155] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.5=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0156] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0157] Y=O, S or Se
[0158] Z=OH, OMe, SH, SMe, NH.sub.2, NHMe, NMe.sub.2, Aryl,
alkyl
[0159] Chiral Rp and Sp isomers, P=S preferably Sp 29
[0160] R=H, OH, OMe, F, O(CH.sub.2).sub.2OCH.sub.3,
O(CH.sub.2).sub.2SCH.sub.3, O(CH.sub.2).sub.2ONMe.sub.2,
O(CH.sub.2).sub.2O(CH.sub.2).sub.2NMe.sub.2, OCH.sub.2CONH.sub.2,
O(CH.sub.2).sub.nXR.sub.3, where X=O, S, NR.sub.4 and R.sub.3,
R.sub.4=H, Me, Et,; OCH.sub.2CONR.sub.5R.sub.6, where R.sub.5,
R.sub.6, where R.sub.6=H, Me, Et and R.sub.6=H, Me, Et or a
ligand
[0161] X=--OH, --SH, -Me, -isopropyl, -tert-Butyl, -phenyl,
--NH-Alkyl, --N(dialkyl), --N(alkyl)(Aryl),
--(CH.sub.2).sub.nXR.sub.7 or --NH(CH.sub.2)XR.sub.7, where X=O, S,
NR.sub.8, R.sub.7=H, Me, Et, and R.sub.8=H, Me, Et, COMe and
n=1-12
[0162] Y=O, S or Se
[0163] Chiral Rp and Sp isomers, P=S preferably Sp 303132
[0164] R=H, OH or OMe, X=O, NH, NMe, S, CH.sub.2, or CF.sub.2; Y=O
or S Q', Q"=H; Q'=H and Q"=Me or Et; or Q', Q"=Me or Et and n=0-5
333435
[0165] R=H, OH or OMe; X=O, NH, NMe, S, CH.sub.2, or CF.sub.2; Y=O
or S Q', Q"=H; Q'=H and Q"=Me or Et; or Q', Q"=Me or Et and n=0-5
3637
[0166] R=H, OH or OMe; X=O, NH, NMe, S, CH.sub.2, or CF.sub.2; Y=O
or S; Q', Q"=H Q'=H and Q"=Me or Et ir Q', Q"=Me or Et; p=0-6 and
q=0-6 3839
[0167] R=H, OH or OMe; X=O, NH, NMe, S, CH.sub.2, or CF.sub.2; Y=O
or S; Q', Q"=H; Q'=H and Q"=Me or Et or Q', Q"=Me or Et; p=0-6 and
q=0-6 4041
[0168] R=H, OH or OMe; X=O, NH, NMe, S, CH.sub.2, or CF.sub.2; Y=O
or S; Q', Q"=H; Q'=H and Q"=Me or Et or Q', Q"=Me or Et; p=0-6 and
q=0-6 424344
[0169] R=H, OH or OMe; X=O, NH, NMe, S, CH.sub.2, or CF.sub.2; Y=O
or S; Q', Q"=H; Q'=H and Q"=Me or Et or Q', Q"=Me or Et; n=0-6;
p=0-6; q=0-6 and r=0-50 454647
[0170] R=H, OH or OMe 48
[0171] R=H, OH or OMe 4950
[0172] R=H, OH or OMe 5152
[0173] R=H, OH or OMe 5354
[0174] R=H, OH or OMe 5556
[0175] R=H, OH or OMe 57
[0176] R=H, OH or OMe
[0177] For structures 27-81, R can also have the values for
structures 1A-26.
[0178] The iRNA agent can include a conjugate (also called a
ligand) on the 5' or 3' terminus of the sense or antisense strand.
Exemplary ligands are shown below. The conjugate can be attached to
the 3' or 5' end. Preferably, a cholesterol conjugate is on the 5'
or 3' end of the sense strand. Preferably, a cholesterol conjugate
is not on the antisense strand.
Preferred Ligands
[0179] 58
[0180] Vitamins
[0181] Peptides
[0182] Carbohydrates
[0183] Terpenes
[0184] Steroids
[0185] Lipids
[0186] Amino acids
[0187] Naproxen
[0188] Ibuprofen
[0189] Amines (mono, di, tri, tetraalkyl or aryl)
[0190] Folate
[0191] Sugar (N-Acetylgalactosamine, galactosamine, galactose,
Mannose)
[0192] --(CH.sub.2).sub.nNQ.sub.1Q.sub.2, where n=0-40, Q.sub.1,
Q.sub.2=H, Me or Et; Q.sub.1=H, Q.sub.2=H, Me, Et or aryl
[0193] (CH.sub.2).sub.pCH=CH(CH.sub.2).sub.qNQ.sub.1Q.sub.2, where
p and/or q=0-40, Q.sub.1, Q.sub.2=H, Me or Et; Q.sub.1=H,
Q.sub.2=H, Me, Et or aryl with E and/or Z configuration
[0194] --(CH.sub.2).sub.pCH=CH(CH.sub.2).sub.qNQ.sub.1Q.sub.2,
where p and/or q=0-40, Q.sub.1, Q.sub.2=H, Me or Et; Q.sub.1=H,
Q.sub.2=H, Me, Et or aryl
[0195]
--(CH.sub.2).sub.pCH=CH(CH.sub.2).sub.qCH=CH(CH.sub.2).sub.rNQ.sub.-
1Q.sub.2, where p, q and/or r=0-40, Q.sub.1, Q.sub.2=H, Me or Et;
Q.sub.1=H, Q.sub.2=H, Me, Et or aryl with E and/or Z
configuration
[0196] --O(CH.sub.2).sub.m(OCH.sub.2CH.sub.2).sub.n--OR, where m,
n=0-40 and R=H, Me, NQ.sub.1Q.sub.2, --C(O)NR'R"--C(S)NR'R"
[0197] --NH(CH.sub.2).sub.m(OCH.sub.2CH.sub.2).sub.n--OR, where m,
n=0-40 and R=H, Me, NQ.sub.1Q.sub.2, --C(O)NR'R"--C(S)NR'R"
[0198] --O(CH.sub.2).sub.m(NHCH.sub.2CH.sub.2).sub.n--R, where m,
n=0-40 and R=H, OH, Me, NQ.sub.1Q.sub.2, --C(O)NR'R"--C(S)NR'R"
[0199] --NH(CH.sub.2).sub.m(NHCH.sub.2CH.sub.2).sub.n--R, where m,
n=0-40 and R=H, OH, Me, NQ.sub.1Q.sub.2, --C(O)NR'R"--C(S)NR'R"
[0200] Dialkylglycerol (sn3, sn1, sn2 and racemic) with number of
methylene varies from 0-40
[0201] Diacylglycerol (sn3, sn1, sn2 and racemic) with number of
methylene varies from 0-40
[0202] Dialkylglycerol (sn3, sn1, sn2 and racemic) with number of
methylene varies from 0-40 and the alkyl chian contains one or more
double bonds with E and/or Z isomers
[0203] Dlacylglycerol (sn3, sn1, sn2 and racemic) with number of
methylene varies from 0-40 and the alkyl chian contains one or more
double bonds with E and/or Z isomers
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] Ligand-Conjugated Monomer Subunits and Monomers for
Oligonucleotide Synthesis
[0209] Definitions
[0210] The term "halo" refers to any radical of fluorine, chlorine,
bromine or iodine.
