U.S. patent application number 13/343426 was filed with the patent office on 2012-06-07 for rnai modulation of aha and therapeutic uses thereof.
This patent application is currently assigned to ALNYLAM PHARMACEUTICALS, INC.. Invention is credited to William Balch, Birgit Bramlage, Rainer Constien, Pamela Tan, Hans-Peter Vornlocher.
Application Number | 20120142757 13/343426 |
Document ID | / |
Family ID | 38724031 |
Filed Date | 2012-06-07 |
United States Patent
Application |
20120142757 |
Kind Code |
A1 |
Constien; Rainer ; et
al. |
June 7, 2012 |
RNAi Modulation of AHA and Therapeutic Uses Thereof
Abstract
The invention relates to a double-stranded ribonucleic acid
(dsRNA) for inhibiting the expression of an Aha gene (Aha1 gene),
comprising an antisense strand having a nucleotide sequence which
is less that 30 nucleotides in length, generally 19-25 nucleotides
in length, and which is substantially complementary to at least a
part of an Aha gene. The invention also relates to a pharmaceutical
composition comprising the dsRNA together with a pharmaceutically
acceptable carrier; methods for treating diseases caused by Aha1
expression and the expression of an Aha gene using the
pharmaceutical composition; and methods for inhibiting the
expression of an Aha gene in a cell.
Inventors: |
Constien; Rainer; (Kulmbach,
DE) ; Bramlage; Birgit; (Kulmbach, DE) ; Tan;
Pamela; (Kulmbach, DE) ; Vornlocher; Hans-Peter;
(Bayreuth, DE) ; Balch; William; (San Diego,
CA) |
Assignee: |
ALNYLAM PHARMACEUTICALS,
INC.
Cambridge
MA
|
Family ID: |
38724031 |
Appl. No.: |
13/343426 |
Filed: |
January 4, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12877025 |
Sep 7, 2010 |
8114984 |
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13343426 |
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11750553 |
May 18, 2007 |
7812150 |
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12877025 |
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60801840 |
May 19, 2006 |
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Current U.S.
Class: |
514/44A ;
435/320.1; 435/325; 435/366; 435/375; 536/24.5 |
Current CPC
Class: |
A61P 35/04 20180101;
A61P 1/18 20180101; C12N 2310/3515 20130101; C12N 15/113 20130101;
A61P 11/00 20180101; A61P 35/00 20180101; A61P 35/02 20180101; C12N
2310/14 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/44.A ;
536/24.5; 435/325; 435/375; 435/320.1; 435/366 |
International
Class: |
A61K 31/713 20060101
A61K031/713; A61P 35/00 20060101 A61P035/00; C12N 15/63 20060101
C12N015/63; C12N 5/10 20060101 C12N005/10; C12N 5/02 20060101
C12N005/02; C12N 15/113 20100101 C12N015/113; A61P 11/00 20060101
A61P011/00 |
Claims
1. A double-stranded ribonucleic acid (dsRNA) for inhibiting the
expression of a human Aha gene in a cell, wherein said dsRNA
comprises at least two sequences that are complementary to each
other and wherein a sense strand comprises a first sequence and an
antisense strand comprises a second sequence comprising a region of
complementarity which is substantially complementary to at least a
part of a mRNA encoding an Aha gene, and wherein said region of
complementarity is less than 30 nucleotides in length and wherein
the dsRNA effects cleavage of an mRNA encoding an Aha gene within
the target sequence of a second dsRNA having a sense strand chosen
from the group of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID
NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19,
SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID
NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID
NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,
SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID
NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,
SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID
NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95,
SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ
ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID
NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, and SEQ ID NO: 183, and an antisense strand
complementary to the latter sense strand and chosen from the group
of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID
NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98,
SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ
ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID
NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, and SEQ ID NO: 184.
2. The dsRNA of claim 1, wherein said Aha gene is an Aha1 gene, and
preferably a Homo sapiens Aha1 gene.
3. The dsRNA of claim 1, wherein, upon contact with a cell
expressing said Aha gene, the dsRNA inhibits expression of said Aha
gene in said cell by at least 20%.
4. The dsRNA of claim 3, wherein said at least 20% inhibition of
expression of an Aha gene is effected in HeLa and/or MLE12
cells.
5. The dsRNA of claim 1, wherein the dsRNA is chosen from the group
of AL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322,
AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329,
AL-DP-7331, AL-DP-7333, AL-DP-7340, AL-DP-7342, AL-DP-7303,
AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316, and AL-DP-7337,
AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344, AL-DP-7306,
AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,
AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328,
AL-DP-7330, AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251,
AL-DP-9252, AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256,
AL-DP-9257, AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9261,
AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265, AL-DP-9266,
AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,
AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276,
AL-DP-9277, AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282,
AL-DP-9283, AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287,
AL-DP-9288, and AL-DP-9289.
6. The dsRNA of claim 1, wherein said dsRNA comprises at least one
modified nucleotide.
7. The dsRNA of claim 6, 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.
8. The dsRNA of claim 6, 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.
9. A cell comprising the dsRNA of claim 1.
10. A pharmaceutical composition for inhibiting the expression of
an Aha gene in an organism, comprising a dsRNA and a
pharmaceutically acceptable carrier, wherein the dsRNA comprises at
least two sequences that are complementary to each other and
wherein a sense strand comprises a first sequence and an antisense
strand comprises a second sequence comprising a region of
complementarity which is substantially complementary to at least a
part of a mRNA encoding an Aha gene, and wherein said region of
complementarity is less than 30 nucleotides in length, and wherein
the dsRNA effects cleavage of an mRNA encoding an Aha gene within
the target sequence of a second dsRNA having a sense strand chosen
from the group of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID
NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19,
SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID
NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID
NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,
SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID
NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,
SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID
NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95,
SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ
ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID
NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, and SEQ ID NO: 183, and an antisense strand
complementary to the latter sense strand and chosen from the group
of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: SO.
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID
NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98,
SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ
ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID
NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, and SEQ ID NO: 184.
11. The pharmaceutical composition of claim 10, wherein said Aha
gene is an Aha1 gene, and preferably a Homo sapiens Aha1 gene.
12. The pharmaceutical composition of claim 10, wherein, upon
contact with a cell expressing said Aha gene, the dsRNA inhibits
expression of said Aha gene in said cell by at least 20%.
13. The pharmaceutical composition of claim 12, wherein said at
least 20% inhibition of expression of an Aha gene is effected in
HeLa and/or MLE12 cells.
14. The pharmaceutical composition of claim 10, wherein the dsRNA
is chosen from the group of AL-DP-7301, AL-DP-7308, AL-DP-7318,
AL-DP-7320, AL-DP-7322, AL-DP-7324, AL-DP-7325, AL-DP-7326,
AL-DP-7327, AL-DP-7329, AL-DP-7331, AL-DP-7333, AL-DP-7340,
AL-DP-7342, AL-DP-7303, AL-DP-7305, AL-DP-7307, AL-DP-7309,
AL-DP-7316, and AL-DP-7337, AL-DP-7304, AL-DP-7312, AL-DP-7339,
AL-DP-7344, AL-DP-7306, AL-DP-7317, AL-DP-7346, AL-DP-7310,
AL-DP-7323, AL-DP-7335, AL-DP-7338, AL-DP-7341, AL-DP-7302,
AL-DP-7315, AL-DP-7328, AL-DP-7330, AL-DP-7336, AL-DP-7345,
AL-DP-9250, AL-DP-9251, AL-DP-9252, AL-DP-9253, AL-DP-9254,
AL-DP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259,
AL-DP-9260, AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9264,
AL-DP-9265, AL-DP-9266, AL-DP-9267, AL-DP-9268, AL-DP-9269,
AL-DP-9270, AL-DP-9271, AL-DP-9272, AL-DP-9273, AL-DP-9274,
AL-DP-9275, AL-DP-9276, AL-DP-9277, AL-DP-9279, AL-DP-9280,
AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-DP-9284, AL-DP-9285,
AL-DP-9286, AL-DP-9287, AL-DP-9288, and AL-DP-9289.
15. The pharmaceutical composition of claim 10 wherein said dsRNA
comprises at least one modified nucleotide.
16. The pharmaceutical composition of claim 15, 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.
17. The pharmaceutical composition of claim 15, 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.
18. A method for inhibiting the expression of an Aha gene in a
cell, the method comprising: (a) introducing into the cell the
dsRNA of claim 1; and (b) maintaining the cell produced in step (a)
for a time sufficient to obtain degradation of the mRNA transcript
of an Aha gene, thereby inhibiting expression of an Aha gene in the
cell.
19. A method of treating, preventing or managing pathological
processes mediated by Aha expression comprising administering to a
patient in need of such treatment, prevention or management a
therapeutically or prophylactically effective amount of the dsRNA
of claim 1.
20. A vector for inhibiting the expression of an Aha gene in a
cell, said vector comprising a regulatory sequence operably linked
to a nucleotide sequence that encodes at least one strand of a
dsRNA, wherein one of the strands of said dsRNA is substantially
complementary to at least a part of a mRNA encoding Aha1 and
wherein said dsRNA is less than 30 base pairs in length and wherein
the dsRNA effects cleavage of an mRNA encoding an Aha gene within
the target sequence of a second dsRNA having a sense strand chosen
from the group of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID
NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19,
SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID
NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
SEQ ID NO: 43. SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID
NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,
SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID
NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,
SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID
NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95,
SHQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ
ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 1 II. SEQ ID
NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, and SEQ ID NO: 183, and an antisense strand
complementary to the latter sense strand and chosen from the group
of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID
NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98,
SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ
ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID
NO: 116, SEQ ID NO. 118. SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO
124, SEQ ID NO. 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, and SEQ ID NO: 184.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. patent application
Ser. No. 12/877,025, filed Sep. 7, 2010, which is a divisional of
U.S. patent application Ser. No. 11/750,553, filed May 18, 2007
(now U.S. Pat. No. 7,812,150), which claims the benefit of U.S.
Provisional Application No. 60/801,840, filed May 19, 2006, each of
which are hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The present invention concerns methods of treatment using
modulators of the gene Activator of Heat Shock Protein 90 ATPase
(Aha). More specifically, the invention concerns methods of
treating disorders associated with undesired Aha activity, by
administering short interfering RNA which down-regulate the
expression of Aha, and agents useful therein.
BACKGROUND OF THE INVENTION
[0003] Activator of Heat Shock Protein 90 ATPase 1 (herein: Aha1)
is an activator of the ATPase-activity of Hsp90 and is able to
stimulate the inherent activity of yeast Hsp90 by 12-fold and human
Hsp90 by 50-fold (Panaretou, B., et al., Mol. Cell. 2002,
10:1307-1318). Biochemical studies have shown that Aha1 binds to
the middle region of Hsp90 (Panaretou et al., 2002, supra, Lotz, G.
P., et al., J. Biol. Chem. 2003, 278:17228-17235), and recent
structural studies of the Aha1-Hsp90 core complex suggest that the
co-chaperone promotes a conformational switch in the middle segment
catalytic loop (370-390) of Hsp90 that releases the catalytic
Arg380 and facilitates its interaction with ATP in the N-terminal
nucleotide-binding domain (Meyer, P., et al., EMBO J. 2004,
23:511-519).
[0004] The molecular chaperone Heat shock protein 90 (Hsp90) is
responsible for the in vivo activation or maturation of specific
client proteins (Picard, D., Cell Mol. Life. Sci. 2002,
59:1640-1648; Pearl, L. H., and Prodromou, C., Adv. Protein Chem.
2002, 59:157-185; Pratt, W. B., and Toft, D. O., Exp. Biol. Med.
2003, 228:111-133; Prodromou, C., and Pearl, L. H., Curr. Cancer
Drug Targets 2003, 3:301-323). Crucial to such activation is the
essential ATPase activity of Hsp90 (Panaretou, B., et al., EMBO J.
1998, 17:4829-4836), which drives a conformational cycle involving
transient association of the N-terminal nucleotide-binding domains
within the Hsp90 dimer (Prodromou, C., et al., EMBO J. 2000,
19:4383-4392).
[0005] As a molecular chaperone, HSP90 promotes the maturation and
maintains the stability of a large number of conformationally
labile client proteins, most of which are involved in biologic
processes that are often deranged within tumor cells, such as
signal transduction, cell-cycle progression and apoptosis. As a
result, and in contrast to other molecular targeted therapeutics,
inhibitors of HSP90 achieve promising anticancer activity through
simultaneous disruption of many oncogenic substrates within cancer
cells (Whitesell L, and Dai C., Future Oncol. 2005; 1:529-540; WO
03/067262). Furthermore, HSP90 has been implicated in the
degradation of Cystic Fibrosis Transmembrane Conductance Regulator
(CFTR). Mutations in the CFTR gene lead to defective folding and
ubiquination of the protein as a consequence of HSP90 ATPase
activity. Following ubiquitination, CFTR is degraded before it can
reach its site of activity. Lack of active CFTR then leads to the
development of cystic fibrosis in human subjects having such
mutation. Therefore, the inhibition of HSP90 activity may be
beneficial for subjects suffering from cancer or Cystic
Fibrosis.
[0006] Hsp90 constitutes about 1-2% of total cellular protein
(Pratt, W. B., Annu Rev. Pharmacol. Toxicol. 1997, 37:297-326), and
the inhibition of such large amounts of protein by means of an
antagonist or inhibitor would potentially require the introduction
of excessive amounts of the inhibitor or antagonist into a cell. An
alternative approach is the inhibition of activators of HSP90's
ATPase activity, such as Aha1, which are present in smaller
amounts. By downregulating the amount of Aha1 present in the cell,
the activity of HSP90 may be lowered substantially.
[0007] Significant sequence homology exists between Homo sapiens
(NM.sub.--012111.1), Mus musculus (NM.sub.--146036.1) and Pan
troglodytes (XM.sub.--510094.1) Aha 1. A clear rattus norvegicus
homologue of Aha 1 has not been identified; however, there is a
Rattus norvegicus (XM.sub.--223680.3) gene which has been termed
activator of heat shock protein ATPase homolog 2 (Ahsa 2) on the
basis of its sequence homology to yeast Ahsa 2. Its sequence is
homologous to mus musculus RIKEN cDNA 1110064P04 gene
(NM.sub.--172391.3), which is in turn similar in sequence to Aus
musculus Aha 1 except for N-terminal truncation. A homo sapiens
Ahsa 2 (NM.sub.--152392.1) has also been predicted, but sequence
homology is limited. The functions of these latter three genes have
not been sufficiently elucidated. However, there exists one region
in which all of the above sequences are identical, and which may be
used as the target for RNAi agents. It may be advantageous to
inhibit the activity of more than one Aha gene.
[0008] 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 genes 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.
[0009] Despite significant advances in the field of RNAi and
advances in the treatment of pathological processes mediated by
HSP90, there remains a need for an agent that can selectively and
efficiently attenuate HSP90 ATPase activity using the cell's own
RNAi machinery. Such agent shall possess both high biological
activity and in vivo stability, and shall effectively inhibit
expression of a target Aha gene, such as Aha1, for use in treating
pathological processes mediated directly or indirectly by Aha
expression, e.g. Aha1 expression.
SUMMARY OF THE INVENTION
[0010] The invention provides double-stranded ribonucleic acid
(dsRNA), as well as compositions and methods for inhibiting the
expression of an Aha gene in a cell or mammal using such dsRNA. The
invention also provides compositions and methods for treating
pathological conditions and diseases mediated by the expression of
an Aha gene, such as in cancer or cystic fibrosis. The dsRNA of the
invention comprises an RNA strand (the antisense strand) having a
region which is less than 30 nucleotides in length, generally 19-24
nucleotides in length, and is substantially complementary to at
least part of an mRNA transcript of an Aha gene.
[0011] In one aspect, the invention provides double-stranded
ribonucleic acid (dsRNA) molecules for inhibiting the expression of
an Aha 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 an Aha gene, and the region of complementarity is
less than 30 nucleotides in length, generally 19-24 nucleotides in
length. The dsRNA effects cleavage of an mRNA encoding an Aha gene
within the target sequence of a second dsRNA having a sense strand
chosen from the group of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,
SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID
NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29,
SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID
NO: 39, SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49,
SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID
NO: 59, SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67,
SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID
NO: 77, SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85,
SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID
NO: 95, SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO:
103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO:
111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO:
119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO:
127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO:
135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO:
143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO:
151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO:
159, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO:
169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO:
177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO: 183, and an
antisense strand complementary to the latter sense strand and
chosen from the group of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,
SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID
NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30,
SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID
NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50,
SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID
NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68,
SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID
NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86,
SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID
NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO:
104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ ID NO:
112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID NO:
120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, and SEQ ID NO: 184 (see Table
1 and Table 2). The Aha gene is preferably an Aha1 gene, and more
preferably a Homo sapiens Aha1 gene. The dsRNA, upon contacting
with a cell expressing the Aha gene, may inhibit the expression of
the Aha gene in said cell by at least 20%, or at least 25%, 30%,
35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 85%, 90% or 95%, e.g. in
HeLa and/or MLE 12 cells. The dsRNA may be different from said
second dsRNA, but may have at least 5, at least 10, at least 15, at
least 18, or at least 20 contiguous nucleotides per strand in
common with one of the above named nucleotide sequences.
[0012] Preferably, the second dsRNA is chosen from the group of
AL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322,
AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329,
AL-DP-7331, AL-DP-7333, AL-DP-7340, AL-DP-7342, AL-DP-7303,
AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316, and AL-DP-7337,
AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344, AL-DP-7306,
AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,
AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328,
AL-DP-7330, AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251,
AL-DP-9252, AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256,
AL-DP-9257, AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9261,
AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265, AL-DP-9266,
AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,
AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276,
AL-DP-9277, AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282,
AL-DP-9283, AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287,
AL-DP-9288, and AL-DP-9289 (see Table 1 and Table 2).
[0013] Alternatively, the dsRNA itself may be chosen from the group
of AL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322,
AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329,
AL-DP-7331, AL-DP-7333, AL-DP-7340, AL-DP-7342, AL-DP-7303,
AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316, and AL-DP-7337,
AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344, AL-DP-7306,
AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,
AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328,
AL-DP-7330, AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251,
AL-DP-9252, AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256,
AL-DP-9257, AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9261,
AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265, AL-DP-9266,
AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,
AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276,
AL-DP-9277, AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282,
AL-DP-9283, AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287,
AL-DP-9288, and AL-DP-9289 (see Table 1 and Table 2).
