U.S. patent application number 16/673865 was filed with the patent office on 2020-07-23 for compositions and methods for inhibiting expression of factor v.
The applicant listed for this patent is Alnylam Pharmaceuticals, Inc.. Invention is credited to John M. Maraganore, Hans-Peter Vornlocher.
Application Number | 20200231968 16/673865 |
Document ID | / |
Family ID | 38023912 |
Filed Date | 2020-07-23 |
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United States Patent
Application |
20200231968 |
Kind Code |
A1 |
Vornlocher; Hans-Peter ; et
al. |
July 23, 2020 |
COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF FACTOR V
Abstract
The invention relates to a double-stranded ribonucleic acid
(dsRNA) for inhibiting the expression of Factor V, comprising an
antisense strand having a nucleotide sequence which is less that 25
nucleotides in length and which is substantially complementary to
at least a part of Factor V. The invention also relates to a
pharmaceutical composition comprising the dsRNA together with a
pharmaceutically acceptable carrier; methods for treating diseases
caused by the expression of Factor V using the pharmaceutical
composition; and methods for inhibiting the expression of Factor V
in a cell.
Inventors: |
Vornlocher; Hans-Peter;
(Bayreuth, DE) ; Maraganore; John M.;
(Charlestown, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Alnylam Pharmaceuticals, Inc. |
Cambridge |
MA |
US |
|
|
Family ID: |
38023912 |
Appl. No.: |
16/673865 |
Filed: |
November 4, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15676874 |
Aug 14, 2017 |
10501740 |
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16673865 |
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15241025 |
Aug 18, 2016 |
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15676874 |
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14155239 |
Jan 14, 2014 |
9441225 |
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15241025 |
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13010300 |
Jan 20, 2011 |
8658782 |
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14155239 |
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12093235 |
Nov 5, 2008 |
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PCT/US2006/043271 |
Nov 7, 2006 |
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13010300 |
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60735759 |
Nov 9, 2005 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 7/02 20180101; A61K
48/00 20130101; A61P 9/00 20180101; C12N 15/113 20130101; C12N
2310/14 20130101; C12N 2320/30 20130101; A61K 47/554 20170801 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 47/54 20060101 A61K047/54; A61K 48/00 20060101
A61K048/00 |
Claims
1. An isolated double-stranded ribonucleic acid (dsRNA) comprising
a sense strand and an antisense strand and wherein the sense strand
consists of the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9,
11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43,
45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, or 75
and the antisense strand consists of the nucleotide sequence of SEQ
ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,
34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66,
68, 70, 72, 74, or 76, respectively.
2. The dsRNA of claim 1, wherein the sense strand consists of the
nucleotide sequence of SEQ ID NO: 27, 29, 31, 33, 35, 37, 39, 41,
43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, or
75 and the antisense strand consists of the nucleotide sequence of
SEQ ID NO: 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54,
56, 58, 60, 62, 64, 66, 68, 70, 72, 74, or 76, respectively.
3. The dsRNA of claim 1, wherein the sense strand consists of the
nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17,
19, 21, 23, or 25 and the antisense strand consists of the
nucleotide sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18,
20, 22, 24, or 26, respectively.
4. The dsRNA of claim 1, wherein the dsRNA comprises at least one
modified nucleotide.
5. The dsRNA of claim 4, wherein said modified nucleotide is chosen
from the group of: a 2'-O-methyl modified nucleotide, a nucleotide
comprising a 5'-phosphorothioate group, and a terminal nucleotide
linked to a cholesteryl derivative or dodecanoic acid bisdecylamide
group.
6. The dsRNA of claim 4, wherein said modified nucleotide is chosen
from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a
2'-deoxy-modified nucleotide, a locked nucleotide, an abasic
nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified
nucleotide, morpholino nucleotide, a phosphoramidate, and a
non-natural base comprising nucleotide.
7. An isolated cell comprising the dsRNA of claim 1.
8. The cell of claim 7, wherein the cell is a mammalian cell.
9. A pharmaceutical composition comprising the dsRNA of claim 1 and
a pharmaceutically acceptable carrier.
10. A method for inhibiting the expression of a Factor V gene in a
cell, the method comprising: (a) introducing into the cell the
dsRNA of claim 1; and (b) maintaining the cell for a time
sufficient to obtain degradation of the mRNA transcript of the
Factor V gene, thereby inhibiting the expression of the Factor V
gene in the cell.
11. The cell of claim 10, wherein the cell is a mammalian cell.
12. A method of treating, preventing or managing thrombophilia
comprising administering to a subject in need of such treatment,
prevention or management a therapeutically or prophylactically
effective amount of the dsRNA of claim 1.
13. A vector comprising a regulatory sequence operably linked to a
nucleotide sequence that encodes at least one strand of the dsRNA
of claim 1.
14. A cell comprising the vector of claim 13.
15. The cell of claim 14, wherein the cell is a mammalian cell.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 15/676,874, filed Aug. 14, 2017, allowed, which is a
continuation of U.S. application Ser. No. 15/241,025, filed on Aug.
18, 2016 (abandoned), which is a continuation of U.S. application
Ser. No. 14/155,239, filed Jan. 14, 2014 now U.S. Pat. No.
9,441,225, issued Sep. 13, 2016, which is a continuation of U.S.
application Ser. No. 13/010,300, filed Jan. 20, 2011, now U.S. Pat.
No. 8,658,782, issued Feb. 25, 2014, which is a continuation of
U.S. application Ser. No. 12/093,235, with a 371c filing date of
Nov. 5, 2008 (abandoned), which is a National Stage under 35 U.S.C.
.sctn. 371 of International Application No. PCT/US2006/043271,
filed Nov. 7, 2006, which claims the benefit of U.S. Provisional
Application No. 60/735,759, filed Nov. 9, 2005, each of which is
incorporated herein by reference, in its entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing with 76
sequences which has been submitted via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 14, 2019, is named AYN030C5_Sequence_Listing.txt, and is
45,056 bytes in size.
FIELD OF THE INVENTION
[0003] This invention relates to double-stranded ribonucleic acid
(dsRNA), and its use in mediating RNA interference to inhibit the
expression of the Factor V Leiden mutant gene and the use of the
dsRNA to treat thrombophilia.
BACKGROUND OF THE INVENTION
[0004] Factor V Leiden thrombophilia is characterized by a poor
anticoagulant response to activated protein C (APC) and an
increased risk of venous thromboembolism. The term "factor V
Leiden" refers to the specific G-to-A substitution at nucleotide
1691 in the gene for factor V that predicts a single amino acid
replacement (Arg506Gln) at one of three APC cleavage sites in the
factor Va molecule. Factor V Leiden is inactivated at a rate
approximately ten times slower than normal factor V and persists
longer in the circulation, resulting in increased thrombin
generation and a mild hypercoagulable state reflected by elevated
levels of prothrombin fragment F1+2 and other activated coagulation
markers. Individuals heterozygous for the factor V Leiden mutation
have a slightly increased risk for venous thrombosis; homozygous
individuals have a much greater thrombotic risk.
[0005] Factor V Leiden is the most common hereditary blood
coagualtion disorder in the United States. It is present 5% of the
in the Caucasian population and 1.2% of the African American
population.
[0006] Factor V Leiden increases the risk of venous thrombosis 3-8
fold for heterozygous (one damaged gene inherited) and
substantially more, 30-140 fold, for homozygous (two damaged genes
inherited) individuals.
[0007] Deep venous thrombosis with the attendant risk of pulmonary
embolism and post phlebitic syndrome is a frequent complication in
older patients who have undergone surgery, suffered trauma or who
have serious illness such as malignancy or sepsis. In any category
patients who are 40 years of age or older are considered to be at
greatest risk. Also the longer the period of immobilization the
greater the risk of DVT. Other factors that have been reported to
contribute to development of DVT are obesity, prior history of DVT
and smoking. While none of these factors alone or in combination
will identify individual patients who will develop DVT, the
incidence of DVT during the postoperative or post-traumatic period
does correlate with the condition.
[0008] DVT has three major risks for the patient, two acute and one
delayed. The acute problems are leg swelling, pain and tenderness
and the risk of pulmonary embolism. In pulmonary embolism part of
the thrombus breaks away and is carried to the lung where it can
block a pulmonary artery causing respiratory distress in proportion
to the amount of blockage, i.e., to the size of the embolus. Large
emboli that block both pulmonary arteries cause immediate death.
The delayed problem is the post phlebitic syndrome in which there
is lower extremity pain or cramps at rest, leg edema, skin changes
and skin breakdown causing chronic ulcers of the lower extremity.
Clinicians have long known that the post phlebitic syndrome
develops in a large percentage of patients who have DVT, especially
those having extensive thrombus formation. Objective studies have
shown that 1-10 years following the occurrence of DVT as much as
80% of patients will have both symptoms and abnormal venous
hemodynamics (Lindner et al, 1986; Markel et at, 1992). While the
post phlebitic syndrome is less dramatic than a major pulmonary
embolus, it is a serious condition for the patients, resulting in
much discomfort and expense.
[0009] In some patients groups DVT and pulmonary embolism are major
causes of morbidity and mortality. Thromboembolism is a major cause
of morbidity and mortality in patients with spinal cord injury. The
prevalence of DVT has been reported to range from 47% (Merli et al,
1988) to 78% (Green et al, 1982). Of these 1 to 2% will die of
pulmonary embolism (Green, 1991). Thrombosis usually occurs 1 to 3
weeks after injury, with a peak between days 7 and 9. The incidence
of thromboembolic complications in patients undergoing surgery for
fractured hip is high, ranging from about 40-60% (Powers et al,
1989; Fordyce and Ling, 1992; Turpie, 1991; Levine et al, 1991;
Hull et al, 1990). In patients undergoing knee arthroplasty the
incidence of DVT ranges from about 50% to 85% (Stulberg et al,
1984; Leclerc et al, 1992; Wilson et al, 1992). In gynecologic
malignancy the incidence of DVT was 35% (Clarke-Person et al,
1984). The incidence of DVT in patients undergoing elective general
abdominal surgery was about 9% in those without malignancy and
about 11% in those with malignancies (Bergqvist et al, Seminars in
Thromb & Hemost 16 Suppl 19-24, 1990).
[0010] For about 50 years efforts to prevent development of DVT and
to treat those that do develop have focused on the judicious use of
anticoagulants, first through full doses of oral anticoagulants and
more recently through low dose heparin prophylaxis (Gallus, 1990).
The aim has been to achieve a helpful degree of anticoagulation
(prolongation of the clotting process) without causing hemorrhage.
Low dose heparin has become the standard of comparison for other
preventive methods since it is relatively safe and simple and
prevents approximately 65% of subclinical thrombi found by leg
scanning after elective general surgery. Postoperative death from
pulmonary emboli may be reduced by 65% also.
[0011] However, there are clinical situations in which low dose
heparin is less effective, most notable after orthopedic surgery
where the use of more complex regimens, including adjusted dose
heparin and various schedules of warfarin prophylaxis are
appropriate. Several studies have shown that higher levels of
anticoagulation are more effective than lower ones. However, if
anticoagulation is too high, bleeding complications result.
[0012] Recently, double-stranded RNA molecules (dsRNA) have been
shown to block gene expression in a highly conserved regulatory
mechanism known as RNA interference (RNAi). WO 99/32619 (Fire et
al.) discloses the use of a dsRNA of at least 25 nucleotides in
length to inhibit the expression of the Factor V Leiden mutant gene
in C. elegans. dsRNA has also been shown to degrade target RNA in
other organisms, including plants (see, e.g., WO 99/53050,
Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila
(see, e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and
mammals (see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et
al.). This natural mechanism has now become the focus for the
development of a new class of pharmaceutical agents for treating
disorders that are caused by the aberrant or unwanted regulation of
a gene.
[0013] Despite significant advances in the field of RNAi and
advances in the treatment of thrombophilia, there remains a need
for an agent that can selectively and efficiently silence the
Factor V Leiden mutant gene using the cell's own RNAi machinery
that has both high biological activity and in vivo stability, and
that can effectively inhibit expression of a target Factor V Leiden
mutant gene for use in treating thrombophilia.
SUMMARY OF THE INVENTION
[0014] The invention provides double-stranded ribonucleic acid
(dsRNA), as well as compositions and methods for inhibiting the
expression of the Factor V Leiden mutant gene in a cell or mammal
using such dsRNA. The invention also provides compositions and
methods for treating pathological conditions and diseases caused by
the expression of the Factor V Leiden mutant gene, such as in
thrombophilia. The dsRNA of the invention comprises an RNA strand
(the antisense strand) having a region which is less than 30
nucleotides in length and is substantially complementary to at
least part of an mRNA transcript of the Factor V Leiden mutant
gene.
[0015] In embodiment, the invention provides double-stranded
ribonucleic acid (dsRNA) molecules for inhibiting the expression of
the Factor V Leiden mutant gene. The dsRNA comprises at least two
sequences that are complementary to each other. The dsRNA comprises
a sense strand comprising a first sequence and an antisense strand
comprising a second sequence. The antisense strand comprises a
nucleotide sequence which is substantially complementary to at
least part of an mRNA encoding Factor V Leiden mutant, and the
region of complementarity is less than 30 nucleotides in length.
The dsRNA, upon contacting with a cell expressing the Factor V
Leiden mutant, inhibits the expression of the Factor V Leiden
mutant gene by at least 40%.