[0211] 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.
[0212] 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 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.
[0213] 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.
[0214] 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.
[0215] 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.
[0216] 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.
[0217] 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.
[0218] 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.
[0219] 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.
[0220] 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.
[0221] 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.
[0222] The term "acyl" refers to an alkylcarbonyl,
cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or
heteroarylcarbonyl substituent, any of which may be further
substituted by substituents.
[0223] 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.naryl (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.
[0224] The terms "adeninyl, cytosinyl, guaninyl, thyminyl, and
uracilyl" and the like refer to radicals of adenine, cytosine,
guanine, thymine, and uracil.
[0225] 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.
[0226] General
[0227] 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.
[0228] 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. 59
[0229] 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.
[0230] 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. 60
[0231] 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: 61
[0232] 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.
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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.
[0237] 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.
[0238] 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.
[0239] 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=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).
[0240] 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.
[0241] Sugar-Based Monomers
[0242] 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. 62
[0243] "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.
[0244] As used herein, an "unusual" nucleobase can include any one
of the following:
[0245] 2-methyladeninyl,
[0246] N6-methyladeninyl,
[0247] 2-methylthio-N-6-methyladeninyl,
[0248] N6-isopentenyladeninyl,
[0249] 2-methylthio-N-6-isopentenyladeninyl,
[0250] N6-(cis-hydroxyisopentenyl)adeninyl,
[0251] 2-methylthio-N-6-(cis-hydroxyisopentenyl)adeninyl,
[0252] N6-glycinylcarbamoyladeninyl,
[0253] N6-threonylcarbamoyladeninyl,
[0254] 2-methylthio-N-6-threonyl carbamoyladeninyl,
[0255] N6-methyl-N-6-threonylcarbamoyladeninyl,
[0256] N6-hydroxynorvalylcarbamoyladeninyl,
[0257] 2-methylthio-N-6-hydroxynorvalyl carbamoyladeninyl,
[0258] N6,N6-dimethyladeninyl,
[0259] 3-methylcytosinyl,
[0260] 5-methylcytosinyl,
[0261] 2-thiocytosinyl,
[0262] 5-formylcytosinyl, 63
[0263] N4-methylcytosinyl,
[0264] 5-hydroxymethylcytosinyl,
[0265] 1-methylguaninyl,
[0266] N2-methylguaninyl,
[0267] 7-methylguaninyl,
[0268] N2,N2-dimethylguaninyl, 646566
[0269] N2,7-dimethylguaninyl,
[0270] N2,N2,7-trimethylguaninyl,
[0271] 1-methylguaninyl,
[0272] 7-cyano-7-deazaguaninyl,
[0273] 7-aminomethyl-7-deazaguaninyl,
[0274] pseudouracilyl,
[0275] dihydrouracilyl,
[0276] 5-methyluracilyl,
[0277] 1-methylpseudouracilyl,
[0278] 2-thiouracilyl,
[0279] 4-thiouracilyl,
[0280] 2-thiothyminyl
[0281] 5-methyl-2-thiouracilyl,
[0282] 3-(3-amino-3-carboxypropyl)uracilyl,
[0283] 5-hydroxyuracilyl,
[0284] 5-methoxyuracilyl,
[0285] uracilyl 5-oxyacetic acid,
[0286] uracilyl 5-oxyacetic acid methyl ester,
[0287] 5-(carboxyhydroxymethyl)uracilyl,
[0288] 5-(carboxyhydroxymethyl)uracilyl methyl ester,
[0289] 5-methoxycarbonylmethyluracilyl,
[0290] 5-methoxycarbonylmethyl-2-thiouracilyl,
[0291] 5-aminomethyl-2-thiouracilyl,
[0292] 5-methylaminomethyluracilyl,
[0293] 5-methylaminomethyl-2-thiouracilyl,
[0294] 5-methylaminomethyl-2-selenouracilyl,
[0295] 5-carbamoylmethyluracilyl,
[0296] 5-carboxymethylaminomethyluracilyl,
[0297] 5-carboxymethylaminomethyl-2-thiouracilyl,
[0298] 3-methyluracilyl,
[0299] 1-methyl-3-(3-amino-3-carboxypropyl) pseudouracilyl,
[0300] 5-carboxymethyluracilyl,
[0301] 5-methyldihydrouracilyl, or
[0302] 3-methylpseudouracilyl.
