U.S. patent application number 12/700188 was filed with the patent office on 2010-07-01 for rnai-mediated inhibition of tumor necrosis factor alpha-related conditions.
This patent application is currently assigned to ALCON RESEARCH, LTD.. Invention is credited to Jon E. Chatterton, Abbot F. Clark, Allan R. Shepard, Martin B. Wax.
Application Number | 20100166676 12/700188 |
Document ID | / |
Family ID | 38702032 |
Filed Date | 2010-07-01 |
United States Patent
Application |
20100166676 |
Kind Code |
A1 |
Shepard; Allan R. ; et
al. |
July 1, 2010 |
RNAi-MEDIATED INHIBITION OF TUMOR NECROSIS FACTOR ALPHA-RELATED
CONDITIONS
Abstract
RNA interference is provided for inhibition of tumor necrosis
factor .alpha. (TNF.alpha.) by silencing TNF.alpha. cell surface
receptor TNF receptor-1 (TNFR1) mRNA expression, or by silencing
TNF.alpha. converting enzyme (TACE/ADAM17) mRNA expression.
Silencing such TNF.alpha. targets, in particular, is useful for
treating patients having a TNF.alpha.-related condition or at risk
of developing a TNF.alpha.-related condition such as the ocular
conditions dry eye, allergic conjunctivitis, or ocular
inflammation, or such as dermatitis, rhinitis, or asthma, for
example.
Inventors: |
Shepard; Allan R.; (Fort
Worth, TX) ; Chatterton; Jon E.; (Fort Worth, TX)
; Clark; Abbot F.; (Arlington, TX) ; Wax; Martin
B.; (Westlake, TX) |
Correspondence
Address: |
ALCON
IP LEGAL, TB4-8, 6201 SOUTH FREEWAY
FORT WORTH
TX
76134
US
|
Assignee: |
ALCON RESEARCH, LTD.
Fort Worth
TX
|
Family ID: |
38702032 |
Appl. No.: |
12/700188 |
Filed: |
February 4, 2010 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
11750262 |
May 17, 2007 |
|
|
|
12700188 |
|
|
|
|
60801788 |
May 19, 2006 |
|
|
|
Current U.S.
Class: |
424/45 ;
514/44A |
Current CPC
Class: |
A61K 31/713 20130101;
C12N 15/1138 20130101; A61P 7/10 20180101; A61P 11/06 20180101;
A61P 27/14 20180101; A61P 27/06 20180101; A61P 29/00 20180101; A61P
43/00 20180101; C12N 15/1137 20130101; A61P 11/02 20180101; A61P
17/04 20180101; C12N 2310/14 20130101; C12N 2310/346 20130101; A61K
31/7088 20130101; A61P 27/02 20180101; C12N 2310/351 20130101; A61P
27/04 20180101; C12N 2320/30 20130101 |
Class at
Publication: |
424/45 ;
514/44.A |
International
Class: |
A61K 9/12 20060101
A61K009/12; A61K 31/7088 20060101 A61K031/7088; A61K 31/7125
20060101 A61K031/7125 |
Claims
1. A method of attenuating expression of TNFR1 mRNA of a subject in
need thereof, comprising: administering to the subject a
composition comprising an effective amount of interfering RNA
having a length of 19 to 49 nucleotides and a pharmaceutically
acceptable carrier, the interfering RNA comprising: a sense
nucleotide strand, an antisense nucleotide strand, and a region of
at least near-perfect contiguous complementarity of at least 19
nucleotides; wherein the antisense strand hybridizes under
physiological conditions to a portion of mRNA corresponding to SEQ
ID NO:2 beginning at nucleotide 124, 328, 387, 391, 393, 395, 406,
421, 423, 444, 447, 455, 459, 460, 467, 469, 470, 471, 475, 479,
513, 517, 531, 543, 556, 576, 587, 588, 589, 595, 601, 602, 611,
612, 651, 664, 667, 668, 669, 677, 678, 785, 786, 788, 791, 792,
804, 813, 824, 838, 843, 877, 884, 929, 959, 960, 961, 963, 964,
965, 970, 973, 974, 1000, 1002, 1013, 1026, 1053, 1056, 1057, 1058,
1161, 1315, 1318, 1357, 1360, 1383, 1393, 1420, 1471, 1573, 1671,
2044, 2045, 2046, 2047, 2048, 2089, 2090, 2091, or, 2092; wherein
the expression of TNFR1 mRNA is attenuated thereby.
2. The method of claim 1, wherein the subject is a human and the
human has dry eye.
3. The method of claim 1, wherein the sense nucleotide strand and
the antisense nucleotide strand are connected by a loop nucleotide
strand.
4. The method of claim 1, wherein the composition is administered
via an aerosol, buccal, dermal, intradermal, inhaling,
intramuscular, intranasal, intraocular, intrapulmonary,
intravenous, intraperitoneal, nasal, ocular, oral, otic,
parenteral, patch, subcutaneous, sublingual, topical, or
transdermal route.
5. The method of claim 1, wherein the interfering RNA is
administered via in vivo expression from an expression vector
capable of expressing the interfering RNA.
6. A method of treating a TNF.alpha.-related ocular condition in a
subject in need thereof, comprising: administering to an eye of the
subject a composition beginning at an effective amount of
interfering RNA having a length of 19 to 49 nucleotides, and a
pharmaceutically acceptable carrier, the interfering RNA comprising
a sense nucleotide strand, an antisense nucleotide strand, and the
sense and antisense strands comprise a region of least near-perfect
contiguous complementarity of at least 19 nucleotides; wherein the
antisense strand hybridizes under physiological conditions to a
portion of mRNA corresponding to SEQ ID NO:2 comprising nucleotide
124, 328, 387, 391, 393, 395, 406, 421, 423, 444, 447, 455, 459,
460, 467, 469, 470, 471, 475, 479, 513, 517, 531, 543, 556, 576,
587, 588, 589, 595, 601, 602, 611, 612, 651, 664, 667, 668, 669,
677, 678, 785, 786, 788, 791, 792, 804, 813, 824, 838, 843, 877,
884, 929, 959, 960, 961, 963, 964, 965, 970, 973, 974, 1000, 1002,
1013, 1026, 1053, 1056, 1057, 1058, 1161, 1315, 1318, 1357, 1360,
1383, 1393, 1420, 1471, 1573, 1671, 2044, 2045, 2046, 2047, 2048,
2089, 2090, 2091, or, 2092; wherein the TNF.alpha.-related ocular
condition is treated thereby, and wherein the TNF.alpha.-related
ocular condition is dry eye, allergic conjunctivitis or ocular
inflammation.
7. The method of claim 6, further comprising administering to the
eye of the subject a second interfering RNA having a length of 19
to 49 nucleotides and comprising: a sense nucleotide strand, an
antisense nucleotide strand, and the sense and antisense strands
comprise a region of least near-perfect contiguous complementarity
of at least 19 nucleotides wherein the antisense strand of the
second interfering RNA hybridizes under physiological conditions to
a portion of mRNA corresponding to SEQ ID NO:1 comprising
nucleotide 297, 333, 334, 335, 434, 470, 493, 547, 570, 573, 618,
649, 689, 755, 842, 844, 846, 860, 878, 894, 900, 909, 910, 913,
942, 970, 984, 1002, 1010, 1053, 1064, 1137, 1162, 1215, 1330,
1334, 1340, 1386, 1393, 1428, 1505, 1508, 1541, 1553, 1557, 1591,
1592, 1593, 1597, 1604, 1605, 1626, 1632, 1658, 1661, 1691, 1794,
1856, 1945, 1946, 1947, 1958, 2022, 2094, 2100, 2121, 2263, 2277,
2347, 2349, 2549, 2578, 2595, 2606, 2608, 2629, 2639, 2764, 2766,
2767, 2769, 3027, 3028, 3261, 3264, 3284, 3313, 3317, 3332, or
3337.
8. The method of claim 6, wherein the sense nucleotide strand and
the antisense nucleotide strand are connected by a loop nucleotide
strand.
9. The method of claim 6, wherein the composition is administered
via an aerosol, buccal, dermal, intradermal, inhaling,
intramuscular, intranasal, intraocular, intrapulmonary,
intravenous, intraperitoneal, nasal, ocular, oral, otic,
parenteral, patch, subcutaneous, sublingual, topical, or
transdermal route.
10. The method of claim 6, wherein the interfering RNA is
administered via in vivo expression from an expression vector
capable of expressing the interfering RNA.
11. The method of claim 6, wherein each strand of the siRNA
molecule is independently about 19 nucleotides to about 25
nucleotides in length.
12. The method of claim 6, wherein each strand of the siRNA
molecule is independently about 19 nucleotides to about 21
nucleotides in length.
13. A method of attenuating expression of TACE mRNA of a subject,
comprising: administering to the subject a composition comprising
an effective amount of interfering RNA having a length of 19 to 49
nucleotides and a pharmaceutically acceptable carrier, the
interfering RNA comprising: a region of at least 13, 14, 15, 16,
17, or 18 contiguous nucleotides having at least 80% sequence
complementarity to, or at least 90% sequence identity with, the
penultimate 13 nucleotides of the 3' end of an mRNA corresponding
to any one of SEQ ID NO:3, SEQ ID NO:14-SEQ ID NO:58, and SEQ ID
NO:155-SEQ ID NO:201, wherein the expression of TACE mRNA is
attenuated thereby.
14. The method of claim 13, wherein the interfering RNA is one of
an shRNA, an miRNA, or an siRNA.
15. The method of claim 13, wherein the composition is administered
via an aerosol, buccal, dermal, intradermal, inhaling,
intramuscular, intranasal, intraocular, intrapulmonary,
intravenous, intraperitoneal, nasal, ocular, oral, otic,
parenteral, patch, subcutaneous, sublingual, topical, or
transdermal route.
16. The method of claim 13, wherein the interfering RNA is
administered via in vivo expression from an expression vector
capable of expressing the interfering RNA.
17. A method of treating a TNF.alpha.-related condition in a
subject in need thereof, comprising: administering to the subject a
composition comprising an effective amount of interfering RNA
having a length of 19 to 49 nucleotides, and a pharmaceutically
acceptable carrier, wherein the interfering RNA comprises a region
of at least 13, 14, 15, 16, 17, or 18 contiguous nucleotides having
at least 80% sequence complementarity to, or at least 90% sequence
identity with, the penultimate 13 nucleotides of the 3' end of an
mRNA corresponding to any one of SEQ ID NO:3, SEQ ID NO:14-SEQ ID
NO:58, and SEQ ID NO:155-SEQ ID NO:201; wherein the
TNF.alpha.-related condition is treated thereby, and wherein the
TNF.alpha.-related ocular condition is dry eye, allergic
conjunctivitis or ocular inflammation.
18. The method of claim 17, wherein the interfering RNA is one of
an shRNA, an miRNA, or an siRNA.
19. The method of claim 17, wherein the composition is administered
via an aerosol, buccal, dermal, intradermal, inhaling,
intramuscular, intranasal, intraocular, intrapulmonary,
intravenous, intraperitoneal, nasal, ocular, oral, otic,
parenteral, patch, subcutaneous, sublingual, topical, or
transdermal route.
20. The method of claim 17, wherein the interfering RNA is
administered via in vivo expression from an expression vector
capable of expressing the interfering RNA.
Description
RELATED APPLICATION
[0001] The present application is a divisional of U.S. patent
application Ser. No. 11/750,262 filed May 17, 2007, which claims
benefit to U.S. Provisional Patent Application Ser. No. 60/801,788
filed on May 19, 2006, titled RNAi-MEDIATED INHIBITION OF TUMOR
NECROSIS FACTOR .alpha.-RELATED CONDITIONS, the text of which is
specifically incorporated by reference herein.
FIELD OF THE INVENTION
[0002] The present invention relates to the field of interfering
RNA compositions for silencing tumor necrosis factor .alpha.
(TNF.alpha.) by silencing the TNF.alpha. cell surface receptor TNF
receptor-1 (TNFR1) mRNA, or the TNF.alpha. converting enzyme
(TACE/ADAM17) mRNA. Silencing such TNF.alpha. targets is useful for
treatment of patients having a TNF.alpha.-related condition or at
risk of developing such a condition.
BACKGROUND OF THE INVENTION
[0003] Inflammation is generally treated with a standard
anti-inflammatory regimen that includes steroids and/or
non-steroidal anti-inflammatory drugs (NSAIDS). Allergic
conjunctivitis, ocular inflammation, dermatitis, rhinitis, and
asthma have historically been treated with a regimen of oral,
intranasal or topical antihistamines in addition to or oral or
intranasal steroids. Systemic treatment typically requires higher
concentrations of the drug compound to be administered to afford an
effective concentration to reach the necessary treatment site.
Antihistamine compounds are known to have central nervous system
activity; drowsiness and drying of mucus membranes are a common
side-effect of antihistamine use. Steroids and NSAIDS have
potential side effects including intraocular pressure increase,
cataract, glaucoma or corneal melting.
[0004] Dry eye, also known as conjunctivitis sicca or
keratoconjunctivitis sicca, is a common opthalmological disorder
involving breakdown of the pre-ocular tear film, resulting in
dehydration of the exposed outer surface of the eye. To date, dry
eye has been treated with topical administration of artificial tear
solutions. Some of these solutions contain mucomimetic substances
to temporarily replace or replenish the mucin layer in mucin
deficient patients. Use of methylprednisolone has been proposed in
a short-term "pulse" treatment to treat exacerbations of dry eye.
The proposed "pulse" therapy is required to avoid complications
associated with traditional steroid therapy for inflammatory
conditions such as increased intraocular pressure and cataract
formation.
[0005] The cytokine TNF.alpha. is a target for anti-inflammatory
therapy of dry eye and uveitis. In a rabbit model of lacrimal gland
inflammation-induced dry eye, inhibition of corneal staining and
restoration of tear breakup time has been achieved by specific
modulation of ocular surface TNF.alpha. levels. Dry eye therapy
resulted by inhibiting TNF.alpha. synthesis (RDP58) or by
specifically neutralizing TNF.alpha. using a monoclonal antibody
(REMICADE.RTM.) or a soluble receptor (ENBREL.RTM.). Each of these
TNF.alpha. directed treatments resulted in levels of efficacy
obtained with topical ocular anti-inflammatory steroids.
[0006] U.S. Patent Publication 2005/0227935, published Oct. 13,
2005, to McSwiggen et al. relates to RNA interference mediated
inhibition of TNF and TNF receptor gene expression. However, said
publication teaches none of the particular target sequences for RNA
interference as provided herein.
[0007] Embodiments of the present invention address the need in the
art for further agents and treatment methods for dry eye and
inflammation and provide alternative therapies therefor.