[0016] For example, the dsRNA molecules of the invention can be
comprised of a first sequence of the dsRNA that is selected from
the group consisting of the sense sequences of Table 1 and the
second sequence is selected from the group consisting of the
antisense sequences of Table 1. The dsRNA molecules of the
invention can be comprised of naturally occurring nucleotides or
can be comprised of at least one modified nucleotide, such as a
2'-O-methyl modified nucleotide, a nucleotide comprising a
5'-phosphorothioate group, and a terminal nucleotide linked to a
cholesteryl derivative or dodecanoic acid bisdecylamide group.
Alternatively, the modified nucleotide may be chosen from the group
of: a 2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified
nucleotide, a locked nucleotide, an abasic nucleotide,
2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide,
morpholino nucleotide, a phosphoramidate, and a non-natural base
comprising nucleotide. Preferably, the first sequence of said dsRNA
is selected from the group consisting of the sense sequences of
Table 1 and the second sequence is selected from the group
consisting of the antisense sequences of Table 1.
[0017] In another embodiment, the invention provides a cell
comprising one of the dsRNAs of the invention. The cell is
preferably a mammalian cell, such as a human cell.
[0018] In another embodiment, the invention provides a
pharmaceutical composition for inhibiting the expression of the
Factor V Leiden mutant gene in an organism, comprising one or more
of the dsRNA of the invention and a pharmaceutically acceptable
carrier.
[0019] In another embodiment, the invention provides a method for
inhibiting the expression of the Factor V Leiden mutant gene in a
cell, comprising the following steps:
[0020] (a) introducing into the cell a double-stranded ribonucleic
acid (dsRNA), wherein the dsRNA comprises at least two sequences
that are complementary to each other. The dsRNA comprises a sense
strand comprising a first sequence and an antisense strand
comprising a second sequence. The antisense strand comprises a
region of complementarity which is substantially complementary to
at least a part of a mRNA encoding Factor V Leiden mutant, and
wherein the region of complementarity is less than 30 nucleotides
in length and wherein the dsRNA, upon contact with a cell
expressing the Factor V Leiden mutant, inhibits expression of the
Factor V Leiden mutant gene by at least 20%; and
[0021] (b) maintaining the cell produced in step (a) for a time
sufficient to obtain degradation of the mRNA transcript of the
Factor V Leiden mutant gene, thereby inhibiting expression of the
Factor V Leiden mutant gene in the cell.
[0022] In another embodiment, the invention provides methods for
treating, preventing or managing thrombophilia comprising
administering to a patient in need of such treatment, prevention or
management a therapeutically or prophylactically effective amount
of one or more of the dsRNAs of the invention.
TABLE-US-00001 TABLE 1 SEQ SEQ Duplex Sense strand sequence ID
Antisense strand ID identifier (5'-3') NO: sequence (5'-3') NO: wt1
cccuggacaggcgaggaauTT 1 auuccucgccuguccagggTT 2 wt2
ccuggacaggcgaggaauaTT 3 uauuccucgccuguccaggTT 4 wt3
cuggacaggcgaggaauacTT 5 guauuccucgccuguccagTT 6 wt4
uggacaggcgaggaauacaTT 7 uguauuccucgccuguccaTT 8 wt5
ggacaggcgaggaauacagTT 9 cuguauuccucgccuguccTT 10 wt6
gacaggcgaggaauacagaTT 11 ucuguauuccucgccugucTT 12 wt7
acaggcgaggaauacagagTT 13 cucuguauuccucgccuguTT 14 wt8
caggcgaggaauacagaggTT 15 ccucuguauuccucgccugTT 16 wt9
aggcgaggaauacagagggTT 17 cccucuguauuccucgccuTT 18 wt10
ggcgaggaauacagagggcTT 19 gcccucuguauuccucgccTT 20 wt11
gcgaggaauacagagggcaTT 21 ugcccucuguauuccucgcTT 22 wt12
cgaggaauacagagggcagTT 23 cugcccucuguauuccucgTT 24 wt13
gaggaauacagagggcagcTT 25 gcugcccucuguauuccucTT 26 wt14
gcagaucccuggacaggcaTT 27 ugccuguccagggaucugcTT 28 wt15
cagaucccuggacaggcaaTT 29 uugccuguccagggaucugTT 30 wt16
agaucccuggacaggcaagTT 31 cuugccuguccagggaucuTT 32 wt17
gaucccuggacaggcaaggTT 33 ccuugccuguccagggaucTT 34 wt18
aucccuggacaggcaaggaTT 35 uccuugccuguccagggauTT 36 wt19
ucccuggacaggcaaggaaTT 37 uuccuugccuguccagggaTT 38 mut1
cccuggacaggcaaggaauTT 39 auuccuugccuguccagggTT 40 mut2
ccuggacaggcaaggaauaTT 41 uauuccuugccuguccaggTT 42 mut3
cuggacaggcaaggaauacTT 43 guauuccuugccuguccagTT 44 mut4
uggacaggcaaggaauacaTT 45 uguauuccuugccuguccaTT 46 mut5
ggacaggcaaggaauacagTT 47 cuguauuccuugccuguccTT 48 mut6
gacaggcaaggaauacagaTT 49 ucuguauuccuugccugucTT 50 mut7
acaggcaaggaauacagagTT 51 cucuguauuccuugccuguTT 52 mut8
caggcaaggaauacagaggTT 53 ccucuguauuccuugccugTT 54 mut9
aggcaaggaauacagagggTT 55 cccucuguauuccuugccuTT 56 mut10
ggcaaggaauacagagggcTT 57 gcccucuguauuccuugccTT 58 mut11
gcaaggaauacagagggcaTT 59 ugcccucuguauuccuugcTT 60 mut12
caaggaauacagagggcagTT 61 cugcccucuguauuccuugTT 62 mut13
aaggaauacagagggcagcTT 63 gcugcccucuguauuccuuTT 64 mut14
acaggcaaggaauacagagtt 65 cucuguauuccuugccugutt 66 mut15
caggcaaggaauacagaggtt 67 ccucuguauuccuugccugtt 68 mut16
aggcaaggaauacagagggtt 69 cccucuguauuccuugccutt 70 mut17
ggcaaggaauacagagggctt 71 gcccucuguauuccuugcctt 72 mut18
gcaaggaauacagagggcatt 73 ugcccucuguauuccuugctt 74 mut19
caaggaauacagagggcagtt 75 cugcccucuguauuccuugtt 76
[0023] In another embodiment, the invention provides vectors for
inhibiting the expression of the Factor V Leiden mutant gene in a
cell, comprising a regulatory sequence operably linked to a
nucleotide sequence that encodes at least one strand of one of the
dsRNA of the invention.
[0024] In another embodiment, the invention provides a cell
comprising a vector for inhibiting the expression of the Factor V
Leiden mutant gene in a cell. The vector comprises a regulatory
sequence operably linked to a nucleotide sequence that encodes at
least one strand of one of the dsRNA of the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0025] No Figures are presented
DETAILED DESCRIPTION OF THE INVENTION
[0026] The invention provides double-stranded ribonucleic acid
(dsRNA), as well as compositions and methods for inhibiting the
expression of the Factor V Leiden mutant gene in a cell or mammal
using the dsRNA. The invention also provides compositions and
methods for treating pathological conditions and diseases in a
mammal caused by the expression of the Factor V Leiden mutant gene
using dsRNA. dsRNA directs the sequence-specific degradation of
mRNA through a process known as RNA interference (RNAi). The
process occurs in a wide variety of organisms, including mammals
and other vertebrates.
[0027] The dsRNA of the invention comprises an RNA strand (the
antisense strand) having a region which is less than 30 nucleotides
in length and is substantially complementary to at least part of an
mRNA transcript of the Factor V Leiden mutant gene. The use of
these dsRNAs enables the targeted degradation of mRNAs of genes
that are implicated in thrombophilia response in mammals. Using
cell-based and animal assays, the present inventors have
demonstrated that very low dosages of these dsRNA can specifically
and efficiently mediate RNAi, resulting in significant inhibition
of expression of the Factor V Leiden mutant gene. Thus, the methods
and compositions of the invention comprising these dsRNAs are
useful for treating thrombophilia.
[0028] The following detailed description discloses how to make and
use the dsRNA and compositions containing dsRNA to inhibit the
expression of a target Factor V Leiden mutant gene, as well as
compositions and methods for treating diseases and disorders caused
by the expression of Factor V Leiden mutant, such as thrombophilia.
The pharmaceutical compositions of the invention comprise a dsRNA
having an antisense strand comprising a region of complementarity
which is less than 30 nucleotides in length and is substantially
complementary to at least part of an RNA transcript of the Factor V
Leiden mutant gene, together with a pharmaceutically acceptable
carrier.
[0029] Accordingly, certain aspects of the invention provide
pharmaceutical compositions comprising the dsRNA of the invention
together with a pharmaceutically acceptable carrier, methods of
using the compositions to inhibit expression of the Factor V Leiden
mutant gene, and methods of using the pharmaceutical compositions
to treat diseases caused by expression of the Factor V Leiden
mutant gene.
I. Definitions
[0030] For convenience, the meaning of certain terms and phrases
used in the specification, examples, and appended claims, are
provided below. If there is an apparent discrepancy between the
usage of a term in other parts of this specification and its
definition provided in this section, the definition in this section
shall prevail.
[0031] "G," "C," "A" and "U" each generally stand for a nucleotide
that contains guanine, cytosine, adenine, and uracil as a base,
respectively. However, it will be understood that the term
"ribonucleotide" or "nucleotide" can also refer to a modified
nucleotide, as further detailed below, or a surrogate replacement
moiety. The skilled person is well aware that guanine, cytosine,
adenine, and uracil may be replaced by other moieties without
substantially altering the base pairing properties of an
oligonucleotide comprising a nucleotide bearing such replacement
moiety. For example, without limitation, a nucleotide comprising
inosine as its base may base pair with nucleotides containing
adenine, cytosine, or uracil. Hence, nucleotides containing uracil,
guanine, or adenine may be replaced in the nucleotide sequences of
the invention by a nucleotide containing, for example, inosine.
Sequences comprising such replacement moieties are embodiments of
the invention.
[0032] By "Factor V Leiden mutant" as used herein is meant, any
mutation in the Factor V gene, protein, peptide, or polypeptide.
The term "factor V Leiden" generally refers to the specific G-to-A
substitution at nucleotide 1691 in the gene for factor V that
predicts a single amino acid replacement (Arg506Gln) at one of
three APC cleavage sites in the factor Va molecule.
[0033] As used herein, "target sequence" refers to a contiguous
portion of the nucleotide sequence of an mRNA molecule formed
during the transcription of the Factor V Leiden mutant gene,
including mRNA that is a product of RNA processing of a primary
transcription product.
[0034] As used herein, the term "strand comprising a sequence"
refers to an oligonucleotide comprising a chain of nucleotides that
is described by the sequence referred to using the standard
nucleotide nomenclature.
[0035] As used herein, and unless otherwise indicated, the term
"complementary," when used to describe a first nucleotide sequence
in relation to a second nucleotide sequence, refers to the ability
of an oligonucleotide or polynucleotide comprising the first
nucleotide sequence to hybridize and form a duplex structure under
certain conditions with an oligonucleotide or polynucleotide
comprising the second nucleotide sequence, as will be understood by
the skilled person. Such conditions can, for example, be stringent
conditions, where stringent conditions may include: 400 mM NaCl, 40
mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. for
12-16 hours followed by washing. Other conditions, such as
physiologically relevant conditions as may be encountered inside an
organism, can apply. The skilled person will be able to determine
the set of conditions most appropriate for a test of
complementarity of two sequences in accordance with the ultimate
application of the hybridized nucleotides.
[0036] This includes base-pairing of the oligonucleotide or
polynucleotide comprising the first nucleotide sequence to the
oligonucleotide or polynucleotide comprising the second nucleotide
sequence over the entire length of the first and second nucleotide
sequence. Such sequences can be referred to as "fully
complementary" with respect to each other herein. However, where a
first sequence is referred to as "substantially complementary" with
respect to a second sequence herein, the two sequences can be fully
complementary, or they may form one or more, but preferably not
more than 4, 3 or 2 mismatched base pairs upon hybridization, while
retaining the ability to hybridize under the conditions most
relevant to their ultimate application. However, where two
oligonucleotides are designed to form, upon hybridization, one or
more single stranded overhangs, such overhangs shall not be
regarded as mismatches with regard to the determination of
complementarity. For example, a dsRNA comprising one
oligonucleotide 21 nucleotides in length and another
oligonucleotide 23 nucleotides in length, wherein the longer
oligonucleotide comprises a sequence of 21 nucleotides that is
fully complementary to the shorter oligonucleotide, may yet be
referred to as "fully complementary" for the purposes of the
invention.
[0037] "Complementary" sequences, as used herein, may also include,
or be formed entirely from, non-Watson-Crick base pairs and/or base
pairs formed from non-natural and modified nucleotides, in as far
as the above requirements with respect to their ability to
hybridize are fulfilled.
[0038] The terms "complementary", "fully complementary" and
"substantially complementary" herein may be used with respect to
the base matching between the sense strand and the antisense strand
of a dsRNA, or between the antisense strand of a dsRNA and a target
sequence, as will be understood from the context of their use.