[0303] 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: 6768
[0304] 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=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.
[0305] X.sup.2 can include "oxy" or "deoxy" substituents in place
of the 2'-OH; or be a ligand or a tethered ligand.
[0306] Examples of "oxy"-substituents include alkoxy or aryloxy
(OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl,
sugar, or protecting group); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.s- ub.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.
[0307] 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. 69
[0308] "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'-OCH.sub.3,2'-O-allyl, 2'-C-- allyl, and 2'-fluoro.
[0309] X.sup.3 is as described for OFG.sup.2 above.
[0310] 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.
[0311] Sugar Replacement-Based Monomers, e.g., Ligand-Conjugated
Monomers (Cyclic)
[0312] 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.4;
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.) 70
[0313] in which,
[0314] X is N(CO)R.sup.7, NR.sup.7 or CH.sub.2;
[0315] Y is NR.sup.8, O, S, CR.sup.9R.sup.10;
[0316] Z is CR.sup.11R.sup.12 or absent;
[0317] 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;
[0318] 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;
[0319] 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;
[0320] R.sup.8 is H or C.sub.1-C.sub.6 alkyl;
[0321] R.sup.13 is hydroxy, C.sub.1-C.sub.4 alkoxy, or halo;
[0322] R.sup.14 is NR.sup.cR.sup.7;
[0323] R.sup.15 is C.sub.1-C.sub.6 alkyl optionally substituted
with cyano, or C.sub.2-C.sub.6 alkenyl;
[0324] R.sup.16 is C.sub.1-C.sub.10 alkyl;
[0325] R.sup.17 is a liquid or solid phase support reagent;
[0326] L is --C(O)(CH.sub.2).sub.qC(O)--, or
--C(O)(CH.sub.2).sub.qS--;
[0327] 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.
[0328] R.sup.b is P(O)(O.sup.-)H, P(OR.sup.15)N(R.sup.6).sub.2 or
L-R.sup.17;
[0329] R.sup.c is H or C.sub.1-C.sub.6 alkyl;
[0330] R.sup.d is H or a ligand;
[0331] Each Ar is, independently, C.sub.6-C.sub.10 aryl optionally
substituted with C.sub.1-C.sub.4 alkoxy;
[0332] n is 1-4; and q is 0-4.
[0333] 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).
[0334] 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 71
[0335] 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: 72
[0336] 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 73
[0337] 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.,
[0338] --(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.
[0339] 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 74
[0340] 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.
[0341] 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, 75
[0342] 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.
[0343] 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 76
[0344] 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.
[0345] Representative cyclic, sugar replacement-based carriers are
shown in FIG. 3.
[0346] Sugar Replacement-Based Monomers (Acyclic)
[0347] 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. 77
[0348] 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.
[0349] Tethers
[0350] 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.n--C(O)--;
TAP-C(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.sub.- 2(LIGAND);
TAP-NR""(CH.sub.2).sub.n--NHNH.sub.2(LIGAND);
TAP-C(O)--(CH.sub.2).sub.n--C(O)(LIGAND);
TAP-C(O)--(CH.sub.2).sub.n--C(O- )O(LIGAND); TAP-C(O)--O(LIGAND);
TAP-C(O)--(CH.sub.2).sub.n--NH--C(O)(LIGA- ND);
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.
[0351] In some embodiments, the tether can terminate with a
mercapto group (i.e., SH) or an olefin (e.g., CH=CH.sub.2). For
example, the tether can be TAP-(CH.sub.2).sub.n--SH,
TAP-C(O)(CH.sub.2).sub.nSH, TAP-(CH.sub.2).sub.n--(CH=CH.sub.2), or
TAP-C(O)(CH.sub.2).sub.n(CH=CH.su- b.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.
[0352] 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.nC(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.
[0353] In other embodiments, it can be desirable for the monomer to
include a phthalimido group (K) at the terminal position of the
tether. 78
[0354] In other embodiments, other protected amino groups can be at
the terminal position of the tether, e.g., alloc, monomethoxy
trityl (MMT), itrifluoroacetyl, Fmoc, or aryl sulfonyl (e.g., the
aryl portion can be ortho-nitrophenyl or ortho,
para-dinitrophenyl).
[0355] 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=CH)--.
[0356] Tethered Ligands
[0357] 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.
[0358] 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.
[0359] 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.
[0360] 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.
[0361] 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,
peptides, polyamines, and peptide mimics.
[0362] The ligand can be a peptide or peptidomimetic. A
peptidomimetic (also referred to herein as an oligopeptidomimetic)
is a molecule capable of folding into a defined three-dimensional
structure similar to a natural peptide. The attachment of peptide
and peptidomimetics to iRNA agents can affect pharmacokinetic
distribution of the iRNA, such as by enhancing cellular recognition
and absorption. The peptide or peptidomimetic moiety can be about
5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40,
45, or 50 amino acids long.
[0363] In some embodiments, the ligand can be one of the following
triterpenes: 79
[0364] In some embodiments, the ligand can be substituted or
unsubstituted cholesterol, or a stereoisomer thereof or one of the
following steroids: 80
[0365] iRNA Agent Structure
[0366] 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.
[0367] 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.
[0368] 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.
[0369] 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.
[0370] 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.
[0371] 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:
[0372] (1) it will be of the Formula 1, 2, 3, or 4 set out in the
RNA Agent section below;
[0373] (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;
[0374] (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;
[0375] (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;
[0376] (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.
[0377] 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).sub.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(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)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)2(O)P-5'-CH.sub.2--),
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.)
[0378] 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 Nykanen 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.
[0379] 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.
[0380] 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.
[0381] 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 0 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.
[0382] 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.
[0383] Modifications and nucleotide surrogates are discussed below.
81
[0384] 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.
[0385] 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.
