U.S. patent application number 15/156430 was filed with the patent office on 2016-09-08 for rnai-mediated inhibition of spleen tyrosine kinase-related inflammatory conditions.
The applicant listed for this patent is Arrowhead Pharmaceuticals, Inc.. Invention is credited to Jon E. Chatterton, Daniel A. Gamache, Steven T. Miller, John M. Yanni.
Application Number | 20160257963 15/156430 |
Document ID | / |
Family ID | 38610402 |
Filed Date | 2016-09-08 |
United States Patent
Application |
20160257963 |
Kind Code |
A1 |
Yanni; John M. ; et
al. |
September 8, 2016 |
RNAi-MEDIATED INHIBITION OF SPLEEN TYROSINE KINASE-RELATED
INFLAMMATORY CONDITIONS
Abstract
RNA interference is provided for inhibition of spleen tyrosine
kinase (Syk) mRNA expression, in particular, for treating patients
having a Syk-related inflammatory condition or at risk of
developing a Syk-related inflammatory condition such as allergic
conjunctivitis, ocular inflammation, dermatitis, rhinitis, asthma,
allergy, or mast-cell disease.
Inventors: |
Yanni; John M.; (Burleson,
TX) ; Chatterton; Jon E.; (Fort Worth, TX) ;
Gamache; Daniel A.; (Arlington, TX) ; Miller; Steven
T.; (Arlington, TX) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Arrowhead Pharmaceuticals, Inc. |
Pasadena |
CA |
US |
|
|
Family ID: |
38610402 |
Appl. No.: |
15/156430 |
Filed: |
May 17, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14270174 |
May 5, 2014 |
9371529 |
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15156430 |
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13172964 |
Jun 30, 2011 |
8865671 |
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14270174 |
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12296621 |
Oct 9, 2008 |
8048863 |
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PCT/US2007/066619 |
Apr 13, 2007 |
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13172964 |
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60791847 |
Apr 13, 2006 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 37/08 20180101;
A61P 11/02 20180101; A61P 29/00 20180101; C12N 2310/531 20130101;
C07H 21/02 20130101; A61P 17/00 20180101; A61P 43/00 20180101; C12N
15/1137 20130101; A61P 27/02 20180101; A61P 27/14 20180101; A61K
31/7105 20130101; C12N 2310/14 20130101; A61K 31/713 20130101; A61P
11/06 20180101; C12N 15/113 20130101; C12N 2320/30 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113; A61K 31/7105 20060101 A61K031/7105; A61K 31/713
20060101 A61K031/713 |
Claims
1. A composition comprising an interfering RNA having a length of
19 to 49 nucleotides, and 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 any one of SEQ ID NO:44-47.
2. The composition of claim 1, wherein the interfering RNA
comprises an shRNA, an siRNA, or an miRNA.
3. The composition of claim 1 wherein the interfering RNA has at
least one 3' overhang.
4. The composition of claim 1 wherein the interfering RNA has at
least one 5' overhang.
5. The composition of claim 1 wherein the interfering RNA has one
or more chemically modified nucleotide.
6. The composition of claim 1 wherein the interfering RNA has one
or more non-phosphodiester linkages.
7. The composition of claim 1 further comprising a pharmaceutically
acceptable carrier.
8. A method of attenuating expression of Syk 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, wherein the
interfering RNA comprises 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 any one of SEQ ID NO:44-47, wherein the expression of
Syk mRNA is attenuated thereby.
9. The method of claim 8, wherein the interfering RNA comprises an
shRNA, an siRNA, or an miRNA.
10. The method of claim 8 wherein the interfering RNA has at least
one 3' overhang.
11. The method of claim 8 wherein the interfering RNA has at least
one 5' overhang.
12. The method of claim 8 wherein the interfering RNA has one or
more chemically modified nucleotide.
13. The method of claim 8 wherein the interfering RNA has one or
more non-phosphodiester linkages.
14. The method of claim 8, 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.
15. A composition comprising an interfering RNA having a length of
19 to 49 nucleotides, and 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 selected from the group consisting of mRNA
corresponding to SEQ ID NO: 1 comprising nucleotide 642, 1009,
1273, or 1533.
16. The composition of claim 15, wherein the interfering RNA
comprises an shRNA, an siRNA, or an miRNA.
17. The composition of claim 15 wherein the interfering RNA has at
least one 3' and/or 5' overhang.
18. The composition of claim 15 wherein the interfering RNA has one
or more chemically modified nucleotide.
19. The composition of claim 15 wherein the interfering RNA has one
or more non-phosphodiester linkages.
20. The composition of claim 15 further comprising a
pharmaceutically acceptable carrier.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
application Ser. No. ______, filed May 5, 2014, which is a
divisional of U.S. application Ser. No. 13/172,964 filed Jun. 30,
2011 and issued as U.S. Pat. No. 8,865,671, which is a divisional
of U.S. application Ser. No. 12/296,621 filed Oct. 9, 2008 and
issued as U.S. Pat. No. 8,048,863, which is the National Stage of
International Application Serial No.: PCT/US2007/066619 filed Apr.
13, 2007, which claims benefit to U.S. Provisional Patent
Application Ser. No. 60/791,847 filed Apr. 13, 2006, 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 spleen tyrosine kinase (Syk) and for
treatment of a Syk-related inflammatory condition. Such conditions
include allergic conjunctivitis, ocular inflammation, dermatitis,
rhinitis, asthma, allergy, or mast-cell disease, for example.
BACKGROUND OF THE INVENTION
[0003] Allergic conjunctivitis, ocular inflammation, dermatitis,
rhinitis, asthma, allergy, and mast-cell disease have historically
been treated with a regimen of oral, intranasal or topical
antihistamines, or oral or intranasal steroids, or, in the case of
allergy, allergen injection treatment. 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.