[0014] The dsRNA may comprise at least one modified nucleotide.
Preferably, the 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.
Alternatively, the 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.
[0015] In another aspect, the invention provides an isolated cell
comprising one of the dsRNAs of the invention. The cell is
generally a mammalian cell, such as a human cell. Other embodiments
of the cell comprising a dsRNA of the invention are as provided for
other aspects of the invention above.
[0016] In yet another aspect, a pharmaceutical composition for
inhibiting the expression of an Aha gene in an organism is
provided, comprising a dsRNA and a pharmaceutically acceptable
carrier, wherein the dsRNA comprises at least two sequences that
are complementary to each other and wherein a sense strand
comprises a first sequence and an antisense strand comprises a
second sequence comprising a region of complementarity which is
substantially complementary to at least a part of a mRNA encoding
an Aha gene, and wherein said region of complementarity is less
than 30 nucleotides in length, and wherein the dsRNA effects
cleavage of an mRNA encoding an Aha gene within the target sequence
of a second dsRNA having a sense strand chosen from the group of
SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO:
13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ
ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO:
33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ
ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO:
53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ
ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO:
71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ
ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO:
89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ
ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID
NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO:
115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:
123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:
131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO:
139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:
147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:
155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO:
165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO:
173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO:
181, and SEQ ID NO: 183, and an antisense strand complementary to
the latter sense strand and chosen from the group of SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46,
SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID
NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64,
SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID
NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82,
SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID
NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO:
100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO:
108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO:
116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, and SEQ ID NO: 184 (see Table 1 and Table 2). Therein, the Aha
gene may be an Aha1 gene, and preferably a Homo sapiens Aha1 gene.
The dsRNA comprised in the pharmaceutical composition may, upon
contact with a cell expressing said Aha gene, inhibit the
expression of said Aha gene in said cell by at least 20%, or at
least 25%, 30%, 35%, 40%, 45%, 50%, 55% 60%, 65%, 70%, 85%, 90% or
95%, e.g. in HeLa and/or MLE 12 cells. The dsRNA may be different
from said second dsRNA, but may have at least 5, at least 10, at
least 15, at least 18, or at least 20 contiguous nucleotides per
strand in common with one of the above named nucleotide
sequences.
[0017] Preferably, the second dsRNA is chosen from the group of
AL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322,
AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329,
AL-DP-7331, AL-DP-7333, AL-DP-7340, AL-DP-7342, AL-DP-7303,
AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316, and AL-DP-7337,
AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344, AL-DP-7306,
AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,
AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328,
AL-DP-7330, AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251,
AL-DP-9252, AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256,
AL-DP-9257, AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9261,
AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265, AL-DP-9266,
AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,
AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276,
AL-DP-9277, AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282,
AL-DP-9283, AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287,
AL-DP-9288, and AL-DP-9289 (see Table 1 and Table 2).
[0018] Alternatively, the dsRNA comprised in the pharmaceutical
composition itself may be chosen from the group of AL-DP-7301,
AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322, AL-DP-7324,
AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329, AL-DP-7331,
AL-DP-7333, AL-DP-7340, AL-DP-7342, AL-DP-7303, AL-DP-7305,
AL-DP-7307, AL-DP-7309, AL-DP-7316, and AL-DP-7337, AL-DP-7304,
AL-DP-7312, AL-DP-7339, AL-DP-7344, AL-DP-7306, AL-DP-7317,
AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335, AL-DP-7338,
AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328, AL-DP-7330,
AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251, AL-DP-9252,
AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256, AL-DP-9257,
AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9261, AL-DP-9262,
AL-DP-9263, AL-DP-9264, AL-DP-9265, AL-DP-9266, AL-DP-9267,
AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271, AL-DP-9272,
AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276, AL-DP-9277,
AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282, AL-DP-9283,
AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287, AL-DP-9288, and
AL-DP-9289 (see Table 1 and Table 2).
[0019] The dsRNA comprised in the pharmaceutical composition may
comprise at least one modified nucleotide. Preferably, 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. Alternatively, 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.
[0020] In yet another aspect, a method for inhibiting the
expression of an Aha gene in a cell is provded, the method
comprising:
(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 and wherein a sense strand
comprises a first sequence and an antisense strand comprises a
second sequence comprising a region of complementarity which is
substantially complementary to at least a part of a mRNA encoding
Aha1, and wherein said region of complementarity is less than 30
nucleotides in length and wherein the dsRNA effects cleavage of an
mRNA encoding an Aha gene within the target sequence of a second
dsRNA having a sense strand chosen from the group of SEQ ID NO: 5,
SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID
NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23,
SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33, SEQ ID
NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID NO: 45,
SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53, SEQ ID
NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID NO: 63,
SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71, SEQ ID
NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID NO: 81,
SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID
NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID NO: 99,
SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO: 107, SEQ
ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO: 115, SEQ ID
NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 123, SEQ ID NO:
125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO: 131, SEQ ID NO:
133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO: 139, SEQ ID NO:
141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO: 147, SEQ ID NO:
149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO: 155, SEQ ID NO:
157, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO: 165, SEQ ID NO:
167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO: 173, SEQ ID NO:
175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO: 181, and SEQ ID NO:
183, and an antisense strand complementary to the latter sense
strand and chosen from the group of SEQ ID NO: 6, SEQ ID NO: 8, SEQ
ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO:
18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 28, SEQ
ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO:
38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ
ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID NO: 56, SEQ ID NO:
58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64, SEQ ID NO: 66, SEQ
ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO:
76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82, SEQ ID NO: 84, SEQ
ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID NO: 92, SEQ ID NO:
94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102,
SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108, SEQ ID NO: 110, SEQ
ID NO: 112, SEQ ID NO: 114, SEQ ID NO: 116, SEQ ID NO: 118, SEQ ID
NO: 120, SEQ ID NO: 122, SEQ ID NO: 124, SEQ ID NO: 126, SEQ ID NO:
128, SEQ ID NO: 130, SEQ ID NO: 132, SEQ ID NO: 134, SEQ ID NO:
136, SEQ ID NO: 138, SEQ ID NO: 140, SEQ ID NO: 142, SEQ ID NO:
144, SEQ ID NO: 146, SEQ ID NO: 148, SEQ ID NO: 150, SEQ ID NO:
152, SEQ ID NO: 154, SEQ ID NO: 156, SEQ ID NO: 158, SEQ ID NO:
160, SEQ ID NO: 164, SEQ ID NO: 166, SEQ ID NO: 168, SEQ ID NO:
170, SEQ ID NO: 172, SEQ ID NO: 174, SEQ ID NO: 176, SEQ ID NO:
178, SEQ ID NO: 180, SEQ ID NO: 182, and SEQ ID NO: 184; and (b)
maintaining the cell produced in step (a) for a time sufficient to
obtain degradation of the mRNA transcript of an Aha gene, thereby
inhibiting expression of an Aha gene in the cell. The Aha gene is
preferably an Aha1 gene, and more preferably a homo sapiens Aha1
gene. The dsRNA may be different from said second dsRNA, but may
have at least 5, at least 10, at least 15, at least 18, or at least
20 contiguous nucleotides per strand in common with one of the
above named nucleotide sequences.
[0021] Preferably, the second dsRNA is chosen from the group of
AL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322,
AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329,
AL-DP-7331, AL-DP-7333, AL-DP-7340, AL-DP-7342, AL-DP-7303,
AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316, and AL-DP-7337,
AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344, AL-DP-7306,
AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,
AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328,
AL-DP-7330, AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251,
AL-DP-9252, AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256,
AL-DP-9257, AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9261,
AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265, AL-DP-9266,
AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,
AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276,
AL-DP-9277, AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282,
AL-DP-9283, AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287,
AL-DP-9288, and AL-DP-9289 (see Table 1 and Table 2).
[0022] Alternatively, the dsRNA itself is chosen from the group of
AL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322,
AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329,
AL-DP-7331, AL-DP-7333, AL-DP-7340, AL-DP-7342, AL-DP-7303,
AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316, and AL-DP-7337,
AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344, AL-DP-7306,
AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,
AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328,
AL-DP-7330, AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251,
AL-DP-9252, AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256,
AL-DP-9257, AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9261,
AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265, AL-DP-9266,
AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,
AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276,
AL-DP-9277, AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282,
AL-DP-9283, AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287,
AL-DP-9288, and AL-DP-9289. Preferably, the method is performed in
vitro. Other embodiments of the method for inhibiting the
expression of an Aha gene in a cell are as provided for other
aspects of the invention above.
[0023] In yet another aspect, a method of treating, preventing or
managing pathological processes mediated by Aha expression is
provided, comprising administering to a patient in need of such
treatment, prevention or management a therapeutically or
prophylactically effective amount of a dsRNA, wherein the dsRNA
comprises at least two sequences that are complementary to each
other and wherein a sense strand comprises a first sequence and an
antisense strand comprises a second sequence comprising a region of
complementarity which is substantially complementary to at least a
part of a mRNA encoding Aha1, and wherein said region of
complementarity is less than 30 nucleotides in length and wherein
the dsRNA effects cleavage of an mRNA encoding an Aha gene within
the target sequence of a second dsRNA having a sense strand chosen
from the group of SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID
NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19,
SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID
NO: 31, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39,
SEQ ID NO: 43, SEQ ID NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID
NO: 51, SEQ ID NO: 53, SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59,
SEQ ID NO: 61, SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID
NO: 69, SEQ ID NO: 71, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77,
SEQ ID NO: 79, SEQ ID NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID
NO: 87, SEQ ID NO: 89, SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95,
SEQ ID NO: 97, SEQ ID NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ
ID NO: 105, SEQ ID NO: 107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID
NO: 113, SEQ ID NO: 115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO:
121, SEQ ID NO: 123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO:
129, SEQ ID NO: 131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO:
137, SEQ ID NO: 139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO:
145, SEQ ID NO: 147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO:
153, SEQ ID NO: 155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO:
163, SEQ ID NO: 165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO:
171, SEQ ID NO: 173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO:
179, SEQ ID NO: 181, and SEQ ID NO: 183, and an antisense strand
complementary to the latter sense strand and chosen from the group
of SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID
NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22,
SEQ ID NO: 24, SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID
NO: 34, SEQ ID NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44,
SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID
NO: 54, SEQ ID NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62,
SEQ ID NO: 64, SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID
NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80,
SEQ ID NO: 82, SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID
NO: 90, SEQ ID NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98,
SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ
ID NO: 108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID
NO: 116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, and SEQ ID NO: 184. The dsRNA may be different from said
second dsRNA, but may have at least 5, at least 10, at least 15, at
least 18, or at least 20 contiguous nucleotides per strand in
common with one of the above named nucleotide sequences.
[0024] Preferably, the second dsRNA is chosen from the group of
AL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322,
AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329,
AL-DP-7331, AL-DP-7333, AL-DP-7340, AL-DP-7342, AL-DP-7303,
AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316, and AL-DP-7337,
AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344, AL-DP-7306,
AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,
AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328,
AL-DP-7330, AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251,
AL-DP-9252, AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256,
AL-DP-9257, AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9261,
AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265, AL-DP-9266,
AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,
AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276,
AL-DP-9277, AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282,
AL-DP-9283, AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287,
AL-DP-9288, and AL-DP-9289 (see Table 1 and Table 2).
[0025] Alternatively, the dsRNA itself is chosen from the group of
AL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322,
AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329,
AL-DP-7331, AL-DP-7333, AL-DP-7340, AL-DP-7342, AL-DP-7303,
AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316, and AL-DP-7337,
AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344, AL-DP-7306,
AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,
AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328,
AL-DP-7330, AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251,
AL-DP-9252, AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256,
AL-DP-9257, AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9261,
AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265, AL-DP-9266,
AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,
AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276,
AL-DP-9277, AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282,
AL-DP-9283, AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287,
AL-DP-9288, and AL-DP-9289. Other embodiments of the method
comprising administering a dsRNA of the invention are as provided
for other aspects of the invention above.
[0026] In yet another aspect, a vector for inhibiting the
expression of an Aha gene in a cell is provided, said vector
comprising a regulatory sequence operably linked to a nucleotide
sequence that encodes at least one strand of a dsRNA, wherein one
of the strands of said dsRNA is substantially complementary to at
least a part of a mRNA encoding Aha1 and wherein said dsRNA is less
than 30 base pairs in length and wherein the dsRNA effects cleavage
of an mRNA encoding an Aha gene within the target sequence of a
second dsRNA having a sense strand chosen from the group of SEQ ID
NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13,
SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID
NO: 23, SEQ ID NO: 27, SEQ ID NO: 29, SEQ ID NO: 31, SEQ ID NO: 33,
SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 39, SEQ ID NO: 43, SEQ ID
NO: 45, SEQ ID NO: 47, SEQ ID NO: 49, SEQ ID NO: 51, SEQ ID NO: 53,
SEQ ID NO: 55, SEQ ID NO: 57, SEQ ID NO: 59, SEQ ID NO: 61, SEQ ID
NO: 63, SEQ ID NO: 65, SEQ ID NO: 67, SEQ ID NO: 69, SEQ ID NO: 71,
SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, SEQ ID NO: 79, SEQ ID
NO: 81, SEQ ID NO: 83, SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89,
SEQ ID NO: 91, SEQ ID NO: 93, SEQ ID NO: 95, SEQ ID NO: 97, SEQ ID
NO: 99, SEQ ID NO: 101, SEQ ID NO: 103, SEQ ID NO: 105, SEQ ID NO:
107, SEQ ID NO: 109, SEQ ID NO: 111, SEQ ID NO: 113, SEQ ID NO:
115, SEQ ID NO: 117, SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO:
123, SEQ ID NO: 125, SEQ ID NO: 127, SEQ ID NO: 129, SEQ ID NO:
131, SEQ ID NO: 133, SEQ ID NO: 135, SEQ ID NO: 137, SEQ ID NO:
139, SEQ ID NO: 141, SEQ ID NO: 143, SEQ ID NO: 145, SEQ ID NO:
147, SEQ ID NO: 149, SEQ ID NO: 151, SEQ ID NO: 153, SEQ ID NO:
155, SEQ ID NO: 157, SEQ ID NO: 159, SEQ ID NO: 163, SEQ ID NO:
165, SEQ ID NO: 167, SEQ ID NO: 169, SEQ ID NO: 171, SEQ ID NO:
173, SEQ ID NO: 175, SEQ ID NO: 177, SEQ ID NO: 179, SEQ ID NO:
181, and SEQ ID NO: 183, and an antisense strand complementary to
the latter sense strand and chosen from the group of SEQ ID NO: 6,
SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID
NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24,
SEQ ID NO: 28, SEQ ID NO: 30, SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID
NO: 36, SEQ ID NO: 38, SEQ ID NO: 40, SEQ ID NO: 44, SEQ ID NO: 46,
SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, SEQ ID NO: 54, SEQ ID
NO: 56, SEQ ID NO: 58, SEQ ID NO: 60, SEQ ID NO: 62, SEQ ID NO: 64,
SEQ ID NO: 66, SEQ ID NO: 68, SEQ ID NO: 70, SEQ ID NO: 72, SEQ ID
NO: 74, SEQ ID NO: 76, SEQ ID NO: 78, SEQ ID NO: 80, SEQ ID NO: 82,
SEQ ID NO: 84, SEQ ID NO: 86, SEQ ID NO: 88, SEQ ID NO: 90, SEQ ID
NO: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO:
100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO:
108, SEQ ID NO: 110, SEQ ID NO: 112, SEQ ID NO: 114, SEQ ID NO:
116, SEQ ID NO: 118, SEQ ID NO: 120, SEQ ID NO: 122, SEQ ID NO:
124, SEQ ID NO: 126, SEQ ID NO: 128, SEQ ID NO: 130, SEQ ID NO:
132, SEQ ID NO: 134, SEQ ID NO: 136, SEQ ID NO: 138, SEQ ID NO:
140, SEQ ID NO: 142, SEQ ID NO: 144, SEQ ID NO: 146, SEQ ID NO:
148, SEQ ID NO: 150, SEQ ID NO: 152, SEQ ID NO: 154, SEQ ID NO:
156, SEQ ID NO: 158, SEQ ID NO: 160, SEQ ID NO: 164, SEQ ID NO:
166, SEQ ID NO: 168, SEQ ID NO: 170, SEQ ID NO: 172, SEQ ID NO:
174, SEQ ID NO: 176, SEQ ID NO: 178, SEQ ID NO: 180, SEQ ID NO:
182, and SEQ ID NO: 184. The dsRNA may be different from said
second dsRNA, but may have at least 5, at least 10, at least 15, at
least 18, or at least 20 contiguous nucleotides per strand in
common with one of the above named nucleotide sequences.
[0027] Preferably, the second dsRNA is chosen from the group of
AL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320, AL-DP-7322,
AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327, AL-DP-7329,
AL-DP-7331, AL-DP-7333, AL-DP-7340, AL-DP-7342, AL-DP-7303,
AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316, and AL-DP-7337,
AL-DP-7304, AL-DP-7312, AL-DP-7339, AL-DP-7344, AL-DP-7306,
AL-DP-7317, AL-DP-7346, AL-DP-7310, AL-DP-7323, AL-DP-7335,
AL-DP-7338, AL-DP-7341, AL-DP-7302, AL-DP-7315, AL-DP-7328,
AL-DP-7330, AL-DP-7336, AL-DP-7345, AL-DP-9250, AL-DP-9251,
AL-DP-9252, AL-DP-9253, AL-DP-9254, AL-DP-9255, AL-DP-9256,
AL-DP-9257, AL-DP-9258, AL-DP-9259, AL-DP-9260, AL-DP-9261,
AL-DP-9262, AL-DP-9263, AL-DP-9264, AL-DP-9265, AL-DP-9266,
AL-DP-9267, AL-DP-9268, AL-DP-9269, AL-DP-9270, AL-DP-9271,
AL-DP-9272, AL-DP-9273, AL-DP-9274, AL-DP-9275, AL-DP-9276,
AL-DP-9277, AL-DP-9279, AL-DP-9280, AL-DP-9281, AL-DP-9282,
AL-DP-9283, AL-DP-9284, AL-DP-9285, AL-DP-9286, AL-DP-9287,
AL-DP-9288, and AL-DP-9289 (see Table 1 and Table 2). Other
embodiments of the vector of the invention are as provided for
other aspects of the invention above.
[0028] In yet another aspect, an isolated cell comprising the above
vector is provided. Other embodiments of the cell comprising a
vector of the invention are as provided for other aspects of the
invention above.