[0386] Modified nucleic acids and nucleotide surrogates can include
one or more of:
[0387] (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.);
[0388] (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;
[0389] (iii) wholesale replacement of the phosphate moiety (bracket
I) with "dephospho" linkers;
[0390] (iv) modification or replacement of a naturally occurring
base;
[0391] (v) replacement or modification of the ribose-phosphate
backbone (bracket II);
[0392] (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.
[0393] 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.
[0394] 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=O and Y=N in the above figure.
[0395] Specific modifications are discussed in more detail
below.
[0396] The Phosphate Group
[0397] 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.
[0398] 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.
[0399] 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.
[0400] Candidate agents can be evaluated for suitability as
described below.
[0401] The Sugar Group
[0402] 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.
[0403] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl,
heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.s- ub.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.
[0404] "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'-OCH.sub.3,2'-O-allyl, 2'-C--
allyl, and 2'-fluoro.
[0405] 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.
[0406] 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.
[0407] 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.
[0408] The modificaton can also entail the wholesale replacement of
a ribose structure with another entity at one or more sites in the
iRNA agent.
[0409] Candidate modifications can be evaluated as described
below.
[0410] Replacement of the Phosphate Group
[0411] 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.
[0412] 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.
[0413] Candidate modifications can be evaluated as described
below.
[0414] Replacement of Ribophosphate Backbone
[0415] 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.
[0416] Examples include the mophilino, cyclobutyl, pyrrolidine and
peptide nucleic acid (PNA) nucleoside surrogates. A preferred
surrogate is a PNA surrogate.
[0417] Candidate modifications can be evaluated as described
below.
[0418] Terminal Modifications
[0419] 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, 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, 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).
[0420] 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)(NH.sub.2)(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'-CH.sub.2--),
5'-alkyletherphosphonat- es (R=alkylether=methoxymethyl
(MeOCH.sub.2--), ethoxymethyl, etc., e.g. RP(OH)(O)--O-5'--).
[0421] 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.
[0422] Candidate modifications can be evaluated as described
below.
[0423] The Bases
[0424] 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.
[0425] 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.
[0426] Candidate modifications can be evaluated as described
below.
[0427] Evaluation of Candidate RNA's
[0428] 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.
[0429] 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.
[0430] 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.
[0431] References
[0432] General References
[0433] 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.
[0434] Modification described in WO 00/44895, WO01/75164, or
WO02/44321 can be used herein.
[0435] The disclosure of all publications, patents, and published
patent applications listed herein are hereby incorporated by
reference.
[0436] Phosphate Group References
[0437] 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.
[0438] Sugar Group References
[0439] 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).
[0440] Replacement of the Phosphate Group References
[0441] 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.
[0442] Replacement of the Phosphate-Ribose Backbone References
[0443] 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.
[0444] Terminal Modification References
[0445] Terminal modifications are described in Manoharan, M. et al.
Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and
references therein.
[0446] Bases References
[0447] 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
[0448] Preferred RNA agents have the following structure (see
Formula 2 below): 82
[0449] 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.
[0450] 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),R.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.2NHR.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.
[0451] A.sup.1 is: 83
[0452] H; OH; OCH.sub.3; W.sup.1; an abasic nucleotide; or
absent;
[0453] (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--O-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'-)).
[0454] A.sup.2 is: 84
[0455] A.sup.3 is: 85
[0456] and
[0457] A.sup.4 is: 86
[0458] H; Z.sup.4; an inverted nucleotide; an a basic nucleotide;
or absent.
[0459] 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.sub- .10, NH(CH.sub.2).sub.nR.sub.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.sub.10,
S(CH.sub.2).sub.nNR.sup.10, S(CH.sub.2).sub.nOR.sup.10,
S(CH.sub.2).sub.nSR.sup.10
O(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.sub.10,
O-Q-R.sup.10 N-Q-R.sup.10, S-Q-R.sub.10 or --O--. W.sup.4 is O,
CH.sub.2, NH, or S.
[0460] X.sup.1, X.sup.2, X.sup.3, and X.sup.4 are each,
independently, O or S.
[0461] 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.
[0462] 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).sub.n OR.sup.10,
(CH.sub.2).sub.n SR.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.10,
O(CH.sub.2CH.sub.2O).sub.mCH.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.
[0463] x is 5-100, chosen to comply with a length for an RNA agent
described herein.
[0464] 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.
[0465] 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.sub.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, 03-(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.
[0466] Preferred RNA agents in which the entire phosphate group has
been replaced have the following structure (see Formula 3 below):
87
[0467] 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.
[0468] 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, N4-acetyl cytosine,
2-thiocytosine, N6-methyladenine, N6-isopentyladenine,
2-methylthio-N-6-isopentenyladenin- e, N-methylguanines, or
O-alkylated bases.
[0469] R.sup.40, R.sup.50, and R.sup.60 are each, independently,
OR.sup.8, O(CH.sub.2CH.sub.2O).sub.mCH.sub.2CH.sub.2OR.sub.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.sub.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.
[0470] x is 5-100 or chosen to comply with a length for an RNA
agent described herein.
[0471] 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.
[0472] 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.
[0473] 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
[0474] 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.
[0475] 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, 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-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.
[0476] x is 5-100, or chosen to comply with a length for an RNA
agent described herein; and g is 0-2.
[0477] Nuclease Resistant Monomers
[0478] 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.
[0479] 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.
[0480] 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.
[0481] 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.
[0482] 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:
[0483] (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;
[0484] (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;
[0485] (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' CH.sub.2--NCH.sub.3--O--CH.sub.2-5' and
3' CH.sub.2--NH--(O.dbd.)--CH.sub.2-5'.;
[0486] (iv) 3'-bridging thiophosphates and 5'-bridging
thiophosphates. Thus, preferred NRM's can included these
structures;
[0487] (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;
[0488] (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;
[0489] (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
[0490] (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.
[0491] 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.