[0004] Signaling through immune receptors such as the IgE receptor
(Fc.epsilon.RI) involves the recruitment and activation of multiple
components of the signaling cascade, including the non-receptor
tyrosine kinase, Syk. Syk activation leads to activation of the
PLC.gamma. and PI3K pathways and ultimately to mast cell
degranulation and activation. Targeting the Syk mRNA would reduce
the levels of Syk protein and interrupt the Fc.epsilon.RI pathway.
This action would interfere with the IgE mediated mast cell
degranulation and release of histamine and other pro-inflammatory
mediators.
[0005] Inhibition of allergic inflammation in the airways using
aerosolized antisense to Syk kinase is reported by Stenton, G. R.
et al., (J Immunol. 169:1028-1036 (2002)). Inhibition of Syk
expression by means of small interfering RNA (siRNA) in a bronchial
epithelial cell line HS-24 and in a rat model of ovalbumin
(OA)-induced asthma reportedly inhibited the expression of
hallmarks of induced inflammatory response (published international
patent application WO 2005007623). Inhibition of expression of
genes involved in IgE production using a short hairpin RNA
targeting FC.epsilon.R1A and treatment of IgE-mediated diseases
such as asthma and allergic rhinitis were reported in published
international patent application WO 2005085443. None of these
documents discloses the interfering RNAs of the present
invention.
[0006] Additional agents and treatment methods would be desirable
for targeting the Syk tyrosine kinase, thereby blocking the actions
of endogenous mast cell degranulation, and release of histamine and
other pro-inflammatory mediators while avoiding the side effects of
systemic antihistamine treatment. Embodiments of the present
invention address the need in the art for such agents and treatment
methods.
SUMMARY OF THE INVENTION
[0007] Embodiments of the present invention overcome these and
other drawbacks of the prior art by providing highly potent and
efficacious treatment, prevention or intervention of a Syk-related
inflammatory condition. In one aspect, methods of the invention
include treating a subject having a Syk-related inflammatory
condition or at risk of developing a Syk-related inflammatory
condition by administering interfering RNAs that silence expression
of Syk mRNA, thus interfering with the PLC.gamma. and PI3K
signaling pathways and preventing a cascade of events related to
mast cell degranulation, and release of histamine and other
pro-inflammatory mediators in a Syk-related inflammatory
condition.
[0008] The present invention is directed to interfering RNAs that
target Syk mRNA and thereby interfere with Syk mRNA expression. The
interfering RNAs of the invention are useful for treating patients
with a Syk-related condition or at risk of developing a Syk-related
condition.
[0009] An embodiment of the invention is a method of attenuating
expression of Syk mRNA of a subject, 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 expression of Syk
mRNA is attenuated thereby.
[0010] Another embodiment of the invention is a method of treating
a Syk-related inflammatory condition in a subject in need thereof.
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 Syk-related inflammatory condition is treated thereby. In one
embodiment, the subject is a human and the human has a Syk-related
inflammatory condition and, in another embodiment, the subject is a
human and the human is at risk of developing a Syk-related
inflammatory condition.
[0011] For the above cited embodiments, 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:2, SEQ ID NO:13-SEQ ID
NO:40, and SEQ ID NO:44-SEQ ID NO:47.
[0012] In further embodiments of the above-cited methods, the
composition further comprises a second interfering RNA having a
length of 19 to 49 nucleotides and 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 a second mRNA
corresponding to any one of SEQ ID NO:2, SEQ ID NO:13-SEQ ID NO:40,
and SEQ ID NO:44-SEQ ID NO:47.
[0013] In yet another embodiment of the invention, a method of
attenuating expression of Syk mRNA of a 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 642, 789,
791, 860, 861, 862, 867, 868, 1009, 1273, 1394, 1436, 1471, 1472,
1533, 1535, 1547, 1680, 1738, 1739, 1766, 1880, 1947, 2022, 2036,
2328, 2329, 2473, 2509, 2520, 2524, 2558, or 2613. The expression
of Syk mRNA is attenuated thereby. In a further embodiment, the
composition comprises an effective amount of interfering RNA having
a length of 19 to 49 nucleotides where the antisense strand
hybridizes under physiological conditions to a portion of mRNA
corresponding to SEQ ID NO:1 comprising nucleotide 1007, 1698, or
1769. In one embodiment, the antisense strand hybridizes to a
portion of mRNA corresponding to SEQ ID NO:1 beginning with
nucleotide 1007, 1698, or 1769 and is 19 or 20 nucleotides
long.
[0014] A method of treating a Syk-related inflammatory 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
642, 789, 791, 860, 861, 862, 867, 868, 1009, 1273, 1394, 1436,
1471, 1472, 1533, 1535, 1547, 1680, 1738, 1739, 1766, 1880, 1947,
2022, 2036, 2328, 2329, 2473, 2509, 2520, 2524, 2558, or 2613. The
Syk-related inflammatory condition is treated thereby. In a further
embodiment, the composition comprises an effective amount of
interfering RNA having a length of 19 to 49 nucleotides where the
antisense strand hybridizes under physiological conditions to a
portion of mRNA corresponding to SEQ ID NO:1 comprising nucleotide
1007, 1698, or 1769. In one embodiment, the antisense strand
hybridizes to a portion of mRNA corresponding to SEQ ID NO:1
beginning with nucleotide 1007, 1698, or 1769 and is 19 or 20
nucleotides long.