[0039] As used herein, a polynucleotide which is "substantially
complementary to at least part of" a messenger RNA (mRNA) refers to
a polynucleotide which is substantially complementary to a
contiguous portion of the mRNA of interest (e.g., encoding Factor V
Leiden mutant). For example, a polynucleotide is complementary to
at least a part of a Factor V Leiden mutant mRNA if the sequence is
substantially complementary to a non-interrupted portion of a mRNA
encoding Factor V Leiden mutant.
[0040] The term "double-stranded RNA" or "dsRNA", as used herein,
refers to a ribonucleic acid molecule, or complex of ribonucleic
acid molecules, having a duplex structure comprising two
anti-parallel and substantially complementary, as defined above,
nucleic acid strands. The two strands forming the duplex structure
may be different portions of one larger RNA molecule, or they may
be separate RNA molecules. Where the two strands are part of one
larger molecule, and therefore are connected by an uninterrupted
chain of nucleotides between the 3'-end of one strand and the 5'
end of the respective other strand forming the duplex structure,
the connecting RNA chain is referred to as a "hairpin loop". Where
the two strands are connected covalently by means other than an
uninterrupted chain of nucleotides between the 3'-end of one strand
and the 5' end of the respective other strand forming the duplex
structure, the connecting structure is referred to as a "linker".
The RNA strands may have the same or a different number of
nucleotides. The maximum number of base pairs is the number of
nucleotides in the shortest strand of the dsRNA. In addition to the
duplex structure, a dsRNA may comprise one or more nucleotide
overhangs.
[0041] As used herein, a "nucleotide overhang" refers to the
unpaired nucleotide or nucleotides that protrude from the duplex
structure of a dsRNA when a 3'-end of one strand of the dsRNA
extends beyond the 5'-end of the other strand, or vice versa.
"Blunt" or "blunt end" means that there are no unpaired nucleotides
at that end of the dsRNA, i.e., no nucleotide overhang. A "blunt
ended" dsRNA is a dsRNA that is double-stranded over its entire
length, i.e., no nucleotide overhang at either end of the
molecule.
[0042] The term "antisense strand" refers to the strand of a dsRNA
which includes a region that is substantially complementary to a
target sequence. As used herein, the term "region of
complementarity" refers to the region on the antisense strand that
is substantially complementary to a sequence, for example a target
sequence, as defined herein. Where the region of complementarity is
not fully complementary to the target sequence, the mismatches are
most tolerated in the terminal regions and, if present, are
preferably in a terminal region or regions, e.g., within 6, 5, 4,
3, or 2 nucleotides of the 5' and/or 3' terminus.
[0043] The term "sense strand," as used herein, refers to the
strand of a dsRNA that includes a region that is substantially
complementary to a region of the antisense strand.
[0044] "Introducing into a cell", when referring to a dsRNA, means
facilitating uptake or absorption into the cell, as is understood
by those skilled in the art. Absorption or uptake of dsRNA can
occur through unaided diffusive or active cellular processes, or by
auxiliary agents or devices. The meaning of this term is not
limited to cells in vitro; a dsRNA may also be "introduced into a
cell", wherein the cell is part of a living organism. In such
instance, introduction into the cell will include the delivery to
the organism. For example, for in vivo delivery, dsRNA can be
injected into a tissue site or administered systemically. In vitro
introduction into a cell includes methods known in the art such as
electroporation and lipofection.
[0045] The terms "silence" and "inhibit the expression of", in as
far as they refer to the Factor V Leiden mutant gene, herein refer
to the at least partial suppression of the expression of the Factor
V Leiden mutant gene, as manifested by a reduction of the amount of
mRNA transcribed from the Factor V Leiden mutant gene which may be
isolated from a first cell or group of cells in which the Factor V
Leiden mutant gene is transcribed and which has or have been
treated such that the expression of the Factor V Leiden mutant gene
is inhibited, as compared to a second cell or group of cells
substantially identical to the first cell or group of cells but
which has or have not been so treated (control cells). The degree
of inhibition is usually expressed in terms of
( mRNA in control cells ) - ( mRNA in treated cells ) ( mRNA in
control cells ) 100 % ##EQU00001##
[0046] Alternatively, the degree of inhibition may be given in
terms of a reduction of a parameter that is functionally linked to
Factor V Leiden mutant gene transcription, e.g. the amount of
protein encoded by the Factor V Leiden mutant gene which is
secreted by a cell, or the number of cells displaying a certain
phenotype, e.g apoptosis. In principle, Factor V Leiden mutant gene
silencing may be determined in any cell expressing the target,
either constitutively or by genomic engineering, and by any
appropriate assay. However, when a reference is needed in order to
determine whether a given siRNA inhibits the expression of the
Factor V Leiden mutant gene by a certain degree and therefore is
encompassed by the instant invention, the assay provided in the
Examples below shall serve as such reference.
[0047] For example, in certain instances, expression of the Factor
V Leiden mutant gene is suppressed by at least about 20%, 25%, 35%,
or 50% by administration of the double-stranded oligonucleotide of
the invention. In a preferred embodiment, the Factor V Leiden
mutant gene is suppressed by at least about 60%, 70%, or 80% by
administration of the double-stranded oligonucleotide of the
invention. In a more preferred embodiment, the Factor V Leiden
mutant gene is suppressed by at least about 85%, 90%, or 95% by
administration of the double-stranded oligonucleotide of the
invention. In a most preferred embodiment, the Factor V Leiden
mutant gene is suppressed by at least about 98%, 99% or more by
administration of the double-stranded oligonucleotide of the
invention.
[0048] The terms "treat", "treatment", and the like, refer to
relief from or alleviation of thrombophilia. In the context of the
present invention insofar as it relates to any of the other
conditions recited herein below (other than thrombophilia), the
terms "treat", "treatment", and the like mean to relieve or
alleviate at least one symptom associated with such condition, or
to slow or reverse the progression of such condition.
[0049] As used herein, the phrases "therapeutically effective
amount" and "prophylactically effective amount" refer to an amount
that provides a therapeutic benefit in the treatment, prevention,
or management of thrombophilia or an overt symptom of
thrombophilia. The specific amount that is therapeutically
effective can be readily determined by ordinary medical
practitioner, and may vary depending on factors known in the art,
such as, e.g. the type of thrombophilia, the patient's history and
age, the stage of thrombophilia, and the administration of other
anti-thrombophilia agents.
[0050] As used herein, a "pharmaceutical composition" comprises a
pharmacologically effective amount of a dsRNA and a
pharmaceutically acceptable carrier. As used herein,
"pharmacologically effective amount," "therapeutically effective
amount" or simply "effective amount" refers to that amount of an
RNA effective to produce the intended pharmacological, therapeutic
or preventive result. For example, if a given clinical treatment is
considered effective when there is at least a 25% reduction in a
measurable parameter associated with a disease or disorder, a
therapeutically effective amount of a drug for the treatment of
that disease or disorder is the amount necessary to effect at least
a 25% reduction in that parameter.
[0051] The term "pharmaceutically acceptable carrier" refers to a
carrier for administration of a therapeutic agent. Such carriers
include, but are not limited to, saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof. The term
specifically excludes cell culture medium. For drugs administered
orally, pharmaceutically acceptable carriers include, but are not
limited to 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.
[0052] As used herein, a "transformed cell" is a cell into which a
vector has been introduced from which a dsRNA molecule may be
expressed.
II. Double-Stranded Ribonucleic Acid (dsRNA)
[0053] In one embodiment, the invention provides double-stranded
ribonucleic acid (dsRNA) molecules for inhibiting the expression of
the Factor V Leiden mutant gene in a cell or mammal, wherein the
dsRNA comprises an antisense strand comprising a region of
complementarity which is complementary to at least a part of an
mRNA formed in the expression of the Factor V Leiden mutant gene,
and wherein the region of complementarity is less than 30
nucleotides in length and wherein said dsRNA, upon contact with a
cell expressing said Factor V Leiden mutant gene, inhibits the
expression of said Factor V Leiden mutant gene by at least 20%. The
dsRNA comprises two RNA strands that are sufficiently complementary
to hybridize to form a duplex structure. One strand of the dsRNA
(the antisense strand) comprises a region of complementarity that
is substantially complementary, and preferably fully complementary,
to a target sequence, derived from the sequence of an mRNA formed
during the expression of the Factor V Leiden mutant gene, the other
strand (the sense strand) comprises a region which is complementary
to the antisense strand, such that the two strands hybridize and
form a duplex structure when combined under suitable conditions.
Preferably, the duplex structure is between 15 and 30, more
preferably between 18 and 25, yet more preferably between 19 and
24, and most preferably between 21 and 23 base pairs in length.
Similarly, the region of complementarity to the target sequence is
between 15 and 30, more preferably between 18 and 25, yet more
preferably between 19 and 24, and most preferably between 21 and 23
nucleotides in length. The dsRNA of the invention may further
comprise one or more single-stranded nucleotide overhang(s). The
dsRNA can be synthesized by standard methods known in the art as
further discussed below, e.g., by use of an automated DNA
synthesizer, such as are commercially available from, for example,
Biosearch, Applied Biosystems, Inc. In a preferred embodiment, the
Factor V Leiden mutant gene is the human Factor V Leiden mutant
gene. In specific embodiments, the antisense strand of the dsRNA
comprises the sense sequences of Table 1 and the second sequence is
selected from the group consisting of the antisense sequences of
Table 1.
[0054] In further embodiments, the dsRNA comprises at least one
nucleotide sequence selected from the groups of sequences provided
in Table 1. In other embodiments, the dsRNA comprises at least two
sequences selected from this group, wherein one of the at least two
sequences is complementary to another of the at least two
sequences, and one of the at least two sequences is substantially
complementary to a sequence of an mRNA generated in the expression
of the Factor V Leiden mutant gene. Preferably, the dsRNA comprises
two oligonucleotides, wherein one oligonucleotide is described by
Table 1 and the second oligonucleotide is described Table 1
[0055] The skilled person is well aware that dsRNAs comprising a
duplex structure of between 20 and 23, but specifically 21, base
pairs have been hailed as particularly effective in inducing RNA
interference (Elbashir et al., EMBO 2001, 20:6877-6888). However,
others have found that shorter or longer dsRNAs can be effective as
well. In the embodiments described above, by virtue of the nature
of the oligonucleotide sequences provided in Table 1, the dsRNAs of
the invention can comprise at least one strand of a length of
minimally 21 nt. It can be reasonably expected that shorter dsRNAs
comprising one of the sequences of Table 1 minus only a few
nucleotides on one or both ends may be similarly effective as
compared to the dsRNAs described above. Hence, dsRNAs comprising a
partial sequence of at least 15, 16, 17, 18, 19, 20, or more
contiguous nucleotides from one of the sequences of Table 1, and
differing in their ability to inhibit the expression of the Factor
V Leiden mutant gene in a FACS assay as described herein below by
not more than 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA
comprising the full sequence, are contemplated by the
invention.
[0056] The dsRNA of the invention can contain one or more
mismatches to the target sequence. In a preferred embodiment, the
dsRNA of the invention contains no more than 3 mismatches. If the
antisense strand of the dsRNA contains mismatches to a target
sequence, it is preferable that the area of mismatch not be located
in the center of the region of complementarity. If the antisense
strand of the dsRNA contains mismatches to the target sequence, it
is preferable that the mismatch be restricted to 5 nucleotides from
either end, for example 5, 4, 3, 2, or 1 nucleotide from either the
5' or 3' end of the region of complementarity. For example, for a
23 nucleotide dsRNA strand which is complementary to a region of
the Factor V Leiden mutant gene, the dsRNA preferably does not
contain any mismatch within the central 13 nucleotides. The methods
described within the invention can be used to determine whether a
dsRNA containing a mismatch to a target sequence is effective in
inhibiting the expression of the Factor V Leiden mutant gene.
Consideration of the efficacy of dsRNAs with mismatches in
inhibiting expression of the Factor V Leiden mutant gene is
important, especially if the particular region of complementarity
in the Factor V Leiden mutant gene is known to have polymorphic
sequence variation within the population.
[0057] In one embodiment, at least one end of the dsRNA has a
single-stranded nucleotide overhang of 1 to 4, preferably 1 or 2
nucleotides. dsRNAs having at least one nucleotide overhang have
unexpectedly superior inhibitory properties than their blunt-ended
counterparts. Moreover, the present inventors have discovered that
the presence of only one nucleotide overhang strengthens the
interference activity of the dsRNA, without affecting its overall
stability. dsRNA having only one overhang has proven particularly
stable and effective in vivo, as well as in a variety of cells,
cell culture mediums, blood, and serum. Preferably, the
single-stranded overhang is located at the 3'-terminal end of the
antisense strand or, alternatively, at the 3'-terminal end of the
sense strand. The dsRNA may also have a blunt end, preferably
located at the 5'-end of the antisense strand. Such dsRNAs have
improved stability and inhibitory activity, thus allowing
administration at low dosages, i.e., less than 5 mg/kg body weight
of the recipient per day. Preferably, the antisense strand of the
dsRNA has a nucleotide overhang at the 3'-end, and the 5'-end is
blunt. In another embodiment, one or more of the nucleotides in the
overhang is replaced with a nucleoside thiophosphate.