SUMMARY OF THE INVENTION
[0008] Embodiments of the present invention provide highly potent
and efficacious treatment, prevention or intervention of a
TNF.alpha.-related condition without side effects associated with
steroids or NSAIDS. In one aspect, methods of the invention include
treating a subject having a TNF.alpha.-related condition or at risk
of developing a TNF.alpha.-related condition by administering
interfering RNAs that silence expression of TACE mRNA or TNFR1
mRNA, thus interfering with proteolytic processing of the precursor
to TNF.alpha., or interfering with binding of TNF.alpha. to its
cell surface receptor, respectively, thereby attenuating activity
of TNF.alpha. and preventing a cascade of events related to
apoptosis and inflammation.
[0009] A TNF.alpha.-related condition includes conditions such as
dry eye and TNF.alpha.-related inflammatory conditions. A
TNF.alpha.-related inflammatory condition includes conditions such
as ocular inflammation, allergic conjunctivitis, dermatitis,
rhinitis, and asthma, for example, and includes those cellular
changes resulting from the activity of TNF.alpha. that leads
directly or indirectly to the TNF.alpha.-related inflammatory
condition. A TNF.alpha.-related condition particularly includes
TNF.alpha.-related ocular conditions such as dry eye, allergic
conjunctivitis, and ocular inflammation. The interfering RNA
provided herein provides for silencing the TNF.alpha. targets TACE
mRNA or TNFR1 mRNA while avoiding undesirable side effects due to
nonspecific agents.
[0010] A method of attenuating expression of TACE mRNA of the
subject is an embodiment of the invention. The method comprises
administering to the subject a composition comprising an effective
amount of interfering RNA having a length of 19 to 49 nucleotides
and a pharmaceutically acceptable carrier, the interfering RNA
comprising a region of at least 13 contiguous nucleotides having at
least 90% sequence complementarity to, or at least 90% sequence
identity with, the penultimate 13 nucleotides of the 3' end of an
mRNA corresponding to any one of SEQ ID NO:3, SEQ ID NO:14-SEQ ID
NO:58, and SEQ ID NO:155-SEQ ID NO:201. The expression of TACE mRNA
is attenuated thereby.
[0011] A method of treating a TNF.alpha.-related condition in a
subject in need thereof is an embodiment of the invention. The
method comprises administering to the subject a composition
comprising an effective amount of interfering RNA having a length
of 19 to 49 nucleotides, and a pharmaceutically acceptable carrier,
wherein the interfering RNA comprises a region of at least 13
contiguous nucleotides having at least 90% sequence complementarity
to, or at least 90% sequence identity with, the penultimate 13
nucleotides of the 3' end of an mRNA corresponding to any one of
SEQ ID NO:3, SEQ ID NO:14-SEQ ID NO:58, and SEQ ID NO:155-SEQ ID
NO:201. The TNF.alpha.-related condition is treated thereby.
[0012] In yet another embodiment of the invention, a method of
attenuating activity of TNF.alpha. of a subject by attenuating
expression of TACE mRNA or TNFR1 mRNA of the subject comprises
administering to the subject a composition comprising an effective
amount of interfering RNA having a length of 19 to 49 nucleotides
and a pharmaceutically acceptable carrier and the interfering RNA
comprises a sense nucleotide strand, an antisense nucleotide
strand, and a region of at least near-perfect contiguous
complementarity of at least 19 nucleotides where the antisense
strand hybridizes under physiological conditions to a portion of
mRNA corresponding to SEQ ID NO:1 comprising nucleotide 297, 333,
334, 335, 434, 470, 493, 547, 570, 573, 618, 649, 689, 755, 842,
844, 846, 860, 878, 894, 900, 909, 910, 913, 942, 970, 984, 1002,
1010, 1053, 1064, 1137, 1162, 1215, 1330, 1334, 1340, 1386, 1393,
1428, 1505, 1508, 1541, 1553, 1557, 1591, 1592, 1593, 1597, 1604,
1605, 1626, 1632, 1658, 1661, 1691, 1794, 1856, 1945, 1946, 1947,
1958, 2022, 2094, 2100, 2121, 2263, 2277, 2347, 2349, 2549, 2578,
2595, 2606, 2608, 2629, 2639, 2764, 2766, 2767, 2769, 3027, 3028,
3261, 3264, 3284, 3313, 3317, 3332, or 3337 or where the antisense
strand hybridizes under physiological conditions to a portion of
mRNA corresponding to SEQ ID NO:2 beginning at nucleotide 124, 328,
387, 391, 393, 395, 406, 421, 423, 444, 447, 455, 459, 460, 467,
469, 470, 471, 475, 479, 513, 517, 531, 543, 556, 576, 587, 588,
589, 595, 601, 602, 611, 612, 651, 664, 667, 668, 669, 677, 678,
785, 786, 788, 791, 792, 804, 813, 824, 838, 843, 877, 884, 929,
959, 960, 961, 963, 964, 965, 970, 973, 974, 1000, 1002, 1013,
1026, 1053, 1056, 1057, 1058, 1161, 1315, 1318, 1324, 1357, 1360,
1383, 1393, 1420, 1471, 1573, 1671, 2044, 2045, 2046, 2047, 2048,
2089, 2090, 2091, 2092, or 2098. The expression of TACE mRNA is
attenuated in those embodiments where the antisense stand
hybridizes to a portion of mRNA corresponding to SEQ ID NO:1 as
cited above. The expression of TNFR1 mRNA is attenuated in those
embodiments where the antisense stand hybridizes to a portion of
mRNA corresponding to SEQ ID NO:2 as cited above.
[0013] A method of treating a TNF.alpha.-related condition in a
subject in need thereof is an embodiment of the invention, the
method comprising administering to the subject a composition
comprising an effective amount of interfering RNA having a length
of 19 to 49 nucleotides, and a pharmaceutically acceptable carrier,
the interfering RNA comprising a sense nucleotide strand, an
antisense nucleotide strand, and a region of at least near-perfect
contiguous complementarity of at least 19 nucleotides; wherein the
antisense strand hybridizes under physiological conditions to a
portion of mRNA corresponding to SEQ ID NO:1 comprising nucleotide
297, 333, 334, 335, 434, 470, 493, 547, 570, 573, 618, 649, 689,
755, 842, 844, 846, 860, 878, 894, 900, 909, 910, 913, 942, 970,
984, 1002, 1010, 1053, 1064, 1137, 1162, 1215, 1330, 1334, 1340,
1386, 1393, 1428, 1505, 1508, 1541, 1553, 1557, 1591, 1592, 1593,
1597, 1604, 1605, 1626, 1632, 1658, 1661, 1691, 1794, 1856, 1945,
1946, 1947, 1958, 2022, 2094, 2100, 2121, 2263, 2277, 2347, 2349,
2549, 2578, 2595, 2606, 2608, 2629, 2639, 2764, 2766, 2767, 2769,
3027, 3028, 3261, 3264, 3284, 3313, 3317, 3332, or 3337. The
TNF.alpha.-related condition is treated thereby.
[0014] A method of treating a TNF.alpha.-related ocular condition
in a subject in need thereof is an embodiment of the invention, the
method comprising administering to the subject a composition
comprising an effective amount of interfering RNA having a length
of 19 to 49 nucleotides, and a pharmaceutically acceptable carrier,
the interfering RNA comprising a sense nucleotide strand, an
antisense nucleotide strand, and a region of at least near-perfect
contiguous complementarity of at least 19 nucleotides; wherein the
antisense strand hybridizes under physiological conditions to a
portion of mRNA corresponding to SEQ ID NO:2 comprising nucleotide
124, 328, 387, 391, 393, 395, 406, 421, 423, 444, 447, 455, 459,
460, 467, 469, 470, 471, 475, 479, 513, 517, 531, 543, 556, 576,
587, 588, 589, 595, 601, 602, 611, 612, 651, 664, 667, 668, 669,
677, 678, 785, 786, 788, 791, 792, 804, 813, 824, 838, 843, 877,
884, 929, 959, 960, 961, 963, 964, 965, 970, 973, 974, 1000, 1002,
1013, 1026, 1053, 1056, 1057, 1058, 1161, 1315, 1318, 1324, 1357,
1360, 1383, 1393, 1420, 1471, 1573, 1671, 2044, 2045, 2046, 2047,
2048, 2089, 2090, 2091, 2092, or 2098. The TNF.alpha.-related
condition is treated thereby.
[0015] A second interfering RNA having a length of 19 to 49
nucleotides could also be administered to the subject in a further
embodiment; the second interfering RNA comprising a sense
nucleotide strand, an antisense nucleotide strand, and a region of
at least near-perfect complementarity of at least 19 nucleotides
wherein the antisense strand of the second interfering RNA
hybridizes under physiological conditions to a portion of mRNA
corresponding to SEQ ID NO:1 comprising a nucleotide as cited
above, or where the antisense strand hybridizes under physiological
conditions to a portion of mRNA corresponding to SEQ ID NO:2
beginning at nucleotide 124, 328, 387, 391, 393, 395, 406, 421,
423, 444, 447, 455, 459, 460, 467, 469, 470, 471, 475, 479, 513,
517, 531, 543, 556, 576, 587, 588, 589, 595, 601, 602, 611, 612,
651, 664, 667, 668, 669, 677, 678, 785, 786, 788, 791, 792, 804,
813, 824, 838, 843, 877, 884, 929, 959, 960, 961, 963, 964, 965,
970, 973, 974, 1000, 1002, 1013, 1026, 1053, 1056, 1057, 1058,
1161, 1315, 1318, 1324, 1357, 1360, 1383, 1393, 1420, 1471, 1573,
1671, 2044, 2045, 2046, 2047, 2048, 2089, 2090, 2091, 2092, or
2098.
[0016] When a first interfering RNA targets SEQ ID NO:1, the second
interfering RNA may target either SEQ ID NO:1 or SEQ ID NO:2, and
conversely, when a first interfering RNA targets SEQ ID NO:2, the
second interfering RNA may target either SEQ ID NO:1 or SEQ ID
NO:2. In further embodiments, a third, fourth, or more interfering
RNAs may be administered.
[0017] A further embodiment of the invention is a method of
treating a TNF.alpha.-related condition in a subject in need
thereof, where the method comprises administering to the subject a
composition comprising a double stranded siRNA molecule that down
regulates expression of a TACE gene via RNA interference, wherein
each strand of the siRNA molecule is independently about 19 to
about 27 nucleotides in length; and one strand of the siRNA
molecule comprises a nucleotide sequence having substantial
complementarity to an mRNA corresponding to the TACE gene, so that
the siRNA molecule directs cleavage of the mRNA via RNA
interference.
[0018] A further embodiment of the invention is a method of
treating a TNF.alpha.-related ocular condition in a subject in need
thereof, where the method comprises administering to the subject a
composition comprising a double stranded siRNA molecule that down
regulates expression of a TNFR1 gene via RNA interference, wherein
each strand of the siRNA molecule is independently about 19 to
about 27 nucleotides in length; and one strand of the siRNA
molecule comprises a nucleotide sequence having substantial
complementarity to an mRNA corresponding to the TNFR1 gene so that
the siRNA molecule directs cleavage of the mRNA via RNA
interference.
[0019] A method of attenuating expression of TACE mRNA of the
subject, comprising administering to the subject a composition
comprising an effective amount of a single-stranded interfering RNA
and a pharmaceutically acceptable carrier is a further embodiment.
The single-stranded interfering RNA has a length of 19 to 49
nucleotides and hybridizes under physiological conditions to a
portion of mRNA corresponding to SEQ ID NO:1 comprising nucleotide
297, 333, 334, 335, 434, 470, 493, 547, 570, 573, 618, 649, 689,
755, 842, 844, 846, 860, 878, 894, 900, 909, 910, 913, 942, 970,
984, 1002, 1010, 1053, 1064, 1137, 1162, 1215, 1330, 1334, 1340,
1386, 1393, 1428, 1505, 1508, 1541, 1553, 1557, 1591, 1592, 1593,
1597, 1604, 1605, 1626, 1632, 1658, 1661, 1691, 1794, 1856, 1945,
1946, 1947, 1958, 2022, 2094, 2100, 2121, 2263, 2277, 2347, 2349,
2549, 2578, 2595, 2606, 2608, 2629, 2639, 2764, 2766, 2767, 2769,
3027, 3028, 3261, 3264, 3284, 3313, 3317, 3332, or 3337, and the
interfering RNA has a region of at least near-perfect contiguous
complementarity with the hybridizing portion of mRNA corresponding
to SEQ ID NO:1. The expression of TACE mRNA is thereby
attenuated.
[0020] The invention includes as a further embodiment a composition
comprising an interfering RNA having a length of 19 to 49
nucleotides, and comprising a nucleotide sequence corresponding to
any one of SEQ ID NO:3, SEQ ID NO:14-SEQ ID NO:58, and SEQ ID
NO:155-SEQ ID NO:201, or a complement thereof; and a
pharmaceutically acceptable carrier.
[0021] The invention includes as a further embodiment a composition
comprising an interfering RNA consisting essentially of a
nucleotide sequence corresponding to any one of SEQ ID NO:59-SEQ ID
NO:69, SEQ ID NO:71-SEQ ID NO:92, and SEQ ID NO:94-SEQ ID NO:154,
or a complement thereof; and a pharmaceutically acceptable
carrier.
[0022] Use of any of the embodiments as described herein in the
preparation of a medicament for attenuating expression of TACE mRNA
or of TNFR1 mRNA as a method of attenuating activity of TNF.alpha.
and thereby treating a TNF.alpha.-related condition as set forth
herein is also an embodiment of the present invention.
BRIEF DESCRIPTION OF THE FIGURES
[0023] In order that the manner in which the above recited and
other advantages and objects of the invention are obtained, a more
particular description of the invention briefly described above
will be rendered by reference to specific embodiments thereof,
which are illustrated, in the appended drawings. Understanding that
these drawings depict only typical embodiments of the invention and
are therefore not to be considered limiting of its scope, the
invention will be described with additional specificity and detail
through the use of the accompanying drawings in which:
[0024] FIG. 1 provides a TNFR1 western blot of GTM-3 cells
transfected with TNFR1 siRNAs #1, #2, #3, and #4, and a RISC-free
control siRNA, each at 10 nM, 1 nM, and 0.1 nM; a non-targeting
control siRNA (NTC2) at 10 nM; and a buffer control (-siRNA). The
arrows indicate the positions of the 55-kDa TNFR1 and 42-kDa actin
bands.
DETAILED DESCRIPTION OF THE INVENTION
[0025] The references cited herein, to the extent that they provide
exemplary procedural or other details supplementary to those set
forth herein, are specifically incorporated by reference.
[0026] Those of skill in the art, in light of the present
disclosure, will appreciate that obvious modifications of the
embodiments disclosed herein can be made without departing from the
spirit and scope of the invention. All of the embodiments disclosed
herein can be made and executed without undue experimentation in
light of the present disclosure. The full scope of the invention is
set out in the disclosure and equivalent embodiments thereof. The
specification should not be construed to unduly narrow the full
scope of protection to which the present invention is entitled.
[0027] The particulars shown herein are by way of example and for
purposes of illustrative discussion of the preferred embodiments of
the present invention only and are presented in the cause of
providing what is believed to be the most useful and readily
understood description of the principles and conceptual aspects of
various embodiments of the invention. In this regard, no attempt is
made to show structural details of the invention in more detail
than is necessary for the fundamental understanding of the
invention, the description taken with the drawings and/or examples
making apparent to those skilled in the art how the several forms
of the invention may be embodied in practice.