[0015] A second interfering RNA having a length of 19 to 49
nucleotides could also be administered to the subject; 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 second portion of mRNA corresponding to SEQ ID NO:1
comprising nucleotide 642, 789, 791, 860, 861, 862, 867, 868, 1009,
1273, 1394, 1436, 1471, 1472, 1533, 1535, 1547, 1680, 1738, 1739,
1766, 1880, 1947, 2022, 2036, 2328, 2329, 2473, 2509, 2520, 2524,
2558, or 2613. A second interfering RNA having a length of 19 to 49
nucleotides could be administered to the subject, where the
antisense strand hybridizes under physiological conditions to a
portion of mRNA corresponding to SEQ ID NO:1 comprising nucleotide
1007, 1698, or 1769. In one embodiment, the antisense strand
hybridizes to a portion of mRNA corresponding to SEQ ID NO:1
beginning with nucleotide 1007, 1698, or 1769 and is 19 or 20
nucleotides long.
[0016] A method of attenuating expression of Syk mRNA of a subject,
comprising administering to the subject a composition comprising an
effective amount of a single-stranded interfering RNA having a
length of 19 to 49 nucleotides, and a pharmaceutically acceptable
carrier, where the single-stranded interfering RNA hybridizes under
physiological conditions to a portion of mRNA corresponding to SEQ
ID NO:1 comprising the nucleotides identified above is a further
embodiment of the invention.
[0017] 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:2, SEQ ID NO:13-SEQ ID NO:40, and SEQ ID
NO:44-SEQ ID NO:47, or a complement thereof; and a pharmaceutically
acceptable carrier. 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:41-SEQ ID
NO:47, or a complement thereof; and a pharmaceutically acceptable
carrier is a further embodiment of the invention.
[0018] A method of treating ocular inflammation or conjunctivitis
in a subject in need thereof is an embodiment of the invention. The
method comprises administering to the subject a composition
comprising a double stranded siRNA molecule that down regulates
expression of a Syk 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 Syk gene so that the siRNA molecule
directs cleavage of the mRNA via RNA interference. In a further
embodiment of this method, each strand of the siRNA molecule is
independently about 19 nucleotides to about 25 nucleotides in
length, or about 19 nucleotides to about 21 nucleotides in
length.
[0019] Use of any of the embodiments as described herein in the
preparation of a medicament for attenuating expression of Syk mRNA
as set forth herein is also an embodiment of the present
invention.
BRIEF DESCRIPTION OF THE FIGURES
[0020] FIG. 1 provides an SYK western blot of 293FT cells
transfected with SYK siRNAs #2, #5, #6, and #8, 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 72-kDa SYK and 42-kDa actin
bands.
DETAILED DESCRIPTION OF THE INVENTION
[0021] 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.
[0022] 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.
[0023] As used herein and unless otherwise indicated, the terms "a"
and "an" are taken to mean "one", "at least one" or "one or
more".
[0024] The term "a Syk-related inflammatory condition" as used
herein, includes histamine and other pro-inflammatory mediated
responses involved in conditions such as allergic conjunctivitis,
ocular inflammation, dermatitis, rhinitis, asthma, allergy, and
mast-cell disease, and includes those cellular changes resulting
from the expression of Syk-mRNA that lead directly or indirectly to
the Syk-related inflammatory condition. The interfering RNA
provided herein provides for such silencing while avoiding
undesirable side effects due to nonspecific agents.
[0025] 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.
[0026] The term "dermatitis," as used herein, refers to
inflammation of the skin and includes, for example, allergic
contact dermatitis, asteatotic dermatitis (dry skin on the lower
legs), atopic dermatitis, cercarial dermatitis, contact dermatitis
including irritant contact dermatitis and urushiol-induced contact
dermatitis, dermatitis herpetiformis, dyshidrotic dermatitis,
eczema, gravitational dermatitis, infective dermatitis, nummular
dermatitis, otitis externa, perioral dermatitis, Pseudomonas
dermatitis (hot tub rash), and seborrhoeic dermatitis.
[0027] The term "rhinitis," as used herein, refers to inflammation
of the mucous membranes of the nose and includes, for example,
allergic rhinitis, atopic rhinitis, infectious rhinitis, irritant
rhinitis, eosinophilic non-allergic rhinitis, rhinitis
medicamentosa, and neutrophilic rhinosinusitis.
[0028] 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.
[0029] The term "allergy," as used herein, refers to an abnormal
reaction of the immune system to a substance that is usually not
harmful and includes, for example, skin allergies such as atopic
dermatitis, hives, and angioedema; respiratory allergies such as
allergic rhinitis, and reactions to dust or mold; food allergies
such as reactions to proteins in cow's milk, egg whites, peanuts,
wheat, soybeans, berries, shellfish, corn, beans, yellow food dye
No. 5 and gum arabic; drug allergies such as reactions to
penicillin, sulfas, barbiturates, anticonvulsants, insulin, local
anesthetics and contrast agents; and insect bite allergies such as
reactions to venom in stings of bees, wasps, hornets, yellow
jackets and fire ants.
[0030] The term "mast-cell disease," as used herein, refers to
systemic mastocytosis which is characterized by mast cell
accumulation in tissues. Such accumulation can affect organs such
as bone marrow, skin, the GI tract, the liver, and the spleen.
Physicians diagnose systemic mastocytosis by finding mast cells in
parts of the body other than the skin and generally treat with
antihistamines.
[0031] 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.
[0032] "Hybridization" 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.
[0033] As used herein, 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.
[0034] Attenuating Expression of an mRNA:
[0035] 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 Syk mRNA is administered. In
other embodiments, two or more interfering RNAs targeting Syk mRNA
are administered.
[0036] "Hybridization" 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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."
[0041] 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.