[0492] 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.
[0493] Chiral S.sub.p Thioates
[0494] 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. 88
[0495] 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.
[0496] 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.).
[0497] 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.
[0498] 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 89
[0499] 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 0, 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.
[0500] 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.
[0501] Cationic Groups
[0502] 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).
[0503] Nonphosphate Linkages
[0504] 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.
[0505] 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
[0506] 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.
[0507] 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.
[0508] Modifications can also include 5'-phosphonates, e.g.,
P(O)(O.sup.-).sub.2--X--C.sup.5'-sugar (X=CH2, CF2, CHF and
5'-phosphate prodrugs, e.g.,
P(O)[OCH2CH2SC(O)R].sub.2CH.sub.2C.sup.5'-sugar. 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.
[0509] 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.
[0510] 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.
[0511] 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.
[0512] 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.)
[0513] 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.
[0514] An iRNA agent can have a first and a second strand chosen
from the following:
[0515] 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;
[0516] 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;
[0517] 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;
[0518] a first strand which does not target a sequence and which
has an NRM modification at the cleavage site or in the cleavage
region;
[0519] 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
[0520] 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;
[0521] 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);
[0522] 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;
[0523] 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;
[0524] 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).
[0525] An iRNA agent can also target two sequences and can have a
first and second strand chosen from:
[0526] 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;
[0527] 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);
[0528] 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;
[0529] 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;
[0530] 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;
[0531] 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);
[0532] 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;
[0533] 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;
[0534] 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
[0535] 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/U.S.04/07070, both of which are hereby incorporated by
reference.
[0536] 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.
[0537] 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.
[0538] 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): 90
[0539] 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.
[0540] 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
0 and Y is S.
[0541] 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.
[0542] 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.sub.4 or
R.sub.6 to form a ring of 5-12 atoms.
[0543] R.sub.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.
[0544] 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.
[0545] 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;
[0546] R.sub.7 is hydrogen, C.sub.1-C.sub.100 alkyl, or
C(O)(CH.sub.2).sub.qC(O)NHR.sup.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.
[0547] Preferred embodiments may include one of more of the
following subsets of iRNA agent delivery modules.
[0548] 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.
[0549] In another subset of RNAi agent delivery modules, X, W, and
Z are 0 and Y is S.
[0550] 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): 91
[0551] 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.
[0552] 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.
[0553] Pharmaceutical Compositions
[0554] 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.
[0555] 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.
[0556] 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.
[0557] 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.
[0558] 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.
[0559] 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.
[0560] 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.
[0561] 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.
[0562] 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.
[0563] 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.
[0564] 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.
[0565] 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.
[0566] 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.
[0567] 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.
[0568] 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.
[0569] Methods for Treating Diseases Caused by Expression of a
Target Gene.
[0570] 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.
[0571] 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): R.sub.776-8; 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, MDR1 gene (Childs,
S., and V. Ling, Imp. Adv. Oncol. (1994) 21-36).
[0572] 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.
[0573] 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.
[0574] 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.
[0575] 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.
[0576] 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, picornaviruses, 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 (Beme 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.
[0577] 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.
[0578] Methods for Inhibiting Expression of a Target Gene.
[0579] 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.
[0580] 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.
[0581] 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 ABLI, BCL1, BCL2, BCL6, CBFA2, CBL, CSFIR, ERBA,
ERBB, EBRB2, ETS 1, ETS 1, 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.
[0582] 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.
[0583] Methods for Identifying iRNA Agent Having Increased
Stability.
[0584] 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.
[0585] 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.
[0586] 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.
[0587] 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.
[0588] 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.
[0589] 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).
[0590] 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).
[0591] 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.
[0592] 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
Example 1
Terminal Modifications Protect Sites of Exonucleolytic Cleavage
[0593] Serum Incubation Assay
[0594] Blood of 8 human volunteers (270 mL) was collected and kept
at room temperature for 3 hours. The blood pool was then
centrifuged at 20.degree. C. and 3000 rcf using Megafuge 1.0
(Heraeus Instruments, Kendro Laboratory Products GmbH,
Langenselbold) to separate the serum from the cellular fraction.
The supernatant was stored in aliquots at -20.degree. C. and used
as needed. Human serum obtained from Sigma (Sigma-Aldrich Chemie
GmbH, Taufkirchen, Germany) was employed in control assays. Assay
results reported herein were consistent among the different serum
sources tested.
[0595] Double stranded RNAs (300 pmol, ca. 4.2 .mu.g) were
incubated at 37.degree. C. in 60 .mu.l 90% human serum/10%
phosphate buffered saline for 0, 15, 30, 60, 120 or 240 minutes, or
8 hours, 16 hours or 24 hours. A 10 .mu.l sample was taken from the
incubation mixture, frozen in liquid nitrogen and stored at
-80.degree. C. until analysis.
[0596] Analysis by Electrophoresis and "Stains all" Detection
[0597] For analysis, samples were thawed, their constituents were
isolated by phenol-extraction and ethanol-precipitation, separated
on denaturing 14% polyacrylamide gels (6M Urea, 20% formamide, Carl
Roth GmbH & Co KG Karlsruhe, Germany) and detected by staining
with the "stains-all" reagent (Sigma-Aldrich Chemie GmbH,
Steinheim, Germany). Reaction mixtures from incubation of selected
dsRNAs were further analysed covering the time points 8 hours, 16
hours and 24 hours.
[0598] Cell Culture
[0599] HeLa SS6 cells were grown at 37.degree. C. in Dulbecco's
modified Eagle medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 100 units/mL penicillin, and 100 .mu.g/mL streptomycin
(Invitrogen, Carlsbad, Calif.). Cells were passaged regularly to
maintain exponential growth. Twenty-four hours prior to siRNA
transfection, cells were seeded on opaque, white 96-well plates
(Costar, Corning, N.Y.) at a concentration of 15,000 cells/well in
150 .mu.L antibiotic-free, phenol red-free DMEM (Invitrogen).