[0028] The following definitions and explanations are meant and
intended to be controlling in any future construction unless
clearly and unambiguously modified in the following examples or
when application of the meaning renders any construction
meaningless or essentially meaningless. In cases where the
construction of the term would render it meaningless or essentially
meaningless, the definition should be taken from Webster's
Dictionary, 3.sup.rd Edition.
[0029] As used herein, all percentages are percentages by weight,
unless stated otherwise.
[0030] As used herein and unless otherwise indicated, the terms "a"
and "an" are taken to mean "one", "at least one" or "one or
more".
[0031] The term "dry eye," also known as conjunctivitis sicca or
keratoconjunctivitis sicca, is a common opthalmological disorder
involving breakdown of the pre-ocular tear film, resulting in
dehydration of the exposed outer surface of the eye.
[0032] The term "ocular inflammation," as used herein, includes
iritis, uveitis, episcleritis, scleritis, keratitis,
endophthalmitis, blepharitis, and iatrogenic inflammatory
conditions, for example.
[0033] The term "allergic conjunctivitis," as used herein, refers
to inflammation of the conjunctiva which is the delicate membrane
that lines the eyelids and covers the exposed surface of the
sclera. The term "allergic conjunctivitis" includes, for example,
atopic keratoconjunctivitis, giant papillary conjunctivitis, hay
fever conjunctivitis, perennial allergic conjunctivitis, and vernal
keratoconjunctivitis.
[0034] The term "dermatitis," as used herein, refers to
inflammation of the skin and includes, for example, allergic
contact dermatitis, urticaria, asteatotic dermatitis (dry skin on
the lower legs), atopic dermatitis, contact dermatitis including
irritant contact dermatitis and urushiol-induced contact
dermatitis, eczema, gravitational dermatitis, nummular dermatitis,
otitis externa, perioral dermatitis, and seborrhoeic
dermatitis.
[0035] The term "rhinitis," as used herein, refers to inflammation
of the mucous membranes of the nose and includes, for example,
allergic rhinitis, atopic rhinitis, irritant rhinitis, eosinophilic
non-allergic rhinitis, rhinitis medicamentosa, and neutrophilic
rhinosinusitis.
[0036] The term "asthma," as used herein, refers to inflammation of
the air passages resulting in narrowing of the airways that
transport air from the nose and mouth to the lungs and includes,
for example, allergic asthma, atopic asthma, atopic bronchial
IgE-mediated asthma, bronchial asthma, bronchiolytis, emphysematous
asthma, essential asthma, exercise-induced asthma, extrinsic asthma
caused by environmental factors, incipient asthma, intrinsic asthma
caused by pathophysiologic disturbances, non-allergic asthma,
non-atopic asthma, and wheezy infant syndrome.
[0037] The phrase "a region of at least 13 contiguous nucleotides
having at least 90% sequence complementarity to, or at least 90%
sequence identity with, the penultimate 13 nucleotides of the 3'
end of an mRNA corresponding to any one of (a sequence identifier)"
allows a one nucleotide substitution. Two nucleotide substitutions
(i.e., 11/13=85% identity/complementarity) are not included in such
a phrase.
[0038] The term "percent identity" describes the percentage of
contiguous nucleotides in a first nucleic acid molecule that is the
same as in a set of contiguous nucleotides of the same length in a
second nucleic acid molecule. The term "percent complementarity"
describes the percentage of contiguous nucleotides in a first
nucleic acid molecule that can base pair in the Watson-Crick sense
with a set of contiguous nucleotides in a second nucleic acid
molecule.
[0039] As used herein, the term "hybridization" means and refers to
a process in which single-stranded nucleic acids with complementary
or near-complementary base sequences interact to form
hydrogen-bonded complexes called hybrids. Hybridization reactions
are sensitive and selective. In vitro, the specificity of
hybridization (i.e., stringency) is controlled by the
concentrations of salt or formamide in prehybridization and
hybridization solutions, for example, and by the hybridization
temperature; such procedures are well known in the art. In
particular, stringency is increased by reducing the concentration
of salt, increasing the concentration of formamide, or raising the
hybridization temperature.
[0040] For example, high stringency conditions could occur at about
50% formamide at 37.degree. C. to 42.degree. C. Reduced stringency
conditions could occur at about 35% to 25% formamide at 30.degree.
C. to 35.degree. C. Examples of stringency conditions for
hybridization are provided in Sambrook, J., 1989, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. Further examples of stringent
hybridization conditions 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, or hybridization at 70.degree. C. in 1.times.SSC or
50.degree. C. in 1.times.SSC, 50% formamide followed by washing at
70.degree. C. in 0.3.times.SSC, or hybridization at 70.degree. C.
in 4.times.SSC or 50.degree. C. in 4.times.SSC, 50% formamide
followed by washing at 67.degree. C. in 1.times.SSC. The
temperature for hybridization is about 5-10.degree. C. less than
the melting temperature (T.sub.m) of the hybrid where T.sub.m is
determined for hybrids between 19 and 49 base pairs in length using
the following calculation: T.sub.m.degree.
C.=81.5+16.6(log.sub.10[Na+])+0.41 (% G+C)-(600/N) where N is the
number of bases in the hybrid, and [Na+] is the concentration of
sodium ions in the hybridization buffer.
[0041] Nucleic acid sequences cited herein are written in a 5' to
3' direction unless indicated otherwise. The term "nucleic acid,"
as used herein, refers to either DNA or RNA or a modified form
thereof comprising the purine or pyrimidine bases present in DNA
(adenine "A," cytosine "C," guanine "G," thymine "T") or in RNA
(adenine "A," cytosine "C," guanine "G," uracil "U"). Interfering
RNAs provided herein may comprise "T" bases, particularly at 3'
ends, even though "T" bases do not naturally occur in RNA. "Nucleic
acid" includes the terms "oligonucleotide" and "polynucleotide" and
can refer to a single-stranded molecule or a double-stranded
molecule. A double-stranded molecule is formed by Watson-Crick base
pairing between A and T bases, C and G bases, and between A and U
bases. The strands of a double-stranded molecule may have partial,
substantial or full complementarity to each other and will form a
duplex hybrid, the strength of bonding of which is dependent upon
the nature and degree of complementarity of the sequence of
bases.
[0042] An mRNA sequence is readily deduced from the sequence of the
corresponding DNA sequence. For example, SEQ ID NO:1 provides the
sense strand sequence of DNA corresponding to the mRNA for TACE.
The mRNA sequence is identical to the DNA sense strand sequence
with the "T" bases replaced with "U" bases. Therefore, the mRNA
sequence of TACE is known from SEQ ID NO:1 and the mRNA of TNFR1 is
known from SEQ ID NO:2.
[0043] RNA interference (RNAi) is a process by which
double-stranded RNA (dsRNA) is used to silence gene expression.
While not wanting to be bound by theory, RNAi begins with the
cleavage of longer dsRNAs into small interfering RNAs (siRNAs) by
an RNaseIII-like enzyme, dicer. SiRNAs are dsRNAs that are usually
about 19 to 28 nucleotides, or 20 to 25 nucleotides, or 21 to 22
nucleotides in length and often contain 2-nucleotide 3' overhangs,
and 5' phosphate and 3' hydroxyl termini. One strand of the siRNA
is incorporated into a ribonucleoprotein complex known as the
RNA-induced silencing complex (RISC). RISC uses this siRNA strand
to identify mRNA molecules that are at least partially
complementary to the incorporated siRNA strand, and then cleaves
these target mRNAs or inhibits their translation. Therefore, the
siRNA strand that is incorporated into RISC is known as the guide
strand or the antisense strand. The other siRNA strand, known as
the passenger strand or the sense strand, is eliminated from the
siRNA and is at least partially homologous to the target mRNA.
Those of skill in the art will recognize that, in principle, either
strand of an siRNA can be incorporated into RISC and function as a
guide strand. However, siRNA design (e.g., decreased siRNA duplex
stability at the 5' end of the antisense strand) can favor
incorporation of the antisense strand into RISC.
[0044] RISC-mediated cleavage of mRNAs having a sequence at least
partially complementary to the guide strand leads to a decrease in
the steady state level of that mRNA and of the corresponding
protein encoded by this mRNA. Alternatively, RISC can also decrease
expression of the corresponding protein via translational
repression without cleavage of the target mRNA. Other RNA molecules
and RNA-like molecules can also interact with RISC and silence gene
expression. Examples of other RNA molecules that can interact with
RISC include short hairpin RNAs (shRNAs), single-stranded siRNAs,
microRNAs (miRNAs), and dicer-substrate 27-mer duplexes. The term
"siRNA" as used herein refers to a double-stranded interfering RNA
unless otherwise noted. Examples of RNA-like molecules that can
interact with RISC include RNA molecules containing one or more
chemically modified nucleotides, one or more deoxyribonucleotides,
and/or one or more non-phosphodiester linkages. For purposes of the
present discussion, all RNA or RNA-like molecules that can interact
with RISC and participate in RISC-mediated changes in gene
expression will be referred to as "interfering RNAs." SiRNAs,
shRNAs, miRNAs, and dicer-substrate 27-mer duplexes are, therefore,
subsets of "interfering RNAs."
[0045] Interfering RNA of embodiments of the invention appear to
act in a catalytic manner for cleavage of target mRNA, i.e.,
interfering RNA is able to effect inhibition of target mRNA in
substoichiometric amounts. As compared to antisense therapies,
significantly less interfering RNA is required to provide a
therapeutic effect under such cleavage conditions.
[0046] The present invention relates to the use of interfering RNA
to inhibit the expression of TNF.alpha. cell surface receptor TNF
receptor-1 (TNFR1), or the TNF.alpha. converting enzyme
(TACE/ADAM17, designated herein "TACE") which inhibition effects
reduction of tumor necrosis factor .alpha. (TNF.alpha.) activity.
Binding of TNF.alpha. to its cell surface receptor, TNF receptor-1
(TNFR1), activates a signaling cascade which affects a variety of
cellular responses including apoptosis and inflammation. TNF.alpha.
itself is initially expressed as an inactive, membrane-bound
precursor. Release of the active form of TNF.alpha. from the cell
surface requires proteolytic processing of the precursor by
TNF.alpha. converting enzyme (TACE/ADAM17), a member of the `A
Disintegrin And Metalloprotease` (ADAM) family.
[0047] According to the present invention, inhibiting the
expression of TNFR1 mRNA, TACE mRNA, or both TNFR1 and TACE mRNAs
effectively reduces the action of TNF.alpha.. Further, interfering
RNAs as set forth herein provided exogenously or expressed
endogenously are particularly effective at silencing TNFR1 mRNA or
TACE mRNA.
[0048] Tumor Necrosis Factor .alpha. Converting Enzyme mRNA
(TACE/ADAM17): The GenBank database provides the DNA sequence for
TACE as accession no. NM.sub.--003183, provided in the "Sequence
Listing" as SEQ ID NO:1. SEQ ID NO:1 provides the sense strand
sequence of DNA that corresponds to the mRNA encoding TACE (with
the exception of "T" bases for "U" bases). The coding sequence for
TACE is from nucleotides 184-2658.
[0049] Equivalents of the above cited TACE mRNA sequence are
alternative splice forms, allelic forms, isozymes, or a cognate
thereof. A cognate is a tumor necrosis factor .alpha. converting
enzyme mRNA from another mammalian species that is homologous to
SEQ ID NO:1 (i.e., an ortholog).
[0050] Tumor Necrosis Factor Receptor-1 mRNA (TNFR1): The GenBank
database provides the DNA sequence for TNFR1 as accession no.
NM.sub.--001065, provided in the "Sequence Listing" as SEQ ID NO:2.
SEQ ID NO:2 provides the sense strand sequence of DNA that
corresponds to the mRNA encoding TNFR1 (with the exception of "T"
bases for "U" bases). The coding sequence for TNFR1 is from
nucleotides 282-1649.
[0051] Equivalents of the above cited TNFR1 mRNA sequence are
alternative splice forms, allelic forms, isozymes, or a cognate
thereof. A cognate is a tumor necrosis factor receptor-1 mRNA from
another mammalian species that is homologous to SEQ ID NO:2 (i.e.,
an ortholog).
[0052] Attenuating expression of an mRNA: The phrase, "attenuating
expression of an mRNA," as used herein, means administering or
expressing an amount of interfering RNA (e.g., an siRNA) to reduce
translation of the target mRNA into protein, either through mRNA
cleavage or through direct inhibition of translation. The reduction
in expression of the target mRNA or the corresponding protein is
commonly referred to as "knock-down" and is reported relative to
levels present following administration or expression of a
non-targeting control RNA (e.g., a non-targeting control siRNA).
Knock-down of expression of an amount including and between 50% and
100% is contemplated by embodiments herein. However, it is not
necessary that such knock-down levels be achieved for purposes of
the present invention. In one embodiment, a single interfering RNA
targeting TACE mRNA or TNFR1 mRNA is administered. In other
embodiments, two or more interfering RNAs targeting TACE mRNA or
TNFR1 mRNA are administered. In further embodiments, interfering
RNAs targeting each of TACE mRNA and TNFR1 mRNA are administered in
combination or in a time interval so as to have overlapping
effects.
[0053] Knock-down is commonly assessed by measuring the mRNA levels
using quantitative polymerase chain reaction (qPCR) amplification
or by measuring protein levels by western blot or enzyme-linked
immunosorbent assay (ELISA). Analyzing the protein level provides
an assessment of both mRNA cleavage as well as translation
inhibition. Further techniques for measuring knock-down include RNA
solution hybridization, nuclease protection, northern
hybridization, gene expression monitoring with a microarray,
antibody binding, radioimmunoassay, and fluorescence activated cell
analysis.
[0054] Inhibition of TACE or TNFR1 may also be determined in vitro
by evaluating target mRNA levels or target protein levels in, for
example, human corneal epithelial cells following transfection of
TACE- or TNFR1-interfering RNA as described infra.
[0055] Inhibition of TNF.alpha. activity due to inhibition of TACE
mRNA expression or of TNFR1 mRNA expression is also inferred in a
human or mammal by observing an improvement in a TNF.alpha.-related
condition symptom such as improvement in symptoms related to dry
eye, allergic conjunctivitis, ocular inflammation, dermatitis,
rhinitis, or asthma. Improvement in any of dry eye symptoms, edema,
itching, inflammation, or tolerance to environmental challenges,
for example, is indicative of inhibition of TNF.alpha.
activity.