[0042] The present invention relates to the use of interfering RNA
to inhibit the expression of Syk mRNA, a non-receptor tyrosine
kinase. Signaling through immune receptors such as the IgE receptor
(Fc.epsilon.RI) involves the recruitment and activation of multiple
components of the signaling cascade, including Syk. Syk activation
leads to activation of the PLC.gamma. and PI3K pathways and
ultimately to mast cell degranulation and activation. Targeting the
Syk mRNA as provided herein reduces the level of Syk protein and
interrupts the Fc.epsilon.RI pathway. This action interferes with
the IgE mediated mast cell degranulation and release of histamine
and other pro-inflammatory mediators. According to the present
invention, interfering RNAs provided exogenously or expressed
endogenously are particularly effective at silencing Syk mRNA.
[0043] 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.
[0044] 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 Syk. The
mRNA sequence is identical to the DNA sense strand sequence with
the "T" bases replaced with "U" bases. Therefore, the mRNA sequence
of Syk is known from SEQ ID NO:1.
[0045] Spleen Tyrosine Kinase (Syk) mRNA:
[0046] The GenBank database provides the DNA sequence for Syk as
accession no. NM_003177, 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 Syk (with the exception of "T"
bases for "U" bases). The coding sequence for Syk is from
nucleotides 148-2055.
[0047] Equivalents of the above cited Syk mRNA sequence are
alternative splice forms, allelic forms, isozymes, or a cognate
thereof. A cognate is a spleen tyrosine kinase mRNA from another
mammalian species that is homologous to SEQ ID NO:1 (i.e., an
ortholog). Syk nucleic acid sequences related to SEQ ID NO:1
include those having GenBank accession numbers BC001645, BC002962,
L28824, X73568, BC011399, and Z29630.
[0048] Inhibition of Syk may also be determined in vitro by
monitoring histamine release in immunologically-challenged mast
cells as follows. Mono-dispersed human mast cell preparations
release pro-inflammatory mediators upon immunological activation.
The degree of activation is determined by quantification of
histamine released into culture supernatant upon stimulation of the
cells with antigen or anti-human IgE. (See Miller et al. Ocular
Immunol Inflamm. 4:39-49, 2006). Immunological challenge of mast
cells transfected with interfering RNA targeting Syk results in
significantly less histamine release than observed with
immunological challenge of non-transfected mast cells or mast cells
transfected with a non-targeting control interfering RNA.
[0049] The inhibition of Syk may also be determined by examining
IgE mediated release of histamine in vitro using human conjunctival
tissue mast cells or in LAD2 mast cells. Human conjunctival tissue
mast cells are obtained using the methodology outlined in U.S. Pat.
No. 5,360,720, for example. LAD2 mast cells are licensed from the
Laboratory of Allergic Diseases, National Institutes of Health,
Bethesda, Md. (Leuk Res. 2003 Aug. 27(8):677-82). Briefly, human
conjunctival tissue mast cells are enzymatically released from
human conjunctival tissues and then partially enriched by density
centrifugation over a PERCOLL.RTM. cushion. A monodispersed cell
suspension is obtained from the resulting pellet, and these cells
are used for a histamine release assay. Cells are transfected with
a Syk interfering RNA or a control interfering RNA 72 h prior to
anti-human IgE stimulation, which triggers mast cell degranulation
and histamine release to the supernatant. Histamine is then
measured in the supernatant by RIA (Beckman Coulter), EIA (Beckman
Coulter) or other method known to one of skill in the art. A
decrease in histamine release in Syk interfering RNA transfected
cells relative to control transfected cells or non-transfected
cells indicates that an interfering RNA is effective at attenuating
Syk and interrupting the FceRI signaling pathway.
[0050] The LAD2 mast cell line is used in much the same way with
the exception that the cells are passively sensitized with human
IgE myeloma prior to stimulation with anti-human IgE to cross-link
receptor bound IgE and trigger degranulation.
[0051] Inhibition of Syk is also inferred in a human or mammal by
observing an improvement in a Syk-related condition symptom such as
improvement in symptoms related to allergic conjunctivitis, ocular
inflammation, dermatitis, rhinitis, asthma, allergy, or mast-cell
disease. Improvement in any of edema, itching, inflammation, or
tolerance to environmental challenges, for example, is indicative
of inhibition of Syk.
[0052] Interfering RNA:
[0053] 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
Syk 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 Syk mRNA. 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.
[0054] 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.
[0055] 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.
[0056] 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 Syk target sequence are then
tested by transfection of cells expressing the target mRNA followed
by assessment of knockdown as described above.
[0057] 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.
[0058] An embodiment of a 19-nucleotide DNA target sequence for Syk
mRNA is present at nucleotides 789 to 807 of SEQ ID NO:1:
TABLE-US-00001 SEQ ID NO: 2 5'- GCACTATCGCATCGACAAA -3'.
[0059] An siRNA of the invention for targeting a corresponding mRNA
sequence of SEQ ID NO:2 and having 21-nucleotide strands and a
2-nucleotide 3' overhang is:
TABLE-US-00002 SEQ ID NO: 3 5'- GCACUAUCGCAUCGACAAANN -3' SEQ ID
NO: 4 3' -NNCGUGAUAGCGUAGCUGUUU -5'.
[0060] 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.
[0061] An siRNA of the invention for targeting a corresponding mRNA
sequence of SEQ ID NO:2 and having 21-nucleotide strands and a 3'UU
overhang on each strand is:
TABLE-US-00003 SEQ ID NO: 5 5'- GCACUAUCGCAUCGACAAAUU -3' SEQ ID
NO: 6 3'- UUCGUGAUAGCGUAGCUGUUU -5'.
[0062] 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:2 and
having 19-nucleotide strands and blunt ends is:
TABLE-US-00004 SEQ ID NO: 7 5'- GCACUAUCGCAUCGACAAA-3' SEQ ID NO: 8
3'- CGUGAUAGCGUAGCUGUUU-5'.
