U.S. patent application number 15/942126 was filed with the patent office on 2019-01-10 for compositions and methods for inhibiting expression of mutant egfr gene.
The applicant listed for this patent is ALNYLAM PHARMACEUTICALS, INC., LUDWIG INSTITUTE FOR CANCER RESEARCH LTD.. Invention is credited to Rudy Bonavia, Webster Cavenee, Frank Furnari, Maria del Mar Inda Perez, Dinah Sah, Pamela Tan.
Application Number | 20190010502 15/942126 |
Document ID | / |
Family ID | 41268470 |
Filed Date | 2019-01-10 |
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United States Patent
Application |
20190010502 |
Kind Code |
A1 |
Sah; Dinah ; et al. |
January 10, 2019 |
COMPOSITIONS AND METHODS FOR INHIBITING EXPRESSION OF MUTANT EGFR
GENE
Abstract
The invention relates to a double-stranded ribonucleic acid
(dsRNA) targeting a mutant Epidermal Growth Factor Receptor (EGFR),
and methods of using the dsRNA to inhibit expression of mutant
EGFR.
Inventors: |
Sah; Dinah; (Hopkinton,
MA) ; Tan; Pamela; (Kulmbach, DE) ; Cavenee;
Webster; (La Jolla, CA) ; Furnari; Frank; (La
Jolla, CA) ; Perez; Maria del Mar Inda; (La Jolla,
CA) ; Bonavia; Rudy; (La Jolla, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ALNYLAM PHARMACEUTICALS, INC.
LUDWIG INSTITUTE FOR CANCER RESEARCH LTD. |
Cambridge
New York |
MA
NY |
US
US |
|
|
Family ID: |
41268470 |
Appl. No.: |
15/942126 |
Filed: |
March 30, 2018 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14936059 |
Nov 9, 2015 |
9957507 |
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15942126 |
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13659315 |
Oct 24, 2012 |
9212364 |
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14936059 |
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13061569 |
Jul 21, 2011 |
8318693 |
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PCT/US2009/055745 |
Sep 2, 2009 |
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13659315 |
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61166488 |
Apr 3, 2009 |
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61147668 |
Jan 27, 2009 |
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61147680 |
Jan 27, 2009 |
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61095487 |
Sep 9, 2008 |
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61093620 |
Sep 2, 2008 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 2310/321 20130101;
C12N 2310/315 20130101; A61P 35/00 20180101; C12N 15/1136 20130101;
A61K 31/713 20130101; C12N 2320/30 20130101; C12N 2320/31 20130101;
C12N 15/1138 20130101; C12N 2310/14 20130101; C12N 2310/3515
20130101; C12N 2310/321 20130101; C12N 2310/3521 20130101 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C12N 15/113 20060101 C12N015/113; A61K 31/713 20060101
A61K031/713 |
Claims
1. A double-stranded ribonucleic acid (dsRNA), wherein said dsRNA
comprises at least two sequences that are complementary to each
other and wherein a sense strand comprises a first sequence and an
antisense strand comprises a second sequence comprising a region of
complementarity which is substantially complementary to at least a
part of a mRNA encoding an delta-Epidermal Growth Factor Receptor
(deltaEGFR), and wherein said region of complementarity is less
than 30 nucleotides in length.
2. The dsRNA of claim 1, wherein said dsRNA comprises at least one
modified nucleotide.
3. The dsRNA of claim 2, wherein at least one of said modified
nucleotides is chosen from the group of: a 2'-O-methyl modified
nucleotide, a nucleotide comprising a 5'-phosphorothioate group,
and a terminal nucleotide linked to a cholesteryl derivative or
dodecanoic acid bisdecylamide group.
4. The dsRNA of claim 2, wherein said modified nucleotide is chosen
from the group of: a 2'-deoxy-2'-fluoro modified nucleotide, a
2'-deoxy-modified nucleotide, a locked nucleotide, an abasic
nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified
nucleotide, morpholino nucleotide, a phosphoramidate, and a
non-natural base comprising nucleotide.
5. The dsRNA of claim 1, wherein the region of complementary is at
least 15 nucleotides in length or is between 19 and 21 nucleotides
in length.