[0056] Interfering RNA: In one embodiment of the invention,
interfering RNA (e.g., siRNA) has a sense strand and an antisense
strand, and the sense and antisense strands comprise a region of at
least near-perfect contiguous complementarity of at least 19
nucleotides. In a further embodiment of the invention, interfering
RNA (e.g., siRNA) has a sense strand and an antisense strand, and
the antisense strand comprises a region of at least near-perfect
contiguous complementarity of at least 19 nucleotides to a target
sequence of TACE mRNA or TNFR1 mRNA, and the sense strand comprises
a region of at least near-perfect contiguous identity of at least
19 nucleotides with a target sequence of TACE mRNA or TNFR1 mRNA,
respectively. In a further embodiment of the invention, the
interfering RNA comprises a region of at least 13, 14, 15, 16, 17,
or 18 contiguous nucleotides having percentages of sequence
complementarity to or, having percentages of sequence identity
with, the penultimate 13, 14, 15, 16, 17, or 18 nucleotides,
respectively, of the 3' end of the corresponding target sequence
within an mRNA.
[0057] The length of each strand of the interfering RNA comprises
19 to 49 nucleotides, and may comprise a length of 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,
40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides.
[0058] The antisense strand of an siRNA is the active guiding agent
of the siRNA in that the antisense strand is incorporated into
RISC, thus allowing RISC to identify target mRNAs with at least
partial complementarity to the antisense siRNA strand for cleavage
or translational repression.
[0059] In embodiments of the present invention, interfering RNA
target sequences (e.g., siRNA target sequences) within a target
mRNA sequence are selected using available design tools.
Interfering RNAs corresponding to a TACE or TNFR1 target sequence
are then tested by transfection of cells expressing the target mRNA
followed by assessment of knockdown as described above.
[0060] Techniques for selecting target sequences for siRNAs are
provided by Tuschl, T. et al., "The siRNA User Guide," revised May
6, 2004, available on the Rockefeller University web site; by
Technical Bulletin #506, "siRNA Design Guidelines," Ambion Inc. at
Ambion's web site; and by other web-based design tools at, for
example, the Invitrogen, Dharmacon, Integrated DNA Technologies,
Genscript, or Proligo web sites. Initial search parameters can
include G/C contents between 35% and 55% and siRNA lengths between
19 and 27 nucleotides. The target sequence may be located in the
coding region or in the 5' or 3' untranslated regions of the
mRNAs.
[0061] An embodiment of a 19-nucleotide DNA target sequence for
TACE mRNA is present at nucleotides 297 to 315 of SEQ ID NO:1:
TABLE-US-00001 5'-GCTCTCAGACTACGATATT-3'. SEQ ID NO: 3
An siRNA of the invention for targeting a corresponding mRNA
sequence of SEQ ID NO:3 and having 21-nucleotide strands and a
2-nucleotide 3' overhang is:
TABLE-US-00002 5'-GCUCUCAGACUACGAUAUUNN-3' SEQ ID NO: 4
3'-NNCGAGAGUCUGAUGCUAUAA-5'. SEQ ID NO: 5
Each "N" residue can be any nucleotide (A, C, G, U, T) or modified
nucleotide. The 3' end can have a number of "N" residues between
and including 1, 2, 3, 4, 5, and 6. The "N" residues on either
strand can be the same residue (e.g., UU, AA, CC, GG, or TT) or
they can be different (e.g., AC, AG, AU, CA, CG, CU, GA, GC, GU,
UA, UC, or UG). The 3' overhangs can be the same or they can be
different. In one embodiment, both strands have a 3'UU
overhang.
[0062] An siRNA of the invention for targeting a corresponding mRNA
sequence of SEQ ID NO:3 and having 21-nucleotide strands and a 3'UU
overhang on each strand is:
TABLE-US-00003 5'-GCUCUCAGACUACGAUAUUUU-3' SEQ ID NO: 6
3'-UUCGAGAGUCUGAUGCUAUAA-5'. SEQ ID NO: 7
[0063] The interfering RNA may also have a 5' overhang of
nucleotides or it may have blunt ends. An siRNA of the invention
for targeting a corresponding mRNA sequence of SEQ ID NO:3 and
having 19-nucleotide strands and blunt ends is:
TABLE-US-00004 5'-GCUCUCAGACUACGAUAUU-3' SEQ ID NO: 8
3'-CGAGAGUCUGAUGCUAUAA-5'. SEQ ID NO: 9
[0064] The strands of a double-stranded interfering RNA (e.g., an
siRNA) may be connected to form a hairpin or stem-loop structure
(e.g., an shRNA). An shRNA of the invention targeting a
corresponding mRNA sequence of SEQ ID NO:3 and having a 19 by
double-stranded stem region and a 3'UU overhang is:
##STR00001##
N is a nucleotide A, T, C, G, U, or a modified form known by one of
ordinary skill in the art. The number of nucleotides N in the loop
is a number between and including 3 to 23, or 5 to 15, or 7 to 13,
or 4 to 9, or 9 to 11, or the number of nucleotides N is 9. Some of
the nucleotides in the loop can be involved in base-pair
interactions with other nucleotides in the loop. Examples of
oligonucleotide sequences that can be used to form the loop include
5'-UUCAAGAGA-3' (Brummelkamp, T. R. et al. (2002) Science 296: 550)
and 5'-UUUGUGUAG-3' (Castanotto, D. et al. (2002) RNA 8:1454). It
will be recognized by one of skill in the art that the resulting
single chain oligonucleotide forms a stem-loop or hairpin structure
comprising a double-stranded region capable of interacting with the
RNAi machinery.
[0065] The siRNA target sequence identified above can be extended
at the 3' end to facilitate the design of dicer-substrate 27-mer
duplexes. Extension of the 19-nucleotide DNA target sequence (SEQ
ID NO:3) identified in the TACE DNA sequence (SEQ ID NO:1) by 6
nucleotides yields a 25-nucleotide DNA target sequence present at
nucleotides 297 to 321 of SEQ ID NO:1:
TABLE-US-00005 5'-GCTCTCAGACTACGATATTCTCTCT-3'. SEQ ID NO: 11
A dicer-substrate 27-mer duplex of the invention for targeting a
corresponding mRNA sequence of SEQ ID NO:11 is:
TABLE-US-00006 5'-GCUCUCAGACUACGAUAUUCUCUCU-3' SEQ ID NO: 12
3'-UUCGAGAGUCUGAUGCUAUAAGAGAGA-5'. SEQ ID NO: 13
The two nucleotides at the 3' end of the sense strand (i.e., the CU
nucleotides of SEQ ID NO:12) may be deoxynucleotides for enhanced
processing. Design of dicer-substrate 27-mer duplexes from 19-21
nucleotide target sequences, such as provided herein, is further
discussed by the Integrated DNA Technologies (IDT) website and by
Kim, D.-H. et al., (February, 2005) Nature Biotechnology 23:2;
222-226.
[0066] When interfering RNAs are produced by chemical synthesis,
phosphorylation at the 5' position of the nucleotide at the 5' end
of one or both strands (when present) can enhance siRNA efficacy
and specificity of the bound RISC complex but is not required since
phosphorylation can occur intracellularly.
[0067] Table 1 lists examples of TACE DNA target sequences of SEQ
ID NO:1 from which siRNAs of the present invention are designed in
a manner as set forth above. TACE encodes tumor necrosis factor
.alpha. converting enzyme, as noted above.
TABLE-US-00007 TABLE 1 TACE Target Sequences for siRNAs # of
Starting Nucleotide with reference to TACE Target Sequence SEQ ID
NO: 1 SEQ ID NO: GCTCTCAGACTACGATATT 297 3 CCAGCAGCATTCGGTAAGA 333
14 CAGCAGCATTCGGTAAGAA 334 15 AGCAGCATTCGGTAAGAAA 335 16
AGAGATCTACAGACTTCAA 355 17 GAAAGCGAGTACACTGTAA 493 18
CCATGAAGAACACGTGTAA 842 19 GAAGAACACGTGTAAATTA 846 20
ATCATCGCTTCTACAGATA 878 21 AGAGCAATTTAGCTTTGAT 1137 22
GGTTTGACGAGCACAAAGA 1330 23 TGATCCGGATGGTCTAGCA 1428 24
GCGATCACGAGAACAATAA 1508 25 GCAGTAAACAATCAATCTA 1541 26
CAATCTATAAGACCATTGA 1553 27 TTTCAAGAACGCAGCAATA 1591 28
TTCAAGAACGCAGCAATAA 1592 29 TCAAGAACGCAGCAATAAA 1593 30
TCATGTATCTGAACAACGA 1661 31 ACAGCGACTGCACGTTGAA 1691 32
GATTAATGCTACTTGCAAA 1794 33 CTGGAGTCCTGTGCATGTA 1945 34
TGGAGTCCTGTGCATGTAA 1946 35 GGAGTCCTGTGCATGTAAT 1947 36
CATGTAATGAAACTGACAA 1958 37 CTATGTCGATGCTGAACAA 2022 38
CAAATGTGAGAAACGAGTA 2100 39 GCATCGGTTCGCATTATCA 2347 40
ATCGGTTCGCATTATCAAA 2349 41 CCAAGTCATTTGAGGATCT 2549 42
CCGGTCACCAGAAGTGAAA 2578 43 AAAGGCTGCCTCCTTTAAA 2595 44
TTTAAACTGCAGCGTCAGA 2608 45 AGATGCTGGTCATGTGTTT 2764 46
ATGCTGGTCATGTGTTTGA 2766 47 TGCTGGTCATGTGTTTGAA 2767 48
CTGGTCATGTGTTTGAACT 2769 49 TGTAATGAACCGCTGAATA 3027 50
GTAATGAACCGCTGAATAT 3028 51 CTAAGACTAATGCTCTCTA 3261 52
AGACTAATGCTCTCTAGAA 3264 53 CCTAACCACCTACCTTACA 3284 54
TACATGGTAGCCAGTTGAA 3313 55 TGGTAGCCAGTTGAATTTA 3317 56
TTTATGGAATCTACCAACT 3332 57 GGAATCTACCAACTGTTTA 3337 58
CATCAAGTACTGAACGTTT 434 155 TCGTGGTGGTGGATGGTAA 470 156
GAAAGCGAGTACACTGTAA 493 157 GAGCCTGACTCTAGGGTTC 547 158
CCACATAAGAGATGATGAT 570 159 CATAAGAGATGATGATGTT 573 160
CGAATATAACATAGAGCCA 618 161 GTTAATGATACCAAAGACA 649 162
CTGAAGATATCAAGAATGT 689 163 ATGAAGAGTTGCTCCCAAA 755 164
ATGAAGAACACGTGTAAAT 844 165 AATTATTGGTGGTAGCAGA 860 166
ATCATCGCTTCTACAGATA 878 167 ATACATGGGCAGAGGGGAA 894 168
GGGCAGAGGGGAAGAGAGT 900 169 GGAAGAGAGTACAACTACA 909 170
GAAGAGAGTACAACTACAA 910 171 GAGAGTACAACTACAAATT 913 172
GCTAATTGACAGAGTTGAT 942 173 CGGAACACTTCATGGGATA 970 174
GGATAATGCAGGTTTTAAA 984 175 AGGCTATGGAATACAGATA 1002 176
GAATACAGATAGAGCAGAT 1010 177 GGTAAAACCTGGTGAAAAG 1053 178
GTGAAAAGCACTACAACAT 1064 179 GAGGAAGCATCTAAAGTTT 1162 180
TATGGGAACTCTTGGATTA 1215 181 TGACGAGCACAAAGAATTA 1334 182
GCACAAAGAATTATGGTAA 1340 183 GGTTACAACTCATGAATTG 1386 184
ACTCATGAATTGGGACATA 1393 185 GTGGCGATCACGAGAACAA 1505 186
CTATAAGACCATTGAAAGT 1557 187 GAACGCAGCAATAAAGTTT 1597 188
GCAATAAAGTTTGTGGGAA 1604 189 CAATAAAGTTTGTGGGAAC 1605 190
GAGGGTGGATGAAGGAGAA 1626 191 GGATGAAGGAGAAGAGTGT 1632 192
GCATCATGTATCTGAACAA 1658 193 CAGGAAATGCTGAAGATGA 1856 194
GAATGGCAAATGTGAGAAA 2094 195 GGATGTAATTGAACGATTT 2121 196
GTGGATAAGAAATTGGATA 2263 197 GGATAAACAGTATGAATCT 2277 198
CCTTTAAACTGCAGCGTCA 2606 199 CGTGTTGACAGCAAAGAAA 2629 200
GCAAAGAAACAGAGTGCTA 2639 201
Table 2 lists examples of TNFR1 DNA target sequences of SEQ ID NO:2
from which siRNAs of the present invention are designed in a manner
as set forth above. TNFR1 encodes tumor necrosis factor .alpha.
receptor-1, as noted above.