[0063] 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:2 and having a 19 bp
double-stranded stem region and a 3'UU overhang is:
##STR00001##
[0064] 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:2) identified in the Syk DNA sequence (SEQ ID NO:1) by 6
nucleotides yields a 25-nucleotide DNA target sequence present at
nucleotides 789 to 813 of SEQ ID NO:1:
TABLE-US-00005 SEQ ID NO: 10 5'- GCACTATCGCATCGACAAAGACAAG-3'.
[0066] A dicer-substrate 27-mer duplex of the invention for
targeting a corresponding mRNA sequence of SEQ ID NO:10 is:
TABLE-US-00006 SEQ ID NO: 11 5'- GCACUAUCGCAUCGACAAAGACAAG -3' SEQ
ID NO: 12 3'- UUCGUGAUAGCGUAGCUGUUUCUGUUC -5'.
[0067] The two nucleotides at the 3' end of the sense strand (i.e.,
the AG nucleotides of SEQ ID NO:11) 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.
[0068] 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.
[0069] Also disclosed herein is a method of attenuating expression
of Syk mRNA of a subject, comprising: [0070] administering to the
subject a composition comprising an effective amount of interfering
RNA (iRNA) having a length of 19 to 49 nucleotides and a
pharmaceutically acceptable carrier, the interfering RNA comprising
a region selected from the group consisting of: [0071] 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:2 and SEQ ID NO:13-SEQ ID NO:40; [0072] 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 any one of SEQ ID NO:2, SEQ ID NO:13-SEQ ID NO:40,
and SEQ ID NO:44-SEQ ID NO:47; and, [0073] 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 15, 16, 17, or 18 nucleotides, respectively, of the 3'
end of an mRNA corresponding to any one of SEQ ID NO:2, SEQ ID
NO:13-SEQ ID NO:40, and SEQ ID NO:44-SEQ ID NO:47, wherein the
expression of Syk mRNA is attenuated thereby.
[0074] Also disclosed is a method of treating a Syk-related
inflammatory condition in a subject in need thereof, comprising:
[0075] 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
[0076] the interfering RNA comprises a region selected from the
group consisting of [0077] 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:2, SEQ
ID NO:13-SEQ ID NO:40, and SEQ ID NO:44-SEQ ID NO:47; [0078] 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 any one of SEQ ID NO:2 and SEQ ID NO:13-SEQ ID
NO:40; and, [0079] 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 15, 16,
17, or 18 nucleotides, respectively, of the 3' end of an mRNA
corresponding to any one of SEQ ID NO:2, SEQ ID NO:13-SEQ ID NO:40,
and SEQ ID NO:44-SEQ ID NO:47, wherein the Syk-related inflammatory
condition is treated thereby.
[0080] Further embodiments comprise a method of attenuating
expression of Syk mRNA of a subject, comprising: [0081]
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: [0082] a sense nucleotide strand, an antisense
nucleotide strand, and a region of at least near-perfect contiguous
complementarity of at least 19 nucleotides; [0083] wherein the
antisense strand hybridizes under physiological conditions to a
portion of mRNA selected from the group consisting of [0084] mRNA
corresponding to SEQ ID NO:1 comprising nucleotide 642, 789, 791,
860, 861, 862, 867, 868, 1009, 1273, 1394, 1436, 1471, 1472, 1533,
1535, 1547, 1680, 1738, 1739, 1766, 1880, 1947, 2022, 2036, 2328,
2329, 2473, 2509, 2520, 2524, 2558, or 2613, and, [0085] mRNA
corresponding to SEQ ID NO:1 comprising nucleotide 1007, 1698, or
1769, [0086] wherein the expression of Syk mRNA is attenuated
thereby.
[0087] Also disclosed herein are methods of treating a Syk-related
inflammatory condition in a subject in need thereof, comprising:
[0088] 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; [0089] wherein the
antisense strand hybridizes under physiological conditions to a
portion of mRNA selected from the group consisting of [0090] mRNA
corresponding to SEQ ID NO:1 comprising nucleotide 642, 789, 791,
860, 861, 862, 867, 868, 1009, 1273, 1394, 1436, 1471, 1472, 1533,
1535, 1547, 1680, 1738, 1739, 1766, 1880, 1947, 2022, 2036, 2328,
2329, 2473, 2509, 2520, 2524, 2558, or 2613, and, [0091] mRNA
corresponding to SEQ ID NO:1 comprising nucleotide 1007, 1698, or
1769, [0092] wherein the Syk-related inflammatory condition is
treated thereby.
[0093] Further embodiments comprise a method of attenuating
expression of Syk mRNA of a subject, comprising: [0094]
administering to the subject a composition comprising an effective
amount of a single-stranded interfering RNA having a length of 19
to 49 nucleotides, and a pharmaceutically acceptable carrier,
[0095] wherein the single-stranded interfering RNA hybridizes under
physiological conditions to a portion of mRNA selected from the
group consisting of [0096] mRNA corresponding to SEQ ID NO:1
comprising nucleotide 642, 789, 791, 860, 861, 862, 867, 868, 1009,
1273, 1394, 1436, 1471, 1472, 1533, 1535, 1547, 1680, 1738, 1739,
1766, 1880, 1947, 2022, 2036, 2328, 2329, 2473, 2509, 2520, 2524,
2558, or 2613, and, [0097] mRNA corresponding to SEQ ID NO:1
comprising nucleotide 1007, 1698, or 1769, [0098] 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, [0099] wherein the expression of Syk mRNA is
thereby attenuated.