[0058] In yet another embodiment, the dsRNA is chemically modified
to enhance stability. The nucleic acids of the invention may be
synthesized and/or modified by methods well established in the art,
such as those described in "Current protocols in nucleic acid
chemistry", Beaucage, S. L. et al. (Edrs.), John Wiley & Sons,
Inc., New York, N.Y., USA, which is hereby incorporated herein by
reference. Chemical modifications may include, but are not limited
to 2' modifications, introduction of non-natural bases, covalent
attachment to a ligand, and replacement of phosphate linkages with
thiophosphate linkages. In this embodiment, the integrity of the
duplex structure is strengthened by at least one, and preferably
two, chemical linkages. Chemical linking may be achieved by any of
a variety of well-known techniques, for example by introducing
covalent, ionic or hydrogen bonds; hydrophobic interactions, van
der Waal s or stacking interactions; by means of metal-ion
coordination, or through use of purine analogues. Preferably, the
chemical groups that can be used to modify the dsRNA include,
without limitation, methylene blue; bifunctional groups, preferably
bis-(2-chloroethyl)amine; N-acetyl-N'-(p-glyoxylbenzoyl)cystamine;
4-thiouracil; and psoralen. In one preferred embodiment, the linker
is a hexa-ethylene glycol linker. In this case, the dsRNA are
produced by solid phase synthesis and the hexa-ethylene glycol
linker is incorporated according to standard methods (e.g.,
Williams, D. J., and K. B. Hall, Biochem. (1996) 35:14665-14670).
In a particular embodiment, the 5'-end of the antisense strand and
the 3'-end of the sense strand are chemically linked via a
hexaethylene glycol linker. In another embodiment, at least one
nucleotide of the dsRNA comprises a phosphorothioate or
phosphorodithioate groups. The chemical bond at the ends of the
dsRNA is preferably formed by triple-helix bonds. Table 1 provides
examples of modified RNAi agents of the invention.
[0059] In certain embodiments, a chemical bond may be formed by
means of one or several bonding groups, wherein such bonding groups
are preferably poly-(oxyphosphinicooxy-1,3-propandiol)-and/or
polyethylene glycol chains. In other embodiments, a chemical bond
may also be formed by means of purine analogs introduced into the
double-stranded structure instead of purines. In further
embodiments, a chemical bond may be formed by azabenzene units
introduced into the double-stranded structure. In still further
embodiments, a chemical bond may be formed by branched nucleotide
analogs instead of nucleotides introduced into the double-stranded
structure. In certain embodiments, a chemical bond may be induced
by ultraviolet light.
[0060] In yet another embodiment, the nucleotides at one or both of
the two single strands may be modified to prevent or inhibit the
activation of cellular enzymes, such as, for example, without
limitation, certain nucleases. Techniques for inhibiting the
activation of cellular enzymes are known in the art including, but
not limited to, 2'-amino modifications, 2'-amino sugar
modifications, 2'-F sugar modifications, 2'-F modifications,
2'-alkyl sugar modifications, uncharged backbone modifications,
morpholino modifications, 2'-O-methyl modifications, and
phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1116-8).
Thus, at least one 2'-hydroxyl group of the nucleotides on a dsRNA
is replaced by a chemical group, preferably by a 2'-amino or a
2'-methyl group. Also, at least one nucleotide may be modified to
form a locked nucleotide. Such locked nucleotide contains a
methylene bridge that connects the 2'-oxygen of ribose with the
4'-carbon of ribose. Oligonucleotides containing the locked
nucleotide are described in Koshkin, A. A., et al., Tetrahedron
(1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett.
(1998), 39: 5401-5404). Introduction of a locked nucleotide into an
oligonucleotide improves the affinity for complementary sequences
and increases the melting temperature by several degrees (Braasch,
D. A. and D. R. Corey, Chem. Biol. (2001), 8:1-7).
[0061] Conjugating a ligand to a dsRNA can enhance its cellular
absorption as well as targeting to a particular tissue. In certain
instances, a hydrophobic ligand is conjugated to the dsRNA to
facilitate direct permeation of the cellular membrane.
Alternatively, the ligand conjugated to the dsRNA is a substrate
for receptor-mediated endocytosis. These approaches have been used
to facilitate cell permeation of antisense oligonucleotides. For
example, cholesterol has been conjugated to various antisense
oligonucleotides resulting in compounds that are substantially more
active compared to their non-conjugated analogs. See M.
ManoharanAntisense & Nucleic Acid Drug Development 2002, 12,
103. Other lipophilic compounds that have been conjugated to
oligonucleotides include 1-pyrene butyric acid,
1,3-bis-O-(hexadecyl)glycerol, and menthol. One example of a ligand
for receptor-mediated endocytosis is folic acid. Folic acid enters
the cell by folate-receptor-mediated endocytosis. dsRNA compounds
bearing folic acid would be efficiently transported into the cell
via the folate-receptor-mediated endocytosis. Li and coworkers
report that attachment of folic acid to the 3'-terminus of an
oligonucleotide resulted in an 8-fold increase in cellular uptake
of the oligonucleotide. Li, S.; Deshmukh, H. M.; Huang, L. Pharm.
Res. 1998, 15, 1540. Other ligands that have been conjugated to
oligonucleotides include polyethylene glycols, carbohydrate
clusters, cross-linking agents, porphyrin conjugates, and delivery
peptides.
[0062] In certain instances, conjugation of a cationic ligand to
oligonucleotides often results in improved resistance to nucleases.
Representative examples of cationic ligands are propylammonium and
dimethylpropylammonium. Interestingly, antisense oligonucleotides
were reported to retain their high binding affinity to mRNA when
the cationic ligand was dispersed throughout the oligonucleotide.
See M. Manoharan Antisense & Nucleic Acid Drug Development
2002, 12, 103 and references therein.
[0063] The ligand-conjugated dsRNA of the invention may be
synthesized by the use of a dsRNA that bears a pendant reactive
functionality, such as that derived from the attachment of a
linking molecule onto the dsRNA. This reactive oligonucleotide may
be reacted directly with commercially-available ligands, ligands
that are synthesized bearing any of a variety of protecting groups,
or ligands that have a linking moiety attached thereto. The methods
of the invention facilitate the synthesis of ligand-conjugated
dsRNA by the use of, in some preferred embodiments, nucleoside
monomers that have been appropriately conjugated with ligands and
that may further be attached to a solid-support material. Such
ligand-nucleoside conjugates, optionally attached to a
solid-support material, are prepared according to some preferred
embodiments of the methods of the invention via reaction of a
selected serum-binding ligand with a linking moiety located on the
5' position of a nucleoside or oligonucleotide. In certain
instances, an dsRNA bearing an aralkyl ligand attached to the
3'-terminus of the dsRNA is prepared by first covalently attaching
a monomer building block to a controlled-pore-glass support via a
long-chain aminoalkyl group. Then, nucleotides are bonded via
standard solid-phase synthesis techniques to the monomer
building-block bound to the solid support. The monomer building
block may be a nucleoside or other organic compound that is
compatible with solid-phase synthesis.
[0064] The dsRNA used in the conjugates of the invention may be
conveniently and routinely made through the well-known technique of
solid-phase synthesis. Equipment for such synthesis is sold by
several vendors including, for example, Applied Biosystems (Foster
City, Calif.). Any other means for such synthesis known in the art
may additionally or alternatively be employed. It is also known to
use similar techniques to prepare other oligonucleotides, such as
the phosphorothioates and alkylated derivatives.
[0065] Teachings regarding the synthesis of particular modified
oligonucleotides may be found in the following U.S. patents: U.S.
Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugated
oligonucleotides; U.S. Pat. No. 5,212,295, drawn to monomers for
the preparation of oligonucleotides having chiral phosphorus
linkages; U.S. Pat. Nos. 5,378,825 and 5,541,307, drawn to
oligonucleotides having modified backbones; U.S. Pat. No.
5,386,023, drawn to backbone-modified oligonucleotides and the
preparation thereof through reductive coupling; U.S. Pat. No.
5,457,191, drawn to modified nucleobases based on the 3-deazapurine
ring system and methods of synthesis thereof; U.S. Pat. No.
5,459,255, drawn to modified nucleobases based on N-2 substituted
purines; U.S. Pat. No. 5,521,302, drawn to processes for preparing
oligonucleotides having chiral phosphorus linkages; U.S. Pat. No.
5,539,082, drawn to peptide nucleic acids; U.S. Pat. No. 5,554,746,
drawn to oligonucleotides having .beta.-lactam backbones; U.S. Pat.
No. 5,571,902, drawn to methods and materials for the synthesis of
oligonucleotides; U.S. Pat. No. 5,578,718, drawn to nucleosides
having alkylthio groups, wherein such groups may be used as linkers
to other moieties attached at any of a variety of positions of the
nucleoside; U.S. Pat. Nos. 5,587,361 and 5,599,797, drawn to
oligonucleotides having phosphorothioate linkages of high chiral
purity; U.S. Pat. No. 5,506,351, drawn to processes for the
preparation of 2'-O-alkyl guanosine and related compounds,
including 2,6-diaminopurine compounds; U.S. Pat. No. 5,587,469,
drawn to oligonucleotides having N-2 substituted purines; U.S. Pat.
No. 5,587,470, drawn to oligonucleotides having 3-deazapurines;
U.S. Pat. No. 5,223,168, and U.S. Pat. No. 5,608,046, both drawn to
conjugated 4'-desmethyl nucleoside analogs; U.S. Pat. Nos.
5,602,240, and 5,610,289, drawn to backbone-modified
oligonucleotide analogs; U.S. Pat. Nos. 6,262,241, and 5,459,255,
drawn to, inter alia, methods of synthesizing
2'-fluoro-oligonucleotides.
[0066] In the ligand-conjugated dsRNA and ligand-molecule bearing
sequence-specific linked nucleosides of the invention, the
oligonucleotides and oligonucleosides may be assembled on a
suitable DNA synthesizer utilizing standard nucleotide or
nucleoside precursors, or nucleotide or nucleoside conjugate
precursors that already bear the linking moiety, ligand-nucleotide
or nucleoside-conjugate precursors that already bear the ligand
molecule, or non-nucleoside ligand-bearing building blocks.
[0067] When using nucleotide-conjugate precursors that already bear
a linking moiety, the synthesis of the sequence-specific linked
nucleosides is typically completed, and the ligand molecule is then
reacted with the linking moiety to form the ligand-conjugated
oligonucleotide. Oligonucleotide conjugates bearing a variety of
molecules such as steroids, vitamins, lipids and reporter
molecules, has previously been described (see Manoharan et al., PCT
Application WO 93/07883). In a preferred embodiment, the
oligonucleotides or linked nucleosides of the invention are
synthesized by an automated synthesizer using phosphoramidites
derived from ligand-nucleoside conjugates in addition to the
standard phosphoramidites and non-standard phosphoramidites that
are commercially available and routinely used in oligonucleotide
synthesis.
[0068] The incorporation of a 2'-O-methyl, 2'-O-ethyl, 2'-O-propyl,
2'-O-allyl, 2'-O-aminoalkyl or 2'-deoxy-2'-fluoro group in
nucleosides of an oligonucleotide confers enhanced hybridization
properties to the oligonucleotide. Further, oligonucleotides
containing phosphorothioate backbones have enhanced nuclease
stability. Thus, functionalized, linked nucleosides of the
invention can be augmented to include either or both a
phosphorothioate backbone or a 2'-O-methyl, 2'-O-ethyl,
2'-O-propyl, 2'-O-aminoalkyl, 2'-O-allyl or 2'-deoxy-2'-fluoro
group.
[0069] In some preferred embodiments, functionalized nucleoside
sequences of the invention possessing an amino group at the
5'-terminus are prepared using a DNA synthesizer, and then reacted
with an active ester derivative of a selected ligand. Active ester
derivatives are well known to those skilled in the art.
Representative active esters include N-hydrosuccinimide esters,
tetrafluorophenolic esters, pentafluorophenolic esters and
pentachlorophenolic esters. The reaction of the amino group and the
active ester produces an oligonucleotide in which the selected
ligand is attached to the 5'-position through a linking group. The
amino group at the 5'-terminus can be prepared utilizing a
5'-Amino-Modifier C6 reagent. In a preferred embodiment, ligand
molecules may be conjugated to oligonucleotides at the 5'-position
by the use of a ligand-nucleoside phosphoramidite wherein the
ligand is linked to the 5'-hydroxy group directly or indirectly via
a linker. Such ligand-nucleoside phosphoramidites are typically
used at the end of an automated synthesis procedure to provide a
ligand-conjugated oligonucleotide bearing the ligand at the
5'-terminus.
[0070] In one preferred embodiment of the methods of the invention,
the preparation of ligand conjugated oligonucleotides commences
with the selection of appropriate precursor molecules upon which to
construct the ligand molecule. Typically, the precursor is an
appropriately-protected derivative of the commonly-used
nucleosides. For example, the synthetic precursors for the
synthesis of the ligand-conjugated oligonucleotides of the
invention include, but are not limited to,
2'-aminoalkoxy-5'-ODMT-nucleosides,
2'-6-aminoalkylamino-5'-ODMT-nucleosides,
5'-6-aminoalkoxy-2'-deoxy-nucleosides,
5'-6-aminoalkoxy-2-protected-nucleosides,
3'-6-aminoalkoxy-5'-ODMT-nucleosides, and
3'-aminoalkylamino-5'-ODMT-nucleosides that may be protected in the
nucleobase portion of the molecule. Methods for the synthesis of
such amino-linked protected nucleoside precursors are known to
those of ordinary skill in the art.