TABLE-US-00008 TABLE 2 TNFR1 Target Sequences for siRNAs TACE
Target Sequences for siRNAs # of Starting Nucleotide with reference
to TACE Target Sequence SEQ ID NO: 2 SEQ ID NO: ACCAGGCCGTGATCTCTAT
124 59 AATTCGATTTGCTGTACCA 444 60 TCGATTTGCTGTACCAAGT 447 61
ACAAAGGAACCTACTTGTA 469 62 GAACCTACTTGTACAATGA 475 63
CTACTTGTACAATGACTGT 479 64 TGTGAGAGCGGCTCCTTCA 531 65
TCAGGTGGAGATCTCTTCT 611 66 CAGGTGGAGATCTCTTCTT 612 67
AGAACCAGTACCGGCATTA 667 68 GAACCAGTACCGGCATTAT 668 69
AACCAGTACCGGCATTATT 669 70 CCGGCATTATTGGAGTGAA 677 71
CGGCATTATTGGAGTGAAA 678 72 AGCCTGGAGTGCACGAAGT 843 73
CTCCTCTTCATTGGTTTAA 960 74 TTGGTTTAATGTATCGCTA 970 75
GTTTAATGTATCGCTACCA 973 76 TTTAATGTATCGCTACCAA 974 77
AGTCCAAGCTCTACTCCAT 1000 78 GAGCTTGAAGGAACTACTA 1053 79
CTTGAAGGAACTACTACTA 1056 80 TTGAAGGAACTACTACTAA 1057 81
ACAAGCCACAGAGCCTAGA 1318 82 TGTACGCCGTGGTGGAGAA 1357 83
CCGTTGCGCTGGAAGGAAT 1383 84 TCTAAGGACCGTCCTGCGA 1671 85
CTAATAGAAACTTGGCACT 2044 86 TAATAGAAACTTGGCACTC 2045 87
AATAGAAACTTGGCACTCC 2046 88 ATAGAAACTTGGCACTCCT 2047 89
TAGAAACTTGGCACTCCTG 2048 90 ATAGCAAGCTGAACTGTCC 2089 91
TAGCAAGCTGAACTGTCCT 2090 92 AGCAAGCTGAACTGTCCTA 2091 93
GCAAGCTGAACTGTCCTAA 2092 94 TGAACTGTCCTAAGGCAGG 2098 95
CAAAGGAACCTACTTGTAC 470 96 GAGCTTGAAGGAACTACTA 1053 97
CACAGAGCCTAGACACTGA 1324 98 TCCAAGCTCTACTCCATTG 1002 99
TGGAGCTGTTGGTGGGAAT 328 100 GACAGGGAGAAGAGAGATA 387 101
GGGAGAAGAGAGATAGTGT 391 102 GAGAAGAGAGATAGTGTGT 393 103
GAAGAGAGATAGTGTGTGT 395 104 GTGTGTGTCCCCAAGGAAA 406 105
GAAAATATATCCACCCTCA 421 106 AAATATATCCACCCTCAAA 423 107
CTGTACCAAGTGCCACAAA 455 108 ACCAAGTGCCACAAAGGAA 459 109
CCAAGTGCCACAAAGGAAC 460 110 CCACAAAGGAACCTACTTG 467 111
CAAAGGAACCTACTTGTAC 470 112 AAAGGAACCTACTTGTACA 471 113
GATACGGACTGCAGGGAGT 513 114 CGGACTGCAGGGAGTGTGA 517 115
TCCTTCACCGCTTCAGAAA 543 116 CAGAAAACCACCTCAGACA 556 117
TGCCTCAGCTGCTCCAAAT 576 118 CTCCAAATGCCGAAAGGAA 587 119
TCCAAATGCCGAAAGGAAA 588 120 CCAAATGCCGAAAGGAAAT 589 121
GCCGAAAGGAAATGGGTCA 595 122 AGGAAATGGGTCAGGTGGA 601 123
GGAAATGGGTCAGGTGGAG 602 124 GTGTGTGGCTGCAGGAAGA 651 125
GGAAGAACCAGTACCGGCA 664 126 CCATGCAGGTTTCTTTCTA 785 127
CATGCAGGTTTCTTTCTAA 786 128 TGCAGGTTTCTTTCTAAGA 788 129
AGGTTTCTTTCTAAGAGAA 791 130 GGTTTCTTTCTAAGAGAAA 792 131
AGAGAAAACGAGTGTGTCT 804 132 GAGTGTGTCTCCTGTAGTA 813 133
CTGTAGTAACTGTAAGAAA 824 134 AGAAAAGCCTGGAGTGCAC 838 135
TTGAGAATGTTAAGGGCAC 877 136 TGTTAAGGGCACTGAGGAC 884 137
GGTCATTTTCTTTGGTCTT 929 138 CCTCCTCTTCATTGGTTTA 959 139
TCCTCTTCATTGGTTTAAT 961 140 CTCTTCATTGGTTTAATGT 963 141
TCTTCATTGGTTTAATGTA 964 142 CTTCATTGGTTTAATGTAT 965 143
TCCAAGCTCTACTCCATTG 1002 144 CTCCATTGTTTGTGGGAAA 1013 145
GGGAAATCGACACCTGAAA 1026 146 TGAAGGAACTACTACTAAG 1058 147
ACCTCCAGCTCCACCTATA 1161 148 CCCACAAGCCACAGAGCCT 1315 149
ACGCCGTGGTGGAGAACGT 1360 150 GGAAGGAATTCGTGCGGCG 1393 151
TGAGCGACCACGAGATCGA 1420 152 GCGAGGCGCAATACAGCAT 1471 153
TGGGCTGCCTGGAGGACAT 1573 154
[0068] As cited in the examples above, one of skill in the art is
able to use the target sequence information provided in Tables 1 or
2 to design interfering RNAs having a length shorter or longer than
the sequences provided in the tables and by referring to the
sequence position in SEQ ID NO:1 or SEQ ID NO:2 and adding or
deleting nucleotides complementary or near complementary to SEQ ID
NO:1 or SEQ ID NO:2, respectively.
[0069] The target RNA cleavage reaction guided by siRNAs and other
forms of interfering RNA is highly sequence specific. In general,
siRNA containing a sense nucleotide strand identical in sequence to
a portion of the target mRNA and an antisense nucleotide strand
exactly complementary to a portion of the target mRNA are siRNA
embodiments for inhibition of mRNAs cited herein. However, 100%
sequence complementarity between the antisense siRNA strand and the
target mRNA, or between the antisense siRNA strand and the sense
siRNA strand, is not required to practice the present invention.
Thus, for example, the invention allows for sequence variations
that might be expected due to genetic mutation, strain
polymorphism, or evolutionary divergence.
[0070] In one embodiment of the invention, the antisense strand of
the siRNA has at least near-perfect contiguous complementarity of
at least 19 nucleotides with the target mRNA. "Near-perfect," as
used herein, means the antisense strand of the siRNA is
"substantially complementary to," and the sense strand of the siRNA
is "substantially identical" to at least a portion of the target
mRNA. "Identity," as known by one of ordinary skill in the art, is
the degree of sequence relatedness between nucleotide sequences as
determined by matching the order and identity of nucleotides
between the sequences. In one embodiment, the antisense strand of
an siRNA having 80% and between 80% up to 100% complementarity, for
example, 85%, 90% or 95% complementarity, to the target mRNA
sequence are considered near-perfect complementarity and may be
used in the present invention. "Perfect" contiguous complementarity
is standard Watson-Crick base pairing of adjacent base pairs. "At
least near-perfect" contiguous complementarity includes "perfect"
complementarity as used herein. Computer methods for determining
identity or complementarity are designed to identify the greatest
degree of matching of nucleotide sequences, for example, BLASTN
(Altschul, S. F., et al. (1990) J. Mol. Biol. 215:403-410).
[0071] The relationship between a target mRNA (sense strand) and
one strand of an siRNA (the sense strand) is that of identity. The
sense strand of an siRNA is also called a passenger strand, if
present. The relationship between a target mRNA (sense strand) and
the other strand of an siRNA (the antisense strand) is that of
complementarity. The antisense strand of an siRNA is also called a
guide strand.
[0072] The penultimate base in a nucleic acid sequence that is
written in a 5' to 3' direction is the next to the last base, i.e.,
the base next to the 3' base. The penultimate 13 bases of a nucleic
acid sequence written in a 5' to 3' direction are the last 13 bases
of a sequence next to the 3' base and not including the 3' base.
Similarly, the penultimate 14, 15, 16, 17, or 18 bases of a nucleic
acid sequence written in a 5' to 3' direction are the last 14, 15,
16, 17, or 18 bases of a sequence, respectively, next to the 3'
base and not including the 3' base.
[0073] In an embodiment of the invention, the region of contiguous
nucleotides is a region of at least 13 contiguous nucleotides
having at least 90% sequence complementarity to, or at least 90%
sequence identity with, the penultimate 13 nucleotides of the 3'
end of an mRNA corresponding to the sequence identified by each
sequence identifier.
[0074] In one embodiment of the invention, the region of contiguous
nucleotides is a region of at least 14 contiguous nucleotides
having at least 85% sequence complementarity to, or at least 85%
sequence identity with, the penultimate 14 nucleotides of the 3'
end of an mRNA corresponding to the sequence identified by each
sequence identifier. Two nucleotide substitutions (i.e., 12/14=86%
identity/complementarity) are included in such a phrase.
[0075] In a further embodiment of the invention, the region of
contiguous nucleotides is a region of at least 15, 16, 17, or 18
contiguous nucleotides having at least 80% sequence complementarity
to, or at least 80% sequence identity with, the penultimate 14
nucleotides of the 3' end of an mRNA corresponding to the sequence
of the sequence identifier. Three nucleotide substitutions are
included in such a phrase.
[0076] The target sequence in the mRNAs corresponding to SEQ ID
NO:1 or SEQ ID NO:2 may be in the 5' or 3' untranslated regions of
the mRNA as well as in the coding region of the mRNA.
[0077] One or both of the strands of double-stranded interfering
RNA may have a 3' overhang of from 1 to 6 nucleotides, which may be
ribonucleotides or deoxyribonucleotides or a mixture thereof. The
nucleotides of the overhang are not base-paired. In one embodiment
of the invention, the interfering RNA comprises a 3' overhang of TT
or UU. In another embodiment of the invention, the interfering RNA
comprises at least one blunt end. The termini usually have a 5'
phosphate group or a 3' hydroxyl group. In other embodiments, the
antisense strand has a 5' phosphate group, and the sense strand has
a 5' hydroxyl group. In still other embodiments, the termini are
further modified by covalent addition of other molecules or
functional groups.
[0078] The sense and antisense strands of the double-stranded siRNA
may be in a duplex formation of two single strands as described
above or may be a single molecule where the regions of
complementarity are base-paired and are covalently linked by a
hairpin loop so as to form a single strand. It is believed that the
hairpin is cleaved intracellularly by a protein termed dicer to
form an interfering RNA of two individual base-paired RNA
molecules.
[0079] Interfering RNAs may differ from naturally-occurring RNA by
the addition, deletion, substitution or modification of one or more
nucleotides. Non-nucleotide material may be bound to the
interfering RNA, either at the 5' end, the 3' end, or internally.
Such modifications are commonly designed to increase the nuclease
resistance of the interfering RNAs, to improve cellular uptake, to
enhance cellular targeting, to assist in tracing the interfering
RNA, to further improve stability, or to reduce the potential for
activation of the interferon pathway. For example, interfering RNAs
may comprise a purine nucleotide at the ends of overhangs.
Conjugation of cholesterol to the 3' end of the sense strand of an
siRNA molecule by means of a pyrrolidine linker, for example, also
provides stability to an siRNA.
[0080] Further modifications include a 3' terminal biotin molecule,
a peptide known to have cell-penetrating properties, a
nanoparticle, a peptidomimetic, a fluorescent dye, or a dendrimer,
for example.
[0081] Nucleotides may be modified on their base portion, on their
sugar portion, or on the phosphate portion of the molecule and
function in embodiments of the present invention. Modifications
include substitutions with alkyl, alkoxy, amino, deaza, halo,
hydroxyl, thiol groups, or a combination thereof, for example.
Nucleotides may be substituted with analogs with greater stability
such as replacing a ribonucleotide with a deoxyribonucleotide, or
having sugar modifications such as 2' OH groups replaced by 2'
amino groups, 2' O-methyl groups, 2' methoxyethyl groups, or a
2'-O, 4'-C methylene bridge, for example. Examples of a purine or
pyrimidine analog of nucleotides include a xanthine, a
hypoxanthine, an azapurine, a methylthioadenine, 7-deaza-adenosine
and O- and N-modified nucleotides. The phosphate group of the
nucleotide may be modified by substituting one or more of the
oxygens of the phosphate group with nitrogen or with sulfur
(phosphorothioates). Modifications are useful, for example, to
enhance function, to improve stability or permeability, or to
direct localization or targeting.
[0082] There may be a region or regions of the antisense
interfering RNA strand that is (are) not complementary to a portion
of SEQ ID NO:1 or SEQ ID NO:2. Non-complementary regions may be at
the 3', 5' or both ends of a complementary region or between two
complementary regions.
[0083] Interfering RNAs may be generated exogenously by chemical
synthesis, by in vitro transcription, or by cleavage of longer
double-stranded RNA with dicer or another appropriate nuclease with
similar activity. Chemically synthesized interfering RNAs, produced
from protected ribonucleoside phosphoramidites using a conventional
DNA/RNA synthesizer, may be obtained from commercial suppliers such
as Ambion Inc. (Austin, Tex.), Invitrogen (Carlsbad, Calif.), or
Dharmacon (Lafayette, Colo.). Interfering RNAs are purified by
extraction with a solvent or resin, precipitation, electrophoresis,
chromatography, or a combination thereof, for example.
Alternatively, interfering RNA may be used with little if any
purification to avoid losses due to sample processing.
[0084] Interfering RNAs can also be expressed endogenously from
plasmid or viral expression vectors or from minimal expression
cassettes, for example, PCR generated fragments comprising one or
more promoters and an appropriate template or templates for the
interfering RNA. Examples of commercially available plasmid-based
expression vectors for shRNA include members of the pSilencer
series (Ambion, Austin, Tex.) and pCpG-siRNA (InvivoGen, San Diego,
Calif.). Viral vectors for expression of interfering RNA may be
derived from a variety of viruses including adenovirus,
adeno-associated virus, lentivirus (e.g., HIV, FIV, and EIAV), and
herpes virus. Examples of commercially available viral vectors for
shRNA expression include pSilencer adeno (Ambion, Austin, Tex.) and
pLenti6/BLOCK-iT.TM.-DEST (Invitrogen, Carlsbad, Calif.). Selection
of viral vectors, methods for expressing the interfering RNA from
the vector and methods of delivering the viral vector are within
the ordinary skill of one in the art. Examples of kits for
production of PCR-generated shRNA expression cassettes include
Silencer Express (Ambion, Austin, Tex.) and siXpress (Mirus,
Madison, Wis.). A first interfering RNA may be administered via in
vivo expression from a first expression vector capable of
expressing the first interfering RNA and a second interfering RNA
may be administered via in vivo expression from a second expression
vector capable of expressing the second interfering RNA, or both
interfering RNAs may be administered via in vivo expression from a
single expression vector capable of expressing both interfering
RNAs.
[0085] Interfering RNAs may be expressed from a variety of
eukaryotic promoters known to those of ordinary skill in the art,
including pol III promoters, such as the U6 or H1 promoters, or pol
II promoters, such as the cytomegalovirus promoter. Those of skill
in the art will recognize that these promoters can also be adapted
to allow inducible expression of the interfering RNA.
[0086] Hybridization under Physiological Conditions: In certain
embodiments of the present invention, an antisense strand of an
interfering RNA hybridizes with an mRNA in vivo as part of the RISC
complex.
[0087] For example, high stringency conditions could occur at about
50% formamide at 37.degree. C. to 42.degree. C. Reduced stringency
conditions could occur at about 35% to 25% formamide at 30.degree.
C. to 35.degree. C. Examples of stringency conditions for
hybridization are provided in Sambrook, J., 1989, Molecular
Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y. Further examples of stringent
hybridization conditions 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, or hybridization at 70.degree. C. in 1.times.SSC or
50.degree. C. in 1.times.SSC, 50% formamide followed by washing at
70.degree. C. in 0.3.times.SSC, or hybridization at 70.degree. C.
in 4.times.SSC or 50.degree. C. in 4.times.SSC, 50% formamide
followed by washing at 67.degree. C. in 1.times.SSC. The
temperature for hybridization is about 5-10.degree. C. less than
the melting temperature (T.sub.m) of the hybrid where T.sub.m is
determined for hybrids between 19 and 49 base pairs in length using
the following calculation: T.sub.m.degree.
C.=81.5+16.6(log.sub.10[Na+])+0.41 (% G+C)-(600/N) where N is the
number of bases in the hybrid, and [Na+] is the concentration of
sodium ions in the hybridization buffer.
[0088] The above-described in vitro hybridization assay provides a
method of predicting whether binding between a candidate siRNA and
a target will have specificity. However, in the context of the RISC
complex, specific cleavage of a target can also occur with an
antisense strand that does not demonstrate high stringency for
hybridization in vitro.