TABLE-US-00001 TABLE 1 RNAi agents for the down-regulation of homo
sapiens (NM_012111.1), mus musculus (N.M_146036.1) and pan
troglodytes (XM_510094.1) Aha 1, and minimal off- target
interactions in rat cells; AL-DP-7561, AL-DP-7562, AL-DP-7563 and
AL-DP-7564 are additionally cross-reactive to mus musculus
(NM_172391.3) and rattus norvegicus (XM_223680.3) Aha 2 SEQ Duplex
ID Antisense strand SEQ ID identifier Sense strand sequence.sup.1
NO: sequence.sup.1 NO: AL-DP-7299 auugguccacggauaagcuTT 1
agcuuauccguggaccaauTT 2 AL-DP-7300 gugaguaagcuugauggagTT 3
cuccaucaagcuuacucacTT 4 AL-DP-7301 agucaaaauccccacuuguTT 5
acaaguggggauuuugacuTT 6 AL-DP-7302 aaaucucguggccuuaaugTT 7
cauuaaggccacgagauuuTT 8 AL-DP-7303 gagauuagugugagccuugTT 9
caaggcucacacuaaucucTT 10 AL-DP-7304 aaucucguggccuuaaugaTT 11
ucauuaaggccacgagauuTT 12 AL-DP-7305 agauuagugugagccuugcTT 13
gcaaggcucacacuaaucuTT 14 AL-DP-7306 cgggcggacgccaccaacgTT 15
cguugguggcguccgcccgTT 16 AL-DP-7307 ggcggacgccaccaacgucTT 17
gacguugguggcguccgccTT 18 AL-DP-7308 gggcggacgccaccaacguTT 19
acguugguggcguccgcccTT 20 AL-DP-7309 caacgucaacaacuggcacTT 21
gugccaguuguugacguugTT 22 AL-DP-7310 gcgggcggacgccaccaacTT 23
guugguggcguccgcccgcTT 24 AL-DP-7311 aucucguggccuuaaugaaTT 25
uucauuaaggccacgagauTT 26 AL-DP-7312 acgucaacaacuggcacugTT 27
cagugccaguuguugacguTT 28 AL-DP-7313 accaacgucaacaacuggcTT 29
gccaguuguugacguugguTT 30 AL-DP-7314 acgcuggaucguggaggagTT 31
cuccuccacgauccagcguTT 32 AL-DP-7315 agacccacgcuggaucgugTT 33
cacgauccagcgugggucuTT 34 AL-DP-7316 gacccacgcuggaucguggTT 35
ccacgauccagcgugggucTT 36 AL-DP-7317 gaauuuacaucagcacccuTT 37
agggugcugauguaaauucTT 38 AL-DP-7318 gggaauuuacaucagcaccTT 39
ggugcugauguaaauucccTT 40 AL-DP-7319 ugggaauuuacaucagcacTT 41
gugcugauguaaauucccaTT 42 AL-DP-7320 ccaacgucaacaacuggcaTT 43
ugccaguuguugacguuggTT 44 AL-DP-7321 aaguggggugagggagaccTT 45
ggucucccucaccccacuuTT 46 AL-DP-7322 acacaaaucucguggccuuTT 47
aaggccacgagauuuguguTT 48 AL-DP-7323 acccacgcuggaucguggaTT 49
uccacgauccagcguggguTT 50 AL-DP-7324 gagucaaaauccccacuugTT 51
caaguggggauuuugacucTT 52 AL-DP-7325 gagcucuauagaguguuuaTT 53
uaaacacucuauagagcucTT 54 AL-DP-7326 ggcagcgguacuacuuugaTT 55
ucaaaguaguaccgcugccTT 56 AL-DP-7327 gacacaaaucucguggccuTT 57
aggccacgagauuugugucTT 58 AL-DP-7328 agcgggcggacgccaccaaTT 59
uugguggcguccgcccgcuTT 60 AL-DP-7329 caaaauccccacuuguaagTT 61
cuuacaaguggggauuuugTT 62 AL-DP-7330 gagacccacgcuggaucguTT 63
acgauccagcgugggucucTT 64 AL-DP-7331 gagccuugccaaagaugagTT 65
cucaucuuuggcaaggcucTT 66 AL-DP-7332 ugacacaaaucucguggccTT 67
ggccacgagauuugugucaTT 68 AL-DP-7333 ggagcucuauagaguguuuTT 69
aaacacucuauagagcuccTT 70 AL-DP-7334 cccacgcuggaucguggagTT 71
cuccacgauccagcgugggTT 72 AL-DP-7335 gauccccaauuugucugauTT 73
aucagacaaauuggggaucTT 74 AL-DP-7336 gagauccccaauuugucugTT 75
cagacaaauuggggaucucTT 76 AL-DP-7337 agccugacacaaaucucguTT 77
acgagauuugugucaggcuTT 78 AL-DP-7338 agauccccaauuugucugaTT 79
ucagacaaauuggggaucuTT 80 AL-DP-7339 agggagacccacgcuggauTT 81
auccagcgugggucucccuTT 82 AL-DP-7340 gagggagacccacgcuggaTT 81
uccagcgugggucucccucTT 84 AL-DP-7341 gccaaguggggugagggagTT 85
cucccucaccccacuuggcTT 86 AL-DP-7342 uggcagcgguacuacuuugTT 87
caaaguaguaccgcugccaTT 88 AL-DP-7343 ugagggagacccacgcuggTT 89
ccagcgugggucucccucaTT 90 AL-DP-7344 aguggagauuagugugagcTT 91
gcucacacuaaucuccacuTT 92 AL-DP-7345 aggagcucuauagaguguuTT 93
aacacucuauagagcuccuTT 94 AL-DP-7346 agcgguacuacuuugagggTT 95
cccucaaaguaguaccgcuTT 96 AL-DP-7561 cgcuggaucguggaggagcTT 97
gcuccuccacgauccagcgTT 98 AL-DP-7562 gcuggaucguggaggagcgTT 99
cgcuccuccacgauccagcTT 100 AL-DP-7563 cuggaucguggaggagcggTT 101
ccgcuccuccacgauccagTT 102 AL-DP-7564 uggaucguggaggagcgggTT 103
cccgcuccuccacgauccaTT 104 .sup.1Capital letters =
desoxyribonucleotides; small letters = ribonucleotides
TABLE-US-00002 TABLE 2 RNAi agents for the down-regulation of homo
sapiens (NM_012111.1), mus musculus (NM_146036.1) and pan
troglodytes (XM_510094.1) Aha 1, and minimal off- target
interactions in human cells SEQ SEQ Duplex ID Antisense strand ID
identifier Sense strand sequence.sup.1 NO: sequence.sup.1 NO:
AL-DP-9250 gccugacacaaaucucgugTT 105 cacgagauuugugucaggcTT 106
AL-DP-9251 ccugacacaaaucucguggTT 107 ccacgagauuugugucaggTT 108
AL-DP-9252 acgccaccaacgucaacaaTT 109 uuguugacguugguggcguTT 110
AL-DP-9253 agcucuauagaguguuuacTT 111 guaaacacucuauagagcuTT 112
AL-DP-9254 gggcuggcagcgguacuacTT 113 guaguaccgcugccagcccTT 114
AL-DP-9255 cuggcagcgguacuacuuuTT 115 aaaguaguaccgcugccagTT 116
AL-DP-9256 ggaugaaguggagauuaguTT 117 acuaaucuccacuucauccTT 118
AL-OP-9257 accagaggagcucuauagaTT 119 ucuauagagcuccucugguTT 120
AL-DP-9258 aaguggagauuagugugagTT 121 cucacacuaaucuccacuuTT 122
AL-DP-9259 gaggagcucuauagaguguTT 123 acacucuauagagcuccucTT 124
AL-DP-9260 gggagacccacgcuggaucTT 125 gauccagcgugggucucccTT 126
AL-DP-9261 ugagccugacacaaaucucTT 127 gagauuugugucaggcucaTT 128
AL-DP-9262 gcggacgccaccaacgucaTT 129 ugacguugguggcguccgcTT 130
AL-DP-9263 cggacgccaccaacgucaaTT 131 uugacguugguggcguccgTT 132
AL-DP-9264 gaaguggagauuagugugaTT 133 ucacacuaaucuccacuucTT 134
AL-DP-9265 cucguggccuuaaugaaggTT 135 ccuucauuaaggccacgagTT 136
AL-DP-9266 ucguggccuuaaugaaggaTT 137 uccuucauuaaggccacgaTT 138
AL-DP-9267 aaugggaauuuacaucagcTT 139 gcugauguaaauucccauuTT 140
AL-DP-9268 ggaauuuacaucagcacccTT 141 gggugcugauguaaauuccTT 142
AL-DP-9269 ggagauuagugugagccuuTT 143 aaggcucacacuaaucuccTT 144
AL-DP-9270 cacaaaucucguggccuuaTT 145 uaaggccacgagauuugugTT 146
AL-DP-9271 acaaaucucguggccuuaaTT 147 uuaaggccacgagauuuguTT 148
AL-DP-9272 ggagacccacgcuggaucgTT 149 cgauccagcgugggucuccTT 150
AL-DP-9273 ggacgccaccaacgucaacTT 151 guugacguugguggcguccTT 152
AL-DP-9274 gaugaaguggagauuagugTT 153 cacuaaucuccacuucaucTT 154
AL-DP-9275 gugagccuugccaaagaugTT 155 caucuuuggcaaggcucacTT 156
AL-DP-9276 caaugaauggagagucaguTT 157 acugacucuccauucauugTT 158
AL-DP-9277 auuagugugagccuugccaTT 159 uggcaaggcucacacuaauTT 160
AL-DP-9278 agaugagccugacacaaauTT 161 auuugugucaggcucaucuTT 162
AL-DP-9279 uagugugagccuugccaaaTT 163 uuuggcaaggcucacacuaTT 164
AL-DP-9280 uuugccaccaucaccuugaTT 165 ucaaggugaugguggcaaaTT 166
AL-DP-9281 acggagagagaugcuucaaTT 167 uugaagcaucucucuccguTT 168
AL-DP-9282 cggagagagaugcuucaaaTT 169 uuugaagcaucucucuccgTT 170
AL-DP-9283 aaaauccccacuuguaagaTT 171 ucuuacaaguggggauuuuTT 172
AL-DP-9284 auccccaauuugucugaugTT 173 caucagacaaauuggggauTT 174
AL-DP-9285 ucaaaauccccacuuguaaTT 175 uuacaaguggggauuuugaTT 176
AL-DP-9286 aaauccccacuuguaagauTT 177 aucuuacaaguggggauuuTT 178
AL-DP-9287 uccccaauuugucugaugaTT 179 ucaucagacaaauuggggaTT 180
AL-DP-9288 auggccaaguggggugaggTT 181 ccucaccccacuuggccauTT 182
AL-DP-9289 ggagucaaaauccccacuuTT 183 aaguggggauuuugacuccTT 184
.sup.1Capital letters = desoxyribonucleotides; small letters =
ribonucleotides
BRIEF DESCRIPTION OF THE FIGURES
[0029] No Figures are presented.
DETAILED DESCRIPTION OF THE INVENTION
[0030] The invention provides double-stranded ribonucleic acid
(dsRNA), as well as compositions and methods for inhibiting the
expression of an Aha 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 an Aha gene using dsRNA. dsRNA directs the
sequence-specific degradation of mRNA through a process known as
RNA interference (RNAi).
[0031] The dsRNA of the invention comprises an RNA strand (the
antisense strand) having a region which is less than 30 nucleotides
in length, generally 19-24 nucleotides in length, and is
substantially complementary to at least part of an mRNA transcript
of an Aha gene. The use of these dsRNAs enables the targeted
degradation of mRNAs of genes that are implicated in replication
and or maintenance of cancer cells in mammals, and/or in the
degradation of misfolded Cystic Fibrosis Transmembrane Conductance
Regulator (CFTR). 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 an Aha gene. Thus, the
methods and compositions of the invention comprising these dsRNAs
are useful for treating pathological processes mediated by Aha
expression, e.g. cancer and/or cystic fibrosis, by targeting a gene
involved in protein degradation.
[0032] The following detailed description discloses how to make and
use the dsRNA and compositions containing dsRNA to inhibit the
expression of an Aha gene, as well as compositions and methods for
treating diseases and disorders caused by the expression of an Aha
gene, such as cancer and/or cystic fibrosis. 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, generally 19-24 nucleotides in length, and
is substantially complementary to at least part of an RNA
transcript of an Aha gene, together with a pharmaceutically
acceptable carrier.
[0033] 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 an Aha gene, and
methods of using the pharmaceutical compositions to treat diseases
caused by expression of an Aha gene.
DEFINITIONS
[0034] 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.
[0035] "G," "C," "A", "T" and "U" (irrespective of whether written
in capital or small letters) each generally stand for a nucleotide
that contains guanine, cytosine, adenine, thymine, 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, thymine, 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.
[0036] As used herein, "Aha gene" refers to Activator of Heat Shock
Protein 90 ATPase genes. "Aha1" refers to Activator of Heat Shock
Protein 90 ATPase 1 genes, non-exhaustive examples of which are
found under Genbank accession numbers NM.sub.--012111.1 (Homo
sapiens), NM.sub.--146036.1 (Mus musculus), and XM.sub.--510094.1
(Pan troglodytes). "Aha2" refers to putative Activator of Heat
Shock Protein 90 ATPase 2 genes, also known Ahsa2, non-exhaustive
examples of which may be found under Genbank accession numbers
NM.sub.--172391.3 (Mus musculus) and XM.sub.--223680.3 (Rattus
norvegicus).
[0037] As used herein, "target sequence" refers to a contiguous
portion of the nucleotide sequence of an mRNA molecule formed
during the transcription of an Aha gene, including mRNA that is a
product of RNA processing of a primary transcription product. The
target sequence of any given RNAi agent of the invention means an
mRNA-sequence of X nucleotides that is targeted by the RNAi agent
by virtue of the complementarity of the antisense strand of the
RNAi agent to such sequence and to which the antisense strand may
hybridize when brought into contact with the mRNA, wherein X is the
number of nucleotides in the antisense strand plus the number of
nucleotides in a single-stranded overhang of the sense strand, if
any.
[0038] 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.
[0039] 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.
[0040] 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 generally 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.
[0041] "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.
[0042] 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.
[0043] 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 Aha1).
For example, a polynucleotide is complementary to at least a part
of an Aha1 mRNA if the sequence is substantially complementary to a
non-interrupted portion of an mRNA encoding Aha1 .
[0044] The term "double-stranded RNA" or "dsRNA", as used herein,
refers to a 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 minus any overhangs that are present in the
duplex. In addition to the duplex structure, a dsRNA may comprise
one or more nucleotide overhangs.
[0045] 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 has no nucleotide overhang at either
end of the molecule.
[0046] 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
generally in a terminal region or regions, e.g., within 6, 5, 4, 3,
or 2 nucleotides of the 5' and/or 3' terminus. Most preferably, the
mismatches are located within 6, 5, 4, 3, or 2 nucleotides of the
5' terminus of the antisense strand and/or the 3' terminus of the
sense strand.
[0047] 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.
[0048] "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.
[0049] The terms "silence" and "inhibit the expression of", in as
far as they refer to an Aha gene, e.g. an Aha1 gene, herein refer
to the at least partial suppression of the expression of an Aha
gene, e.g. an Aha1 gene, as manifested by a reduction of the amount
of mRNA transcribed from an Aha gene which may be isolated from a
first cell or group of cells in which an Aha gene is transcribed
and which has or have been treated such that the expression of an
Aha 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).
Preferably, the cells are HeLa or MLE 12 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##
[0050] Alternatively, the degree of inhibition may be given in
terms of a reduction of a parameter that is functionally linked to
Aha gene transcription, e.g. the amount of protein encoded by an
Aha gene which is secreted by a cell, or found in solution after
lysis of such cells, or the number of cells displaying a certain
phenotype, e.g. apoptosis or cell surface CFTR. In principle, Aha
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 dsRNA inhibits the expression of an Aha
gene by a certain degree and therefore is encompassed by the
instant invention, the assays provided in the Examples below shall
serve as such reference.
[0051] For example, in certain instances, expression of an Aha
gene, e.g. an Aha1 gene, is suppressed by at least about 20%, 25%,
35%, or 50% by administration of the double-stranded
oligonucleotide of the invention. In some embodiment, an Aha gene,
e.g. an Aha1 gene, is suppressed by at least about 60%, 70%, or 80%
by administration of the double-stranded oligonucleotide of the
invention. In some embodiments, an Aha gene, e.g. an Aha1 gene, is
suppressed by at least about 85%, 90%, or 95% by administration of
the double-stranded oligonucleotide of the invention. Table 6
provides values for inhibition of Aha1 expression using various
dsRNA molecules of the invention.
[0052] As used herein in the context of Aha expression, e.g. Aha1
expression, the terms "treat", "treatment", and the like, refer to
relief from or alleviation of pathological processes mediated by
Aha expression. In the context of the present invention insofar as
it relates to any of the other conditions recited herein below
(other than pathological processes mediated by Aha expression), 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.
[0053] 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 pathological processes mediated by Aha expression
or an overt symptom of pathological processes mediated by Aha
expression. 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 pathological processes mediated by Aha expression, the patient's
history and age, the stage of pathological processes mediated by
Aha expression, and the administration of other anti-pathological
processes mediated by Aha expression agents.
[0054] 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.
[0055] 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.
[0056] As used herein, a "transformed cell" is a cell into which a
vector has been introduced from which a dsRNA molecule may be
expressed.
[0057] Double-Stranded Ribonucleic Acid (dsRNA)
[0058] In one embodiment, the invention provides double-stranded
ribonucleic acid (dsRNA) molecules for inhibiting the expression of
an Aha gene, e.g. an Aha1 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 an Aha gene, e.g. an Aha1 gene,
and wherein the region of complementarity is less than 30
nucleotides in length, generally 19-24 nucleotides in length. The
dsRNA may be identical to one of the dsRNAs shown in Table 1 and
Table 2, or it may effect cleavage of an mRNA encoding an Aha gene
within the target sequence of one of the dsRNAs shown in Table 1
and Table 2. Preferably, the dsRNA has at least 5, at least 10, at
least 15, at least 18, or at least 20 contiguous nucleotides per
strand in common with at least one strand, but preferably both
strands, of one of the dsRNAs shown in Table 1 and Table 2.