[0600] Dual Luciferase Gene Silencing Assays
[0601] In vitro activity of siRNAs was determined using a
high-throughput 96-well plate format luciferase silencing assay.
Cells were first transiently transfected with plasmids encoding
firefly (target) and renilla (control) luciferase. DNA
transfections were performed using Lipofectamine 2000 (Invitrogen)
(0.5 .mu.L/.mu.g total DNA) and the plasmids gWiz-Luc (Aldevron,
Fargo, N.Dak.) (200 ng/well) and pRL-CMV (Promega, Madison, Wis.)
(200 ng/well). After 2 h, the plasmid transfection medium was
removed, and the firefly luciferase targeting siRNAs were added to
the cells at 100 nM concentration. siRNA transfections were
performed using TransIT-TKO (Mirus, Madison, Wis.) (0.3 mL/well).
After 24 h, cells were analyzed for both firefly and renilla
luciferase expression using a plate luminometer (VICTOR.sub.2,
PerkinElmer, Boston, Mass.) and the Dual-Glo Luciferase Assay kit
(Promega). Firefly/renilla luciferase expression ratios were used
to determine percent gene silencing relative to mock-treated (no
siRNA) controls.
[0602] Results
[0603] dsRNAs including phosphorothioate modifications were
incubated in 90% human serum at 37.degree. C. as described above.
Gel electropheresis of the RNA products revealed that the
phosphorothioate modifications enhanced stability (FIG. 1). Similar
results were observed when 3'-Naproxen, 5-Nitroindole,
alkyamino-dT, abasic pyrrolidine, and cationic C6 amino dT were
conjugated to the 3' end of dsRNAs (FIGS. 2, 3, and 4).
[0604] The dual luciferase assay revealed that modifications than
enhanced resistance to exonuclease activity retained silencing
activity. FIGS. 5, 6, and 7 show the silencing activity of 3'
conjugated Naproxen, abasic pyrrolidine, and cationic C6 amino dT,
respectively.
[0605] FIGS. 8 and 9 show that the 3'Naproxen conjugated dsRNAs had
enhanced exonuclease resistance in mouse serum and liver tissue, as
well as in human serum (see FIG. 2).
Example 2
siRNA Modifications Enhanced Duplex Stability
[0606] Radiolabel method for monitoring serum stability of siRNA
duplexes: siRNA duplexes were prepared at a stock concentration of
1 .mu.M in which either the sense (S) or antisense strand (AS)
contained a trace amount of 5'-.sup.32P labeled material (e.g.
.sup.32P-S/AS and S/.sup.32P-AS). The presence of the end-labeled
sense or antisense strand allowed for monitoring of the individual
strand within the context of the siRNA duplex. Therefore, two
duplex preparations were made for each siRNA sequence tested. siRNA
duplexes were incubated in 90% human serum at a final concentration
of 100 nM duplex. Samples were removed and quenched in a stop mix
at appropriate times. For a typical time course, 10 seconds, 15
minutes, 30 minutes, 1 hour, 2 hours and 4 hours time points were
taken. Samples were analyzed by denaturing polyacrylamide gel
electrophoresis along with a control sample (4 hour buffer-alone
incubation) and a partial alkaline hydrolysis ladder of the labeled
sense or antisense strand as a marker. The gel was imaged using a
Fuji phosphorimager to detect the full length sense and antisense
strands along with any degradation fragments that were generated by
serum nucleases during incubation.
[0607] Since there is the possibility of losing the 5' phosphate
label due to phosphatase activity in the serum, an alternative to
5' end labeling is to place an internal .sup.32P or .sup.33P label
within either the sense or antisense strand. This labeling method
is much more laborious than 5' end labeling and currently we have
no evidence that dephosphorylation occurs during serum
incubation.
[0608] A series of chemical modifications that fall into the
following categories; backbone modification, sugar modification,
nucleobase modification and 3' conjugate, were tested and showed
enhanced serum stability as compared to a unmodified siRNA duplex.
A description of each modification, its location within the siRNA
duplex, and the serum stability data follows.
[0609] Serum stability of unmodified parent duplex: The unmodified
parent duplex, AL-DUP-1000, was used to establish the serum
stability baseline for evaluating the effect of chemical
modifications on nuclease resistance.
1 AL-DUP-1000 5'-CUUACGCUGAGUACUUCGAdTdT-3' ALN-SEQ-1000 SEQ ID NO:
3' dTdTGAAUGCGACUCAUGAAGCU-5' ALN-SEQ-1001 SEQ ID NO:
[0610] AL-DUP-1000 was subjected to the serum stability assay to
evaluate its inherent nuclease resistance and to define its
degradation pattern (FIG. 21). Denaturing gel electrophoresis was
used analyze AL-DUP-1000 in a human serum stability assay. An siRNA
duplex containing 5' end-labeled sense RNA (*s/as) and a duplex
containing 5' end-labeled antisense RNA (as/s*) were each incubated
in 90% human serum and time points were assayed at 10 seconds, 5
min, 15 min, 30 min, 1 hour, 2 hours and 4 hours. The control was a
4 hour time point for siRNA duplex incubated in PBS buffer alone,
OH-was the partial alkaline hydrolysis marker. This unmodified
duplex was observed to be degraded by both 3'-5' exonucleases and
endonucleases (FIG. 21).
[0611] Cleavage of the 3' end of both the sense and antisense
strands by 3'-5' exonucleases occurs within the first 5 minutes of
incubation resulting in the loss of the 3' terminal dT residues
(top vertical lines in s*/as and s/as* panels of FIG. 21). In
addition to exonuclease degradation, both strands were cleaved by
endonucleases. There was a major endonuclease site at position
sixteen of the antisense strand (bottom vertical lines in s*/as and
s/as* panels of FIG. 21) that appears as early as 10 seconds. Very
little full length sense or antisense strand was remaining after 1
hour in human serum. Chemical modifications were introduced within
the context of the parent duplex to evaluate their effect on
nuclease resistance. These chemical modifications fall within one
of the following classes: backbone modification, sugar
modification, nucleobase modification, cationic modification and
conjugate.