[0100] Also disclosed are method of treating ocular inflammation or
conjunctivitis in a subject in need thereof, comprising: [0101]
administering to the subject a composition comprising a double
stranded siRNA molecule that down regulates expression of a Syk
gene via RNA interference, [0102] wherein: [0103] each strand of
the siRNA molecule is independently about 19 to about 27
nucleotides in length; and [0104] one strand of the siRNA molecule
comprises a nucleotide sequence having substantial complementarity
to an mRNA corresponding to the Syk gene so that the siRNA molecule
directs cleavage of the mRNA via RNA interference.
[0105] Also disclosed are various compositions 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:2, SEQ ID NO:13-SEQ ID NO:40, and SEQ ID NO:44-SEQ ID NO:47, or
a complement thereof; and a pharmaceutically acceptable carrier.
Various other compositions comprise an interfering RNA having a
length of 19 to 49 nucleotides, and consisting essentially of a
nucleotide sequence corresponding to any one of SEQ ID NO:41-SEQ ID
NO:47, or a complement thereof; and a pharmaceutically acceptable
carrier.
[0106] 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.
[0107] 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.
Examples
[0108] Table 1 lists examples of Syk DNA target sequences of SEQ ID
NO:1 from which siRNAs of the present invention are designed in a
manner as set forth above. Syk encodes spleen tyrosine kinase, as
noted above.
TABLE-US-00007 TABLE 1 Syk Target Sequences for siRNAs # of
Starting Nucleotide with SEQ reference to ID Syk Target Sequence
SEQ ID NO: 1 NO GCACTATCGCATCGACAAA 789 2 ACTATCGCATCGACAAAGA 791
13 GGCAGCTAGTCGAGCATTA 860 14 GCAGCTAGTCGAGCATTAT 861 15
CAGCTAGTCGAGCATTATT 862 16 AGTCGAGCATTATTCTTAT 867 17
GTCGAGCATTATTCTTATA 868 18 AGTTATTAGCAGAAGCAAA 1394 19
CGTACATCGTGCGCATGAT 1436 20 GAGTCCTGGATGCTAGTTA 1471 21
AGTCCTGGATGCTAGTTAT 1472 22 ACAGACATGTCAAGGATAA 1535 23
AGGATAAGAACATCATAGA 1547 24 TGATTTCGGACTCTCCAAA 1680 25
CATGGAAAGTGGCCTGTCA 1738 26 ATGGAAAGTGGCCTGTCAA 1739 27
CTCCGGAATGCATCAACTA 1766 28 GAAGTGAAGTCACCGCTAT 1880 29
GATGTACGATCTCATGAAT 1947 30 GCTGCGCAATTACTACTAT 2022 31
ACTATGACGTGGTGAACTA 2036 32 TTACGATCTGTTTCCAAAT 2328 33
TACGATCTGTTTCCAAATC 2329 34 CTTAGCATGTGACTCCTGA 2473 35
AGGAATTTGGCTGCTTCTA 2509 36 TGCTTCTACGGCCATGAGA 2520 37
TCTACGGCCATGAGACTGA 2524 38 AAGCTTTCCTGACAATAAA 2558 39
CCAGTGCAGTTTCTAAGCA 2613 40
TABLE-US-00008 TABLE 2 Further Syk Target Sequences for siRNAs # of
Starting Nucleotide with reference to SEQ ID Syk Target Sequence
SEQ ID NO: 1 NO: GTGGAATAATCTCAAGAAT 1007 41 AGCACTGCGTGCTGATGAA
1698 42 CGGAATGCATCAACTACTA 1769 43 AAUGCCUUGGUUCCAUGGA 642 44
GGAAUAAUCUCAAGAAUCA 1009 45 GAACUGGGCUCUGGUAAUU 1273 46
GAACAGACAUGUCAAGGAU 1533 47
[0109] As cited in the examples above, one of skill in the art is
able to use the target sequence information provided in Table 1 to
design interfering RNAs having a length shorter or longer than the
sequences provided in Table 1 by referring to the sequence position
in SEQ ID NO:1 and adding or deleting nucleotides complementary or
near complementary to SEQ ID NO:1.
[0110] 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.
[0111] 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).
[0112] 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.
[0113] 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.
[0114] 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.
[0115] 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.
[0116] The target sequence in the mRNAs corresponding to SEQ ID
NO:1 may be in the 5' or 3' untranslated regions of the mRNA as
well as in the coding region of the mRNA.
[0117] 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.
[0118] 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.
[0119] 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.
[0120] 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.
[0121] 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.
[0122] 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. Non-complementary regions may be at the 3', 5' or
both ends of a complementary region or between two complementary
regions.
[0123] 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.
[0124] 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.
[0125] 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.
[0126] 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.
[0127] Hybridization Under Physiological Conditions:
[0128] 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.
[0129] 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.
[0130] Single-Stranded Interfering RNA:
[0131] 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 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. The ss interfering RNA of Table 1 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.
[0132] 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.
[0133] Hairpin Interfering RNA:
[0134] 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.
[0135] Mode of Administration:
[0136] 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.
[0137] 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. Further
modes of administration include tablets, pills, and capsules.
[0138] 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.
[0139] Administration may be directly to the lungs, via, for
example, an aerosolized preparation, and by inhalation via an
inhaler or a nebulizer, for example
[0140] Subject:
[0141] A subject in need of treatment for a Syk-related
inflammatory condition or at risk for developing a Syk-related
inflammatory condition is a human or other mammal having a
Syk-related inflammatory condition or at risk of developing a
Syk-related inflammatory condition, such as allergic
conjunctivitis, ocular inflammation, dermatitis, rhinitis, asthma,
allergy, or mast-cell disease for example, associated with
undesired or inappropriate expression or activity of Syk as cited
herein.
[0142] Ocular structures associated with such disorders 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.
[0143] 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.
[0144] 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.
[0145] A subject may also be an otic cell, a lung cell, an ocular
cell, cell culture, organ or an ex vivo organ or tissue.