Alternative dsRNAs that target elsewhere in the target sequence of
one of the dsRNAs provided in Table 1 and Table 2 can readily be
determined using the target sequence and the flanking Aha1
sequence.
[0059] 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 generally
fully complementary, to a target sequence, derived from the
sequence of an mRNA formed during the expression of an Aha 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. Generally, the duplex structure is between 15 and 30,
more generally between 18 and 25, yet more generally between 19 and
24, and most generally between 19 and 21 base pairs in length.
Similarly, the region of complementarity to the target sequence is
between 15 and 30, more generally between 18 and 25, yet more
generally between 19 and 24, and most generally between 19 and 21
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, an
Aha gene is the human Aha1 gene. In specific embodiments, the first
strand of the dsRNA comprises the sense sequences of the RNAi
agents AL-DP-7301-AL-DP-7346 and AL-DP-7561-AL-DP-7564 (Table 1),
and AL-DP-9250-AL-DP-9289 (Table 2), and the second sequence is
selected from the group consisting of the antisense sequences of
AL-DP-7301-AL-DP-7346 and AL-DP-7561-AL-DP-7564 (Table 1), and
AL-DP-9250-AL-DP-9289 (Table 2).
[0060] In further embodiments, the dsRNA comprises at least one
nucleotide sequence selected from the groups of sequences provided
above for the RNAi agents AL-DP-7301-AL-DP-7346 and
AL-DP-7561-AL-DP-7564 (Table 1), and AL-DP-9250-AL-DP-9289 (Table
2). 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 an Aha gene, e.g. an Aha1 gene. Generally, the dsRNA comprises
two oligonucleotides, wherein one oligonucleotide may be described
as the sense strand in one of the RNAi agents AL-DP-7301-AL-DP-7346
and AL-DP-7561-AL-DP-7564 (Table 1), and AL-DP-9250-AL-DP-9289
(Table 2), and the second oligonucleotide may be described as the
antisense strand in one of the RNAi agents AL-DP-7301-AL-DP-7346
and AL-DP-7561-AL-DP-7564 (Table 1), and AL-DP-9250-AL-DP-9289
(Table 2).
[0061] 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 for the RNAi agents
AL-DP-7301-AL-DP-7346 and AL-DP-7561-AL-DP-7564 (Table 1), and
AL-DP-9250-AL-DP-9289 (Table 2), 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 provided herein for the RNAi agents AL-DP-7301-AL-DP-7346
and AL-DP-7561-AL-DP-7564 (Table 1), and AL-DP-9250-AL-DP-9289
(Table 2), 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 the RNAi agents AL-DP-7301-AL-DP-7346 and
AL-DP-7561-AL-DP-7564 (Table 1), and AL-DP-9250-AL-DP-9289 (Table
2), and differing in their ability to inhibit the expression of an
Aha gene, e.g. an Aha1 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.
[0062] Further dsRNAs that cleave within the target sequence of the
RNAi agents AL-DP-7301-AL-DP-7346 and AL-DP-7561-AL-DP-7564 (Table
1), and AL-DP-9250-AL-DP-9289 (Table 2), can readily be made using
the Aha1 gene sequence and the respective target sequence. The RNAi
agents provided in Table 1 and Table 2 identify a site in the Aha1
mRNA that is susceptible to RNAi based cleavage. As such the
present invention includes RNAi agents that target within the
sequence targeted by one of the agents of the present invention. As
used herein a dsRNA is said to target within the sequence of a
second dsRNA if the dsRNA cleaves the message anywhere within the
mRNA that is complementary to the antisense strand of the second
dsRNA. Such a dsRNA will generally have least 5, at least 10, at
least 15, at least 18, or at least 20 contiguous nucleotides from
one of the sequences provided in Table 1 and Table 2 coupled to
additional nucleotide sequences taken from the region contiguous to
the selected sequence in an mRNA encoding an Aha gene. For example,
the 3'-most 15 nucleotides of the target sequence of AL-DP-7301
combined with the next 6 nucleotides from the target Aha1 gene
produces a single strand agent of 21 nucleotides that is based on
one of the sequences provided in Table 1 and Table 2.
[0063] Preferably, the second dsRNA is chosen from the group of
dsRNAs having a certain activity in inhibiting the expression of an
Aha gene in a suitable assay, such as the assays described herein.
Consequently, in certain preferred ambodiments, the second dsRNA is
chosen from the group of AL-DP-7301, AL-DP-7308, AL-DP-7318,
AL-DP-7320, AL-DP-7322, AL-DP-7324, AL-DP-7325, AL-DP-7326,
AL-DP-7327, AL-DP-7329, AL-DP-7331, AL-DP-7333, AL-DP-7340,
AL-DP-7342, AL-DP-7303, AL-DP-7305, AL-DP-7307, AL-DP-7309,
AL-DP-7316, and AL-DP-7337, AL-DP-7304, AL-DP-7312, AL-DP-7339,
AL-DP-7344, AL-DP-7306, AL-DP-7317, AL-DP-7346, AL-DP-7310,
AL-DP-7323, AL-DP-7335, AL-DP-7338, AL-DP-7341, AL-DP-7302,
AL-DP-7315, AL-DP-7328, AL-DP-7330, AL-DP-7336, AL-DP-7345,
AL-DP-9250, AL-DP-9251, AL-DP-9252, AL-DP-9253, AL-DP-9254,
AL-DP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259,
AL-DP-9260, AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9264,
AL-DP-9265, AL-DP-9266, AL-DP-9267, AL-DP-9268, AL-DP-9269,
AL-DP-9270, AL-DP-9271, AL-DP-9272, AL-DP-9273, AL-DP-9274,
AL-DP-9275, AL-DP-9276, AL-DP-9277, AL-DP-9279, AL-DP-9280,
AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-DP-9284, AL-DP-9285,
AL-DP-9286, AL-DP-9287, AL-DP-9288, and AL-DP-9289.
[0064] 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, and preferably from
the 5'-end. For example, for a 23 nucleotide dsRNA strand which is
complementary to a region of an Aha gene, the dsRNA generally does
not contain any mismatch within the central 13 nucleotides. In
another embodiment, the antisense strand of the dsRNA does not
contain any mismatch in the region from positions 1, or 2, to
positions 9, or 10, of the antisense strand (counting 5'-3'). 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 an Aha gene.
Consideration of the efficacy of dsRNAs with mismatches in
inhibiting expression of an Aha gene is important, especially if
the particular region of complementarity in an Aha gene is known to
have polymorphic sequence variation within the population.
[0065] In one embodiment, at least one end of the dsRNA has a
single-stranded nucleotide overhang of 1 to 4, generally 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. Generally, 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, generally
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. Generally, 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.
[0066] 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. Specific examples of preferred dsRNA compounds useful in
this invention include dsRNAs containing modified backbones or no
natural internucleoside linkages. As defined in this specification,
dsRNAs having modified backbones 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 this
specification, and as sometimes referenced in the art, modified
dsRNAs that do not have a phosphorus atom in their internucleoside
backbone can also be considered to be oligonucleosides.
[0067] Preferred modified dsRNA 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.
[0068] Representative U.S. patents that teach the preparation of
the above phosphorus-containing linkages include, but are not
limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,195; 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; 5,541,316; 5,550,111;
5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is
herein incorporated by reference
[0069] Preferred modified dsRNA backbones that do not include a
phosphorus atom therein have backbones that are formed by short
chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatoms and alkyl or cycloalkyl internucleoside linkages, or
one or more short chain heteroatomic or heterocyclic
internucleoside linkages. These include those having morpholino
linkages (formed in part from the sugar portion of a nucleoside);
siloxane backbones; sulfide, sulfoxide and sulfone backbones;
formacetyl and thioformacetyl backbones; methylene formacetyl and
thioformacetyl backbones; alkene containing backbones; sulfamate
backbones; methyleneimino and methylenehydrazino backbones;
sulfonate and sulfonamide backbones; amide backbones; and others
having mixed N, O, S and CH.sub.2 component parts.
[0070] Representative U.S. patents that teach the preparation of
the above oligonucleosides include, but are not limited to, U.S.
Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 5,64,562; 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;
5,608,046; 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.
[0071] In other preferred dsRNA mimetics, both the sugar and the
internucleoside linkage, i.e., the backbone, of the nucleotide
units are replaced with novel groups. The base units are maintained
for hybridization with an appropriate nucleic acid target compound.
One such oligomeric compound, an dsRNA 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 dsRNA is replaced with an amide containing backbone, in
particular an aminoethylglycine backbone. The nucleobases are
retained and are bound directly or indirectly to aza nitrogen atoms
of the amide portion of the backbone. Representative U.S. 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, 1497-1500.
[0072] Most preferred embodiments of the invention are dsRNAs with
phosphorothioate backbones and oligonucleosides with heteroatom
backbones, and in particular --CH.sub.2--NH--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--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 dsRNAs having morpholino backbone structures of the
above-referenced U.S. Pat. No. 5,034,506.
[0073] Modified dsRNAs may also contain one or more substituted
sugar moieties. Preferred dsRNAs comprise one of the following at
the 2' position: OH; F; O--, S--, or N-alkyl; O--, S--, or
N-alkenyl; O--, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the
alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.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.nO[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. Other preferred dsRNAs 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.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an dsRNA, or a group for improving
the pharmacodynamic properties of an dsRNA, 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, 486-504) i.e., an alkoxy-alkoxy group. A further
preferred modification includes 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O_CH.sub.2--O_CH.sub.2--N(CH.sub.2).sub.2, also described in
examples hereinbelow.
[0074] Other preferred modifications include 2'-methoxy
(2'-OCH.sub.3), 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
dsRNA, particularly the 3' position of the sugar on the 3' terminal
nucleotide or in 2'-5' linked dsRNAs and the 5'position of 5'
terminal nucleotide. DsRNAs may also have sugar mimetics such as
cyclobutyl moieties in place of the pentofuranosyl sugar.
Representative U.S. patents that teach the preparation of such
modified sugar structures include, but are not limited to, U.S.
Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;
5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;
5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;
5,658,873; 5,670,633; and 5,700,920, certain of which are commonly
owned with the instant application, and each of which is herein
incorporated by reference in its entirety.
[0075] DsRNAs may also include 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
anal 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-daazaadenine
and 3-deazaguanine and 3-deazaadenine. 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. L, ed. John Wiley & Sons, 1990, these
disclosed by Englisch et al., Angewandte Chemie, International
Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S.,
Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,
S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these
nucleobases are particularly useful for increasing the binding
affinity of the oligomeric compounds of the invention. These
include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6
and 0-6 substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and
Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca
Raton, 1993, pp. 276-278) and are presently preferred base
substitutions, even more particularly when combined with
2'-O-methoxyethyl sugar modifications.
[0076] Representative U.S. patents that teach 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;
5,130,30; 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; and 5,681,941, each of which is
herein incorporated by reference, and U.S. Pat. No. 5,750,692, also
herein incorporated by reference.
[0077] Another modification of the dsRNAs of the invention involves
chemically linking to the dsRNA one or more moieties or conjugates
which enhance the activity, cellular distribution or cellular
uptake of the dsRNA. Such moieties include but are not limited to
lipid moieties such as a cholesterol moiety (Letsinger et al.,
Proc. Natl. Acid. Sci. USA, 199, 86, 6553-6556), cholic acid
(Manoharan et al., Biorg. Med. Chem. Let., 1994 4 1053-1060), a
thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y.
Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg. Med. Chem.
Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al.,
Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,
dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J,
1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,
327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937).
[0078] Representative U.S. patents that teach the preparation of
such dsRNA 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; 5,591,584; 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.
[0079] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within an dsRNA. The
present invention also includes dsRNA compounds which are chimeric
compounds. "Chimeric" dsRNA compounds or "chimeras," in the context
of this invention, are dsRNA compounds, particularly dsRNAs, which
contain two or more chemically distinct regions, each made up of at
least one monomer unit, i.e., a nucleotide in the case of an dsRNA
compound. These dsRNAs typically contain at least one region
wherein the dsRNA is modified so as to confer upon the dsRNA
increased resistance to nuclease degradation, increased cellular
uptake, and/or increased binding affinity for the target nucleic
acid. An additional region of the dsRNA may serve as a substrate
for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way
of example, RNase H is a cellular endonuclease which cleaves the
RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore,
results in cleavage of the RNA target, thereby greatly enhancing
the efficiency of dsRNA inhibition of gene expression.
Consequently, comparable results can often be obtained with shorter
dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate
deoxydsRNAs hybridizing to the same target region. Cleavage of the
RNA target can be routinely detected by gel electrophoresis and, if
necessary, associated nucleic acid hybridization techniques known
in the art.
[0080] In certain instances, the dsRNA may be modified by a
non-ligand group. A number of non-ligand molecules have been
conjugated to dsRNAs in order to enhance the activity, cellular
distribution or cellular uptake of the dsRNA, 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-5-tritylthiol
(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan
et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol
(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic
chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et
al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990,
259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res.,
1990, 18:3777), a polyamine or a polyethylene glycol chain
(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or
adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,
36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,
1995, 1264:229), or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277:923). Representative United States
patents that teach the preparation of such dsRNA conjugates have
been listed above. Typical conjugation protocols involve the
synthesis of dsRNAs 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 dsRNA
still bound to the solid support or following cleavage of the dsRNA
in solution phase. Purification of the dsRNA conjugate by HPLC
typically affords the pure conjugate.
[0081] Vector Encoded RNAi Agents
[0082] The dsRNA of the invention can also be expressed from
recombinant viral vectors intracellularly in vivo. The recombinant
viral vectors of the invention comprise sequences encoding the
dsRNA of the invention and any suitable promoter for expressing the
dsRNA sequences. Suitable promoters include, for example, the U6 or
H1 RNA pol III promoter sequences and the cytomegalovirus promoter.
Selection of other suitable promoters is within the skill in the
art. The recombinant viral vectors of the invention can also
comprise inducible or regulatable promoters for expression of the
dsRNA in a particular tissue or in a particular intracellular
environment. The use of recombinant viral vectors to deliver dsRNA
of the invention to cells in vivo is discussed in more detail
below.
[0083] dsRNA of the invention can be expressed from a recombinant
viral vector either as two separate, complementary RNA molecules,
or as a single RNA molecule with two complementary regions.
[0084] Any viral vector capable of accepting the coding sequences
for the dsRNA molecule(s) to be expressed can be used, for example
vectors derived from adenovirus (AV); adeno-associated virus (AAV);
retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine
leukemia virus); herpes virus, and the like. The tropism of viral
vectors can be modified by pseudotyping the vectors with envelope
proteins or other surface antigens from other viruses, or by
substituting different viral capsid proteins, as appropriate.
[0085] For example, lentiviral vectors of the invention can be
pseudotyped with surface proteins from vesicular stomatitis virus
(VSV), rabies, Ebola, Mokola, and the like. AAV vectors of the
invention can be made to target different cells by engineering the
vectors to express different capsid protein serotypes. For example,
an AAV vector expressing a serotype 2 capsid on a serotype 2 genome
is called AAV 2/2. This serotype 2 capsid gene in the AAV 2/2
vector can be replaced by a serotype 5 capsid gene to produce an
AAV 2/5 vector. Techniques for constructing AAV vectors which
express different capsid protein serotypes are within the skill in
the art; see, e.g., Rabinowitz J E et al. (2002), J Virol
76:791-801, the entire disclosure of which is herein incorporated
by reference.
[0086] Selection of recombinant viral vectors suitable for use in
the invention, methods for inserting nucleic acid sequences for
expressing the dsRNA into the vector, and methods of delivering the
viral vector to the cells of interest are within the skill in the
art. See, for example, Dornburg R (1995), Gene Therap. 2: 301-310;
Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990),
Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30;
and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire
disclosures of which are herein incorporated by reference.
[0087] Preferred viral vectors are those derived from AV and AAV.
In a particularly preferred embodiment, the dsRNA of the invention
is expressed as two separate, complementary single-stranded RNA
molecules from a recombinant AAV vector comprising, for example,
either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV)
promoter.
[0088] A suitable AV vector for expressing the dsRNA of the
invention, a method for constructing the recombinant AV vector, and
a method for delivering the vector into target cells, are described
in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
[0089] Suitable AAV vectors for expressing the dsRNA of the
invention, methods for constructing the recombinant AV vector, and
methods for delivering the vectors into target cells are described
in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et
al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J.
Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat. No.
5,139,941; International Patent Application No. WO 94/13788; and
International Patent Application No. WO 93/24641, the entire
disclosures of which are herein incorporated by reference.
[0090] Pharmaceutical Compositions Comprising dsRNA
[0091] 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 an Aha gene, such as
pathological processes mediated by Aha1 expression. Such
pharmaceutical compositions are formulated based on the mode of
delivery. One example is compositions that are formulated for
systemic administration via parenteral delivery.
[0092] The pharmaceutical compositions of the invention are
administered in dosages sufficient to inhibit expression of an Aha
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 an Aha gene.
[0093] In general, a suitable dose of dsRNA will be in the range of
0.01 microgram to 5.0 milligrams per kilogram body weight of the
recipient per day, generally in the range of 1 microgram to 1 mg
per kilogram body weight per day. The pharmaceutical composition
may be administered once daily, or the dsRNA may be administered as
two, three, or more sub-doses at appropriate intervals throughout
the day or even using continuous infusion or delivery through a
controlled release formulation. 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 and are particularly useful
for vaginal delivery of agents, such as could be used with the
agents of the present invention. In this embodiment, the dosage
unit contains a corresponding multiple of the daily dose.
[0094] 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.
[0095] Advances in mouse genetics have generated a number of mouse
models for the study of various human diseases, such as
pathological processes mediated by Aha expression. Such models are
used for in vivo testing of dsRNA, as well as for determining a
therapeutically effective dose.
[0096] The present invention also includes pharmaceutical
compositions and formulations which include the dsRNA compounds of
the invention. The pharmaceutical compositions of the present
invention may be administered in a number of ways depending upon
whether local or systemic treatment is desired and upon the area to
be treated. Administration may be topical, pulmonary, e.g., by
inhalation or insufflation of powders or aerosols, including by
nebulizer; intratracheal, intranasal, epidermal and transdermal,
oral or parenteral. Parenteral administration includes intravenous,
intraarterial, subcutaneous, intraperitoneal or intramuscular
injection or infusion; or intracranial, e.g., intrathecal or
intraventricular, administration.