[0071] In many cases, protecting groups are used during the
preparation of the compounds of the invention. As used herein, the
term "protected" means that the indicated moiety has a protecting
group appended thereon. In some preferred embodiments of the
invention, compounds contain one or more protecting groups. A wide
variety of protecting groups can be employed in the methods of the
invention. In general, protecting groups render chemical
functionalities inert to specific reaction conditions, and can be
appended to and removed from such functionalities in a molecule
without substantially damaging the remainder of the molecule.
[0072] Representative hydroxyl protecting groups, for example, are
disclosed by Beaucage et al. (Tetrahedron, 1992, 48:2223-2311).
Further hydroxyl protecting groups, as well as other representative
protecting groups, are disclosed in Greene and Wuts, Protective
Groups in Organic Synthesis, Chapter 2, 2d ed., John Wiley &
Sons, New York, 1991, and Oligonucleotides And Analogues A
Practical Approach, Ekstein, F. Ed., IRL Press, N.Y, 1991.
[0073] Examples of hydroxyl protecting groups include, but are not
limited to, t-butyl, t-butoxymethyl, methoxymethyl,
tetrahydropyranyl, 1-ethoxyethyl, 1-(2-chloroethoxy)ethyl,
2-trimethyl silyl ethyl, p-chlorophenyl, 2,4-dinitrophenyl, benzyl,
2, 6-dichlorobenzyl, diphenylmethyl, p,p'-dinitrobenzhydryl,
p-nitrobenzyl, triphenylmethyl, trimethylsilyl, triethylsilyl,
t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl,
benzoylformate, acetate, chloroacetate, trichloroacetate,
trifluoroacetate, pivaloate, benzoate, p-phenylbenzoate,
9-fluorenylmethyl carbonate, mesylate and tosylate.
[0074] Amino-protecting groups stable to acid treatment are
selectively removed with base treatment, and are used to make
reactive amino groups selectively available for substitution.
Examples of such groups are the Fmoc (E. Atherton and R. C.
Sheppard in The Peptides, S. Udenfriend, J. Meienhofer, Eds.,
Academic Press, Orlando, 1987, volume 9, p.1) and various
substituted sulfonylethyl carbamates exemplified by the Nsc group
(Samukov et al., Tetrahedron Lett., 1994, 35:7821; Verhart and
Tesser, Rec. Tray. Chico. Pays-Bas, 1987, 107:621).
[0075] Additional amino-protecting groups include, but are not
limited to, carbamate protecting groups, such as
2-trimethylsilylethoxycarbonyl (Teoc),
1-methyl-1-(4-biphenylyl)ethoxycarbonyl (Bpoc), t-butoxycarbonyl
(BOC), allyloxycarbonyl (Alloc), 9-fluorenylmethyloxycarbonyl
(Fmoc), and benzyloxycarbonyl (Cbz); amide protecting groups, such
as formyl, acetyl, trihaloacetyl, benzoyl, and nitrophenylacetyl;
sulfonamide protecting groups, such as 2-nitrobenzenesulfonyl; and
imine and cyclic imide protecting groups, such as phthalimido and
dithiasuccinoyl. Equivalents of these amino-protecting groups are
also encompassed by the compounds and methods of the invention.
[0076] Many solid supports are commercially available and one of
ordinary skill in the art can readily select a solid support to be
used in the solid-phase synthesis steps. In certain embodiments, a
universal support is used. A universal support allows for
preparation of oligonucleotides having unusual or modified
nucleotides located at the 3'-terminus of the oligonucleotide.
Universal Support 500 and Universal Support II are universal
supports that are commercially available from Glen Research, 22825
Davis Drive, Sterling, Va. For further details about universal
supports see Scott et al., Innovations and Perspectives in
solid-phase Synthesis, 3rd International Symposium, 1994, Ed. Roger
Epton, Mayflower Worldwide, 115-124]; Azhayev, A. V. Tetrahedron
1999, 55, 787-800; and Azhayev and Antopolsky Tetrahedron 2001, 57,
4977-4986. In addition, it has been reported that the
oligonucleotide can be cleaved from the universal support under
milder reaction conditions when oligonucleotide is bonded to the
solid support via a syn-1,2-acetoxyphosphate group which more
readily undergoes basic hydrolysis. See Guzaev, A. I.; Manoharan,
M. J. Am. Chem. Soc. 2003, 125, 2380.
[0077] The nucleosides are linked by phosphorus-containing or
non-phosphorus-containing covalent internucleoside linkages. For
the purposes of identification, such conjugated nucleosides can be
characterized as ligand-bearing nucleosides or ligand-nucleoside
conjugates. The linked nucleosides having an aralkyl ligand
conjugated to a nucleoside within their sequence will demonstrate
enhanced dsRNA activity when compared to like dsRNA compounds that
are not conjugated.
[0078] The aralkyl-ligand-conjugated oligonucleotides of the
invention also include conjugates of oligonucleotides and linked
nucleosides wherein the ligand is attached directly to the
nucleoside or nucleotide without the intermediacy of a linker
group. The ligand may preferably be attached, via linking groups,
at a carboxyl, amino or oxo group of the ligand. Typical linking
groups may be ester, amide or carbamate groups.
[0079] Specific examples of preferred modified oligonucleotides
envisioned for use in the ligand-conjugated oligonucleotides of the
invention include oligonucleotides containing modified backbones or
non-natural internucleoside linkages. As defined here,
oligonucleotides having modified backbones or internucleoside
linkages include those that retain a phosphorus atom in the
backbone and those that do not have a phosphorus atom in the
backbone. For the purposes of the invention, modified
oligonucleotides that do not have a phosphorus atom in their
intersugar backbone can also be considered to be
oligonucleosides.
[0080] Specific oligonucleotide chemical modifications are
described below. It is not necessary for all positions in a given
compound to be uniformly modified. Conversely, more than one
modifications may be incorporated in a single dsRNA compound or
even in a single nucleotide thereof.
[0081] Preferred modified internucleoside linkages or backbones
include, for example, phosphorothioates, chiral phosphorothioates,
phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters,
methyl and other alkyl phosphonates including 3'-alkylene
phosphonates and chiral phosphonates, phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates, thionophosphoramidates,
thionoalkylphosphonates, thionoalkylphosphotriesters, and
boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs
of these, and those having inverted polarity wherein the adjacent
pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to
5'-2'. Various salts, mixed salts and free-acid forms are also
included.
[0082] Representative United States Patents relating to the
preparation of the above phosphorus-atom-containing linkages
include, but are not limited to, U.S. Pat. Nos. 3,687,808;
4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423;
5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939;
5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;
5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050;
and 5,697,248, each of which is herein incorporated by
reference.
[0083] Preferred modified internucleoside linkages or backbones
that do not include a phosphorus atom therein (i.e.,
oligonucleosides) have backbones that are formed by short chain
alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl
or cycloalkyl intersugar linkages, or one or more short chain
heteroatomic or heterocyclic intersugar linkages. These include
those having morpholino linkages (formed in part from the sugar
portion of a nucleoside); siloxane backbones; sulfide, sulfoxide
and sulfone backbones; formacetyl and thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; alkene
containing backbones; sulfamate backbones; methyleneimino and
methylenehydrazino backbones; sulfonate and sulfonamide backbones;
amide backbones; and others having mixed N, O, S and CH2 component
parts.
[0084] Representative United States patents relating to the
preparation of the above oligonucleosides include, but are not
limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;
5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;
5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;
5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and
5,677,439, each of which is herein incorporated by reference.
[0085] In other preferred oligonucleotide mimetics, both the sugar
and the internucleoside linkage, i.e., the backbone, of the
nucleoside units are replaced with novel groups. The nucleobase
units are maintained for hybridization with an appropriate nucleic
acid target compound. One such oligonucleotide, an oligonucleotide
mimetic, that has been shown to have excellent hybridization
properties, is referred to as a peptide nucleic acid (PNA). In PNA
compounds, the sugar-backbone of an oligonucleotide is replaced
with an amide-containing backbone, in particular an
aminoethylglycine backbone. The nucleobases are retained and are
bound directly or indirectly to atoms of the amide portion of the
backbone. Representative United States patents that teach the
preparation of PNA compounds include, but are not limited to, U.S.
Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is
herein incorporated by reference. Further teaching of PNA compounds
can be found in Nielsen et al., Science, 1991, 254, 1497.
[0086] Some preferred embodiments of the invention employ
oligonucleotides with phosphorothioate linkages and
oligonucleosides with heteroatom backbones, and in particular
--CH.sub.2--NH--O--CH.sub.2--CH.sub.2--N(CH.sub.3)--O--CH.sub.2--
[known as a methylene (methylimino) or MIMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2--, and
--O--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2-- ]
of the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0087] The oligonucleotides employed in the ligand-conjugated
oligonucleotides of the invention may additionally or alternatively
comprise nucleobase (often referred to in the art simply as "base")
modifications or substitutions. As used herein, "unmodified" or
"natural" nucleobases include the purine bases adenine (A) and
guanine (G), and the pyrimidine bases thymine (T), cytosine (C),
and uracil (U). Modified nucleobases include other synthetic and
natural nucleobases, such as 5-methylcytosine (5-me-C),
5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,
6-methyl and other alkyl derivatives of adenine and guanine,
2-propyl and other alkyl derivatives of adenine and guanine,
2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and
cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine
and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,
8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted
adenines and guanines, 5-halo particularly 5-bromo,
5-trifluoromethyl and other 5-substituted uracils and cytosines,
7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,
7-deazaguanine and 7-deazaadenine and 3-deazaguanine and
3-deazaadenine.
[0088] Further nucleobases include those disclosed in U.S. Pat. No.
3,687,808, those disclosed in the Concise Encyclopedia Of Polymer
Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John
Wiley & Sons, 1990, those disclosed by Englisch et al.,
Angewandte Chemie, International Edition, 1991, 30, 613, and those
disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and
Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC
Press, 1993. Certain of these nucleobases are particularly useful
for increasing the binding affinity of the oligonucleotides of the
invention. These include 5-substituted pyrimidines,
6-azapyrimidines and N-2, N-6 and O-6 substituted purines,
including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-Methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2 .degree. C.
(Id., pages 276-278) and are presently preferred base
substitutions, even more particularly when combined with
2'-methoxyethyl sugar modifications.
[0089] Representative United States patents relating to the
preparation of certain of the above-noted modified nucleobases as
well as other modified nucleobases include, but are not limited to,
the above noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.
4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and
5,808,027; all of which are hereby incorporated by reference.
[0090] In certain embodiments, the oligonucleotides employed in the
ligand-conjugated oligonucleotides of the invention may
additionally or alternatively comprise one or more substituted
sugar moieties. Preferred oligonucleotides comprise one of the
following at the 2' position: OH; F; O-, S-, or N-alkyl, O-, S-, or
N-alkenyl, or O, S- or N-alkynyl, wherein the alkyl, alkenyl and
alkynyl may be substituted or unsubstituted C.sub.1 to C.sub.10
alkyl or C.sub.2 to C.sub.10 alkenyl and alkynyl. Particularly
preferred are O[(CH.sub.2).sub.nO].sub.mCH.sub.3,
O(CH.sub.2).sub.nOCH.sub.3, O(CH.sub.2).sub.nNH.sub.2,
O(CH.sub.2).sub.nCH.sub.3, O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.nON[(CH.sub.2).sub.nCH.sub.3)]2, where n and m are
from 1 to about 10. Other preferred oligonucleotides comprise one
of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or
O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3,
SOCH.sub.3, SO.sub.2 CH.sub.3,ONO.sub.2, NO.sub.2, N.sub.3,
NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. a preferred
modification includes 2'-methoxyethoxy
[2'-O--CH.sub.2CH.sub.2OCH.sub.3, also known as
2'-O--(2-methoxyethyl) or 2'-MOE] (Martin et al., Helv. Chim. Acta,
1995, 78, 486), i.e., an alkoxyalkoxy group. A further preferred
modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3)2 group, also known as 2'-DMAOE, as
described in U.S. Pat. No. 6,127,533, filed on Jan. 30, 1998, the
contents of which are incorporated by reference.
[0091] Other preferred modifications include 2'-methoxy
(2'-O--CH3), 2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2)
and 2'-fluoro (2'-F). Similar modifications may also be made at
other positions on the oligonucleotide, particularly the 3'
position of the sugar on the 3' terminal nucleotide or in 2'-5'
linked oligonucleotides.
[0092] As used herein, the term "sugar substituent group" or
"2'-substituent group" includes groups attached to the 2'-position
of the ribofuranosyl moiety with or without an oxygen atom. Sugar
substituent groups include, but are not limited to, fluoro,
O-alkyl, O -alkylamino, O-alkylalkoxy, protected O-alkylamino,
O-alkylaminoalkyl, O-alkyl imidazole and polyethers of the formula
(O-alkyl).sub.m, wherein m is 1 to about 10. Preferred among these
polyethers are linear and cyclic polyethylene glycols (PEGs), and
(PEG)-containing groups, such as crown ethers and those which are
disclosed by Ouchi et al. (Drug Design and Discovery 1992, 9:93);
Ravasio et al. (J. Org. Chem. 1991, 56:4329); and Delgardo et. al.
(Critical Reviews in Therapeutic Drug Carrier Systems 1992, 9:249),
each of which is hereby incorporated by reference in its entirety.