[0146] Formulations and Dosage:
[0147] 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.
[0148] Interfering RNAs of the present invention are administered
as solids, 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%
[0149] 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 pM 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.
[0150] Therapeutic treatment of patients with siRNAs directed
against Syk 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.
[0151] An effective amount of a formulation may depend on factors
such as the age, race, and sex of the subject, the severity of the
Syk-related inflammatory 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 spleen tyrosine kinase containing tissue at a therapeutic
dose thereby ameliorating a Syk-related process.
[0152] 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 (Mirus
Corporation, Madison, Wis.), LIPOFECTIN.RTM., Lipofectamine,
OLIGOFECTAMINE.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.
[0153] 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.
[0154] For ophthalmic, otic, or pulmonary delivery, an interfering
RNA may be combined with ophthalmologically, 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.
[0155] 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.
[0156] Kits:
[0157] 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.,
anon-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.
[0158] 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.
[0159] The ability of Syk interfering RNA to knock-down the levels
of endogenous Syk 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 Ophthalmol 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 Syk 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 Syk interfering RNA is used that produces the desired level of
knock-down in target gene expression.
[0160] 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.
[0161] 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.
[0162] Interfering RNA for Specifically Silencing SYK in 293FT
Cells
[0163] The present study examines the ability of SYK-interfering
RNA to knock down the levels of endogenous SYK protein expression
in cultured 293FT cells.
[0164] Transfection of 293FT cells (Invitrogen, Carlsbad, Calif.)
was accomplished using standard in vitro concentrations (0.1-10 nM)
of SYK 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 MgCl2. 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-SYK
antibody (Santa Cruz Biotechnology, Santa Cruz, Calif.) were
performed to assess SYK protein expression. The SYK siRNAs are
double-stranded interfering RNAs having specificity for the
following targets: siSYK #2 targets the sequence
AAUGCCUUGGUUCCAUGGA (SEQ ID NO: 44); siSYK #5 targets the sequence
GGAAUAAUCUCAAGAAUCA (SEQ ID NO: 45); siSYK #6 targets the sequence
GAACUGGGCUCUGGUAAUU (SEQ ID NO: 46); siSYK #8 targets the sequence
GAACAGACAUGUCAAGGAU (SEQ ID NO: 47). As shown by the data of FIG.
1, siSYK #5, siSYK #6, and siSYK #8 siRNAs reduced SYK 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 siSYK #6, and siSYK #8 siRNAs were particularly
effective. The siSYK #2 siRNA was ineffective at all three
concentrations.
Sequence CWU 1
1
4712672DNAHomo sapiens 1aggaagagcc gcgggcccgg cggctgaggc caccccggcg
gcggctggag agcgaggagg 60agcgggtggc cccgcgctgc gcccgccctc gcctcacctg
gcgcaggtgg acacctgcgc 120aggtgtgtgc cctccggccc ctgaagcatg
gccagcagcg gcatggctga cagcgccaac 180cacctgccct tctttttcgg
caacatcacc cgggaggagg cagaagatta cctggtccag 240gggggcatga
gtgatgggct ttatttgctg cgccagagcc gcaactacct gggtggcttc
300gccctgtccg tggcccacgg gaggaaggca caccactaca ccatcgagcg
ggagctgaat 360ggcacctacg ccatcgccgg tggcaggacc catgccagcc
ccgccgacct ctgccactac 420cactcccagg agtctgatgg cctggtctgc
ctcctcaaga agcccttcaa ccggccccaa 480ggggtgcagc ccaagactgg
gccctttgag gatttgaagg aaaacctcat cagggaatat 540gtgaagcaga
catggaacct gcagggtcag gctctggagc aggccatcat cagtcagaag
600cctcagctgg agaagctgat cgctaccaca gcccatgaaa aaatgccttg
gttccatgga 660aaaatctctc gggaagaatc tgagcaaatt gtcctgatag
gatcaaagac aaatggaaag 720ttcctgatcc gagccagaga caacaacggc
tcctacgccc tgtgcctgct gcacgaaggg 780aaggtgctgc actatcgcat
cgacaaagac aagacaggga agctctccat ccccgaggga 840aagaagttcg
acacgctctg gcagctagtc gagcattatt cttataaagc agatggtttg
900ttaagagttc ttactgtccc atgtcaaaaa atcggcacac agggaaatgt
taattttgga 960ggccgtccac aacttccagg ttcccatcct gcgacttggt
cagcgggtgg aataatctca 1020agaatcaaat catactcctt cccaaagcct
ggccacagaa agtcctcccc tgcccaaggg 1080aaccggcaag agagtactgt
gtcattcaat ccgtatgagc cagaacttgc accctgggct 1140gcagacaaag