[0097] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful. Preferred
topical formulations include those in which the dsRNAs of the
invention are in admixture with a topical delivery agent such as
lipids, liposomes, fatty acids, fatty acid esters, steroids,
chelating agents and surfactants. Preferred lipids and liposomes
include neutral (e.g. dioleoylphosphatidyl ethanolamine=DOPE,
dimyristoylphosphatidyl choline=DMPC, distearolyphosphatidyl
choline) negative (e.g. dimyristoylphosphatidyl glycerol=DMPG) and
cationic (e.g. dioleoyltetramethylaminopropyl=DOTAP and
dioleoylphosphatidyl ethanolamine=DOTMA). DsRNAs of the invention
may be encapsulated within liposomes or may form complexes thereto,
in particular to cationic liposomes. Alternatively, dsRNAs may be
complexed to lipids, in particular to cationic lipids. Preferred
fatty acids and esters include but are not limited arachidonic
acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid,
capric acid, myristic acid, palmitic acid, stearic acid, linoleic
acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,
glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an
acylcarnitine, an acylcholine, or a C.sub.1-10 alkyl ester (e.g.
isopropylmyristate IPM), monoglyceride, diglyceride or
pharmaceutically acceptable salt thereof. Topical formulations are
described in detail in U.S. patent application Ser. No. 09/315,298
filed on May 20, 1999 which is incorporated herein by reference in
its entirety.
[0098] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Preferred oral formulations are those in which
dsRNAs of the invention are administered in conjunction with one or
more penetration enhancers, surfactants, and chelators. Preferred
surfactants include fatty acids and/or esters or salts thereof,
bile acids and/or salts thereof. Preferred bile acids/salts include
chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid
(UDCA), cholic acid, dehydrocholic acid, deoxycholic acid,
glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic
acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate
and sodium glycodihydrofusidate. Preferred fatty acids include
arachidonic acid, undecanoic acid, oleic acid, lauric acid,
caprylic acid, capric acid, myristic acid, palmitic acid, stearic
acid, linoleic acid, linolenic acid, dicaprate, tricaprate,
monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a monoglyceride, a diglyceride or a pharmaceutically acceptable
salt thereof (e.g. sodium). Also preferred are combinations of
penetration enhancers, for example, fatty acids/salts in
combination with bile acids/salts. A particularly preferred
combination is the sodium salt of lauric acid, capric acid and
UDCA. Further penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
DsRNAs of the invention may be delivered orally, in granular form
including sprayed dried particles, or complexed to form micro or
nanoparticles. DsRNA complexing agents include poly-amino acids;
polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,
polyalkylcyanoacrylates; cationized gelatins, albumins, starches,
acrylates, polyethyleneglycols (PEG) and starches;
polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,
celluloses and starches. Particularly preferred complexing agents
include chitosan, N-trimethylchitosan, poly-L-lysine,
polyhistidine, polyornithine, polyspermines, protamine,
polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE),
polyaminostyrene (e.g. p-amino), poly(methylcyanoacrylate),
poly(ethylcyanoacrylate), poly(butylcyanoacrylate),
poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate),
DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide,
DEAE-albumin and DEAE-dextran, polymethylacrylate,
polyhexylacrylate, poly(D,L-lactic acid),
poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol (PEG). Oral formulations for dsRNAs and their
preparation are described in detail in U.S. application. Ser. No.
08/886,829 (filed Jul. 1, 1997), Ser. No. 09/108,673 (filed Jul. 1,
1998), Ser. No. 09/256,515 (filed Feb. 23, 1999), Ser. No.
09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298 (filed May
20, 1999), each of which is incorporated herein by reference in
their entirety.
[0099] Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
[0100] Pharmaceutical compositions of the present invention
include, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids.
[0101] The pharmaceutical formulations of the present invention,
which may conveniently be presented in unit dosage form, may be
prepared according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
[0102] The compositions of the present invention may be formulated
into any of many possible dosage forms such as, but not limited to,
tablets, capsules, gel capsules, liquid syrups, soft gels,
suppositories, and enemas. The compositions of the present
invention may also be formulated as suspensions in aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain
substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
[0103] In one embodiment of the present invention the
pharmaceutical compositions may be formulated and used as foams.
Pharmaceutical foams include formulations such as, but not limited
to, emulsions, microemulsions, creams, jellies and liposomes. While
basically similar in nature these formulations vary in the
components and the consistency of the final product. The
preparation of such compositions and formulations is generally
known to those skilled in the pharmaceutical and formulation arts
and may be applied to the formulation of the compositions of the
present invention.
[0104] Emulsions
[0105] The compositions of the present invention may be prepared
and formulated as emulsions. Emulsions are typically heterogenous
systems of one liquid dispersed in another in the form of droplets
usually exceeding 0.1 .mu.m in diameter (Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p.
335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often
biphasic systems comprising two immiscible liquid phases intimately
mixed and dispersed with each other. In general, emulsions may be
of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
When an aqueous phase is finely divided into and dispersed as
minute droplets into a bulk oily phase, the resulting composition
is called a water-in-oil (w/o) emulsion. Alternatively, when an
oily phase is finely divided into and dispersed as minute droplets
into a bulk aqueous phase, the resulting composition is called an
oil-in-water (o/w) emulsion. Emulsions may contain additional
components in addition to the dispersed phases, and the active drug
which may be present as a solution in either the aqueous phase,
oily phase or itself as a separate phase. Pharmaceutical excipients
such as emulsifiers, stabilizers, dyes, and anti-oxidants may also
be present in emulsions as needed. Pharmaceutical emulsions may
also be multiple emulsions that are comprised of more than two
phases such as, for example, in the case of oil-in-water-in-oil
(o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex
formulations often provide certain advantages that simple binary
emulsions do not. Multiple emulsions in which individual oil
droplets of an o/w emulsion enclose small water droplets constitute
a w/o/w emulsion. Likewise a system of oil droplets enclosed in
globules of water stabilized in an oily continuous phase provides
an o/w/o emulsion.
[0106] Emulsions are characterized by little or no thermodynamic
stability. Often, the dispersed or discontinuous phase of the
emulsion is well dispersed into the external or continuous phase
and maintained in this form through the means of emulsifiers or the
viscosity of the formulation. Either of the phases of the emulsion
may be a semisolid or a solid, as is the case of emulsion-style
ointment bases and creams. Other means of stabilizing emulsions
entail the use of emulsifiers that may be incorporated into either
phase of the emulsion. Emulsifiers may broadly be classified into
four categories: synthetic surfactants, naturally occurring
emulsifiers, absorption bases, and finely dispersed solids (Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
[0107] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (Rieger, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
Surfactants are typically amphiphilic and comprise a hydrophilic
and a hydrophobic portion. The ratio of the hydrophilic to the
hydrophobic nature of the surfactant has been termed the
hydrophile/lipophile balance (HLB) and is a valuable tool in
categorizing and selecting surfactants in the preparation of
formulations. Surfactants may be classified into different classes
based on the nature of the hydrophilic group: nonionic, anionic,
cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 285).
[0108] Naturally occurring emulsifiers used in emulsion
formulations include lanolin, beeswax, phosphatides, lecithin and
acacia. Absorption bases possess hydrophilic properties such that
they can soak up water to form w/o emulsions yet retain their
semisolid consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, nonswelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
[0109] A large variety of non-emulsifying materials are also
included in emulsion formulations and contribute to the properties
of emulsions. These include fats, oils, waxes, fatty acids, fatty
alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and antioxidants (Block, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0110] Hydrophilic colloids or hydrocolloids include naturally
occurring gums and synthetic polymers such as polysaccharides (for
example, acacia, agar, alginic acid, carrageenan, guar gum, karaya
gum, and tragacanth), cellulose derivatives (for example,
carboxymethylcellulose and carboxypropylcellulose), and synthetic
polymers (for example, carbomers, cellulose ethers, and
carboxyvinyl polymers). These disperse or swell in water to form
colloidal solutions that stabilize emulsions by forming strong
interfacial films around the dispersed-phase droplets and by
increasing the viscosity of the external phase.
[0111] Since emulsions often contain a number of ingredients such
as carbohydrates, proteins, sterols and phosphatides that may
readily support the growth of microbes, these formulations often
incorporate preservatives. Commonly used preservatives included in
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Antioxidants are also
commonly added to emulsion formulations to prevent deterioration of
the formulation. Antioxidants used may be free radical scavengers
such as tocopherols, alkyl gallates, butylated hydroxyanisole,
butylated hydroxytoluene, or reducing agents such as ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric
acid, tartaric acid, and lecithin.
[0112] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture have
been reviewed in the literature (Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for
oral delivery have been very widely used because of ease of
formulation, as well as efficacy from an absorption and
bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base
laxatives, oil-soluble vitamins and high fat nutritive preparations
are among the materials that have commonly been administered orally
as o/w emulsions.
[0113] In one embodiment of the present invention, the compositions
of dsRNAs and nucleic acids are formulated as microemulsions. A
microemulsion may be defined as a system of water, oil and
amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution (Rosoff, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically
microemulsions are systems that are prepared by first dispersing an
oil in an aqueous surfactant solution and then adding a sufficient
amount of a fourth component, generally an intermediate
chain-length alcohol to form a transparent system. Therefore,
microemulsions have also been described as thermodynamically
stable, isotropically clear dispersions of two immiscible liquids
that are stabilized by interfacial films of surface-active
molecules (Leung and Shah, in: Controlled Release of Drugs:
Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH
Publishers, New York, pages 185-215). Microemulsions commonly are
prepared via a combination of three to five components that include
oil, water, surfactant, cosurfactant and electrolyte. Whether the
microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w)
type is dependent on the properties of the oil and surfactant used
and on the structure and geometric packing of the polar heads and
hydrocarbon tails of the surfactant molecules (Schott, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 1985, p. 271).
[0114] The phenomenological approach utilizing phase diagrams has
been extensively studied and has yielded a comprehensive knowledge,
to one skilled in the art, of how to formulate microemulsions
(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger
and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
volume 1, p. 335). Compared to conventional emulsions,
microemulsions offer the advantage of solubilizing water-insoluble
drugs in a formulation of thermodynamically stable droplets that
are formed spontaneously.
[0115] Surfactants used in the preparation of microemulsions
include, but are not limited to, ionic surfactants, non-ionic
surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol
monooleate (M0310), hexaglycerol monooleate (PO310), hexaglycerol
pentaoleate (PO500), decaglycerol monocaprate (MCA750),
decaglycerol monooleate (MO0750), decaglycerol sequioleate (SO750),
decaglycerol decaoleate (DAO750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules. Microemulsions may, however,
be prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase may typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase may include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C.sub.8-C.sub.12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized
C.sub.8-C.sub.10 glycerides, vegetable oils and silicone oil.
[0116] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both o/w and w/o) have been
proposed to enhance the oral bioavailability of drugs, including
peptides (Constantinides et al., Pharmaceutical Research, 1994, 11,
1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13,
205). Microemulsions afford advantages of improved drug
solubilization, protection of drug from enzymatic hydrolysis,
possible enhancement of drug absorption due to surfactant-induced
alterations in membrane fluidity and permeability, ease of
preparation, ease of oral administration over solid dosage forms,
improved clinical potency, and decreased toxicity (Constantinides
et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.
Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form
spontaneously when their components are brought together at ambient
temperature. This may be particularly advantageous when formulating
thermolabile drugs, peptides or dsRNAs. Microemulsions have also
been effective in the transdermal delivery of active components in
both cosmetic and pharmaceutical applications. It is expected that
the microemulsion compositions and formulations of the present
invention will facilitate the increased systemic absorption of
dsRNAs and nucleic acids from the gastrointestinal tract, as well
as improve the local cellular uptake of dsRNAs and nucleic acids
within the gastrointestinal tract, vagina, buccal cavity and other
areas of administration.
[0117] Microemulsions of the present invention may also contain
additional components and additives such as sorbitan monostearate
(Grill 3), Labrasol, and penetration enhancers to improve the
properties of the formulation and to enhance the absorption of the
dsRNAs and nucleic acids of the present invention. Penetration
enhancers used in the microemulsions of the present invention may
be classified as belonging to one of five broad
categories_surfactants, fatty acids, bile salts, chelating agents,
and non-chelating non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these
classes has been discussed above.
[0118] Liposomes
[0119] There are many organized surfactant structures besides
microemulsions that have been studied and used for the formulation
of drugs. These include monolayers, micelles, bilayers and
vesicles. Vesicles, such as liposomes, have attracted great
interest because of their specificity and the duration of action
they offer from the standpoint of drug delivery. As used in the
present invention, the term "liposome" means a vesicle composed of
amphiphilic lipids arranged in a spherical bilayer or bilayers.
[0120] Liposomes are unilamellar or multilamellar vesicles which
have a membrane formed from a lipophilic material and an aqueous
interior. The aqueous portion contains the composition to be
delivered. Cationic liposomes possess the advantage of being able
to fuse to the cell wall. Non-cationic liposomes, although not able
to fuse as efficiently with the cell wall, are taken up by
macrophages in vivo.
[0121] In order to cross intact mammalian skin, lipid vesicles must
pass through a series of fine pores, each with a diameter less than
50 nm, under the influence of a suitable transdermal gradient.
Therefore, it is desirable to use a liposome which is highly
deformable and able to pass through such fine pores.
[0122] Further advantages of liposomes include; liposomes obtained
from natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; liposomes can protect encapsulated drugs in their internal
compartments from metabolism and degradation (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
Important considerations in the preparation of liposome
formulations are the lipid surface charge, vesicle size and the
aqueous volume of the liposomes.
[0123] Liposomes are useful for the transfer and delivery of active
ingredients to the site of action. Because the liposomal membrane
is structurally similar to biological membranes, when liposomes are
applied to a tissue, the liposomes start to merge with the cellular
membranes and as the merging of the liposome and cell progresses,
the liposomal contents are emptied into the cell where the active
agent may act.
[0124] Liposomal formulations have been the focus of extensive
investigation as the mode of delivery for many drugs. There is
growing evidence that for topical administration, liposomes present
several advantages over other formulations. Such advantages include
reduced side-effects related to high systemic absorption of the
administered drug, increased accumulation of the administered drug
at the desired target, and the ability to administer a wide variety
of drugs, both hydrophilic and hydrophobic, into the skin.
[0125] Several reports have detailed the ability of liposomes to
deliver agents including high-molecular weight DNA into the skin.
Compounds including analgesics, antibodies, hormones and
high-molecular weight DNAs have been administered to the skin. The
majority of applications resulted in the targeting of the upper
epidermis
[0126] Liposomes fall into two broad classes. Cationic liposomes
are positively charged liposomes which interact with the negatively
charged DNA molecules to form a stable complex. The positively
charged DNA/liposome complex binds to the negatively charged cell
surface and is internalized in an endosome. Due to the acidic pH
within the endosome, the liposomes are ruptured, releasing their
contents into the cell cytoplasm (Wang et al., Biochem. Biophys.
Res. Commun., 1987, 147, 980-985).
[0127] Liposomes which are pH-sensitive or negatively-charged,
entrap DNA rather than complex with it. Since both the DNA and the
lipid are similarly charged, repulsion rather than complex
formation occurs. Nevertheless, some DNA is entrapped within the
aqueous interior of these liposomes. pH-sensitive liposomes have
been used to deliver DNA encoding the thymidine kinase gene to cell
monolayers in culture. Expression of the exogenous gene was
detected in the target cells (Zhou et al., Journal of Controlled
Release, 1992, 19, 269-274).
[0128] One major type of liposomal composition includes
phospholipids other than naturally-derived phosphatidylcholine.
Neutral liposome compositions, for example, can be formed from
dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine (DPPC). Anionic liposome compositions generally
are formed from dimyristoyl phosphatidylglycerol, while anionic
fusogenic liposomes are formed primarily from dioleoyl
phosphatidylethanolamine (DOPE). Another type of liposomal
composition is formed from phosphatidylcholine (PC) such as, for
example, soybean PC, and egg PC. Another type is formed from
mixtures of phospholipid and/or phosphatidylcholine and/or
cholesterol.
[0129] Several studies have assessed the topical delivery of
liposomal drug formulations to the skin. Application of liposomes
containing interferon to guinea pig skin resulted in a reduction of
skin herpes sores while delivery of interferon via other means
(e.g. as a solution or as an emulsion) were ineffective (Weiner et
al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an
additional study tested the efficacy of interferon administered as
part of a liposomal formulation to the administration of interferon
using an aqueous system, and concluded that the liposomal
formulation was superior to aqueous administration (du Plessis et
al., Antiviral Research, 1992, 18, 259-265).
[0130] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasome.TM. I
(glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether)
and Novasome.TM. II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et al. S. T. P. Pharma. Sci., 1994, 4, 6, 466).
[0131] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome (A) comprises one or more glycolipids, such
as monosialoganglioside G.sub.m1, or (B) is derivatized with one or
more hydrophilic polymers, such as a polyethylene glycol (PEG)
moiety. While not wishing to be bound by any particular theory, it
is thought in the art that, at least for sterically stabilized
liposomes containing gangliosides, sphingomyelin, or
PEG-derivatized lipids, the enhanced circulation half-life of these
sterically stabilized liposomes derives from a reduced uptake into
cells of the reticuloendothelial system (RES) (Allen et al., FEBS
Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53,
3765).
[0132] Various liposomes comprising one or more glycolipids are
known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci.,
1987, 507, 64) reported the ability of monosialoganglioside
G.sub.m1, galactocerebroside sulfate and phosphatidylinositol to
improve blood half-lives of liposomes. These findings were
expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A.,
1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to
Allen et al., disclose liposomes comprising (1) sphingomyelin and
(2) the ganglioside G.sub.m1 or a galactocerebroside sulfate ester.
U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes
comprising sphingomyelin. Liposomes comprising
1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499
(Lim et al).
[0133] Many liposomes comprising lipids derivatized with one or
more hydrophilic polymers, and methods of preparation thereof, are
known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53,
2778) described liposomes comprising a nonionic detergent,
2C.sub.1215G, that contains a PEG moiety. Illum et al. (FEBS Lett.,
1984, 167, 79) noted that hydrophilic coating of polystyrene
particles with polymeric glycols results in significantly enhanced
blood half-lives. Synthetic phospholipids modified by the
attachment of carboxylic groups of polyalkylene glycols (e.g., PEG)
are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899).
Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments
demonstrating that liposomes comprising phosphatidylethanolamine
(PE) derivatized with PEG or PEG stearate have significant
increases in blood circulation half-lives. Blume et al. (Biochimica
et Biophysica Acta, 1990, 1029, 91) extended such observations to
other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from
the combination of distearoylphosphatidylethanolamine (DSPE) and
PEG. Liposomes having covalently bound PEG moieties on their
external surface are described in European Patent No. EP 0 445 131
B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20
mole percent of PE derivatized with PEG, and methods of use
thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556
and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and
European Patent No. EP 0 496 813 B1). Liposomes comprising a number
of other lipid-polymer conjugates are disclosed in WO 91/05545 and
U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073
(Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids
are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935
(Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.)
describe PEG-containing liposomes that can be further derivatized
with functional moieties on their surfaces.
[0134] A limited number of liposomes comprising nucleic acids are
known in the art. WO 96/40062 to Thierry et al. discloses methods
for encapsulating high molecular weight nucleic acids in liposomes.
U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded
liposomes and asserts that the contents of such liposomes may
include dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes
certain methods of encapsulating oligodeoxynucleotides in
liposomes. WO 97/04787 to Love et al. discloses liposomes
comprising dsRNAs targeted to the raf gene.
[0135] Transfersomes are yet another type of liposomes, and are
highly deformable lipid aggregates which are attractive candidates
for drug delivery vehicles. Transfersomes may be described as lipid
droplets which are so highly deformable that they are easily able
to penetrate through pores which are smaller than the droplet.
Transfersomes are adaptable to the environment in which they are
used, e.g. they are self-optimizing (adaptive to the shape of pores
in the skin), self-repairing, frequently reach their targets
without fragmenting, and often self-loading. To make transfersomes
it is possible to add surface edge-activators, usually surfactants,
to a standard liposomal composition. Transfersomes have been used
to deliver serum albumin to the skin. The transfersome-mediated
delivery of serum albumin has been shown to be as effective as
subcutaneous injection of a solution containing serum albumin.
[0136] Surfactants find wide application in formulations such as
emulsions (including microemulsions) and liposomes. The most common
way of classifying and ranking the properties of the many different
types of surfactants, both natural and synthetic, is by the use of
the hydrophile/lipophile balance (HLB). The nature of the
hydrophilic group (also known as the "head") provides the most
useful means for categorizing the different surfactants used in
formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel
Dekker, Inc., New York, N.Y., 1988, p. 285).
[0137] If the surfactant molecule is not ionized, it is classified
as a nonionic surfactant. Nonionic surfactants find wide
application in pharmaceutical and cosmetic products and are usable
over a wide range of pH values. In general their HLB values range
from 2 to about 18 depending on their structure. Nonionic
surfactants include nonionic esters such as ethylene glycol esters,
propylene glycol esters, glyceryl esters, polyglyceryl esters,
sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic
alkanolamides and ethers such as fatty alcohol ethoxylates,
propoxylated alcohols, and ethoxylated/propoxylated block polymers
are also included in this class. The polyoxyethylene surfactants
are the most popular members of the nonionic surfactant class.
[0138] If the surfactant molecule carries a negative charge when it
is dissolved or dispersed in water, the surfactant is classified as
anionic. Anionic surfactants include carboxylates such as soaps,
acyl lactylates, acyl amides of amino acids, esters of sulfuric
acid such as alkyl sulfates and ethoxylated alkyl sulfates,
sulfonates such as alkyl benzene sulfonates, acyl isethionates,
acyl taurates and sulfosuccinates, and phosphates. The most
important members of the anionic surfactant class are the alkyl
sulfates and the soaps.
[0139] If the surfactant molecule carries a positive charge when it
is dissolved or dispersed in water, the surfactant is classified as
cationic. Cationic surfactants include quaternary ammonium salts
and ethoxylated amines. The quaternary ammonium salts are the most
used members of this class.
[0140] If the surfactant molecule has the ability to carry either a
positive or negative charge, the surfactant is classified as
amphoteric. Amphoteric surfactants include acrylic acid
derivatives, substituted alkylamides, N-alkylbetaines and
phosphatides.
[0141] The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
[0142] Penetration Enhancers
[0143] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly dsRNAs, to the skin of animals. Most drugs are
present in solution in both ionized and nonionized forms. However,
usually only lipid soluble or lipophilic drugs readily cross cell
membranes. It has been discovered that even non-lipophilic drugs
may cross cell membranes if the membrane to be crossed is treated
with a penetration enhancer. In addition to aiding the diffusion of
non-lipophilic drugs across cell membranes, penetration enhancers
also enhance the permeability of lipophilic drugs.
[0144] Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.
92). Each of the above mentioned classes of penetration enhancers
are described below in greater detail.
[0145] Surfactants: In connection with the present invention,
surfactants (or "surface-active agents") are chemical entities
which, when dissolved in an aqueous solution, reduce the surface
tension of the solution or the interfacial tension between the
aqueous solution and another liquid, with the result that
absorption of dsRNAs through the mucosa is enhanced. In addition to
bile salts and fatty acids, these penetration enhancers include,
for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether
and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews
in Therapeutic Drug Carrier Systems, 1991, p. 92); and
perfluorochemical emulsions, such as FC-43 (Takahashi et al., J.
Pharm. Pharmacol., 1988, 40, 252).
[0146] Fatty acids: Various fatty acids and their derivatives which
act as penetration enhancers include, for example, oleic acid,
lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,
caprylic acid, arachidonic acid, glycerol 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines,
C.sub.1-C.sub.10 alkyl esters thereof (e.g., methyl, isopropyl and
t-butyl), and mono- and di-glycerides thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carryier Systems,
1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug
Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm.
Pharmacol., 1992, 44, 651-654).
[0147] Bile salts: The physiological role of bile includes the
facilitation of dispersion and absorption of lipids and fat-soluble
vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.
Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural
bile salts, and their synthetic derivatives, act as penetration
enhancers. Thus the term "bile salts" includes any of the naturally
occurring components of bile as well as any of their synthetic
derivatives. The bile salts of the invention include, for example,
cholic acid (or its pharmaceutically acceptable sodium salt, sodium
cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic
acid (sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic
acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm.
Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990,
79, 579-583).
[0148] Chelating Agents: Chelating agents, as used in connection
with the present invention, can be defined as compounds that remove
metallic ions from solution by forming complexes therewith, with
the result that absorption of dsRNAs through the mucosa is
enhanced. With regards to their use as penetration enhancers in the
present invention, chelating agents have the added advantage of
also serving as DNase inhibitors, as most characterized DNA
nucleases require a divalent metal ion for catalysis and are thus
inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,
315-339). Chelating agents of the invention include but are not
limited to disodium ethylenediaminetetraacetate (EDTA), citric
acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and
homovanilate), N-acyl derivatives of collagen, laureth-9 and
N-amino acyl derivatives of beta-diketones (enamines) (Lee et al.,
Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page
92; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14,
43-51).
[0149] Non-chelating non-surfactants: As used herein, non-chelating
non-surfactant penetration enhancing compounds can be defined as
compounds that demonstrate insignificant activity as chelating
agents or as surfactants but that nonetheless enhance absorption of
dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews
in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of
penetration enhancers include, for example, unsaturated cyclic
ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621-626).
[0150] Agents that enhance uptake of dsRNAs at the cellular level
may also be added to the pharmaceutical and other compositions of
the present invention. For example, cationic lipids, such as
lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic
glycerol derivatives, and polycationic molecules, such as
polylysine (Lollo et al., PCT Application WO 97/30731), are also
known to enhance the cellular uptake of dsRNAs.
[0151] Other agents may be utilized to enhance the penetration of
the administered nucleic acids, including glycols such as ethylene
glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and
terpenes such as limonene and menthone.
[0152] Carriers
[0153] Certain compositions of the present invention also
incorporate carrier compounds in the formulation. As used herein,
"carrier compound" or "carrier" can refer to a nucleic acid, or
analog thereof, which is inert (i.e., does not possess biological
activity per se) but is recognized as a nucleic acid by in vivo
processes that reduce the bioavailability of a nucleic acid having
biological activity by, for example, degrading the biologically
active nucleic acid or promoting its removal from circulation. The
coadministration of a nucleic acid and a carrier compound,
typically with an excess of the latter substance, can result in a
substantial reduction of the amount of nucleic acid recovered in
the liver, kidney or other extracirculatory reservoirs, presumably
due to competition between the carrier compound and the nucleic
acid for a common receptor. For example, the recovery of a
partially phosphorothioate dsRNA in hepatic tissue can be reduced
when it is coadministered with polyinosinic acid, dextran sulfate,
polycytidic acid or 4-acetamido-4'
isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et al., Antisense
Res. Dev., 1995, 5, 115-121; Takakura et al., Antisense & Nucl.
Acid Drug Dev., 1996, 6, 177-183.
[0154] Excipients
[0155] In contrast to a carrier compound, a "pharmaceutical
carrier" or "excipient" is a pharmaceutically acceptable solvent,
suspending agent or any other pharmacologically inert vehicle for
delivering one or more nucleic acids to an animal. The excipient
may be liquid or solid and is selected, with the planned manner of
administration in mind, so as to provide for the desired bulk,
consistency, etc., when combined with a nucleic acid and the other
components of a given pharmaceutical composition. Typical
pharmaceutical carriers include, but are not limited to, binding
agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and
other sugars, microcrystalline cellulose, pectin, gelatin, calcium
sulfate, ethyl cellulose, polyacrylates or calcium hydrogen
phosphate, etc.); lubricants (e.g., magnesium stearate, talc,
silica, colloidal silicon dioxide, stearic acid, metallic
stearates, hydrogenated vegetable oils, corn starch, polyethylene
glycols, sodium benzoate, sodium acetate, etc.); disintegrants
(e.g., starch, sodium starch glycolate, etc.); and wetting agents
(e.g., sodium lauryl sulphate, etc).
[0156] Pharmaceutically acceptable organic or inorganic excipient
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can also be used to
formulate the compositions of the present invention. Suitable
pharmaceutically acceptable carriers include, but are not limited
to, water, salt solutions, alcohols, polyethylene glycols, gelatin,
lactose, amylose, magnesium stearate, talc, silicic acid, viscous
paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the
like.
[0157] Formulations for topical administration of nucleic acids may
include sterile and non-sterile aqueous solutions, non-aqueous
solutions in common solvents such as alcohols, or solutions of the
nucleic acids in liquid or solid oil bases. The solutions may also
contain buffers, diluents and other suitable additives.
Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can be used.
[0158] Suitable pharmaceutically acceptable excipients include, but
are not limited to, water, salt solutions, alcohol, polyethylene
glycols, gelatin, lactose, amylose, magnesium stearate, talc,
silicic acid, viscous paraffin, hydroxymethylcellulose,
polyvinylpyrrolidone and the like.
[0159] Pharmaceutical Compositions for the Delivery to the
Respiratory Tract
[0160] Another aspect of the invention provides for the delivery of
IRNA agents to the respiratory tract, particularly for the
treatment of cystic fibrosis. The respiratory tract includes the
upper airways, including the oropharynx and larynx, followed by the
lower airways, which include the trachea followed by bifurcations
into the bronchi and bronchioli. The upper and lower airways are
called the conductive airways. The terminal bronchioli then divide
into respiratory bronchioli which then lead to the ultimate
respiratory zone, the alveoli, or deep lung. The deep lung, or
alveoli, are the primary target of inhaled therapeutic aerosols for
systemic delivery of iRNA agents.
[0161] Pulmonary delivery compositions can be delivered by
inhalation by the patient of a dispersion so that the composition,
preferably the iRNA agent, within the dispersion can reach the lung
where it can, for example, be readily absorbed through the alveolar
region directly into blood circulation. Pulmonary delivery can be
effective both for systemic delivery and for localized delivery to
treat diseases of the lungs.
[0162] Pulmonary delivery can be achieved by different approaches,
including the use of nebulized, aerosolized, micellular and dry
powder-based formulations; administration by inhalation may be oral
and/or nasal. Delivery can be achieved with liquid nebulizers,
aerosol-based inhalers, and dry powder dispersion devices.
Metered-dose devices are preferred. One of the benefits of using an
atomizer or inhaler is that the potential for contamination is
minimized because the devices are self contained. Dry powder
dispersion devices, for example, deliver drugs that may be readily
formulated as dry powders. An iRNA composition may be stably stored
as lyophilized or spray-dried powders by itself or in combination
with suitable powder carriers. The delivery of a composition for
inhalation can be mediated by a dosing timing element which can
include a timer, a dose counter, time measuring device, or a time
indicator which when incorporated into the device enables dose
tracking, compliance monitoring, and/or dose triggering to a
patient during administration of the aerosol medicament.
[0163] Examples of pharmaceutical devices for aerosol delivery
include metered dose inhalers (MDIs), dry powder inhalers (DPIs),
and air jet nebulizers. Exemplary delivery systems by inhalation
which can be readily adapted for delivery of the subject iRNA
agents are described in, for example, U.S. Pat. Nos. 5,756,353;
5,858,784; and PCT applications WO98/31346; WO98/10796; WO00/27359;
WO01/54664; WO02/060412. Other aerosol formulations that may be
used for delivering the iRNA agents are described in U.S. Pat. Nos.
6,294,153; 6,344,194; 6,071,497, and PCT applications WO02/066078;
WO02/053190; WO01/60420; WO00/66206. Further, methods for
delivering iRNA agents can be adapted from those used in delivering
other oligonucleotides (e.g., an antisense oligonucleotide) by
inhalation, such as described in Templin et al., Antisense Nucleic
Acid Drug Dev, 2000, 10:359-68; Sandrasagra et al., Expert Opin
Biol Ther, 2001, 1:979-83; Sandrasagra et al., Antisense Nucleic
Acid Drug Dev, 2002, 12:177-81.
[0164] The delivery of the inventive agents may also involve the
administration of so called "pro-drugs", i.e. formulations or
chemical modifications of a therapeutic substance that require some
form of processing or transport by systems innate to the subject
organism to release the therapeutic substance, preferably at the
site where its action is desired; this latter embodiment may be
used in conjunction with delivery of the respiratory tract, but
also together with other embodiments of the present invention. For
example, the human lungs can remove or rapidly degrade
hydrolytically cleavable deposited aerosols over periods ranging
from minutes to hours. In the upper airways, ciliated epithelia
contribute to the "mucociliary excalator" by which particles are
swept from the airways toward the mouth. Pavia, D., "Lung
Mucociliary Clearance," in Aerosols and the Lung: Clinical and
Experimental Aspects, Clarke, S. W. and Pavia, D., Eds.,
Butterworths, London, 1984. In the deep lungs, alveolar macrophages
are capable of phagocytosing particles soon after their deposition.
Warheit et al. Microscopy Res. Tech., 26: 412-422 (1993); and
Brain, J. D., "Physiology and Pathophysiology of Pulmonary
Macrophages," in The Reticuloendothelial System, S. M. Reichard and
J. Filkins, Eds., Plenum, New. York., pp. 315-327, 1985.
[0165] In preferred embodiments, particularly where systemic dosing
with the iRNA agent is desired, the aerosoled iRNA agents are
formulated as microparticles. Microparticles having a diameter of
between 0.5 and ten microns can penetrate the lungs, passing
through most of the natural barriers. A diameter of less than ten
microns is required to bypass the throat; a diameter of 0.5 microns
or greater is required to avoid being exhaled.
[0166] Other Components
[0167] The compositions of the present invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions, at their art-established usage levels.
Thus, for example, the compositions may contain additional,
compatible, pharmaceutically-active materials such as, for example,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the compositions of the present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the present invention. The formulations can be
sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the nucleic acid(s) of the
formulation.
[0168] Aqueous suspensions may contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0169] Certain embodiments of the invention provide pharmaceutical
compositions containing (a) one or more dsRNA agents and (b) one or
more other chemotherapeutic agents which function by a non-RNA
interference mechanism. Examples of such chemotherapeutic agents
include but are not limited to daunorubicin, daunomycin,
dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,
bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,
bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,
mithramycin, prednisone, hydroxyprogesterone, testosterone,
tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,
pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,
methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,
taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate,
irinotecan, topotecan, gemcitabine, teniposide, cisplatin and
diethylstilbestrol (DES). See, generally, The Merck Manual of
Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al.,
eds., Rahway, N.J. When used with the compounds of the invention,
such chemotherapeutic agents may be used individually (e.g., 5-FU
and oligonucleotide), sequentially (e.g., 5-FU and oligonucleotide
for a period of time followed by MTX and oligonucleotide), or in
combination with one or more other such chemotherapeutic agents
(e.g., 5-FU, MTX and oligonucleotide, or 5-FU, radiotherapy and
oligonucleotide). Anti-inflammatory drugs, including but not
limited to nonsteroidal anti-inflammatory drugs and
corticosteroids, and antiviral drugs, including but not limited to
ribivirin, vidarabine, acyclovir and ganciclovir, may also be
combined in compositions of the invention. See, generally, The
Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al.,
eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively).
Other non-dsRNA chemotherapeutic agents are also within the scope
of this invention. Two or more combined compounds may be used
together or sequentially.
[0170] 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.
[0171] 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 generally
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.
[0172] 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 pathological processes mediated by Aha expression. 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.
[0173] Methods for Treating Diseases Caused by Expression of an Aha
Gene
[0174] The invention relates in particular to the use of a dsRNA or
a pharmaceutical composition prepared therefrom for the treatment
of Cystic Fibrosis. Owing to the inhibitory effect on Aha1
expression, an dsRNA according to the invention or a pharmaceutical
composition prepared therefrom can enhance the quality of life of
Cystic Fibrosis patients.
[0175] Furthermore, the invention relates to the use of a dsRNA or
a pharmaceutical composition of the invention aimed at the
treatment of cancer, e.g., for inhibiting tumor growth and tumor
metastasis. For example, the dsRNA or a pharmaceutical composition
prepared therefrom may be used for the treatment of solid tumors,
like breast cancer, lung cancer, head and neck cancer, brain
cancer, abdominal cancer, colon cancer, colorectal cancer,
esophagus cancer, gastrointestinal cancer, glioma, liver cancer,
tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer,
pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor,
multiple myeloma and for the treatment of skin cancer, like
melanoma, for the treatment of lymphomas and blood cancer. The
invention further relates to the use of an dsRNA according to the
invention or a pharmaceutical composition prepared therefrom for
inhibiting Aha1 expression and/or for inhibiting accumulation of
ascites fluid and pleural effusion in different types of cancer,
e.g., breast cancer, lung cancer, head cancer, neck cancer, brain
cancer, abdominal cancer, colon cancer, colorectal cancer,
esophagus cancer, gastrointestinal cancer, glioma, liver cancer,
tongue cancer, neuroblastoma, osteosarcoma, ovarian cancer,
pancreatic cancer, prostate cancer, retinoblastoma, Wilm's tumor,
multiple myeloma, skin cancer, melanoma, lymphomas and blood
cancer. Owing to the inhibitory effect on Aha1 expression, an dsRNA
according to the invention or a pharmaceutical composition prepared
therefrom can enhance the quality of life of cancer patients.