[0089] Single-stranded interfering RNA: As cited above, interfering
RNAs ultimately function as single strands. Single-stranded (ss)
interfering RNA has been found to effect mRNA silencing, albeit
less efficiently than double-stranded RNA. Therefore, embodiments
of the present invention also provide for administration of a ss
interfering RNA that hybridizes under physiological conditions to a
portion of SEQ ID NO:1 or SEQ ID NO:2 and has a region of at least
near-perfect contiguous complementarity of at least 19 nucleotides
with the hybridizing portion of SEQ ID NO:1 or SEQ ID NO:2,
respectively. The ss interfering RNA of Table 1 or Table 2 has a
length of 19 to 49 nucleotides as for the ds interfering RNA cited
above. The ss interfering RNA has a 5' phosphate or is
phosphorylated in situ or in vivo at the 5' position. The term "5'
phosphorylated" is used to describe, for example, polynucleotides
or oligonucleotides having a phosphate group attached via ester
linkage to the C5 hydroxyl of the sugar (e.g., ribose, deoxyribose,
or an analog of same) at the 5' end of the polynucleotide or
oligonucleotide.
[0090] SS interfering RNAs are synthesized chemically or by in
vitro transcription or expressed endogenously from vectors or
expression cassettes as for ds interfering RNAs. 5' Phosphate
groups may be added via a kinase, or a 5' phosphate may be the
result of nuclease cleavage of an RNA. Delivery is as for ds
interfering RNAs. In one embodiment, ss interfering RNAs having
protected ends and nuclease resistant modifications are
administered for silencing. SS interfering RNAs may be dried for
storage or dissolved in an aqueous solution. The solution may
contain buffers or salts to inhibit annealing or for
stabilization.
[0091] Hairpin interfering RNA: A hairpin interfering RNA is a
single molecule (e.g., a single oligonucleotide chain) that
comprises both the sense and antisense strands of an interfering
RNA in a stem-loop or hairpin structure (e.g., a shRNA). For
example, shRNAs can be expressed from DNA vectors in which the DNA
oligonucleotides encoding a sense interfering RNA strand are linked
to the DNA oligonucleotides encoding the reverse complementary
antisense interfering RNA strand by a short spacer. If needed for
the chosen expression vector, 3' terminal T's and nucleotides
forming restriction sites may be added. The resulting RNA
transcript folds back onto itself to form a stem-loop
structure.
[0092] Mode of administration: Interfering RNA may be delivered via
aerosol, buccal, dermal, intradermal, inhaling, intramuscular,
intranasal, intraocular, intrapulmonary, intravenous,
intraperitoneal, nasal, ocular, oral, otic, parenteral, patch,
subcutaneous, sublingual, topical, or transdermal administration,
for example.
[0093] Administration may be directly to the eye by ocular tissue
administration such as periocular, conjunctival, subtenon,
intracameral, intravitreal, intraocular, subretinal,
subconjunctival, retrobulbar, intracanalicular, or suprachoroidal
administration; by injection, by direct application to the eye
using a catheter or other placement device such as a retinal
pellet, intraocular insert, suppository or an implant comprising a
porous, non-porous, or gelatinous material; by topical ocular drops
or ointments; or by a slow release device in the cul-de-sac or
implanted adjacent to the sclera (transscleral) or within the eye.
Intracameral injection may be through the cornea into the anterior
chamber to allow the agent to reach the trabecular meshwork.
Intracanalicular injection may be into the venous collector
channels draining Schlemm's canal or into Schlemm's canal.
[0094] Administration may be directly to the ear via, for example,
topical otic drops or ointments, slow release devices in the ear or
implanted adjacent to the ear. Local administration includes otic
intramuscular, intratympanic cavity and intracochlear injection
routes of administration. Furthermore, agents can be administered
to the inner ear by placement of a gelfoam, or similar absorbent
and adherent product, soaked with the interfering RNA against the
window membrane of the middle/inner ear or adjacent structure.
[0095] Administration may be directly to the lungs, via, for
example, an aerosolized preparation, and by inhalation via an
inhaler or a nebulizer, for example.
[0096] Further modes of administration include tablets, pills, and
capsules, all of which are capable of formulation by one of
ordinary skill in the art.
[0097] Subject: A subject in need of treatment for a
TNF.alpha.-related condition or at risk for developing a
TNF.alpha.-related condition is a human or other mammal having a
TNF.alpha.-related inflammatory condition or having dry eye or at
risk of developing a TNF.alpha.-related inflammatory condition or
dry eye. A TNF.alpha.-related inflammatory condition includes, for
example, allergic conjunctivitis, ocular inflammation, dermatitis,
rhinitis, or asthma associated with undesired or inappropriate
activity of TNF.alpha. as cited herein.
[0098] Ocular structures associated with a TNF.alpha.-related
condition may include the eye, retina, choroid, lens, cornea,
trabecular meshwork, iris, optic nerve, optic nerve head, sclera,
aqueous chamber, vitreous chamber, ciliary body, or posterior
segment, for example.
[0099] Otic structures associated with such disorders may include
the inner ear, middle ear, outer ear, tympanic cavity or membrane,
cochlea, or Eustachian tube, for example.
[0100] Pulmonary structures associated with such disorders may
include the nose, mouth, pharynx, larynx, bronchial tubes, trachea,
carina (the ridge separating the opening of the right and left main
bronchi), and lungs, particularly the lower lungs, such as
bronchioli and alveoli.
[0101] A subject may also be an otic cell, a lung cell, an ocular
cell, cell culture, organ or an ex vivo organ or tissue.
[0102] Formulations and Dosage: Pharmaceutical formulations
comprise interfering RNAs, or salts thereof, of the invention up to
99% by weight mixed with a physiologically acceptable carrier
medium such as water, buffer, saline, glycine, hyaluronic acid,
mannitol, and the like.
[0103] Interfering RNAs of the present invention are administered
as solutions, suspensions, or emulsions. The following are examples
of possible formulations embodied by this invention.
TABLE-US-00009 Amount in weight % Interfering RNA up to 99; 0.1-99;
0.1-50; 0.5-10.0 Hydroxypropylmethylcellulose 0.5 Sodium chloride
0.8 Benzalkonium Chloride 0.01 EDTA 0.01 NaOH/HCl Qs pH 7.4
Purified water (RNase-free) Qs 100 Ml
TABLE-US-00010 Amount in weight % Interfering RNA up to 99; 0.1-99;
0.1-50; 0.5-10.0 Phosphate Buffered Saline 1.0 Benzalkonium
Chloride 0.01 Polysorbate 80 0.5 Purified water (RNase-free) q.s.
to 100%
TABLE-US-00011 Amount in weight % Interfering RNA up to 99; 0.1-99;
0.1-50; 0.5-10.0 Monobasic sodium phosphate 0.05 Dibasic sodium
phosphate 0.15 (anhydrous) Sodium chloride 0.75 Disodium EDTA 0.05
Cremophor EL 0.1 Benzalkonium chloride 0.01 HCl and/or NaOH pH
7.3-7.4 Purified water (RNase-free) q.s. to 100%
TABLE-US-00012 Amount in weight % Interfering RNA up to 99; 0.1-99;
0.1-50; 0.5-10.0 Phosphate Buffered Saline 1.0
Hydroxypropyl-.beta.-cyclodextrin 4.0 Purified water (RNase-free)
q.s. to 100%
[0104] Generally, an effective amount of the interfering RNAs of
embodiments of the invention results in an extracellular
concentration at the surface of the target cell of from 100 .mu.M
to 100 nM, or from 1 nM to 50 nM, or from 5 nM to about 10 nM, or
about 25 nM. The dose required to achieve this local concentration
will vary depending on a number of factors including the delivery
method, the site of delivery, the number of cell layers between the
delivery site and the target cell or tissue, whether delivery is
local or systemic, etc. The concentration at the delivery site may
be considerably higher than it is at the surface of the target cell
or tissue. Topical compositions are delivered to the surface of the
target organ one to four times per day, or on an extended delivery
schedule such as daily, weekly, bi-weekly, monthly, or longer,
according to the routine discretion of a skilled clinician. The pH
of the formulation is about pH 4-9, or pH 4.5 to pH 7.4.
[0105] Therapeutic treatment of patients with siRNAs directed
against TACE mRNA or TNFR1 mRNA is expected to be beneficial over
small molecule treatments by increasing the duration of action,
thereby allowing less frequent dosing and greater patient
compliance.
[0106] An effective amount of a formulation may depend on factors
such as the age, race, and sex of the subject, the severity of the
TNF.alpha.-related condition, the rate of target gene
transcript/protein turnover, the interfering RNA potency, and the
interfering RNA stability, for example. In one embodiment, the
interfering RNA is delivered topically to a target organ and
reaches TACE mRNA- or TNFR1 mRNA-containing tissue at a therapeutic
dose thereby ameliorating a TNF.alpha.-related process.
[0107] Acceptable carriers: An acceptable carrier refers to those
carriers that cause at most, little to no ocular irritation,
provide suitable preservation if needed, and deliver one or more
interfering RNAs of the present invention in a homogenous dosage.
An acceptable carrier for administration of interfering RNA of
embodiments of the present invention include the cationic
lipid-based transfection reagents TransIT.RTM.-TKO (Minis
Corporation, Madison, Wis.), LIPOFECTIN.RTM., Lipofectamine,
OLIGOFECTAMINET.TM. (Invitrogen, Carlsbad, Calif.), or
DHARMAFECT.TM. (Dharmacon, Lafayette, Colo.); polycations such as
polyethyleneimine; cationic peptides such as Tat, polyarginine, or
Penetratin (Antp peptide); or liposomes. Liposomes are formed from
standard vesicle-forming lipids and a sterol, such as cholesterol,
and may include a targeting molecule such as a monoclonal antibody
having binding affinity for endothelial cell surface antigens, for
example. Further, the liposomes may be PEGylated liposomes.
[0108] The interfering RNAs may be delivered in solution, in
suspension, or in bioerodible or non-bioerodible delivery devices.
The interfering RNAs can be delivered alone or as components of
defined, covalent conjugates. The interfering RNAs can also be
complexed with cationic lipids, cationic peptides, or cationic
polymers; complexed with proteins, fusion proteins, or protein
domains with nucleic acid binding properties (e.g., protamine); or
encapsulated in nanoparticles. Tissue- or cell-specific delivery
can be accomplished by the inclusion of an appropriate targeting
moiety such as an antibody or antibody fragment.
[0109] For ophthalmic, otic, or pulmonary delivery, an interfering
RNA may be combined with opthalmologically, optically, or pulmonary
acceptable preservatives, co-solvents, surfactants, viscosity
enhancers, penetration enhancers, buffers, sodium chloride, or
water to form an aqueous, sterile suspension or solution. Solution
formulations may be prepared by dissolving the interfering RNA in a
physiologically acceptable isotonic aqueous buffer. Further, the
solutions may include an acceptable surfactant to assist in
dissolving the inhibitor. Viscosity building agents, such as
hydroxymethyl cellulose, hydroxyethyl cellulose, methylcellulose,
polyvinylpyrrolidone, or the like may be added to the compositions
of the present invention to improve the retention of the
compound.
[0110] In order to prepare a sterile ointment formulation, the
interfering RNA is combined with a preservative in an appropriate
vehicle, such as mineral oil, liquid lanolin, or white petrolatum.
Sterile gel formulations may be prepared by suspending the
interfering RNA in a hydrophilic base prepared from the combination
of, for example, CARBOPOL.RTM.-940 (BF Goodrich, Charlotte, N.C.),
or the like, according to methods known in the art. VISCOAT.RTM.
(Alcon Laboratories, Inc., Fort Worth, Tex.) may be used for
intraocular injection, for example. Other compositions of the
present invention may contain penetration enhancing agents such as
cremephor and TWEEN.RTM. 80 (polyoxyethylene sorbitan monolaureate,
Sigma Aldrich, St. Louis, Mo.), in the event the interfering RNA is
less penetrating in the organ or tissue of interest.
[0111] Kits: Embodiments of the present invention provide a kit
that includes reagents for attenuating the expression of an mRNA as
cited herein in a cell. The kit contains an siRNA or an shRNA
expression vector. For siRNAs and non-viral shRNA expression
vectors the kit also may contain a transfection reagent or other
suitable delivery vehicle. For viral shRNA expression vectors, the
kit may contain the viral vector and/or the necessary components
for viral vector production (e.g., a packaging cell line as well as
a vector comprising the viral vector template and additional helper
vectors for packaging). The kit may also contain positive and
negative control siRNAs or shRNA expression vectors (e.g., a
non-targeting control siRNA or an siRNA that targets an unrelated
mRNA). The kit also may contain reagents for assessing knockdown of
the intended target gene (e.g., primers and probes for quantitative
PCR to detect the target mRNA and/or antibodies against the
corresponding protein for western blots). Alternatively, the kit
may comprise an siRNA sequence or an shRNA sequence and the
instructions and materials necessary to generate the siRNA by in
vitro transcription or to construct an shRNA expression vector.
[0112] A pharmaceutical combination in kit form is further provided
that includes, in packaged combination, a carrier means adapted to
receive a container means in close confinement therewith and a
first container means including an interfering RNA composition and
an acceptable carrier. Such kits can further include, if desired,
one or more of various conventional pharmaceutical kit components,
such as, for example, containers with one or more pharmaceutically
acceptable carriers, additional containers, etc., as will be
readily apparent to those skilled in the art. Printed instructions,
either as inserts or as labels, indicating quantities of the
components to be administered, guidelines for administration,
and/or guidelines for mixing the components, can also be included
in the kit.
[0113] The ability of TACE- or TNFR1-interfering RNA to knock-down
the levels of endogenous TACE or TNFR1 expression in, for example,
human corneal epithelial cells is evaluated in vitro as follows.
Transformed human corneal epithelial cells, for example, the
CEPI-17 cell line (Offord et al. (1999) Invest Opthalmol Vis Sci.
40:1091-1101), are plated 24 h prior to transfection in KGM
keratinocyte medium (Cambrex, East Rutherford, N.J.). Transfection
is performed using DharmaFECT.TM. 1 (Dharmacon, Lafayette, Colo.)
according to the manufacturer's instructions at TACE- or
TNFR1-interfering RNA concentrations ranging from 0.1 nM-100 nM.
Non-targeting control interfering RNA and lamin A/C interfering RNA
(Dharmacon) are used as controls. Target mRNA levels are assessed
by qPCR 24 h post-transfection using, for example, TAQMAN.RTM.
forward and reverse primers and a probe set that encompasses the
target site (Applied Biosystems, Foster City, Calif.). Target
protein levels may be assessed approximately 72 h post-transfection
(actual time dependent on protein turnover rate) by western blot,
for example. Standard techniques for RNA and/or protein isolation
from cultured cells are well-known to those skilled in the art. To
reduce the chance of non-specific, off-target effects, the lowest
possible concentration of TACE- or TNFR1 interfering RNA is used
that produces the desired level of knock-down in target gene
expression.
[0114] Those of skill in the art, in light of the present
disclosure, will appreciate that obvious modifications of the
embodiments disclosed herein can be made without departing from the
spirit and scope of the invention. All of the embodiments disclosed
herein can be made and executed without undue experimentation in
light of the present disclosure. The full scope of the invention is
set out in the disclosure and equivalent embodiments thereof. The
specification should not be construed to unduly narrow the full
scope of protection to which the present invention is entitled.