6. (canceled)
7. The dsRNA of claim 1, wherein the dsRNA comprises a sense strand
consisting of a sense strand sequence selected from Tables 2 and 3,
and an antisense strand consisting of an antisense sequence
selected from Tables 2 and 3.
8. The dsRNA of claim 1, wherein the sense strand and the antisense
strand are also substantially complementary to at least a part of
an mRNA encoding a wildtype Epidermal Gro The dsRNA of claim 1,
wherein the sense strand comprises a nucleotide sequence of SEQ ID
NO:2, and the antisense strand comprises a nucleotide sequence of
SEQ ID NO:3.
9. The dsRNA of claim 1, wherein the sense strand comprises a
nucleotide sequence of SEQ ID NO:2, and the antisense strand
comprises a nucleotide sequence of SEQ ID NO:3.
10. A cell containing the dsRNA of claim 1.
11. A pharmaceutical composition for inhibiting expression of a
deltaEGFR gene comprising the dsRNA of claim 1.
12. The pharmaceutical composition of claim 11, further comprising
a dsRNA comprising at least two sequences that are complementary to
each other and wherein a sense strand comprises a first sequence
and an antisense strand comprises a second sequence which is
substantially complementary to at least a part of an mRNA encoding
an Interleukin-6 (IL6), and wherein said region of complementarity
is less than 30 nucleotides in length.
13. The pharmaceutical composition of claim 12, wherein the IL6
dsRNA comprises a sense strand comprising a sequence selected from
Tables 5-8, and an antisense sequence comprising an antisense
sequence selected from Tables 5-8.
14. A method of inhibiting deltaEGFR expression in a cell, the
method comprising: (a) introducing into the cell the dsRNA of claim
1; and (b) maintaining the cell produced in step (a) for a time
sufficient to obtain degradation of the mRNA transcript of the
deltaEGFR gene, thereby inhibiting expression of the deltaEGFR gene
in the cell.
15. A method of treating a disorder mediated by deltaEGFR
expression comprising administering to a human in need of such
treatment a therapeutically effective amount of the dsRNA of claim
1.
16. The method of claim 15, wherein the human has cancer, a tumor,
an astrocytic tumor, or a glioma.
17. (canceled)
18. (canceled)
19. (canceled)
20. The method of claim 15, further comprising administering a
therapeutically effective amount of a dsRNA comprising at least two
sequences that are complementary to each other and wherein a sense
strand comprises a first sequence and an antisense strand comprises
a second sequence which is substantially complementary to at least
a part of an mRNA encoding an IL6, and wherein said region of
complementarity is less than 30 nucleotides in length.
21. A vector comprising a nucleotide sequence that encodes at least
one strand of a dsRNA, wherein one of the strands of said dsRNA is
substantially complementary to at least a part of an mRNA encoding
a deltaEGFR and wherein said dsRNA is less than 30 base pairs in
length.
22. The vector of claim 21, wherein the region of complementarity
is at least 15 nucleotides in length or is 19 to 21 nucleotides in
length.
23. (canceled)
24. A cell comprising the vector of claim 20.
25. A double-stranded ribonucleic acid (dsRNA), wherein said dsRNA
comprises at least two sequences that are complementary to each
other and wherein a sense strand comprises a first sequence and an
antisense strand comprises a second sequence comprising a region of
complementarity which is substantially complementary to at least a
part of a mRNA encoding IL-6, and wherein said region of
complementarity is less than 30 nucleotides in length.
26. The dsRNA of claim 25, wherein the IL6 dsRNA comprises a sense
strand comprising a sequence selected from Tables 5-8, and an
antisense sequence comprising an antisense sequence selected from
Tables 5-8.
27. A method of treating a disorder mediated by IL-6 expression
comprising administering to a human in need of such treatment a
therapeutically effective amount of the dsRNA of claim 25.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 14/936,059, filed Nov. 9, 2015, now allowed, which is a
continuation of U.S. application Ser. No. 13/659,315, filed Oct.
24, 2012, now U.S. Pat. No. 9,212,364, issued Dec. 15, 2015, which
is a continuation of U.S. application Ser. No. 13/061,569, filed
Jul. 21, 2011, now U.S. Pat. No. 8,318,693, issued Nov. 27, 2012,
which is a national phase application under 35 U.S.C. .sctn. 371 of
PCT International Application No. PCT/US2009/055745, filed Sep. 2,
2009, which claims the benefit of U.S. Provisional Application No.