[0176] The invention furthermore relates to the use of an dsRNA or
a pharmaceutical composition thereof, e.g., for treating Cystic
Fibrosis or cancer or for preventing tumor metastasis, in
combination with other pharmaceuticals and/or other therapeutic
methods, e.g., with known pharmaceuticals and/or known therapeutic
methods, such as, for example, those which are currently employed
for treating Cystic Fibrosis or cancer and/or for preventing tumor
metastasis. Where the pharmaceutical composition aims for the
treatment of Cystic fibrosis, preference is given to a combination
with daily chest physiotherapy, orally applied pancreatic enzymes,
daily oral or inhaled antibiotics to counter lung infection,
inhaled anti-asthma therapy, corticosteroid tablets, dietary
vitamin supplements, especially A and D, inhalation of
Pulmozyme.TM., medicines to relieve constipation or to improve the
activity of the enzyme supplements, insulin for CF-related
diabetes, medication for CF-associated liver disease, and oxygen to
help with breathing.
[0177] Where the pharmaceutical composition aims for the treatment
of cancer and/or for preventing tumor metastasis, preference is
given to a combination with radiation therapy and chemotherapeutic
agents, such as cisplatin, cyclophosphamide, 5-fluorouracil,
adriamycin, daunorubicin or tamoxifen.
[0178] The invention can also be practiced by including with a
specific RNAi agent another anti-cancer chemotherapeutic agent,
such as any conventional chemotherapeutic agent. The combination of
a specific binding agent with such other agents can potentiate the
chemotherapeutic protocol. Numerous chemotherapeutic protocols will
present themselves in the mind of the skilled practitioner as being
capable of incorporation into the method of the invention. Any
chemotherapeutic agent can be used, including alkylating agents,
antimetabolites, hormones and antagonists, radioisotopes, as well
as natural products. For example, the compound of the invention can
be administered with antibiotics such as doxorubicin and other
anthracycline analogs, nitrogen mustards such as cyclophosphamide,
pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea,
taxol and its natural and synthetic derivatives, and the like. As
another example, in the case of mixed tumors, such as
adenocarcinoma of the breast, where the tumors include
gonadotropin-dependent and gonadotropin-independent cells, the
compound can be administered in conjunction with leuprolide or
goserelin (synthetic peptide analogs of LH-RH). Other
antineoplastic protocols include the use of a tetracycline compound
with another treatment modality, e.g., surgery, radiation, etc.,
also referred to herein as "adjunct antineoplastic modalities."
Thus, the method of the invention can be employed with such
conventional regimens with the benefit of reducing side effects and
enhancing efficacy.
[0179] Methods for Inhibiting Expression of an Aha Gene
[0180] In yet another aspect, the invention provides a method for
inhibiting the expression of an Aha gene in a mammal. The method
comprises administering a composition of the invention to the
mammal such that expression of the target Aha gene, e.g. Aha1, is
silenced. Because of their high specificity, the dsRNAs of the
invention specifically target RNAs (primary or processed) of the
target Aha gene. Compositions and methods for inhibiting the
expression of these Aha genes using dsRNAs can be performed as
described elsewhere herein.
[0181] 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 an Aha gene, e.g. Aha1, 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, subcutaneous, transdermal,
airway (aerosol), nasal, rectal, vaginal and topical (including
buccal and sublingual) administration. In preferred embodiments,
the compositions are administered by intravenous infusion or
injection.
[0182] 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
[0183] Gene Walking of an Aha Gene
[0184] siRNA design was carried out to identify siRNAs targeting
Aha1. The mRNA sequences of Homo sapiens (NM.sub.--012111.1), mus
musculus (NM.sub.--146036.1) and pan troglodytes
(XM.sub.--510094.1) Aha 1 were examined by computer analysis to
identify homologous sequences of 19 or 21 nucleotides that yield
RNAi agents cross-reactive between these three species. Of those
identified, 48 such sequences were selected for minimal off-target
interactions in rats (at least 3 mismatches to any other gene in
the rat genome, or at least two mismatches to any other gene in the
rat genome, wherein one of said at least two mismatches is located
in a position complementary to position 9 or 10 of the antisense
strand of the corresponding RNAi agent, counting 5' to 3') and the
corresponding dsRNAs synthesized for screening
(AL-DP-7301-AL-DP-7346, see Table 1). AL-DP-7561, AL-DP-7562,
AL-DP-7563 and AL-DP-7564 which are additionally cross-reactive to
mus musculus (NM.sub.--172391.3) and rattus norvegicus
(XM.sub.--223680.3) Aha 2, were also synthesized and screened. In
addition, a further 40 sequences were selected for minimal
predicted off-target interactions in humans (at least 3 mismatches
to any other gene in the human genome, or at least two mismatches
to any other gene in the human genome, wherein one of said at least
two mismatches is located in a position complementary to position 9
or 10 of the antisense strand of the corresponding RNAi agent,
counting 5' to 3') and the corresponding dsRNAs synthesized for
screening (AL-DP-9250-AL-DP-9289, see Table 2). 17 sequences were
identified as belonging to both sets (AL-DP-7301, AL-DP-7304,
AL-DP-7305, AL-DP-7307, AL-DP-7310, AL-DP-7312, AL-DP-7315,
AL-DP-7316, AL-DP-7317, AL-DP-7323, AL-DP-7324, AL-DP-7332,
AL-DP-7336, AL-DP-7337, AL-DP-7338, AL-DP-7342, and AL-DP-7344.
[0185] dsRNA Synthesis
[0186] Source of Reagents
[0187] 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.
[0188] siRNA Synthesis
[0189] 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).
[0190] 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, Unterschlei.beta.heim, 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.
[0191] For the synthesis of 3'-cholesterol-conjugated siRNAs
(herein referred to as -Choi-3'), an appropriately modified solid
support was used for RNA synthesis. The modified solid support was
prepared as follows:
Diethyl-2-azabutane-1,4-dicarboxylate AA
##STR00001##
[0193] 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%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl-
]-amino}-propionic acid ethyl ester AB
##STR00002##
[0195] 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.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid
ethyl ester AC
##STR00003##
[0197]
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.
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,1-
5,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-h-
exanoyl}ethoxycarbonylmethyl-amino)-propionic acid ethyl ester
AD
##STR00004##
[0199] 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%).
[0200]
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-yloxycarbonylam-
ino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylic acid ethyl ester
AE
##STR00005##
[0201] 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%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic
acid
17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1-
7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AF
##STR00006##
[0203] 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%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-
-yl}-6-oxo-hexyl)-carbamic acid
17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1-
7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl ester AG
##STR00007##
[0205] 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%).
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
##STR00008##
[0207] 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.
Cholesterol Derivatised CPG AI
##STR00009##
[0209] 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).
[0210] 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.
[0211] Nucleic acid sequences are represented below using standard
nomenclature, and specifically the abbreviations of Table 2.
TABLE-US-00003 TABLE 3 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)
[0212] dsRNA Expression Vectors
[0213] In another aspect of the invention, Aha1 specific dsRNA
molecules that modulate Aha1 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).
[0214] 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.
[0215] The recombinant dsRNA expression vectors are generally 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), 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. Natl. Acad. Sci. USA 85:3014-3018; Armentano et
al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al.,
1991, Proc. Natl. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991,
Proc. Natl. 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, J. Immunol. 150:4104-4115; U.S.
Pat. No. 4,868,116; U.S. Pat. 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.
[0216] 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
generally 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)).
[0217] 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.
[0218] Generally, 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.
[0219] 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 Aha1 gene or
multiple Aha1 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.
[0220] The Aha1 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.
[0221] Aha1 siRNA In Vitro Screening
[0222] Single Dose Screen in HeLa and MLE 12 Cells
[0223] HeLa cells were obtained from American Type Culture
Collection (Rockville, Md., cat. No. HB-8065) and cultured in Ham's
F12 (Biochrom AG, Berlin, Germany, cat. No. FG0815) supplemented to
contain 10% fetal calf serum (FCS) (Biochrom AG, Berlin, Germany,
cat. No. S0115), Penicillin 100 U/ml, Streptomycin 100 .mu.g/ml
(Biochrom AG, Berlin, Germany, cat. No. A2213) at 37.degree. C. in
an atmosphere with 5% CO.sub.2 in a humidified incubator (Heraeus
HERAcell, Kendro Laboratory Products, Langenselbold, Germany).
[0224] MLE 12 cells were obtained from American Type Culture
Collection (Rockville, Md., cat. No. CRL-2110) and cultured in
HITES Medium (1:1 mix Dulbecco's MEM (Biochrom AG, Berlin, Germany,
cat. No: F0435)+Ham's F12 (Biochrom AG, Berlin, Germany, cat. No:
FG0815)) supplemented to contain 2% fetal calf serum (FCS)
(Biochrom AG, Berlin, Germany, cat. No. 50115), Penicillin 100
U/ml, Streptomycin 100 .mu.g/ml (Biochrom AG, Berlin, Germany, cat.
No. A2213), 4 mM L-Glutamin (Biochrom AG, Berlin, Germany, cat. No:
K0282), 1.times. Insulin/Transferrin/Na-Selenit (Gibco: 51500-056),
10 nM Hydrocortisone (Sigma Munich, Germany, cat. No: H6909), 10 nM
.beta.-Estradiol (Sigma Munich, Germany, cat. No: E2257), and 10 mM
HEPES (USB Europe GmBH, Staufen, Germany cat. No.: 16926) at
37.degree. C. in an atmosphere with 5% CO.sub.2 in a humidified
incubator (Heraeus HERAcell, Kendro Laboratory Products,
Langenselbold, Germany).
[0225] Transfection and mRNA Quantification
[0226] For transfection with siRNA, HeLa and MLE12 cells were
seeded at a density of 2.0.times.10.sup.4 cells/well in 96-well
plates and transfected directly. Transfection of siRNA (30 nM) was
carried out with lipofectamine 2000 (Invitrogen GmbH, Karlsruhe,
Germany, cat. No. 11668-019) as described by the manufacturer. 24
hours after transfection cells were lysed and Aha1 mRNA levels were
quantified with the Quantigene Explore Kit (Genosprectra, Dumbarton
Circle Fremont, USA, cat. No. QG-000-02) according to the
manufacturer's protocol. Aha1 mRNA levels were normalized to GAPDH
mRNA. Readings were obtained in quadruplicates for each siRNA.
siRNA duplexes unrelated to Aha1 gene were used as control. The
activity of a given Aha1 specific siRNA duplex was expressed as
percent Aha1 mRNA concentration in treated cells relative to Aha1
mRNA concentration in cells treated with the control siRNA
duplex.
TABLE-US-00004 TABLE 4 Probe sequences used with Quantigene Explore
Kit (Genospectra) in quantification of Homo sapiens (hs) Aha1 FPL
Name Function Sequence hsAha1 001 CE
GATGTAAATTCCCATTGCTTCTCTTTTTTCTCTTGGAAAGAAAGT hsAha1 002 CE
TGAACTCTGTTTTGAGGGTGCTTTTTTCTCTTGGAAAGAAAGT hsAha1 003 CE
GGGTCTACTGACTCTCCATTCATTGTTTTTCTCTTGGAAAGAAAGT hsAha1 004 CE
CCTTGCGCTCCTCAGTTTTCTTTTTCTCTTGGAAAGAAAGT hsAha1 005 CE
GGTTTTTGAAGGAGCAGGCTTAGTTTTTCTCTTGGAAAGAAAGT hsAha1 006 LE
ACGCTGTTTTCATCAGACAAATTTTTTTAGGCATAGGACCCGTGTCT hsAha1 007 LE
GCTCACACTAATCTCCACTTCATCCTTTTTAGGCATAGGACCCGTGTCT hsAha1 008 LE
TCATTAAGGCCACGAGATTTGTTTTTTAGGCATAGGACCCGTGTCT hsAha1 009 LE
TAGGTAAGATCATGCCCTGGGTTTTTAGGCATAGGACCCGTGTCT hsAha1 010 LE
ACTCCAACAGGTCTGGCCTGTTTTTAGGCATAGGACCCGTGTCT hsAha1 011 BL
GTCAGGCTCATCTTTGGCAAG hsAha1 012 BL TAGAAGTTTCACCCCTTCTTCCT hsAha1
013 BL AGTGCTGGCTGCCCCACT
TABLE-US-00005 TABLE 5 Probe sequences used with Quantigene Explore
Kit (Genospectra) in quantification of Mus musculus (mm) Aha1 FPL
Name Function Sequence mmAhsa 1001 CE
CTCGAACGGCCAGGAACATTTTTCTCTTGGAAAGAAAGT mmAhsa 1002 CE
GCACTTGCCCTCTTCATTTTCTATTTTTCTCTTGGAAAGAAAGT mmAhsa 1003 CE
TTGATGGATGCCTCCCCATTTTTTCTCTTGGAAAGAAAGT mmAhsa 1004 CE
AACTCTGTCTTGAGGGTGCTGATTTTTTCTCTTGGAAAGAAAGT CE
TTTGGCCTGGCTTTTTGAATTTTTCTCTTGGAAAGAAAGT LE
CAAGCTTGTTCACTTCGGTCACCTCTTTTTAGGCATAGGACCCGTGTCT LE
CCTGACTTAGAGGTACCTGTCCAGTTTTTTTAGGCATAGGACCCGTGTCT LE
GATTTCCACATGTCCTTTGTACTGCACTTTTTTAGGCATAGGACCCGTGTCT LE
ATTTTCATCAGACAAATTGGGTTTTTAGGCATAGGACCCGTGTCT LE
TAATCTCCACTTCATCCACGCTTTTTTAGGCATAGGACCCGTGTCT LE
TTTCACCCCGTCTTCCTTCATTTTTAGGCATAGGACCCGTGTCT LE
ACTGTGGGCAAGATCATGCCCTGAGTATTTTTAGGCATAGGACCCGTGTCT BL
AAGATAAGTTTGCCTTTCCTGTTG BL CAGTTTGATGGTCCACTCATAGAAG BL
CATCTTTGGCAAGGCTCACAC BL TTAAGGCCACGAGATTTGTGTCAGGCT BL
GTAAATTCCCACTGCTTCTCTCAGAAG BL CACTGGATCTACTGACTCTCCATTC BL
CTCAGTCTTTAGTGCTGGCTGGCC BL GGAGCAGACTTAGCCTTGCAAGT
[0227] Dose-Response Curves in HeLa Cells
[0228] Transfection and mRNA quantification: For transfection with
siRNA, HeLa cells were seeded at a density of 2.0.times.10.sup.4
cells/well in 96-well plates and transfected directly. Transfection
of siRNA was carried out with lipofectamine 2000 (Invitrogen GmbH,
Karlsruhe, Germany, cat. No. 11668-019) as described by the
manufacturer. siRNAs were concentrated from 30 nM in 3 fold
dilutions to 14 pM. 24 hours after transfection Hela cells were
lysed and Aha1 mRNA levels were quantified with the Quantigene
Explore Kit (Genosprectra, Dumbarton Circle Fremont, USA, cat. No.
QG-000-02) according to the protocol. Aha1 mRNA levels were
normalized to GAP-DH mRNA. For each siRNA four individual
datapoints were collected. siRNA duplexes unrelated to Aha1 gene
were used as control. The activity of a given Aha1 specific siRNA
duplex was expressed as percent Aha1 mRNA concentration in treated
cells relative to Aha1 mRNA concentration in cells treated with the
control siRNA duplex. XL-fit was used to calculate IC.sub.50
values.
[0229] Table 6 provides values for inhibition of Aha1 expression
using various dsRNA molecules of the invention.
TABLE-US-00006 TABLE 6 Residual Aha1 mRNA in % of control in HeLa
and MLE12 cells treated with 30 nM solutions of various RNAi agents
specific for Aha1, and IC.sub.50 for selected RNAi agents
determined in HeLa cells IC.sub.50 in MLE12 cells, HeLa cells,
residual HeLa cells residual Duplex identifier mRNA [%] [nM] mRNA
[%] AL-DP-7299 19 .+-. 3 105 .+-. 17 AL-DP-7300 7 .+-. 2 94 .+-. 13
AL-DP-7301 3 .+-. 1 0.035 14 .+-. 3 AL-DP-7302 17 .+-. 5 61 .+-. 16
AL-DP-7303 5 .+-. 2 23 .+-. 5 AL-DP-7304 7 .+-. 3 30 .+-. 7
AL-DP-7305 6 .+-. 2 26 .+-. 6 AL-DP-7306 27 .+-. 8 45 .+-. 11
AL-DP-7307 16 .+-. 6 1.1 27 .+-. 8 AL-DP-7308 13 .+-. 5 0.21 20
.+-. 7 AL-DP-7309 10 .+-. 3 0.36 22 .+-. 8 AL-DP-7310 51 .+-. 13 59
.+-. 13 AL-DP-7311 4 .+-. 3 0.07 16 .+-. 4 AL-DP-7312 14 .+-. 3 40
.+-. 8 AL-DP-7313 63 .+-. 14 80 .+-. 10 AL-DP-7314 97 .+-. 21 88
.+-. 10 AL-DP-7315 76 .+-. 24 77 .+-. 10 AL-DP-7316 7 .+-. 2 0.34
23 .+-. 6 AL-DP-7317 11 .+-. 3 44 .+-. 12 AL-DP-7318 4 .+-. 2 0.29
16 .+-. 3 AL-DP-7319 38 .+-. 7 73 .+-. 21 AL-DP-7320 16 .+-. 4 0.07
15 .+-. 5 AL-DP-7321 130 .+-. 36 81 .+-. 16 AL-DP-7322 4 .+-. 2
0.045 10 .+-. 3 AL-DP-7323 24 .+-. 6 55 .+-. 11 AL-DP-7324 3 .+-. 2
0.089 12 .+-. 4 AL-DP-7325 5 .+-. 2 0.3 12 .+-. 4 AL-DP-7326 3 .+-.