[0612] Backbone modifications enhanced nuclease resistance:
Specific phophodiester linkages of the siRNA duplex were replaced
by either phosphorothioate or methylphosphonate and their stability
was evaluated in the human serum stability assay. Table 1 contains
the sequences of the duplexes tested. Substitution of the
phosphodiester linkage at the 3' end of both the sense and
antisense strands inhibited exonucleolytic degradation of the 3'
overhangs (FIGS. 22A and 22B) as compared to the unmodified parent
duplex (refer to FIG. 21). Full length starting material was
present for four hours for both the sense and antisense strands.
The endonucleolytic cleavage pattern seen in the unmodified duplex
was unchanged. Similar results were obtained for duplexes that
contained additional phosphorothioates at their 3' ends (data not
shown). The placement of phosphorothioates at the endonucleolytic
cleavage sites (duplexes 1419, 1420 and 1421) did not inhibit
endonucleolytic cleavage at these sites (data not shown). In
summary, a single phosphorothioate or methylphosphonate between the
two 3' terminal nucleotides was sufficient to protect the 3' ends
from exonuclease degradation. Additional phosphorothioates at the
3' ends appear to enhance this effect, which may be necessary for
long term exposure to serum nucleases.
2TABLE 1 siRNA duplexes containing backbone modifications. (* =
phosphorothioate, mp = methylphosphonate) Alnylam Duplex Duplex
Sequence Alnylam Sequence AL-DUP-1393
5'-CUUACGCUGAGUACUUCGAdT*dT-3' AL-SEQ-1026
3'-dT*dTGAAUGCGACUCAUGAAGCU-5' AL-SEQ-1027 AL-DUP-1394
5'-CUUACGCUGAGUACUUCGA*dT*dT-3' AL-SEQ-1028
3'-dT*dT*GAAUGCGACUCAUGAAGCU-5' AL-SEQ-1029 AL-DUP-1395
5'-CUUACGCUGAGUACUUCG*A*dT*dT-3' AL-SEQ-1030
3'-dT*dT*G*AAUGCGACUCAUGAAGCU-5' AL-SEQ-1031 AL-DUP-1396
5'-CUUACGCUGAGUACUUC*G*A*dT*dT-3' AL-SEQ-1032
3'-dT*dT*G*A*AUGCGACUCAUGAAGCU-5' AL-SEQ-1033 AL-DUP-1419
5'-CUUACGCUGAGU*ACUUCGAdTdT-3' AL-SEQ-2182
3'-dTdTGAAUGCGACUCA*UGAAGCU-5' AL-SEQ-2184 AL-DUP-1420
5'-CUU*ACGCUGAGU*ACUUCGAdTdT-3' AL-SEQ-2183
3'-dTdTGAA*UGCGACUCA*UGAAGCU-5' AL-SEQ-2185 AL-DUP-1421
5'-CUU*ACGCUGAGU*ACUUCGAdT*dT-3' AL-SEQ-2186
3'-dT*dTGAA*UGCGACUCA*UGAAGCU-5' AL-SEQ-2188 AL-DUP-1329
5'-CUUACGCUGAGUACUUCGAdTmpdT-3' AL-SEQ-1038
3'-dTmpdTGAAUGCGACUCAUGAAGCU-5' AL-SEQ-1039
[0613] Sugar modifications enhanced nuclease resistance: The effect
of replacing the 2'OH with 2'OMe was evaluated at the sites of
endonucleolytic cleavage as well as at the 3' ends of the siRNA
duplex. The duplexes tested in the human serum stability assay are
shown in Table 2. Some of these duplexes also contained
phosphorothioate linkages to evaluate whether the combination of
the two modifications enhance nuclease resistance more
significantly. Substitution of the terminal dT residues with
2'OMe-U (AL-DUP-1027) reduced 3'-5' exonuclease degradation
slightly over the unmodified parent duplex (data not shown);
however, the extent of exonuclease protection by 2'OMe-U was far
less than that achieved by placing a
3TABLE 2 siRNA duplexes containing 2'OMe substitutions. (U =
2'OMe-uridine, * = phosphorothioate) Alnylam Duplex Duplex Sequence
Alnylam Sequence AL-DUP-1027 5'-CUUACGCUGAGUACUUCGAUU-3'
AL-SEQ-1006 3'-UUGAAUGCGACUCAUGAAGCU-5' AL-SEQ-1007 AL-DUP-1036
5'-C*UUACGCUGAGUACUUCGAU*U-3' AL-SEQ-1008 3'-U*UGAAUGCGACUCAUGAAG-
C*U-5' AL-SEQ-1009 AL-DUP-13ff 5'-C*UUACGCUGAGUACUUCGAU- *U-3'
AL-SEQ-gggg 3'-U*UGAAUGCGACUCAUGAAGC*U-5' AL-SEQ-hhhh AL-DUP-1363
5'-C*UUACGCUGAGUACUUCGAU*U-3' AL-SEQ-1162
3'-U*UGAAUGCGACUCAUGAAGC*U-5' AL-SEQ-1163
[0614] phosphorothioate between the two terminal dT residues (see
FIG. 22A). Addition of a single phosphorothioate between the two
terminal 2'OMe-uridine residues effectively inhibited 3'-5'
exonucleolytic cleavage as seen in FIG. 23 for duplexes
AL-DUP-1036, AL-DUP-13ff, and AL-DUP-1363. 2'OMe substitution on
its own was much more effective at protecting from endonucleolytic
cleavage when placed at the internal cleavage sites. The parent
duplex was cleaved 3' of U at two UpA sites within the duplex. Both
strands are cleaved due to the symmetry of this dinucleotide repeat
and mapping data was used to confirm the sites of cleavage (data
not shown). Placement of 2'OMe at the strong endonucleolytic site
((FIG. 23, star in s/*as gel, AL-DUP-13ff) resulted in inhibition
of cleavage at this site. The second, weaker endonucleolytic site
(FIG. 23, black star in *s/as), however, was slightly enhanced when
the strong site was protected with 2'OMe (FIG. 23, compare
AL-DUP-13ff to AL-DUP-1036). Protection of both sites with 2'OMe
(AL-DUP-1363) resulted in reduced endonucleolytic cleavage at both
sites (FIG. 23). The inhibitory effect of the 2'OMe substitution is
consistent with the mechanism of endonucleolytic cleavage, which
requires the 2'OH as a nucleophile in the cleavage reaction. 2'OMe
modification will also be an effective means to protect the 3'
overhang of single overhang siRNA duplexes where the 3' overhang is
composed of ribonucleotides. In this situation, 2'OMe substitution
can be used to block the possible loss of the terminal two
nucleotides by endonucleolytic cleavage and phosphorothioate can be
used to protect from exonuclease degradation.