61/093,620, filed Sep. 2, 2008, U.S. Provisional Application No.
61/095,487, filed Sep. 9, 2008, U.S. Provisional Application No.
61/147,668, filed Jan. 27, 2009, U.S. Provisional Application No.
61/147,680, filed Jan. 27, 2009, and U.S. Provisional Application
No. 61/166,488, filed Apr. 3, 2009. Each of these prior
applications is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0002] The invention relates to a double-stranded ribonucleic acid
(dsRNA) targeting a mutant Epidermal Growth Factor Receptor (EGFR),
and methods of using the dsRNA to inhibit expression of mutant
EGFR.
BACKGROUND OF THE INVENTION
[0003] The Epidermal Growth Factor Receptor (EGFR) gene is
frequently upregulated in carcinomas of the breast, kidney, ovary,
cervix, and in squamous cells. The upregulation is typically due to
gene amplification or overexpression. EGFR upregulation in gliomas
is most often associated with the rearrangement of the EGFR gene
resulting in alterations of its transcript so that such gliomas
express both wild-type endogenous EGFR as well as the episomal
mutant form. The most common of the rearrangements are genomic
alterations leading to deletion of exons 2-7 in the EGFR mRNA
(called ds 2-7 EGFR, deltaEGFR, EGFR-de2-7, or EGFRvIII), which
causes an in-frame truncation of 801 bp in the extracellular domain
of the molecule. The EGFR gene is amplified in >50% of
glioblastomas. This amplification is often associated with
expression of deltaEGFR, which conveys enhanced tumor
aggressiveness.
[0004] Double-stranded RNA molecules (dsRNA) have been shown to
block gene expression in a highly conserved regulatory mechanism
known as RNA interference (RNAi). WO 99/32619 (Fire et al.)
disclosed the use of a dsRNA of at least 25 nucleotides in length
to inhibit the expression of genes in C. elegans. dsRNA has also
been shown to degrade target RNA in other organisms, including
plants (see, e.g., WO 99/53050, Waterhouse et al.; and WO 99/61631,
Heifetz et al.), Drosophila (see, e.g., Yang, D., et al., Curr.
Biol. (2000) 10:1191-1200), and mammals (see WO 00/44895, Limmer;
and DE 101 00 586.5, Kreutzer et al.).
SUMMARY OF THE INVENTION
[0005] The invention provides compositions containing
double-stranded ribonucleic acid (dsRNA) and methods for inhibiting
the expression of a mutant EGFR gene, such as a deltaEGFR gene, in
a cell or mammal. The invention also provides compositions and
methods for treating pathological conditions and diseases caused by
the expression of deltaEGFR gene, such as cancer, including glioma.
The dsRNAs included in the compositions featured herein include a
dsRNA having an RNA strand (the antisense strand) having a region
which is less than 30 nucleotides in length, generally 19-24
nucleotides in length, and is substantially complementary to at
least part of an mRNA transcript of the deltaEGFR gene. In some
embodiments, the dsRNA also targets a wildtype mRNA transcript of
the EGFR gene.
[0006] In one embodiment, a dsRNA for inhibiting expression of a
deltaEGFR gene includes at least two sequences that are
complementary to each other. The dsRNA includes a sense strand
having a first sequence and an antisense strand having a second
sequence. The antisense strand includes a nucleotide sequence that
is substantially complementary to at least part of an mRNA encoding
deltaEGFR, and the region of complementarity is less than 30
nucleotides in length, and at least 15 nucleotides in length.
Generally, the dsRNA is 19 to 24, e.g., 19 to 21 nucleotides in
length. In some embodiments the dsRNA is from about 10 to about 15
nucleotides in length, and in other embodiments the dsRNA is from
about 25 to about 30 nucleotides in length. The dsRNA, upon
contacting with a cell expressing deltaEGFR, inhibits the
expression of the deltaEGFR gene by at least 20%, at least 25%, at
least 30%, at least 35%, or at least 40%, such as when assayed by a
method as described herein. In one embodiment, the deltaEGFR dsRNA
is formulated in a stable nucleic acid particle (SNALP).