Further sugar modifications are disclosed by Cook
(Anti-thrombophilia Drug Design, 1991, 6:585-607). Fluoro, O-alkyl,
O-alkylamino, O-alkyl imidazole, O-alkylaminoalkyl, and alkyl amino
substitution is described in U.S. Pat. No. 6,166,197, entitled
"Oligomeric Compounds having Pyrimidine Nucleotide(s) with 2' and
5' Substitutions," hereby incorporated by reference in its
entirety.
[0093] Additional sugar substituent groups amenable to the
invention include 2'-SR and 2'-NR2 groups, wherein each R is,
independently, hydrogen, a protecting group or substituted or
unsubstituted alkyl, alkenyl, or alkynyl. 2'-SR Nucleosides are
disclosed in U.S. Pat. No. 5,670,633, hereby incorporated by
reference in its entirety. The incorporation of 2'-SR monomer
synthons is disclosed by Hamm et al. (J. Org. Chem., 1997,
62:3415-3420). 2'-NR nucleosides are disclosed by Goettingen, M., J
Org. Chem., 1996, 61, 6273-6281; and Polushin et al., Tetrahedron
Lett., 1996, 37, 3227-3230. Further representative 2'-substituent
groups amenable to the invention include those having one of
formula I or II:
##STR00001##
[0094] wherein,
[0095] E is C.sub.1-C.sub.10 alkyl, N(Q.sub.3)(Q.sub.4) or N.dbd.C
(Q.sub.3)(Q.sub.4); each Q.sub.3 and Q.sub.4 is, independently, H,
C.sub.1-C.sub.10 alkyl, dialkylaminoalkyl, a nitrogen protecting
group, a tethered or untethered conjugate group, a linker to a
solid support; or Q.sub.3 and Q.sub.4, together, form a nitrogen
protecting group or a ring structure optionally including at least
one additional heteroatom selected from N and O;
[0096] q.sub.1 is an integer from 1 to 10;
[0097] q.sub.2 is an integer from 1 to 10;
[0098] q.sub.3 is 0 or 1;
[0099] q.sub.4 is 0, 1 or 2;
[0100] each Z.sub.1, Z.sub.2 and Z.sub.3 is, independently,
C.sub.4-C.sub.7 cycloalkyl, C.sub.5-C.sub.14 aryl or
C.sub.3-C.sub.15 heterocyclyl, wherein the heteroatom in said
heterocyclyl group is selected from oxygen, nitrogen and
sulfur;
[0101] Z.sub.4 is OM.sub.1, SM.sub.1, or N(M.sub.1).sub.2; each
M.sub.1 is, independently, H, C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.8 haloalkyl, C(.dbd.NH)N(H)M.sub.2,
C(.dbd.O)N(H)M.sub.2 or OC(.dbd.O)N(H)M.sub.2; M.sub.2 is H or
C.sub.1-C.sub.8 alkyl; and
[0102] Z.sub.5 is C.sub.1-C.sub.10 alkyl, C.sub.1-C.sub.10
haloalkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl,
C.sub.6-C.sub.14 aryl, N(Q.sub.3)(Q.sub.4), OQ.sub.3, halo,
SQ.sub.3 or CN.
[0103] Representative 2'-O-sugar substituent groups of formula I
are disclosed in U.S. Pat. No. 6,172,209, entitled "Capped
2'-Oxyethoxy Oligonucleotides," hereby incorporated by reference in
its entirety. Representative cyclic 2'-O-sugar substituent groups
of formula II are disclosed in U.S. Pat. No. 6,271,358, entitled
"RNA Targeted 2'-Modified Oligonucleotides that are
Conformationally Preorganized," hereby incorporated by reference in
its entirety.
[0104] Sugars having O-substitutions on the ribosyl ring are also
amenable to the invention. Representative substitutions for ring O
include, but are not limited to, S, CH.sub.2, CHF, and CF.sub.2.
See, e.g., Secrist et al., Abstract 21, Program & Abstracts,
Tenth International Roundtable, Nucleosides, Nucleotides and their
Biological Applications, Park City, Utah, Sep. 16-20, 1992.
[0105] Oligonucleotides may also have sugar mimetics, such as
cyclobutyl moieties, in place of the pentofuranosyl sugar.
Representative United States patents relating to the preparation of
such modified sugars include, but are not limited to, U.S. Pat.
Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;
5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;
5,591,722; 5,597,909; 5,610,300; 5,627,0531 5,639,873; 5,646,265;
5,658,873; 5,670,633; 5,700,920; and 5,859,221, all of which are
hereby incorporated by reference.
[0106] Additional modifications may also be made at other positions
on the oligonucleotide, particularly the 3' position of the sugar
on the 3' terminal nucleotide. For example, one additional
modification of the ligand-conjugated oligonucleotides of the
invention involves chemically linking to the oligonucleotide one or
more additional non-ligand moieties or conjugates which enhance the
activity, cellular distribution or cellular uptake of the
oligonucleotide. Such moieties include but are not limited to lipid
moieties, such as a cholesterol moiety (Letsinger et al., Proc.
Natl. Acad. Sci. USA, 1989, 86, 6553), cholic acid (Manoharan et
al., Bioorg. Med. Chem. Lett., 1994, 4, 1053), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660, 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,
1992, 20, 533), an aliphatic chain, e.g., dodecandiol or undecyl
residues (Saison-Behmoaras et al., EMBO 1, 1991, 10, 111; Kabanov
et al., FEBS Lett., 1990, 259, 327; Svinarchuk et al., Biochimie,
1993, 75, 49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate
(Manoharan et al., Tetrahedron Lett., 1995, 36, 3651; Shea et al.,
Nucl. Acids Res., 1990, 18, 3777), a polyamine or a polyethylene
glycol chain (Manoharan et al., Nucleosides & Nucleotides,
1995, 14, 969), or adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651), a palmityl moiety (Mishra et
al., Biochim. Biophys. Acta, 1995, 1264, 229), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923).
[0107] Representative United States patents relating to the
preparation of such oligonucleotide conjugates include, but are not
limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;
5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;
4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;
5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;
5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928; and 5,688,941, each of which is herein incorporated by
reference.
[0108] The invention also includes compositions employing
oligonucleotides that are substantially chirally pure with regard
to particular positions within the oligonucleotides. Examples of
substantially chirally pure oligonucleotides include, but are not
limited to, those having phosphorothioate linkages that are at
least 75% Sp or Rp (Cook et al., U.S. Pat. No. 5,587,361) and those
having substantially chirally pure (Sp or Rp) alkylphosphonate,
phosphoramidate or phosphotriester linkages (Cook, U.S. Pat. Nos.
5,212,295 and 5,521,302).
[0109] In certain instances, the oligonucleotide may be modified by
a non-ligand group. A number of non-ligand molecules have been
conjugated to oligonucleotides in order to enhance the activity,
cellular distribution or cellular uptake of the oligonucleotide,
and procedures for performing such conjugations are available in
the scientific literature. Such non-ligand moieties have included
lipid moieties, such as cholesterol (Letsinger et al., Proc. Natl.
Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al.,
Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765),
a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992,
20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues
(Saison-Behmoaras et al., EMBO 1, 1991, 10:111; Kabanov et al.,
FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993,
75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or
triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate
(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al.,
Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene
glycol chain (Manoharan et al., Nucleosides & Nucleotides,
1995, 14:969), or adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et
al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277:923). Representative United States
patents that teach the preparation of such oligonucleotide
conjugates have been listed above. Typical conjugation protocols
involve the synthesis of oligonucleotides bearing an aminolinker at
one or more positions of the sequence. The amino group is then
reacted with the molecule being conjugated using appropriate
coupling or activating reagents. The conjugation reaction may be
performed either with the oligonucleotide still bound to the solid
support or following cleavage of the oligonucleotide in solution
phase. Purification of the oligonucleotide conjugate by HPLC
typically affords the pure conjugate. The use of a cholesterol
conjugate is particularly preferred since sucha moiety can increase
targeting to tissues in the liver, a site of Factor V protein
production.
[0110] Alternatively, the molecule being conjugated may be
converted into a building block, such as a phosphoramidite, via an
alcohol group present in the molecule or by attachment of a linker
bearing an alcohol group that may be phosphitylated.
[0111] Importantly, each of these approaches may be used for the
synthesis of ligand conjugated oligonucleotides. Aminolinked
oligonucleotides may be coupled directly with ligand via the use of
coupling reagents or following activation of the ligand as an NHS
or pentfluorophenolate ester. Ligand phosphoramidites may be
synthesized via the attachment of an aminohexanol linker to one of
the carboxyl groups followed by phosphitylation of the terminal
alcohol functionality. Other linkers, such as cysteamine, may also
be utilized for conjugation to a chloroacetyl linker present on a
synthesized oligonucleotide.
III. Pharmaceutical Compositions Comprising dsRNA
[0112] In one embodiment, the invention provides pharmaceutical
compositions comprising a dsRNA, as described herein, and a
pharmaceutically acceptable carrier. The pharmaceutical composition
comprising the dsRNA is useful for treating a disease or disorder
associated with the expression or activity of the Factor V Leiden
mutant gene, such as thrombophilia.
[0113] The pharmaceutical compositions of the invention are
administered in dosages sufficient to inhibit expression of the
Factor V Leiden mutant gene. The present inventors have found that,
because of their improved efficiency, compositions comprising the
dsRNA of the invention can be administered at surprisingly low
dosages. A maximum dosage of 5 mg dsRNA per kilogram body weight of
recipient per day is sufficient to inhibit or completely suppress
expression of the Factor V Leiden mutant gene.
[0114] In general, a suitable dose of dsRNA will be in the range of
0.01 to 5.0 milligrams per kilogram body weight of the recipient
per day, preferably in the range of 0.1 to 200 micrograms per
kilogram body weight per day, more preferably in the range of 0.1
to 100 micrograms per kilogram body weight per day, even more
preferably in the range of 1.0 to 50 micrograms per kilogram body
weight per day, and most preferably in the range of 1.0 to 25
micrograms per kilogram body weight per day. The pharmaceutical
composition may be administered once daily, or the dsRNA may be
administered as two, three, four, five, six or more sub-doses at
appropriate intervals throughout the day or even using continuous
infusion. In that case, the dsRNA 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 dsRNA 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.
[0115] 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 disease
or disorder, 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
dsRNAs 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.
[0116] Advances in mouse genetics have generated a number of mouse
models for the study of various human diseases, such as
thrombophilia. Such models are used for in vivo testing of dsRNA,
as well as for determining a therapeutically effective dose.
[0117] 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), rectal, vaginal and topical (including buccal and
sublingual) administration. In preferred embodiments, the
pharmaceutical compositions are administered intraveneously.
[0118] For intramuscular, 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 dsRNA in the cells that express the Factor V
Leiden mutant gene. Such substances include, for example, micellar
structures, such as liposomes or capsids, as described below.
Surprisingly, the present inventors have discovered that
compositions containing only naked dsRNA and a physiologically
acceptable solvent are taken up by cells, where the dsRNA
effectively inhibits expression of the Factor V Leiden mutant gene.
Although microinjection, lipofection, viruses, viroids, capsids,
capsoids, or other auxiliary agents are required to introduce dsRNA
into cell cultures, surprisingly these methods and agents are not
necessary for uptake of dsRNA in vivo. 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.
[0119] The pharmaceutical compositions useful according to the
invention also include encapsulated formulations to protect the
dsRNA 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.
[0120] The present invention further provides devices containing
the RNAi agents of the present invention, such as devices that come
into contact with the blood. Examples of devices that come into
contact with blood include vascular grafts, stents, orthopedic
prosthesis, cardiac prosthesis, and extracorporeal circulation
systems.
[0121] Toxicity and therapeutic efficacy of such compounds 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. Compounds which exhibit
high therapeutic indices are preferred.
[0122] The data obtained from cell culture assays and animal
studies can be used in formulation a range of dosage for use in
humans. The dosage of compositions of the invention lies 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 compound 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 compound 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 compound 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.
[0123] In addition to their administration individually or as a
plurality, as discussed above, the dsRNAs of the invention can be
administered in combination with other known agents effective in
treatment of thrombophilia. In any event, the administering
physician can adjust the amount and timing of dsRNA administration
on the basis of results observed using standard measures of
efficacy known in the art or described herein.
[0124] The RNAi agents of the present invention can also be
co-administered with suitable anti-platelet agents, including, but
not limited to, fibrinogen receptor antagonists (e.g. to treat or
prevent unstable angina or to prevent reocclusion after angioplasty
and restenosis), anticoagulants such as aspirin, thrombolytic
agents such as plasminogen activators or streptokinase to achieve
synergistic effects in the treatment of various vascular
pathologies, or lipid lowering agents including
antihypercholesterolemics (e.g. HMG CoA reductase inhibitors such
as lovastatin and simvastatin, HMG CoA synthase inhibitors, etc.)
to treat or prevent atherosclerosis. For example, patients
suffering from coronary artery disease, and patients subjected to
angioplasty procedures, would benefit from coadministration of
fibrinogen receptor antagonists and present RNAi agents.
Methods for Treating Diseases Caused by Expression of the Factor V
Leiden Mutant Gene
[0125] In one embodiment, the invention provides a method for
treating a subject having a pathological condition mediated by the
expression of the Factor V Leiden mutant gene, such as
thrombophilia. In this embodiment, the dsRNA acts as a therapeutic
agent for controlling the expression of the Factor V Leiden mutant
protein. The method comprises administering a pharmaceutical
composition of the invention to the patient (e.g., human), such
that expression of the Factor V Leiden mutant gene is silenced.