[0115] While a particular embodiment of the invention has been
shown and described, numerous variations and alternate embodiments
will occur to those skilled in the art. Accordingly, it is intended
that the invention be limited only in terms of the appended
claims.
[0116] The invention may be embodied in other specific forms
without departing from its spirit or essential characteristics. The
described embodiments are to be considered in all respects only as
illustrative and not restrictive. The scope of the invention is,
therefore, indicated by the appended claims rather than by the
foregoing description. All changes to the claims that come within
the meaning and range of equivalency of the claims are to be
embraced within their scope. Further, all published documents,
patents, and applications mentioned herein are hereby incorporated
by reference, as if presented in their entirety.
Example 1
Interfering RNA for Specifically Silencing TNFR1 in GTM-3 Cells
[0117] The present study examines the ability of TNFR1 interfering
RNA to knock down the levels of endogenous TNFR1 protein expression
in cultured GTM-3 cells.
[0118] Transfection of GTM-3 cells (Pang, I. H. et al., 1994. Curr.
Eye Res. 13:51-63) was accomplished using standard in vitro
concentrations (0.1-10 nM) of TNFR1 siRNAs, siCONTROL RISC-free
siRNA #1, or siCONTROL Non-targeting siRNA #2 (NTC2) and
DHARMAFECT.RTM. #1 transfection reagent (Dharmacon, Lafayette,
Colo.). All siRNAs were dissolved in 1.times.siRNA buffer, an
aqueous solution of 20 mM KCl, 6 mM HEPES (pH 7.5), 0.2 mM
MgCl.sub.2. Control samples included a buffer control in which the
volume of siRNA was replaced with an equal volume of 1.times.siRNA
buffer (siRNA). Western blots using an anti-TNFR1 antibody (Santa
Cruz Biotechnology, Santa Cruz, Calif.) were performed to assess
TNFR1 protein expression. The TNFR1 siRNAs are double-stranded
interfering RNAs having specificity for the following targets:
siTNFR1 #1 targets the sequence CAAAGGAACCUACUUGUAC (SEQ ID NO:
202); siTNFR1 #2 targets the sequence GAGCUUGAAGGAACUACUA (SEQ ID
NO: 203); siTNFR1 #3 targets the sequence CACAGAGCCUAGACACUGA (SEQ
ID NO: 204); siTNFR1 #4 targets the sequence UCCAAGCUCUACUCCAUUG
(SEQ ID NO: 205). As shown by the data in FIG. 1, siTNFR1 #1,
siTNFR1 #2, and siTNFR1 #3 siRNAs reduced TNFR1 protein expression
significantly at the 10 nM and 1 nM concentrations relative to the
control siRNAs, but exhibited reduced efficacy at 0.1 nM. The
siTNFR1 #2 and siTNFR1 #3 siRNAs were particularly effective. The
siTNFR1 #4 siRNA also showed a concentration dependent reduction in
TNFR1 protein expression as expected.
Sequence CWU 1
1
20513572DNAHomo sapiens 1acctgcactt ctgggggcgt cgagcctggc
ggtagaatct tcccagtagg cggcgcggga 60gggaaaagag gattgagggg ctaggccggg
cggatcccgt cctcccccga tgtgagcagt 120tttccgaaac cccgtcaggc
gaaggctgcc cagagaggtg gagtcggtag cggggccggg 180aacatgaggc
agtctctcct attcctgacc agcgtggttc ctttcgtgct ggcgccgcga
240cctccggatg acccgggctt cggcccccac cagagactcg agaagcttga
ttctttgctc 300tcagactacg atattctctc tttatctaat atccagcagc
attcggtaag aaaaagagat 360ctacagactt caacacatgt agaaacacta
ctaacttttt cagctttgaa aaggcatttt 420aaattatacc tgacatcaag
tactgaacgt ttttcacaaa atttcaaggt cgtggtggtg 480gatggtaaaa
acgaaagcga gtacactgta aaatggcagg acttcttcac tggacacgtg
540gttggtgagc ctgactctag ggttctagcc cacataagag atgatgatgt
tataatcaga 600atcaacacag atggggccga atataacata gagccacttt
ggagatttgt taatgatacc 660aaagacaaaa gaatgttagt ttataaatct
gaagatatca agaatgtttc acgtttgcag 720tctccaaaag tgtgtggtta
tttaaaagtg gataatgaag agttgctccc aaaagggtta 780gtagacagag
aaccacctga agagcttgtt catcgagtga aaagaagagc tgacccagat
840cccatgaaga acacgtgtaa attattggtg gtagcagatc atcgcttcta
cagatacatg 900ggcagagggg aagagagtac aactacaaat tacttaatag
agctaattga cagagttgat 960gacatctatc ggaacacttc atgggataat
gcaggtttta aaggctatgg aatacagata 1020gagcagattc gcattctcaa
gtctccacaa gaggtaaaac ctggtgaaaa gcactacaac 1080atggcaaaaa
gttacccaaa tgaagaaaag gatgcttggg atgtgaagat gttgctagag
1140caatttagct ttgatatagc tgaggaagca tctaaagttt gcttggcaca
ccttttcaca 1200taccaagatt ttgatatggg aactcttgga ttagcttatg
ttggctctcc cagagcaaac 1260agccatggag gtgtttgtcc aaaggcttat
tatagcccag ttgggaagaa aaatatctat 1320ttgaatagtg gtttgacgag
cacaaagaat tatggtaaaa ccatccttac aaaggaagct 1380gacctggtta
caactcatga attgggacat aattttggag cagaacatga tccggatggt
1440ctagcagaat gtgccccgaa tgaggaccag ggagggaaat atgtcatgta
tcccatagct 1500gtgagtggcg atcacgagaa caataagatg ttttcaaact
gcagtaaaca atcaatctat 1560aagaccattg aaagtaaggc ccaggagtgt
tttcaagaac gcagcaataa agtttgtggg 1620aactcgaggg tggatgaagg
agaagagtgt gatcctggca tcatgtatct gaacaacgac 1680acctgctgca
acagcgactg cacgttgaag gaaggtgtcc agtgcagtga caggaacagt
1740ccttgctgta aaaactgtca gtttgagact gcccagaaga agtgccagga
ggcgattaat 1800gctacttgca aaggcgtgtc ctactgcaca ggtaatagca
gtgagtgccc gcctccagga 1860aatgctgaag atgacactgt ttgcttggat
cttggcaagt gtaaggatgg gaaatgcatc 1920cctttctgcg agagggaaca
gcagctggag tcctgtgcat gtaatgaaac tgacaactcc 1980tgcaaggtgt
gctgcaggga cctttctggc cgctgtgtgc cctatgtcga tgctgaacaa
2040aagaacttat ttttgaggaa aggaaagccc tgtacagtag gattttgtga
catgaatggc 2100aaatgtgaga aacgagtaca ggatgtaatt gaacgatttt
gggatttcat tgaccagctg 2160agcatcaata cttttggaaa gtttttagca
gacaacatcg ttgggtctgt cctggttttc 2220tccttgatat tttggattcc
tttcagcatt cttgtccatt gtgtggataa gaaattggat 2280aaacagtatg
aatctctgtc tctgtttcac cccagtaacg tcgaaatgct gagcagcatg
2340gattctgcat cggttcgcat tatcaaaccc tttcctgcgc cccagactcc
aggccgcctg 2400cagcctgccc ctgtgatccc ttcggcgcca gcagctccaa
aactggacca ccagagaatg 2460gacaccatcc aggaagaccc cagcacagac
tcacatatgg acgaggatgg gtttgagaag 2520gaccccttcc caaatagcag
cacagctgcc aagtcatttg aggatctcac ggaccatccg 2580gtcaccagaa
gtgaaaaggc tgcctccttt aaactgcagc gtcagaatcg tgttgacagc
2640aaagaaacag agtgctaatt tagttctcag ctcttctgac ttaagtgtgc
aaaatatttt 2700tatagatttg acctacaaat caatcacagc ttgtattttg
tgaagactgg gaagtgactt 2760agcagatgct ggtcatgtgt ttgaacttcc
tgcaggtaaa cagttcttgt gtggtttggc 2820ccttctcctt ttgaaaaggt
aaggtgaagg tgaatctagc ttattttgag gctttcaggt 2880tttagttttt
aaaatatctt ttgacctgtg gtgcaaaagc agaaaataca gctggattgg
2940gttatgaata tttacgtttt tgtaaattaa tcttttatat tgataacagc
actgactagg 3000gaaatgatca gttttttttt atacactgta atgaaccgct
gaatatgagg catttggcat 3060ttatttgtga tgacaactgg aatagttttt
tttttttttt tttttttttg ccttcaacta 3120aaaacaaagg agataaatct
agtatacatt gtctctaaat tgtgggtcta tttctagtta 3180ttacccagag
tttttatgta gcagggaaaa tatatatcta aatttagaaa tcatttgggt
3240taatatggct cttcataatt ctaagactaa tgctctctag aaacctaacc
acctacctta 3300cagtgagggc tatacatggt agccagttga atttatggaa
tctaccaact gtttagggcc 3360ctgatttgct gggcagtttt tctgtatttt
ataagtatct tcatgtatcc ctgttactga 3420tagggataca tgctcttaga
aaattcacta ttggctggga gtggtggctc atgcctgtaa 3480tcccagcact
tggagaggct gaggttgcgc cactacactc cagcctgggt gacagagtga
3540gactctgcct caaaaaaaaa aaaaaaaaaa aa 357222236DNAHomo sapiens
2gctgttgcaa cactgcctca ctcttcccct cccaccttct ctcccctcct ctctgcttta
60attttctcag aattctctgg actgaggctc cagttctggc ctttggggtt caagatcact
120gggaccaggc cgtgatctct atgcccgagt ctcaaccctc aactgtcacc
ccaaggcact 180tgggacgtcc tggacagacc gagtcccggg aagccccagc
actgccgctg ccacactgcc 240ctgagcccaa atgggggagt gagaggccat
agctgtctgg catgggcctc tccaccgtgc 300ctgacctgct gctgccactg
gtgctcctgg agctgttggt gggaatatac ccctcagggg 360ttattggact
ggtccctcac ctaggggaca gggagaagag agatagtgtg tgtccccaag
420gaaaatatat ccaccctcaa aataattcga tttgctgtac caagtgccac
aaaggaacct 480acttgtacaa tgactgtcca ggcccggggc aggatacgga
ctgcagggag tgtgagagcg 540gctccttcac cgcttcagaa aaccacctca
gacactgcct cagctgctcc aaatgccgaa 600aggaaatggg tcaggtggag
atctcttctt gcacagtgga ccgggacacc gtgtgtggct 660gcaggaagaa
ccagtaccgg cattattgga gtgaaaacct tttccagtgc ttcaattgca
720gcctctgcct caatgggacc gtgcacctct cctgccagga gaaacagaac
accgtgtgca 780cctgccatgc aggtttcttt ctaagagaaa acgagtgtgt
ctcctgtagt aactgtaaga 840aaagcctgga gtgcacgaag ttgtgcctac
cccagattga gaatgttaag ggcactgagg 900actcaggcac cacagtgctg
ttgcccctgg tcattttctt tggtctttgc cttttatccc 960tcctcttcat
tggtttaatg tatcgctacc aacggtggaa gtccaagctc tactccattg
1020tttgtgggaa atcgacacct gaaaaagagg gggagcttga aggaactact
actaagcccc 1080tggccccaaa cccaagcttc agtcccactc caggcttcac
ccccaccctg ggcttcagtc 1140ccgtgcccag ttccaccttc acctccagct
ccacctatac ccccggtgac tgtcccaact 1200ttgcggctcc ccgcagagag
gtggcaccac cctatcaggg ggctgacccc atccttgcga 1260cagccctcgc
ctccgacccc atccccaacc cccttcagaa gtgggaggac agcgcccaca
1320agccacagag cctagacact gatgaccccg cgacgctgta cgccgtggtg
gagaacgtgc 1380ccccgttgcg ctggaaggaa ttcgtgcggc gcctagggct
gagcgaccac gagatcgatc 1440ggctggagct gcagaacggg cgctgcctgc
gcgaggcgca atacagcatg ctggcgacct 1500ggaggcggcg cacgccgcgg
cgcgaggcca cgctggagct gctgggacgc gtgctccgcg 1560acatggacct
gctgggctgc ctggaggaca tcgaggaggc gctttgcggc cccgccgccc
1620tcccgcccgc gcccagtctt ctcagatgag gctgcgcccc tgcgggcagc
tctaaggacc 1680gtcctgcgag atcgccttcc aaccccactt ttttctggaa
aggaggggtc ctgcaggggc 1740aagcaggagc tagcagccgc ctacttggtg
ctaacccctc gatgtacata gcttttctca 1800gctgcctgcg cgccgccgac
agtcagcgct gtgcgcgcgg agagaggtgc gccgtgggct 1860caagagcctg
agtgggtggt ttgcgaggat gagggacgct atgcctcatg cccgttttgg
1920gtgtcctcac cagcaaggct gctcgggggc ccctggttcg tccctgagcc
tttttcacag 1980tgcataagca gttttttttg tttttgtttt gttttgtttt
gtttttaaat caatcatgtt 2040acactaatag aaacttggca ctcctgtgcc
ctctgcctgg acaagcacat agcaagctga 2100actgtcctaa ggcaggggcg
agcacggaac aatggggcct tcagctggag ctgtggactt 2160ttgtacatac
actaaaattc tgaagttaaa gctctgctct tggaaaaaaa aaaaaaaaaa
2220aaaaaaaaaa aaaaaa 2236319DNAArtificialTarget Sequence
3gctctcagac tacgatatt 19421DNAArtificialSense Strand with 3'NN
4gcucucagac uacgauauun n 21521DNAArtificialAntisense strand with
3'NN 5aauaucguag ucugagagcn n 21621RNAArtificialSense Strand
6gcucucagac uacgauauuu u 21721RNAArtificialAntisense Strand
7aauaucguag ucugagagcu u 21819RNAArtificialSense Strand 8gcucucagac
uacgauauu 19919RNAArtificialAntisense Strand 9aauaucguag ucugagagc
191048DNAArtificialHairpin duplex with loop 10gcucucagac uacgauauun
nnnnnnnaau aucguagucu gagagcuu 481125DNAArtificialSense Strand
11gcucucagac uacgauauuc ucucu 251225RNAArtificialSense Strand
12gcucucagac uacgauauuc ucucu 251327RNAArtificialAntisense Strand
13agagagaaua ucguagucug agagcuu 271419DNAArtificialTarget Sequence
14ccagcagcat tcggtaaga 191519DNAArtificialTarget Sequence
15cagcagcatt cggtaagaa 191619DNAArtificialTarget Sequence
16agcagcattc ggtaagaaa 191719DNAArtificialTarget Sequence
17agagatctac agacttcaa 191819DNAArtificialTarget Sequence
18gaaagcgagt acactgtaa 191919DNAArtificialTarget Sequence
19ccatgaagaa cacgtgtaa 192019DNAArtificialTarget Sequence
20gaagaacacg tgtaaatta 192119DNAArtificialTarget Sequence
21atcatcgctt ctacagata 192219DNAArtificialTarget Sequence
22agagcaattt agctttgat 192319DNAArtificialTarget Sequence
23ggtttgacga gcacaaaga 192419DNAArtificialTarget Sequence