1 0.27 19 .+-. 7 AL-DP-7327 3 .+-. 1 0.08 13 .+-. 7 AL-DP-7328 49
.+-. 14 67 .+-. 10 AL-DP-7329 6 .+-. 2 0.2 18 .+-. 5 AL-DP-7330 64
.+-. 19 78 .+-. 10 AL-DP-7331 5 .+-. 2 0.55 13 .+-. 5 AL-DP-7332 95
.+-. 20 82 .+-. 15 AL-DP-7333 2 .+-. 1 0.27 9 .+-. 3 AL-DP-7334 94
.+-. 17 83 .+-. 19 AL-DP-7335 11 .+-. 5 57 .+-. 11 AL-DP-7336 22
.+-. 4 63 .+-. 12 AL-DP-7337 6 .+-. 2 0.29 20 .+-. 5 AL-DP-7338 39
.+-. 6 56 .+-. 10 AL-DP-7339 10 .+-. 1 35 .+-. 6 AL-DP-7340 8 .+-.
2 0.61 19 .+-. 6 AL-DP-7341 17 .+-. 4 55 .+-. 16 AL-DP-7342 6 .+-.
4 0.5 15 .+-. 3 AL-DP-7343 26 .+-. 4 103 .+-. 19 AL-DP-7344 5 .+-.
2 38 .+-. 11 AL-DP-7345 53 .+-. 22 63 .+-. 15 AL-DP-7346 22 .+-. 4
44 .+-. 11 AL-DP-9250 4 .+-. 1 AL-DP-9251 51 .+-. 9 AL-DP-9252 19
.+-. 2 AL-DP-9253 11 .+-. 1 AL-DP-9254 7 .+-. 1 AL-DP-9255 5 .+-. 0
AL-DP-9256 5 .+-. 0 AL-DP-9257 7 .+-. 0 AL-DP-9258 9 .+-. 1
AL-DP-9259 7 .+-. 1 AL-DP-9260 15 .+-. 3 AL-DP-9261 21 .+-. 2
AL-DP-9262 24 .+-. 4 AL-DP-9263 25 .+-. 6 AL-DP-9264 7 .+-. 2
AL-DP-9265 8 .+-. 1 AL-DP-9266 11 .+-. 2 AL-DP-9267 45 .+-. 4
AL-DP-9268 9 .+-. 1 AL-DP-9269 5 .+-. 1 AL-DP-9270 6 .+-. 1
AL-DP-9271 6 .+-. 2 AL-DP-9272 26 .+-. 8 AL-DP-9273 11 .+-. 1
AL-DP-9274 7 .+-. 1 AL-DP-9275 8 .+-. 1 AL-DP-9276 4 .+-. 1
AL-DP-9277 10 .+-. 1 AL-DP-9278 2 .+-. 0 AL-DP-9279 3 .+-. 0
AL-DP-9280 12 .+-. 1 AL-DP-9281 8 .+-. 2 AL-DP-9282 3 .+-. 0
AL-DP-9283 6 .+-. 1 AL-DP-9284 39 .+-. 2 AL-DP-9285 4 .+-. 1
AL-DP-9286 61 .+-. 11 AL-DP-9287 3 .+-. 1 AL-DP-9288 27 .+-. 5
AL-DP-9289 6 .+-. 1
[0230] In summary, AL-DP-7301, AL-DP-7308, AL-DP-7318, AL-DP-7320,
AL-DP-7322, AL-DP-7324, AL-DP-7325, AL-DP-7326, AL-DP-7327,
AL-DP-7329, AL-DP-7331, AL-DP-7333, AL-DP-7340, and AL-DP-7342
inhibited Aha1 expression by at least 80% in both HeLa and MLE12
cells, AL-DP-7303, AL-DP-7305, AL-DP-7307, AL-DP-7309, AL-DP-7316,
and AL-DP-7337 inhibited Aha1 expression by at least 80% in HeLa
cells and by at least 70% in MLE12 cells, AL-DP-7304, AL-DP-7312,
AL-DP-7339, and AL-DP-7344 inhibited Aha1 expression by at least
80% in HeLa cells and by at least 60% in MLE12 cells, AL-DP-7306,
AL-DP-7317, and AL-DP-7346 inhibited Aha1 expression by at least
70% in HeLa cells and by at least 50% in MLE12 cells, AL-DP-7310,
AL-DP-7323, AL-DP-7335, AL-DP-7338, and AL-DP-7341 inhibited Aha1
expression by at least 40% in both HeLa and MLE12 cells, and
AL-DP-7302, AL-DP-7315, AL-DP-7328, AL-DP-7330, AL-DP-7336, and
AL-DP-7345, inhibited Aha1 expression by at least 20% in both HeLa
and MLE12 cells.
[0231] In addition, AL-DP-9250, AL-DP-9252, AL-DP-9253, AL-DP-9254,
AL-DP-9255, AL-DP-9256, AL-DP-9257, AL-DP-9258, AL-DP-9259,
AL-DP-9260, AL-DP-9264, AL-DP-9265, AL-DP-9266, AL-DP-9268,
AL-DP-9269, AL-DP-9270, AL-DP-9271, AL-DP-9273, AL-DP-9274,
AL-DP-9275, AL-DP-9276, AL-DP-9277, AL-DP-9279, AL-DP-9280,
AL-DP-9281, AL-DP-9282, AL-DP-9283, AL-DP-9285, AL-DP-9287, and
AL-DP-9289 inhibited Aha1 expression by at least 80% in HeLa cells,
AL-DP-9261, AL-DP-9262, AL-DP-9263, AL-DP-9272, and AL-DP-9288
inhibited Aha1 expression by at least 70% in HeLa cells, AL-DP-9263
inhibited Aha1 expression by at least 60% in HeLa cells, AL-DP-9267
inhibited Aha1 expression by at least 50% in HeLa cells, AL-DP-9251
inhibited Aha1 expression by at least 40% in HeLa cells, and
AL-DP-9286 inhibited Aha1 expression by at least 30% in HeLa cells.
Sequence CWU 1
1
217121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 1auugguccac ggauaagcut t
21221DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 2agcuuauccg uggaccaaut t
21321DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 3gugaguaagc uugauggagt t
21421DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 4cuccaucaag cuuacucact t
21521DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 5agucaaaauc cccacuugut t
21621DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 6acaagugggg auuuugacut t
21721DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 7aaaucucgug gccuuaaugt t
21821DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 8cauuaaggcc acgagauuut t
21921DNAArtificial SequenceDescription of Combined DNA/RNA Molecule
Synthetic oligonucleotide 9gagauuagug ugagccuugt t
211021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 10caaggcucac acuaaucuct t
211121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 11aaucucgugg ccuuaaugat t
211221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 12ucauuaaggc cacgagauut t
211321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 13agauuagugu gagccuugct t
211421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 14gcaaggcuca cacuaaucut t
211521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 15cgggcggacg ccaccaacgt t
211621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 16cguugguggc guccgcccgt t
211721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 17ggcggacgcc accaacguct t
211821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 18gacguuggug gcguccgcct t
211921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 19gggcggacgc caccaacgut t
212021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 20acguuggugg cguccgccct t
212121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 21caacgucaac aacuggcact t
212221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 22gugccaguug uugacguugt t
212321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 23gcgggcggac gccaccaact t
212421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 24guugguggcg uccgcccgct t
212521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 25aucucguggc cuuaaugaat t
212621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 26uucauuaagg ccacgagaut t
212721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 27acgucaacaa cuggcacugt t
212821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 28cagugccagu uguugacgut t
212921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 29accaacguca acaacuggct t
213021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 30gccaguuguu gacguuggut t
213121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 31acgcuggauc guggaggagt t
213221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 32cuccuccacg auccagcgut t
213321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 33agacccacgc uggaucgugt t
213421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 34cacgauccag cgugggucut t
213521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 35gacccacgcu ggaucguggt t
213621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 36ccacgaucca gcguggguct t
213721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 37gaauuuacau cagcacccut t
213821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 38agggugcuga uguaaauuct t
213921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 39gggaauuuac aucagcacct t
214021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 40ggugcugaug uaaauuccct t
214121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 41ugggaauuua caucagcact t
214221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 42gugcugaugu aaauucccat t
214321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 43ccaacgucaa caacuggcat t
214421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 44ugccaguugu ugacguuggt t
214521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 45aaguggggug agggagacct t
214621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 46ggucucccuc accccacuut t
214721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 47acacaaaucu cguggccuut t
214821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 48aaggccacga gauuugugut t
214921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 49acccacgcug gaucguggat t
215021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 50uccacgaucc agcgugggut t
215121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 51gagucaaaau ccccacuugt t
215221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 52caagugggga uuuugacuct t
215321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 53gagcucuaua gaguguuuat t
215421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 54uaaacacucu auagagcuct t
215521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 55ggcagcggua cuacuuugat t
215621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 56ucaaaguagu accgcugcct t
215721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 57gacacaaauc ucguggccut t
215821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 58aggccacgag auuuguguct t
215921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 59agcgggcgga cgccaccaat t
216021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 60uugguggcgu ccgcccgcut t
216121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 61caaaaucccc acuuguaagt t
216221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 62cuuacaagug gggauuuugt t
216321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 63gagacccacg cuggaucgut t
216421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 64acgauccagc gugggucuct t
216521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 65gagccuugcc aaagaugagt t
216621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 66cucaucuuug gcaaggcuct t
216721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 67ugacacaaau cucguggcct t
216821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 68ggccacgaga uuugugucat t
216921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 69ggagcucuau agaguguuut t
217021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 70aaacacucua uagagcucct t
217121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 71cccacgcugg aucguggagt t
217221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 72cuccacgauc cagcgugggt t
217321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 73gauccccaau uugucugaut t
217421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 74aucagacaaa uuggggauct t
217521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 75gagaucccca auuugucugt t
217621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 76cagacaaauu ggggaucuct t
217721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 77agccugacac aaaucucgut t
217821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 78acgagauuug ugucaggcut t
217921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 79agauccccaa uuugucugat t
218021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 80ucagacaaau uggggaucut t
218121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 81agggagaccc acgcuggaut t
218221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 82auccagcgug ggucucccut t
218321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 83gagggagacc cacgcuggat t
218421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 84uccagcgugg gucucccuct t
218521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 85gccaaguggg gugagggagt t
218621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 86cucccucacc ccacuuggct t
218721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 87uggcagcggu acuacuuugt t
218821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 88caaaguagua ccgcugccat t
218921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 89ugagggagac ccacgcuggt t
219021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 90ccagcguggg ucucccucat t
219121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 91aguggagauu agugugagct t
219221DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 92gcucacacua aucuccacut t
219321DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 93aggagcucua uagaguguut t
219421DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 94aacacucuau agagcuccut t
219521DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 95agcgguacua cuuugagggt t
219621DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 96cccucaaagu aguaccgcut t
219721DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 97cgcuggaucg uggaggagct t
219821DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 98gcuccuccac gauccagcgt t
219921DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 99gcuggaucgu ggaggagcgt t
2110021DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic oligonucleotide 100cgcuccucca cgauccagct t
2110121DNAArtificial SequenceDescription of Combined DNA/RNA
Molecule Synthetic
oligonucleotide 101cuggaucgug gaggagcggt t 2110221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 102ccgcuccucc acgauccagt t 2110321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 103uggaucgugg aggagcgggt t 2110421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 104cccgcuccuc cacgauccat t 2110521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 105gccugacaca aaucucgugt t 2110621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 106cacgagauuu gugucaggct t 2110721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 107ccugacacaa aucucguggt t 2110821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 108ccacgagauu ugugucaggt t 2110921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 109acgccaccaa cgucaacaat t 2111021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 110uuguugacgu ugguggcgut t 2111121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 111agcucuauag aguguuuact t 2111221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 112guaaacacuc uauagagcut t 2111321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 113gggcuggcag cgguacuact t 2111421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 114guaguaccgc ugccagccct t 2111521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 115cuggcagcgg uacuacuuut t 2111621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 116aaaguaguac cgcugccagt t 2111721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 117ggaugaagug gagauuagut t 2111821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 118acuaaucucc acuucaucct t 2111921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 119accagaggag cucuauagat t 2112021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 120ucuauagagc uccucuggut t 2112121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 121aaguggagau uagugugagt t 2112221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 122cucacacuaa ucuccacuut t 2112321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 123gaggagcucu auagagugut t 2112421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 124acacucuaua gagcuccuct t 2112521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 125gggagaccca cgcuggauct t 2112621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 126gauccagcgu gggucuccct t 2112721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 127ugagccugac acaaaucuct t 2112821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 128gagauuugug ucaggcucat t 2112921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 129gcggacgcca ccaacgucat t 2113021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 130ugacguuggu ggcguccgct t 2113121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 131cggacgccac caacgucaat t 2113221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 132uugacguugg uggcguccgt t 2113321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 133gaaguggaga uuagugugat t 2113421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 134ucacacuaau cuccacuuct t 2113521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 135cucguggccu uaaugaaggt t 2113621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 136ccuucauuaa ggccacgagt t 2113721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 137ucguggccuu aaugaaggat t 2113821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 138uccuucauua aggccacgat t 2113921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 139aaugggaauu uacaucagct t 2114021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 140gcugauguaa auucccauut t 2114121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 141ggaauuuaca ucagcaccct t 2114221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 142gggugcugau guaaauucct t 2114321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 143ggagauuagu gugagccuut t 2114421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 144aaggcucaca cuaaucucct t 2114521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 145cacaaaucuc guggccuuat t 2114621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 146uaaggccacg agauuugugt t 2114721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 147acaaaucucg uggccuuaat t 2114821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 148uuaaggccac gagauuugut t 2114921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 149ggagacccac gcuggaucgt t 2115021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 150cgauccagcg ugggucucct t 2115121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 151ggacgccacc aacgucaact t 2115221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 152guugacguug guggcgucct t 2115321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 153gaugaagugg agauuagugt t 2115421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 154cacuaaucuc cacuucauct t 2115521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 155gugagccuug ccaaagaugt t 2115621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 156caucuuuggc aaggcucact t 2115721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 157caaugaaugg agagucagut t 2115821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 158acugacucuc cauucauugt t 2115921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 159auuaguguga gccuugccat t 2116021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 160uggcaaggcu cacacuaaut t 2116121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 161agaugagccu gacacaaaut t 2116221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 162auuuguguca ggcucaucut t 2116321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 163uagugugagc cuugccaaat t 2116421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 164uuuggcaagg cucacacuat t 2116521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 165uuugccacca ucaccuugat t 2116621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 166ucaaggugau gguggcaaat t 2116721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 167acggagagag augcuucaat t 2116821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 168uugaagcauc ucucuccgut t 2116921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 169cggagagaga ugcuucaaat t 2117021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 170uuugaagcau cucucuccgt t 2117121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 171aaaaucccca cuuguaagat t 2117221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 172ucuuacaagu ggggauuuut t 2117321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 173auccccaauu ugucugaugt t 2117421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 174caucagacaa auuggggaut t 2117521DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 175ucaaaauccc cacuuguaat t 2117621DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 176uuacaagugg ggauuuugat t 2117721DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 177aaauccccac uuguaagaut t 2117821DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 178aucuuacaag uggggauuut t 2117921DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 179uccccaauuu gucugaugat t 2118021DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 180ucaucagaca aauuggggat t 2118121DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 181auggccaagu ggggugaggt t 2118221DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 182ccucacccca cuuggccaut t 2118321DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 183ggagucaaaa uccccacuut t 2118421DNAArtificial
SequenceDescription of Combined DNA/RNA Molecule Synthetic
oligonucleotide 184aaguggggau uuugacucct t 2118545DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
185gatgtaaatt cccattgctt ctcttttttc tcttggaaag aaagt
4518643DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 186tgaactctgt tttgagggtg cttttttctc ttggaaagaa agt
4318746DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 187gggtctactg actctccatt cattgttttt ctcttggaaa
gaaagt 4618841DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 188ccttgcgctc ctcagttttc tttttctctt
ggaaagaaag t 4118944DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 189ggtttttgaa ggagcaggct tagtttttct
cttggaaaga aagt 4419047DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 190acgctgtttt catcagacaa
atttttttag gcataggacc cgtgtct 4719149DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
191gctcacacta atctccactt catccttttt aggcatagga cccgtgtct
4919246DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 192tcattaaggc cacgagattt gttttttagg cataggaccc
gtgtct 4619345DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 193taggtaagat catgccctgg gtttttaggc
ataggacccg tgtct 4519444DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 194actccaacag gtctggcctg
tttttaggca taggacccgt gtct 4419521DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 195gtcaggctca tctttggcaa g
2119623DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 196tagaagtttc accccttctt cct 2319718DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
197agtgctggct gccccact 1819839DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 198ctcgaacggc caggaacatt
tttctcttgg aaagaaagt 3919944DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 199gcacttgccc tcttcatttt
ctatttttct cttggaaaga aagt 4420040DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 200ttgatggatg cctccccatt
ttttctcttg gaaagaaagt 4020144DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 201aactctgtct tgagggtgct
gattttttct cttggaaaga aagt 4420240DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 202tttggcctgg ctttttgaat
ttttctcttg gaaagaaagt 4020349DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 203caagcttgtt cacttcggtc
acctcttttt aggcatagga
cccgtgtct 4920450DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 204cctgacttag aggtacctgt ccagtttttt
taggcatagg acccgtgtct 5020552DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 205gatttccaca tgtcctttgt
actgcacttt tttaggcata ggacccgtgt ct 5220645DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
206attttcatca gacaaattgg gtttttaggc ataggacccg tgtct
4520746DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 207taatctccac ttcatccacg cttttttagg cataggaccc
gtgtct 4620844DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 208tttcaccccg tcttccttca tttttaggca
taggacccgt gtct 4420951DNAArtificial SequenceDescription of
Artificial Sequence Synthetic probe 209actgtgggca agatcatgcc
ctgagtattt ttaggcatag gacccgtgtc t 5121024DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
210aagataagtt tgcctttcct gttg 2421125DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
211cagtttgatg gtccactcat agaag 2521221DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
212catctttggc aaggctcaca c 2121327DNAArtificial SequenceDescription
of Artificial Sequence Synthetic probe 213ttaaggccac gagatttgtg
tcaggct 2721427DNAArtificial SequenceDescription of Artificial
Sequence Synthetic probe 214gtaaattccc actgcttctc tcagaag
2721525DNAArtificial SequenceDescription of Artificial Sequence
Synthetic probe 215cactggatct actgactctc cattc 2521624DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
216ctcagtcttt agtgctggct ggcc 2421723DNAArtificial
SequenceDescription of Artificial Sequence Synthetic probe
217ggagcagact tagccttgca agt 23
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