gcccccagag agaagcccta cccatggaca cagaggtgta cgagagcccc
1200tacgcggacc ctgaggagat caggcccaag gaggtttacc tggaccgaaa
gctgctgacg 1260ctggaagaca aagaactggg ctctggtaat tttggaactg
tgaaaaaggg ctactaccaa 1320atgaaaaaag ttgtgaaaac cgtggctgtg
aaaatactga aaaacgaggc caatgacccc 1380gctcttaaag atgagttatt
agcagaagca aatgtcatgc agcagctgga caacccgtac 1440atcgtgcgca
tgatcgggat atgcgaggcc gagtcctgga tgctagttat ggagatggca
1500gaacttggtc ccctcaataa gtatttgcag cagaacagac atgtcaagga
taagaacatc 1560atagaactgg ttcatcaggt ttccatgggc atgaagtact
tggaggagag caattttgtg 1620cacagagatc tggctgcaag aaatgtgttg
ctagttaccc aacattatgc caagatcagt 1680gatttcggac tctccaaagc
actgcgtgct gatgaaaact actacaaggc ccagacccat 1740ggaaagtggc
ctgtcaagtg gtacgctccg gaatgcatca actactacaa gttctccagc
1800aaaagcgatg tctggagctt tggagtgttg atgtgggaag cattctccta
tgggcagaag 1860ccatatcgag ggatgaaagg aagtgaagtc accgctatgt
tagagaaagg agagcggatg 1920gggtgccctg cagggtgtcc aagagagatg
tacgatctca tgaatctgtg ctggacatac 1980gatgtggaaa acaggcccgg
attcgcagca gtggaactgc ggctgcgcaa ttactactat 2040gacgtggtga
actaaccgct cccgcacctg tcggtggctg cctttgatca caggagcaat
2100cacaggaaaa tgtatccaga ggaattgatt gtcagccacc tccctctgcc
agtcgggaga 2160gccaggcttg gatggaacat gcccacaact tgtcacccaa
agcctgtccc aggactcacc 2220ctccacaaag caaaggcagt cccgggagaa
aagacggatg gcaggatcca aggggctagc 2280tggatttgtt tgttttcttg
tctgtgtgat tttcatacag gttattttta cgatctgttt 2340ccaaatccct
ttcatgtctt tccacttctc tgggtcccgg ggtgcatttg ttactcatcg
2400ggcccaggga cattgcagag tggcctagag cactctcacc ccaagcggcc
ttttccaaat 2460gcccaaggat gccttagcat gtgactcctg aagggaaggc
aaaggcagag gaatttggct 2520gcttctacgg ccatgagact gatccctggc
cactgaaaag ctttcctgac aataaaaatg 2580ttttgaggct ttaaaaagaa
aatcaagttt gaccagtgca gtttctaagc atgtagccag 2640ttaaggaaag
aaagaaaaaa aaaaaaaaaa aa 2672219DNAArtificialTarget Sequence
2gcactatcgc atcgacaaa 19321DNAArtificialSense Strand with 3'NN
3gcacuaucgc aucgacaaan n 21421DNAArtificialAntisense Strand with
3'NN 4uuugucgaug cgauagugcn n 21521RNAArtificialSense Strand
5gcacuaucgc aucgacaaau u 21621RNAArtificialAntisense Strand
6uuugucgaug cgauagugcu u 21719RNAArtificialSense Strand 7gcacuaucgc
aucgacaaa 19819RNAArtificialAntisense Strand 8uuugucgaug cgauagugc
19948DNAArtificialHairpin duplex with loop 9gcacuaucgc aucgacaaan
nnnnnnnuuu gucgaugcga uagugcuu 481025DNAArtificialSense Strand
10gcactatcgc atcgacaaag acaag 251125RNAArtificialSense Strand
11gcacuaucgc aucgacaaag acaag 251227RNAArtificialAntisense Strand
12cuugucuuug ucgaugcgau agugcuu 271319DNAArtificialTarget Sequence
13actatcgcat cgacaaaga 191419DNAArtificialTarget Sequence
14ggcagctagt cgagcatta 191519DNAArtificialTarget Sequence
15gcagctagtc gagcattat 191619DNAArtificialTarget Sequence
16cagctagtcg agcattatt 191719DNAArtificialTarget Sequence
17agtcgagcat tattcttat 191819DNAArtificialTarget Sequence
18gtcgagcatt attcttata 191919DNAArtificialTarget Sequence
19agttattagc agaagcaaa 192019DNAArtificialTarget Sequence
20cgtacatcgt gcgcatgat 192119DNAArtificialTarget Sequence
21gagtcctgga tgctagtta 192219DNAArtificialTarget Sequence
22agtcctggat gctagttat 192319DNAArtificialTarget Sequence
23acagacatgt caaggataa 192419DNAArtificialTarget Sequence
24aggataagaa catcataga 192519DNAArtificialTarget Sequence
25tgatttcgga ctctccaaa 192619DNAArtificialTarget Sequence
26catggaaagt ggcctgtca 192719DNAArtificialTarget Sequence
27atggaaagtg gcctgtcaa 192819DNAArtificialTarget Sequence
28ctccggaatg catcaacta 192919DNAArtificialTarget Sequence
29gaagtgaagt caccgctat 193019DNAArtificialTarget Sequence
30gatgtacgat ctcatgaat 193119DNAArtificialTarget Sequence
31gctgcgcaat tactactat 193219DNAArtificialTarget Sequence
32actatgacgt ggtgaacta 193319DNAArtificialTarget Sequence
33ttacgatctg tttccaaat 193419DNAArtificialTarget Sequence
34tacgatctgt ttccaaatc 193519DNAArtificialTarget Sequence
35cttagcatgt gactcctga 193619DNAArtificialTarget Sequence
36aggaatttgg ctgcttcta 193719DNAArtificialTarget Sequence
37tgcttctacg gccatgaga 193819DNAArtificialTarget Sequence
38tctacggcca tgagactga 193919DNAArtificialTarget Sequence
39aagctttcct gacaataaa 194019DNAArtificialTarget Sequence
40ccagtgcagt ttctaagca 194119DNAArtificialTarget Sequence
41gtggaataat ctcaagaat 194219DNAArtificialTarget Sequence
42agcactgcgt gctgatgaa 194319DNAArtificialTarget Sequence
43cggaatgcat caactacta 194419RNAArtificialsynthetic sequence
44aaugccuugg uuccaugga 194519RNAArtificialsynthetic sequence
45ggaauaaucu caagaauca 194619RNAArtificialsynthetic sequence
46gaacugggcu cugguaauu 194719RNAArtificialsynthetic seuqence
47gaacagacau gucaaggau 19
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