Because of their high specificity, the dsRNAs of the invention
specifically target mRNAs of the Factor V Leiden mutant gene.
Thrombophilia
[0126] The compounds of the invention are useful in those
conditions where anticoagulant therapy or prophylaxis is indicated,
including the following.
[0127] Compounds of the invention are useful for treating or
preventing venous thromboembolism (e.g. obstruction or occlusion of
a vein by a detached thrombus; obstruction or occlusion of a lung
artery by a detached thrombus), cardiogenic thromboembolism (e.g.
obstruction or occlusion of the heart by a detached thrombus),
arterial thrombosis (e.g. formation of a thrombus within an artery
that may cause infarction of tissue supplied by the artery),
atherosclerosis (e.g. arteriosclerosis characterized by irregularly
distributed lipid deposits) in mammals, and for lowering the
propensity of devices that come into contact with blood to clot
blood.
[0128] Examples of venous thromboembolism which may be treated or
prevented with compounds of the invention include obstruction of a
vein, obstruction of a lung artery (pulmonary embolism), deep vein
thrombosis, thrombosis associated with cancer and cancer
chemotherapy, thrombosis inherited with thrombophilic diseases such
as Factor V Leiden, and thrombosis resulting from acquired
thrombophilic disorders such as systemic lupus erythematosus
(inflammatory connective tissue disease). Also with regard to
venous thromboembolism, compounds of the invention are useful for
maintaining patency of indwelling catheters.
[0129] Examples of cardiogenic thromboembolism which may be treated
or prevented with compounds of the invention include thromboembolic
stroke (detached thrombus causing neurological affliction related
to impaired cerebral blood supply), cardiogenic thromboembolism
associated with atrial fibrillation (rapid, irregular twitching of
upper heart chamber muscular fibrils), cardiogenic thromboembolism
associated with prosthetic heart valves such as mechanical heart
valves, and cardiogenic thromboembolism associated with heart
disease.
[0130] Examples of arterial thrombosis include unstable angina
(severe constrictive pain in chest of coronary origin), myocardial
infarction (heart muscle cell death resulting from insufficient
blood supply), ischemic heart disease (local anemia due to
obstruction (such as by arterial narrowing) of blood supply),
reocclusion during or after percutaneous transluminal coronary
angioplasty, restenosis after percutaneous transluminal coronary
angioplasty, occlusion of coronary artery bypass grafts, and
occlusive cerebrovascular disease. Also with regard to arterial
thrombosis, compounds of the invention are useful for maintaining
patency in arteriovenous cannulas.
[0131] Examples of atherosclerosis include arteriosclerosis.
[0132] The invention thus provides the use of an anti-Factor V
Leiden mutant dsRNA administered to a human, particularly by
intraveneous administration, for the treatment of thrombophilia
[0133] 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), nasal, rectal, vaginal and topical (including buccal and
sublingual) administration, and epidural administration. In
preferred embodiments, the pharmaceutical compositions are
administered intraveneously by infusion or injection.
Methods for Inhibiting Expression of the Factor V Leiden Mutant
Gene
[0134] In yet another aspect, the invention provides a method for
inhibiting the expression of the Factor V Leiden mutant gene in a
mammal. The method comprises administering a composition of the
invention to the mammal such that expression of the target Factor V
Leiden mutant gene is silenced. Because of their high specificity,
the dsRNAs of the invention specifically target RNAs (primary or
processed) of the target Factor V Leiden mutant gene. Compositions
and methods for inhibiting the expression of these Factor V Leiden
mutant genes using dsRNAs can be performed as described elsewhere
herein.
[0135] In one embodiment, the method comprises administering a
composition comprising a dsRNA, wherein the dsRNA comprises a
nucleotide sequence which is complementary to at least a part of an
RNA transcript of the Factor V Leiden mutant gene of the mammal to
be treated. When the organism to be treated is a mammal such as a
human, the composition may be administered by any means known in
the art including, but not limited to oral or parenteral routes,
including intravenous, intramuscular, intracranial, subcutaneous,
transdermal, airway (aerosol), nasal, rectal, vaginal and topical
(including buccal and sublingual) administration. In preferred
embodiments, the compositions are administered by intraveneous
infusion or injection.
[0136] 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 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
Gene Walking of the Factor V Leiden Mutant Gene
[0137] siRNAs were identified in a multi step sequence analysis
process in order to design siRNAs targeting the Factor V Leiden
mutant gene.
[0138] The in silico selected siRNAs are provided in Table 1.
dsRNA Synthesis
Source of Reagents
[0139] Where the source of a reagent is not specifically given
herein, such reagent may be obtained from any supplier of reagents
for molecular biology at a quality/purity standard for application
in molecular biology.
siRNA Synthesis
[0140] Single-stranded RNAs were produced by solid phase synthesis
on a scale of 1 .mu.mole using an Expedite 8909 synthesizer
(Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany)
and controlled pore glass (CPG, 500 .ANG., Proligo Biochemie GmbH,
Hamburg, Germany) as solid support. RNA and RNA containing
2'-O-methyl nucleotides were generated by solid phase synthesis
employing the corresponding phosphoramidites and 2'-O-methyl
phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg,
Germany). These building blocks were incorporated at selected sites
within the sequence of the oligoribonucleotide chain using standard
nucleoside phosphoramidite chemistry such as described in Current
protocols in nucleic acid chemistry, Beaucage, S. L. et al.
(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA.
Phosphorothioate linkages were introduced by replacement of the
iodine oxidizer solution with a solution of the Beaucage reagent
(Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further
ancillary reagents were obtained from Mallinckrodt Baker
(Griesheim, Germany).
[0141] Deprotection and purification of the crude
oligoribonucleotides by anion exchange HPLC were carried out
according to established procedures. Yields and concentrations were
determined by UV absorption of a solution of the respective RNA at
a wavelength of 260 nm using a spectral photometer (DU 640B,
Beckman Coulter GmbH, UnterschleiBheim, Germany). Double stranded
RNA was generated by mixing an equimolar solution of complementary
strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM
sodium chloride), heated in a water bath at 85-90.degree. C. for 3
minutes and cooled to room temperature over a period of 3-4 hours.
The annealed RNA solution was stored at -20.degree. C. until
use.
[0142] For the synthesis of 3'-cholesterol-conjugated siRNAs
(herein referred to as -Chol-3'), an appropriately modified solid
support was used for RNA synthesis. The modified solid support was
prepared as follows:
[0143] Diethyl-2-azabutane-1,4-dicarboxylate AA
##STR00002##
[0144] A 4.7 M aqueous solution of sodium hydroxide (50 mL) was
added into a stirred, ice-cooled solution of ethyl glycinate
hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl
acrylate (23.1 g, 0.23 mole) was added and the mixture was stirred
at room temperature until completion of the reaction was
ascertained by TLC. After 19 h the solution was partitioned with
dichloromethane (3.times.100 mL). The organic layer was dried with
anhydrous sodium sulfate, filtered and evaporated. The residue was
distilled to afford AA (28.8 g, 61%).
[0145]
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-h-
exanoyl]-amino}-propionic acid ethyl ester AB
##STR00003##
[0146] Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) was
dissolved in dichloromethane (50 mL) and cooled with ice.
Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) was added to
the solution at 0.degree. C. It was then followed by the addition
of Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and
dimethylamino pyridine (0.305 g, 2.5 mmol). The solution was
brought to room temperature and stirred further for 6 h. Completion
of the reaction was ascertained by TLC. The reaction mixture was
concentrated under vacuum and ethyl acetate was added to
precipitate diisopropyl urea. The suspension was filtered. The
filtrate was washed with 5% aqueous hydrochloric acid, 5% sodium
bicarbonate and water. The combined organic layer was dried over
sodium sulfate and concentrated to give the crude product which was
purified by column chromatography (50% EtOAC/Hexanes) to yield
11.87 g (88%) of AB.
[0147] 3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic
acid ethyl ester AC
##STR00004##
[0148]
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-he-
xanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol)
was dissolved in 20% piperidine in dimethylformamide at 0.degree.
C. The solution was continued stirring for 1 h. The reaction
mixture was concentrated under vacuum, water was added to the
residue, and the product was extracted with ethyl acetate. The
crude product was purified by conversion into its hydrochloride
salt.
[0149]
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,-
13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-pr-
opionic acid ethyl ester AD
##STR00005##
[0150] The hydrochloride salt of
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid
ethyl ester AC (4.7 g, 14.8 mmol) was taken up in dichloromethane.
The suspension was cooled to 0.degree. C. on ice. To the suspension
diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) was added. To the
resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol)
was added. The reaction mixture was stirred overnight. The reaction
mixture was diluted with dichloromethane and washed with 10%
hydrochloric acid. The product was purified by flash chromatography
(10.3 g, 92%).
[0151]
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,1-
3,14,15,16,17-tetradecahydro-1H-cyclopenta[a] phenanthren-3
-yloxycarbonylamino]-hexanoyl}-4 -oxo-pyrrolidine-3-carboxylic acid
ethyl ester AE
##STR00006##
[0152] Potassium t-butoxide (1.1 g, 9.8 mmol) was slurried in 30 mL
of dry toluene. The mixture was cooled to 0.degree. C. on ice and 5
g (6.6 mmol) of diester AD was added slowly with stirring within 20
mins. The temperature was kept below 5.degree. C. during the
addition. The stirring was continued for 30 mins at 0.degree. C.
and 1 mL of glacial acetic acid was added, immediately followed by
4 g of NaH.sub.2PO.sub.4.H.sub.2O in 40 mL of water The resultant
mixture was extracted twice with 100 mL of dichloromethane each and
the combined organic extracts were washed twice with 10 mL of
phosphate buffer each, dried, and evaporated to dryness. The
residue was dissolved in 60 mL of toluene, cooled to 0.degree. C.
and extracted with three 50 mL portions of cold pH 9.5 carbonate
buffer. The aqueous extracts were adjusted to pH 3 with phosphoric
acid, and extracted with five 40 mL portions of chloroform which
were combined, dried and evaporated to dryness. The residue was
purified by column chromatography using 25% ethylacetate/hexane to
afford 1.9 g of b-ketoester (39%).
[0153]
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbami-
c acid 17-(1,5 -dimethyl-hexyl)-10,13 -dimethyl-2,3,4,7,8,9,
10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl
ester AF
##STR00007##
[0154] Methanol (2 mL) was added dropwise over a period of 1 h to a
refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium
borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring
was continued at reflux temperature for 1 h. After cooling to room
temperature, 1 N HCl (12.5 mL) was added, the mixture was extracted
with ethylacetate (3.times.40 mL). The combined ethylacetate layer
was dried over anhydrous sodium sulfate and concentrated under
vacuum to yield the product which was purified by column
chromatography (10% MeOH/CHCl.sub.3) (89%).
[0155]
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrro-
lidin-1-yl}-6-oxo-hexyl)-carbamic acid
17-(1,5-dimethyl-hexyl)-10,13
-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopent-
a[a]phenanthren-3-yl ester AG
##STR00008##
[0156] Diol AF (1.25 gm 1.994 mmol) was dried by evaporating with
pyridine (2.times.5 mL) in vacuo. Anhydrous pyridine (10 mL) and
4,4'-dimethoxytritylchloride (0.724 g, 2.13 mmol) were added with
stirring. The reaction was carried out at room temperature
overnight. The reaction was quenched by the addition of methanol.
The reaction mixture was concentrated under vacuum and to the
residue dichloromethane (50 mL) was added. The organic layer was
washed with 1M aqueous sodium bicarbonate. The organic layer was
dried over anhydrous sodium sulfate, filtered and concentrated. The
residual pyridine was removed by evaporating with toluene. The
crude product was purified by column chromatography (2%
MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl.sub.3) (1.75 g, 95%).
[0157] Succinic acid
mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimet-
hyl-hexyl)-10,13 -dimethyl
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H
cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}
-pyrrolidin-3-yl) ester AH
##STR00009##
[0158] Compound AG (1.0 g, 1.05 mmol) was mixed with succinic
anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and
dried in a vacuum at 40.degree. C. overnight. The mixture was
dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318
g, 0.440 mL, 3.15 mmol) was added and the solution was stirred at
room temperature under argon atmosphere for 16 h. It was then
diluted with dichloromethane (40 mL) and washed with ice cold
aqueous citric acid (5 wt %, 30 mL) and water (2.times.20 mL). The
organic phase was dried over anhydrous sodium sulfate and
concentrated to dryness. The residue was used as such for the next
step.
[0159] Cholesterol derivatised CPG AI
##STR00010##
[0160] Succinate AH (0.254 g, 0.242 mmol) was dissolved in a
mixture of dichloromethane/acetonitrile (3:2, 3 mL). To that
solution DMAP (0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL),
2,2'-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in
acetonitrile/dichloroethane (3:1, 1.25 mL) were added successively.
To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol)
in acetonitrile (0.6 ml) was added. The reaction mixture turned
bright orange in color. The solution was agitated briefly using a
wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG)
(1.5 g, 61 mM) was added. The suspension was agitated for 2 h. The
CPG was filtered through a sintered funnel and washed with
acetonitrile, dichloromethane and ether successively. Unreacted
amino groups were masked using acetic anhydride/pyridine. The
achieved loading of the CPG was measured by taking UV measurement
(37 mM/g).