24tgatccggat ggtctagca 192519DNAArtificialTarget Sequence
25gcgatcacga gaacaataa 192619DNAArtificialTarget Sequence
26gcagtaaaca atcaatcta 192719DNAArtificialTarget Sequence
27caatctataa gaccattga 192819DNAArtificialTarget Sequence
28tttcaagaac gcagcaata 192919DNAArtificialTarget Sequence
29ttcaagaacg cagcaataa 193019DNAArtificialTarget Sequence
30tcaagaacgc agcaataaa 193119DNAArtificialTarget Sequence
31tcatgtatct gaacaacga 193219DNAArtificialTarget Sequence
32acagcgactg cacgttgaa 193319DNAArtificialTarget Sequence
33gattaatgct acttgcaaa 193419DNAArtificialTarget Sequence
34ctggagtcct gtgcatgta 193519DNAArtificialTarget Sequence
35tggagtcctg tgcatgtaa 193619DNAArtificialTarget Sequence
36ggagtcctgt gcatgtaat 193719DNAArtificialTarget Sequence
37catgtaatga aactgacaa 193819DNAArtificialTarget Sequence
38ctatgtcgat gctgaacaa 193919DNAArtificialTarget Sequence
39caaatgtgag aaacgagta 194019DNAArtificialTarget Sequence
40gcatcggttc gcattatca 194119DNAArtificialTarget Sequence
41atcggttcgc attatcaaa 194219DNAArtificialTarget Sequence
42ccaagtcatt tgaggatct 194319DNAArtificialTarget Sequence
43ccggtcacca gaagtgaaa 194419DNAArtificialTarget Sequence
44aaaggctgcc tcctttaaa 194519DNAArtificialTarget Sequence
45tttaaactgc agcgtcaga 194619DNAArtificialTarget Sequence
46agatgctggt catgtgttt 194719DNAArtificialTarget Sequence
47atgctggtca tgtgtttga 194819DNAArtificialTarget Sequence
48tgctggtcat gtgtttgaa 194919DNAArtificialTarget Sequence
49ctggtcatgt gtttgaact 195019DNAArtificialTarget Sequence
50tgtaatgaac cgctgaata 195119DNAArtificialTarget Sequence
51gtaatgaacc gctgaatat 195219DNAArtificialTarget Sequence
52ctaagactaa tgctctcta 195319DNAArtificialTarget Sequence
53agactaatgc tctctagaa 195419DNAArtificialTarget Sequence
54cctaaccacc taccttaca 195519DNAArtificialTarget Sequence
55tacatggtag ccagttgaa 195619DNAArtificialTarget Sequence
56tggtagccag ttgaattta 195719DNAArtificialTarget Sequence
57tttatggaat ctaccaact 195819DNAArtificialTarget Sequence
58ggaatctacc aactgttta 195919DNAArtificialTarget Sequence
59accaggccgt gatctctat 196019DNAArtificialTarget Sequence
60aattcgattt gctgtacca 196119DNAArtificialTarget Sequence
61tcgatttgct gtaccaagt 196219DNAArtificialTarget Sequence
62acaaaggaac ctacttgta 196319DNAArtificialTarget Sequence
63gaacctactt gtacaatga 196419DNAArtificialTarget Sequence
64ctacttgtac aatgactgt 196519DNAArtificialTarget Sequence
65tgtgagagcg gctccttca 196619DNAArtificialTarget Sequence
66tcaggtggag atctcttct 196719DNAArtificialTarget Sequence
67caggtggaga tctcttctt 196819DNAArtificialTarget Sequence
68agaaccagta ccggcatta 196919DNAArtificialTarget Sequence
69gaaccagtac cggcattat 197019DNAArtificialTarget Sequence
70aaccagtacc ggcattatt 197119DNAArtificialTarget Sequence
71ccggcattat tggagtgaa 197219DNAArtificialTarget Sequence
72cggcattatt ggagtgaaa 197319DNAArtificialTarget Sequence
73agcctggagt gcacgaagt 197419DNAArtificialTarget Sequence
74ctcctcttca ttggtttaa 197519DNAArtificialTarget Sequence
75ttggtttaat gtatcgcta 197619DNAArtificialTarget Sequence
76gtttaatgta tcgctacca 197719DNAArtificialTarget Sequence
77tttaatgtat cgctaccaa 197819DNAArtificialTarget Sequence
78agtccaagct ctactccat 197919DNAArtificialTarget Sequence
79gagcttgaag gaactacta 198019DNAArtificialTarget Sequence
80cttgaaggaa ctactacta 198119DNAArtificialTarget Sequence
81ttgaaggaac tactactaa 198219DNAArtificialTarget Sequence
82acaagccaca gagcctaga 198319DNAArtificialTarget Sequence
83tgtacgccgt ggtggagaa 198419DNAArtificialTarget Sequence
84ccgttgcgct ggaaggaat 198519DNAArtificialTarget Sequence
85tctaaggacc gtcctgcga 198619DNAArtificialTarget Sequence
86ctaatagaaa cttggcact 198719DNAArtificialTarget Sequence
87taatagaaac ttggcactc 198819DNAArtificialTarget Sequence
88aatagaaact tggcactcc 198919DNAArtificialTarget Sequence
89atagaaactt ggcactcct 199019DNAArtificialTarget Sequence
90tagaaacttg gcactcctg 199119DNAArtificialTarget Sequence
91atagcaagct gaactgtcc 199219DNAArtificialTarget Sequence
92tagcaagctg aactgtcct 199319DNAArtificialTarget Sequence
93agcaagctga actgtccta 199419DNAArtificialTarget Sequence
94gcaagctgaa ctgtcctaa 199519DNAArtificialTarget Sequence
95tgaactgtcc taaggcagg 199619DNAArtificialTarget Sequence
96caaaggaacc tacttgtac 199719DNAArtificialTarget Sequence
97gagcttgaag gaactacta 199819DNAArtificialTarget Sequence
98cacagagcct agacactga 199919DNAArtificialTarget Sequence
99tccaagctct actccattg 1910019DNAArtificialTarget Sequence
100tggagctgtt ggtgggaat 1910119DNAArtificialTarget Sequence
101gacagggaga agagagata 1910219DNAArtificialTarget Sequence
102gggagaagag agatagtgt 1910319DNAArtificialTarget Sequence
103gagaagagag atagtgtgt 1910419DNAArtificialTarget Sequence
104gaagagagat agtgtgtgt
1910519DNAArtificialTarget Sequence 105gtgtgtgtcc ccaaggaaa
1910619DNAArtificialTarget Sequence 106gaaaatatat ccaccctca
1910719DNAArtificialTarget Sequence 107aaatatatcc accctcaaa
1910819DNAArtificialTarget Sequence 108ctgtaccaag tgccacaaa
1910919DNAArtificialTarget Sequence 109accaagtgcc acaaaggaa
1911019DNAArtificialTarget Sequence 110ccaagtgccacaaaggaac
1911119DNAArtificialTarget Sequence 111ccacaaaggaacctacttg
1911219DNAArtificialTarget Sequence 112caaaggaacc tacttgtac
1911319DNAArtificialTarget Sequence 113aaaggaacct acttgtaca
1911419DNAArtificialTarget Sequence 114gatacggact gcagggagt
1911519DNAArtificialTarget Sequence 115cggactgcag ggagtgtga
1911619DNAArtificialTarget Sequence 116tccttcaccg cttcagaaa
1911719DNAArtificialTarget Sequence 117cagaaaacca cctcagaca
1911819DNAArtificialTarget Sequence 118tgcctcagct gctccaaat
1911919DNAArtificialTarget Sequence 119ctccaaatgc cgaaaggaa
1912019DNAArtificialTarget Sequence 120tccaaatgccgaaaggaaa
1912119DNAArtificialTarget Sequence 121ccaaatgccg aaaggaaat
1912219DNAArtificialTarget Sequence 122gccgaaagga aatgggtca
1912319DNAArtificialTarget Sequence 123aggaaatggg tcaggtgga
1912419DNAArtificialTarget Sequence 124ggaaatgggt caggtggag
1912519DNAArtificialTarget Sequence 125gtgtgtggct gcaggaaga
1912619DNAArtificialTarget Sequence 126ggaagaacca gtaccggca
1912719DNAArtificialTarget Sequence 127ccatgcaggt ttctttcta
1912819DNAArtificialTarget Sequence 128catgcaggtt tctttctaa
1912919DNAArtificialTarget Sequence 129tgcaggtttc tttctaaga
1913019DNAArtificialTarget Sequence 130aggtttcttt ctaagagaa
1913119DNAArtificialTarget Sequence 131ggtttctttc taagagaaa
1913219DNAArtificialTarget Sequence 132agagaaaacg agtgtgtct
1913319DNAArtificialTarget Sequence 133gagtgtgtct cctgtagta
1913419DNAArtificialTarget Sequence 134ctgtagtaac tgtaagaaa
1913519DNAArtificialTarget Sequence 135agaaaagcct ggagtgcac
1913619DNAArtificialTarget Sequence 136ttgagaatgt taagggcac
1913719DNAArtificialTarget Sequence 137tgttaagggc actgaggac
1913819DNAArtificialTarget Sequence 138ggtcattttc tttggtctt
1913919DNAArtificialTarget Sequence 139cctcctcttc attggttta
1914019DNAArtificialTarget Sequence 140tcctcttcat tggtttaat
1914119DNAArtificialTarget Sequence 141ctcttcattg gtttaatgt
1914219DNAArtificialTarget Sequence 142tcttcattgg tttaatgta
1914319DNAArtificialTarget Sequence 143cttcattggt ttaatgtat
1914419DNAArtificialTarget Sequence 144tccaagctct actccattg
1914519DNAArtificialTarget Sequence 145ctccattgtt tgtgggaaa
1914619DNAArtificialTarget Sequence 146gggaaatcga cacctgaaa
1914719DNAArtificialTarget Sequence 147tgaaggaact actactaag
1914819DNAArtificialTarget Sequence 148acctccagct ccacctata
1914919DNAArtificialTarget Sequence 149cccacaagcc acagagcct
1915019DNAArtificialTarget Sequence 150acgccgtggt ggagaacgt
1915119DNAArtificialTarget Sequence 151ggaaggaatt cgtgcggcg
1915219DNAArtificialTarget Sequence 152tgagcgacca cgagatcga
1915319DNAArtificialTarget Sequence 153gcgaggcgca atacagcat
1915419DNAArtificialTarget Sequence 154tgggctgcct ggaggacat
1915519DNAArtificialTarget Sequence 155catcaagtac tgaacgttt
1915619DNAArtificialTarget Sequence 156tcgtggtggt ggatggtaa
1915719DNAArtificialTarget Sequence 157gaaagcgagt acactgtaa
1915819DNAArtificialTarget Sequence 158gagcctgact ctagggttc
1915919DNAArtificialTarget Sequence 159ccacataaga gatgatgat
1916019DNAArtificialTarget Sequence 160cataagagat gatgatgtt
1916119DNAArtificialTarget Sequence 161cgaatataac atagagcca
1916219DNAArtificialTarget Sequence 162gttaatgata ccaaagaca
1916319DNAArtificialTarget Sequence 163ctgaagatat caagaatgt
1916419DNAArtificialTarget Sequence 164atgaagagtt gctcccaaa
1916519DNAArtificialTarget Sequence 165atgaagaaca cgtgtaaat
1916619DNAArtificialTarget Sequence 166aattattggt ggtagcaga
1916719DNAArtificialTarget Sequence 167atcatcgctt ctacagata
1916819DNAArtificialTarget Sequence 168atacatgggc agaggggaa
1916919DNAArtificialTarget Sequence 169gggcagaggg gaagagagt
1917019DNAArtificialTarget Sequence 170ggaagagagt acaactaca
1917119DNAArtificialTarget Sequence 171gaagagagta caactacaa
1917219DNAArtificialTarget Sequence 172gagagtacaa ctacaaatt
1917319DNAArtificialTarget Sequence 173gctaattgac agagttgat
1917419DNAArtificialTarget Sequence 174cggaacactt catgggata
1917519DNAArtificialTarget Sequence 175ggataatgca ggttttaaa
1917619DNAArtificialTarget Sequence 176aggctatgga atacagata
1917719DNAArtificialTarget Sequence 177gaatacagat agagcagat
1917819DNAArtificialTarget Sequence 178ggtaaaacct ggtgaaaag
1917919DNAArtificialTarget Sequence 179gtgaaaagca ctacaacat
1918019DNAArtificialTarget Sequence 180gaggaagcat ctaaagttt
1918119DNAArtificialTarget Sequence 181tatgggaact cttggatta
1918219DNAArtificialTarget Sequence 182tgacgagcac aaagaatta
1918319DNAArtificialTarget Sequence 183gcacaaagaa ttatggtaa
1918419DNAArtificialTarget Sequence 184ggttacaact catgaattg
1918519DNAArtificialTarget Sequence 185actcatgaat tgggacata
1918619DNAArtificialTarget Sequence 186gtggcgatca cgagaacaa
1918719DNAArtificialTarget Sequence 187ctataagacc attgaaagt
1918819DNAArtificialTarget Sequence 188gaacgcagca ataaagttt
1918919DNAArtificialTarget Sequence 189gcaataaagt ttgtgggaa
1919019DNAArtificialTarget Sequence 190caataaagtt tgtgggaac
1919119DNAArtificialTarget Sequence 191gagggtggat gaaggagaa
1919219DNAArtificialTarget Sequence 192ggatgaagga gaagagtgt
1919319DNAArtificialTarget Sequence 193gcatcatgta tctgaacaa
1919419DNAArtificialTarget Sequence 194caggaaatgc tgaagatga
1919519DNAArtificialTarget Sequence 195gaatggcaaa tgtgagaaa
1919619DNAArtificialTarget Sequence 196ggatgtaatt gaacgattt
1919719DNAArtificialTarget Sequence 197gtggataaga aattggata
1919819DNAArtificialTarget Sequence 198ggataaacag tatgaatct
1919919DNAArtificialTarget Sequence 199cctttaaact gcagcgtca
1920019DNAArtificialTarget Sequence 200cgtgttgaca gcaaagaaa
1920119DNAArtificialTarget Sequence 201gcaaagaaac agagtgcta
1920219RNAArtificialTarget Sequence 202caaaggaacc uacuuguac
1920319RNAArtificialTarget Sequence 203gagcuugaag gaacuacua
1920419RNAArtificialTarget Sequence 204cacagagccu agacacuga
1920519RNAArtificialTarget Sequence 205uccaagcucu acuccauug 19
* * * * *