[0615] Cationic modifications enhanced nuclease resistance: The
effect of three different cationic chemical modifications on
nuclease resistance was tested and compared to the parent
unmodified duplex. The structures of the three cationic
modifications tested are shown below. 92
4TABLE 3 siRNA duplexes containing cationic substitutions. (aadT =
alkylamine-dT, AbP = abasic pyrrolidine cationic, aaU =
allylamino-U, * = phosphorothioate, U = 2'OMe-U) Alnylam Duplex
Duplex Sequence Alnylam Sequence AL-DUP-10aa
5'-CUUACGCUGAGUACUUCGAdTaadT-3' AL-SEQ-1017
3'-aadTdTGAAUGCGACUCAUGAAGCU-5' AL-SEQ-1018 AL-DUP-10bb
5'-CUUACGCUGAGUACUUCGAaadTaadT-3' AL-SEQ-1015
3'-aadTaadTGAAUGCGACUCAUGAAGCU-5' AL-SEQ-1016 AL-DUP-1ccc
5'-CUUACGCUGAGUACUUCGAdTdTAbP-3' AL-SEQ-dddd
3'-AbPdTdTGAAUGCGACUCAUGAAGCU-5' AL-SEQ-eeee AL-DUP-1403
5'-C*UaaUACGCUGAGUACUUCGAU*U-3' AL-SEQ-2080
3'-U*UGAAaaUGCGACUCAUGAAGC*U-5' AL-SEQ-2081 AL-DUP-1406
5'-C*UaaUACGCUGAGaaUACUUCGAU*U-3' AL-SEQ-2082
3'-U*UGAAaaUGCGACUCAaaUGAAGC*U-5' AL-SEQ-2083
[0616] The sequences of the duplexes assayed in the human serum
stability assay are shown in Table 3. Both alkylamino-dT and abasic
pyrrolidine cationic modifications were placed at the 3' terminal
overhang to evaluate their effect on 3'-5' exonuclease degradation.
Allylamino-uridines were placed at the internal endonucleolytic
cleavage sites to evaluate their ability to inhibit endonucleolytic
cleavage. As seen in FIG. 24, replacing the 3' terminal dT residue
with a single alkylamino-dT efficiently inhibited 3'-5'
exonucleolytic degradation (FIG. 24, AL-DUP-10aa, left gel image).
Replacement of both dT residues in the overhang with alkylamino-dT
resulted in a similar extent of inhibition (data not shown).
Addition of an abasic pyrrolidine cationic modification at the 3'
terminus of each strand also protected against exonucleolytic
degradation (FIG. 24, middle gel image). Both the alkylamino-dT and
abasic pyrrolidine modifications protected from 3'-5'
exonucleolytic cleavage up to 23 hours (data not shown). Placement
of allylamino-U at the internal cleavage sites inhibited
endonucleolytic cleavage as shown in FIG. 24 for duplex
AL-DUP-1403. The ends of this duplex were stabilized from
exonucleolytic degradation by 2'OMe-U and phosphorothioate
substitutions in order to separate the two different cleavage
events. Endonucleolytic cleavage was inhibited at both internal
cleavage sites by allylamino-U substitution for AL-DUP-1406 (data
not shown).
[0617] 3' conjugates enhanced nuclease resistance: Conjugation of
naproxen and ibuprofen to the 3' end of the siRNA were tested for
their ability to inhibit 3'-5' exonucleolytic degradation. The
structure of naproxen is shown in below: 93
[0618] Table 4 lists the siRNAs that were tested in the human serum
stability assay. Conjugation of either naproxen or ibuprofen to the
3' end inhibited exonucleolytic degradation. FIG. 18 shows the
serum stability data for the naproxen modified duplex (AL-DUP-1069)
and similar results were obtained for AL-DUP1413. Presumably the
conjugates inhibit exonucleolytic cleavage by sterically blocking
the exonuclease from binding to the 3' end of the siRNA duplex.
Similar data was also obtained for AL-DUP-1069 in pooled mouse
serum.
5TABLE 4 siRNA duplexes containing 3 conjugates. (Nap = Naproxen,
Ibu = Ibuprofen) Alnylam Duplex Duplex Sequence SEQ ID NO:
AL-DUP-1069 5'-CUUACGCUGAGUACUUCGAdTdTNap-3'
3'-NapdTdTGAAUGCGACUCAUGAAGCU-- 5' AL-DUP-1413
5'-CUUACGCUGAGUACUUCGAdTdTIbu-3'
3'-NapdTdTGAAUGCGACUCAUGAAGCU-5'
[0619] 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.
* * * * *