[0161] The synthesis of siRNAs bearing a 5'-12-dodecanoic acid
bisdecylamide group (herein referred to as "5'-C32-") or a
5'-cholesteryl derivative group (herein referred to as "5'-Chol-")
was performed as described in WO 2004/065601, except that, for the
cholesteryl derivative, the oxidation step was performed using the
Beaucage reagent in order to introduce a phosphorothioate linkage
at the 5'-end of the nucleic acid oligomer.
[0162] Nucleic acid sequences are represented below using standard
nomenclature, and specifically the abbreviations of Table 2.
TABLE-US-00002 TABLE 2 Abbreviations of nucleotide monomers used in
nucleic acid sequence representation. It will be understood that
these monomers, when present in an oligonucleotide, are mutually
linked by 5'-3'-phosphodiester bonds. Abbreviation.sup.a
Nucleotide(s) A, a 2'-deoxy-adenosine-5'-phosphate,
adenosine-5'-phosphate C, c 2'-deoxy-cytidine-5'-phosphate,
cytidine-5'-phosphate G, g 2'-deoxy-guanosine-5'-phosphate,
guanosine-5'-phosphate T, t 2'-deoxy-thymidine-5'-phosphate,
thymidine-5'-phosphate U, u 2'-deoxy-uridine-5'-phosphate,
uridine-5'-phosphate N, n any 2'-deoxy-nucleotide/nucleotide (G, A,
C, or T, g, a, c or u) Am 2'-O-methyladenosine-5'-phosphate Cm
2'-O-methylcytidine-5'-phosphate Gm
2'-O-methylguanosine-5'-phosphate Tm
2'-O-methyl-thymidine-5'-phosphate Um
2'-O-methyluridine-5-'phosphate Af
2'-fluoro-2'-deoxy-adenosine-5'-phosphate Cf
2'-fluoro-2'-deoxy-cytidine-5'-phosphate Gf
2'-fluoro-2'-deoxy-guanosine-5'-phosphate Tf
2'-fluoro-2'-deoxy-thymidine-5'-phosphate Uf
2'-fluoro-2'-deoxy-uridine-5'-phosphate A, C, G, T, U, underlined:
nucleoside-5'-phosphorothioate a, c, g, t, u am, cm, gm,
underlined: 2-O-methyl-nucleoside-5'-phosphorothioate tm, um
.sup.acapital letters represent 2'-deoxyribonucleotides (DNA),
lower case letters represent ribonucleotides (RNA)
dsRNA Expression Vectors
[0163] In another aspect of the invention, Factor V Leiden mutant
specific dsRNA molecules that modulate Factor V Leiden mutant gene
expression activity are expressed from transcription units inserted
into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG.
(1996), 12:5-10; Skillern, A., et al., International PCT
Publication No. WO 00/22113, Conrad, International PCT Publication
No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). These
transgenes can be introduced as a linear construct, a circular
plasmid, or a viral vector, which can be incorporated and inherited
as a transgene integrated into the host genome. The transgene can
also be constructed to permit it to be inherited as an
extrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci.
USA (1995) 92:1292).
[0164] The individual strands of a dsRNA can be transcribed by
promoters on two separate expression vectors and co-transfected
into a target cell. Alternatively each individual strand of the
dsRNA can be transcribed by promoters both of which are located on
the same expression plasmid. In a preferred embodiment, a dsRNA is
expressed as an inverted repeat joined by a linker polynucleotide
sequence such that the dsRNA has a stem and loop structure.
[0165] The recombinant dsRNA expression vectors are preferably DNA
plasmids or viral vectors. dsRNA expressing viral vectors can be
constructed based on, but not limited to, adeno-associated virus
(for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol.
(1992) 158:97-129)); adenovirus (see, for example, Berkner, et al.,
BioTechniques (1998) 6:616),
[0166] Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld
et al. (1992), Cell 68:143-155)); or alphavirus as well as others
known in the art. Retroviruses have been used to introduce a
variety of genes into many different cell types, including
epithelial cells, in vitro and/or in vivo (see, e.g., Eglitis, et
al., Science (1985) 230:1395-1398; Danos and Mulligan, Proc. Natl.
Acad. Sci. USA (1998) 85:6460-6464; Wilson et al., 1988, Proc.
NatI. Acad. Sci. USA 85:3014-3018; Armentano et al., 1990, Proc.
NatI. Acad. Sci. USA 87:61416145; Huber et al., 1991, Proc. NatI.
Acad. Sci. USA 88:8039-8043; Ferry et al., 1991, Proc. NatI. Acad.
Sci. USA 88:8377-8381; Chowdhury et al., 1991, Science
254:1802-1805; van Beusechem. et al., 1992, Proc. Nad. Acad. Sci.
USA 89:7640-19 ; Kay et al., 1992, Human Gene Therapy 3:641-647;
Dai et al., 1992, Proc. Natl.Acad. Sci. USA 89:10892-10895; Hwu et
al., 1993,1 Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S.
Patent No. 4,980,286; PCT Application WO 89/07136; PCT Application
WO 89/02468; PCT Application WO 89/05345; and PCT Application WO
92/07573). Recombinant retroviral vectors capable of transducing
and expressing genes inserted into the genome of a cell can be
produced by transfecting the recombinant retroviral genome into
suitable packaging cell lines such as PA317 and Psi-CRIP (Comette
et al., 1991, Human Gene Therapy 2:5-10; Cone et al., 1984, Proc.
Natl. Acad. Sci. USA 81:6349). Recombinant adenoviral vectors can
be used to infect a wide variety of cells and tissues in
susceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et
al., 1992, J. Infectious Disease, 166:769), and also have the
advantage of not requiring mitotically active cells for
infection.
[0167] The promoter driving dsRNA expression in either a DNA
plasmid or viral vector of the invention may be a eukaryotic RNA
polymerase I (e.g. ribosomal RNA promoter), RNA polymerase II (e.g.
CMV early promoter or actin promoter or Ul snRNA promoter) or
preferably RNA polymerase III promoter (e.g. U6 snRNA or 7SK RNA
promoter) or a prokaryotic promoter, for example the T7 promoter,
provided the expression plasmid also encodes T7 RNA polymerase
required for transcription from a T7 promoter. The promoter can
also direct transgene expression to the pancreas (see, e.g. the
insulin regulatory sequence for pancreas (Bucchini et al., 1986,
Proc. Natl. Acad. Sci. USA 83:2511-2515)).
[0168] In addition, expression of the transgene can be precisely
regulated, for example, by using an inducible regulatory sequence
and expression systems such as a regulatory sequence that is
sensitive to certain physiological regulators, e.g., circulating
glucose levels, or hormones (Docherty et al., 1994, FASEB J.
8:20-24). Such inducible expression systems, suitable for the
control of transgene expression in cells or in mammals include
regulation by ecdysone, by estrogen, progesterone, tetracycline,
chemical inducers of dimerization, and isopropyl-beta-D1
-thiogalactopyranoside (EPTG). A person skilled in the art would be
able to choose the appropriate regulatory/promoter sequence based
on the intended use of the dsRNA transgene.
[0169] Preferably, recombinant vectors capable of expressing dsRNA
molecules are delivered as described below, and persist in target
cells. Alternatively, viral vectors can be used that provide for
transient expression of dsRNA molecules. Such vectors can be
repeatedly administered as necessary. Once expressed, the dsRNAs
bind to target RNA and modulate its function or expression.
Delivery of dsRNA expressing vectors can be systemic, such as by
intravenous or intramuscular administration, by administration to
target cells ex-planted from the patient followed by reintroduction
into the patient, or by any other means that allows for
introduction into a desired target cell.
[0170] dsRNA expression DNA plasmids are typically transfected into
target cells as a complex with cationic lipid carriers (e.g.
Oligofectamine) or non-cationic lipid-based carriers (e.g.
Transit-TKO.TM.). Multiple lipid transfections for dsRNA-mediated
knockdowns targeting different regions of a single Factor V Leiden
mutant gene or multiple Factor V Leiden mutant genes over a period
of a week or more are also contemplated by the invention.
Successful introduction of the vectors of the invention into host
cells can be monitored using various known methods. For example,
transient transfection. can be signaled with a reporter, such as a
fluorescent marker, such as Green Fluorescent Protein (GFP). Stable
transfection. of ex vivo cells can be ensured using markers that
provide the transfected cell with resistance to specific
environmental factors (e.g., antibiotics and drugs), such as
hygromycin B resistance.
[0171] The Factor V Leiden mutant specific dsRNA molecules can also
be inserted into vectors and used as gene therapy vectors for human
patients. Gene therapy vectors can be delivered to a subject by,
for example, intravenous injection, local administration (see U.S.
Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et
al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The
pharmaceutical preparation of the gene therapy vector can include
the gene therapy vector in an acceptable diluent, or can comprise a
slow release matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system.
Sequence CWU 1
1
76121DNAArtificial SequenceDescription of Artificial Sequence
Synthetic RNAi agent strand sequenceDescription of Combined DNA/RNA
Molecule Synthetic RNAi agent strand sequence5'-terminal nucleic
acid is a nucleoside, i.e. base + sugar but lacking
5'-phosphatemodified_base(1)..(19)2'-hydroxy corresponding base
1cccuggacag gcgaggaaut t 21221DNAArtificial SequenceDescription of
Artificial Sequence Synthetic RNAi agent strand sequenceDescription
of Combined DNA/RNA Molecule Synthetic RNAi agent strand
sequence5'-terminal nucleic acid is a nucleoside, i.e. base + sugar
but lacking 5'-phosphatemodified_base(1)..(19)2'-hydroxy
corresponding base 2auuccucgcc uguccagggt t 21321DNAArtificial
SequenceDescription of Artificial Sequence Synthetic RNAi agent
strand sequenceDescription of Combined DNA/RNA Molecule Synthetic
RNAi agent strand sequence5'-terminal nucleic acid is a nucleoside,
i.e. base + sugar but lacking
5'-phosphatemodified_base(1)..(19)2'-hydroxy corresponding base
3ccuggacagg cgaggaauat t 21421DNAArtificial SequenceDescription of
Artificial Sequence Synthetic RNAi agent strand sequenceDescription
of Combined DNA/RNA Molecule Synthetic RNAi agent strand
sequence5'-terminal nucleic acid is a nucleoside, i.e. base + sugar
but lacking 5'-phosphatemodified_base(1)..(19)2'-hydroxy
corresponding base 4uauuccucgc cuguccaggt t 21521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic RNAi agent
strand sequenceDescription of Combined DNA/RNA Molecule Synthetic
RNAi agent strand sequence5'-terminal nucleic acid is a nucleoside,
i.e. base + sugar but lacking
5'-phosphatemodified_base(1)..(19)2'-hydroxy corresponding base
5cuggacaggc gaggaauact t 21621DNAArtificial SequenceDescription of
Artificial Sequence Synthetic RNAi agent strand sequenceDescription
of Combined DNA/RNA Molecule Synthetic RNAi agent strand
sequence5'-terminal nucleic acid is a nucleoside, i.e. base + sugar
but lacking 5'-phosphatemodified_base(1)..(19)2'-hydroxy
corresponding base 6guauuccucg ccuguccagt t 21721DNAArtificial
SequenceDescription of Artificial Sequence Synthetic RNAi agent
strand sequenceDescription of Combined DNA/RNA Molecule Synthetic
RNAi agent strand sequence5'-terminal nucleic acid is a nucleoside,
i.e. base + sugar but lacking
5'-phosphatemodified_base(1)..(19)2'-hydroxy corresponding base
7uggacaggcg aggaauacat t 21821DNAArtificial SequenceDescription of
Artificial Sequence Synthetic RNAi agent strand sequenceDescription
of Combined DNA/RNA Molecule Synthetic RNAi agent strand
sequence5'-terminal nucleic acid is a nucleoside, i.e. base + sugar
but lacking 5'-phosphatemodified_base(1)..(19)2'-hydroxy
corresponding base 8uguauuccuc gccuguccat t 21921DNAArtificial
SequenceDescription of Artificial Sequence Synthetic RNAi agent
strand sequenceDescription of Combined DNA/RNA Molecule Synthetic
RNAi agent strand sequence5'-terminal nucleic acid is a nucleoside,
i.e. base + sugar but lacking
5'-phosphatemodified_base(1)..(19)2'-hydroxy corresponding base
9ggacaggcga ggaauacagt t 211021DNAArtificial SequenceDescription of
Artificial Sequence Synthetic RNAi agent strand sequenceDescription
of Combined DNA/RNA Molecule Synthetic RNAi agent strand
sequence5'-terminal nucleic acid is a nucleoside, i.e. base + sugar
but lacking 5'-phosphatemodified_base(1)..(19)2'-hydroxy
corresponding base 10cuguauuccu cgccugucct t 211121DNAArtificial
SequenceDescription of Artificial Sequence Synthetic RNAi agent
strand sequenceDescription of Combined DNA/RNA Molecule Synthetic
RNAi agent strand sequence5'-terminal nucleic acid is a nucleoside,
i.e. base + sugar but lacking
5'-phosphatemodified_base(1)..(19)2'-hydroxy corresponding base
11gacaggcgag gaauacagat t 211221DNAArtificial SequenceDescription
of Artificial Sequence Synthetic RNAi agent strand
sequenceDescription of Combined DNA/RNA Molecule Synthetic RNAi
agent strand sequence5'-terminal nucleic acid is a nucleoside,
i.