[0007] For example, the dsRNA molecules featured herein can include
a first sequence of the dsRNA that is selected from the group
consisting of the sense sequences of Tables 2, 3 and 4, and a
second sequence that is selected from the group consisting of the
antisense sequences of Tables 2, 3 and 4. The dsRNA molecules
featured herein can include naturally occurring nucleotides or can
include at least one modified nucleotide, such as a 2'-O-methyl
modified nucleotide, a nucleotide having a 5'-phosphorothioate
group, and a terminal nucleotide linked to a conjugate group, such
as a cholesteryl derivative or a vitamin E group. Alternatively,
the modified nucleotide may be chosen from the group of: a
2'-deoxy-2'-fluoro modified nucleotide, a 2'-deoxy-modified
nucleotide, a locked nucleotide, an abasic nucleotide,
2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide,
morpholino nucleotide, a phosphoramidate, and a non-natural base
comprising nucleotide. Generally, such modified sequence will be
based on a first sequence of said dsRNA selected from the group
consisting of the sense sequences of Tables 2, 3 and 4 and a second
sequence selected from the group consisting of the antisense
sequences of Tables 2, 3 and 4.
[0008] In one aspect, an interleukin-6 (IL6) dsRNA is also featured
in the invention, and the IL-6 dsRNA is capable of decreasing
levels of IL6 protein secretion in cultured cells, e.g., human
cultured cells. In one embodiment, the cultured cells are
U87-.DELTA.EGFR cells. In another embodiment, the IL-6 dsRNA is
capable of decreasing IL6 secretion into culture supernatant by
20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or more. In yet another
embodiment, the IL6 dsRNA is capable of reducing tumor volume in an
animal model, such as in a mouse, rat, or primate model. In another
embodiment, a first sequence of an IL6 dsRNA is selected from the
group consisting of the sense sequences of Tables 5, 6, 7, and 8, a
second sequence is selected from the group consisting of the
antisense sequences of Tables 5, 6, 7, and 8.
[0009] In another aspect, the invention provides a cell containing
at least one of the dsRNAs featured in the invention. The cell is
generally a mammalian cell, such as a human cell.
[0010] In yet another aspect, the invention provides a
pharmaceutical composition for inhibiting the expression of a
deltaEGFR gene in an organism, generally a human subject. The
composition typically includes one or more of the dsRNAs described
herein and a pharmaceutically acceptable carrier or delivery
vehicle. In one embodiment, the composition is used for treating
cancer, e.g., a glioma.
[0011] In another embodiment, the pharmaceutical composition is
formulated for administration of a dosage regimen described herein,
e.g., not more than once every four weeks, not more than once every
three weeks, not more than once every two weeks, or not more than
once every week. In another embodiment, the pharmaceutical
composition can be maintained for a month or longer, e.g., one,
two, three, or six months, or one year or longer.
[0012] In another embodiment, a composition containing a dsRNA
featured in the invention, e.g., a dsRNA targeting deltaEGFR, is
administered with a non-dsRNA therapeutic agent, such as an agent
known to treat a cancer, such as a glioma. For example, a dsRNA
featured in the invention can be administered with, e.g, a
chemotherapeutic agent, such as temozolomide, or with radiation
therapy.
[0013] In one embodiment, the composition further includes a dsRNA
having at least two sequences that are complementary to each other,
and where a sense strand includes a region of complementarity that
is substantially complementary to at least a part of an mRNA
encoding an IL6 protein, and where the region of complementarity is
less than 30 nucleotides in length and at least 15 nucleotides in
length. Generally, the IL6 dsRNA is 19 to 24, e.g., 19 to 21
nucleotides in length. In some embodiments, the dsRNA is from about
10 to about 15 nucleotides in length, and in other embodiments the
dsRNA is from about 25 to about 30 nucleotides in length. In
another embodiment, a first sequence of the IL6 dsRNA is selected
from the group consisting of the sense sequences of Tables 5, 6, 7,
and 8, a second sequence is selected from the group consisting of
the antisense sequences of Tables 5, 6, 7, and 8.
[0014] In another aspect, the deltaEGFR dsRNA is administered to a
patient, and then a non-dsRNA agent is administered to the patient
(or vice versa). In one embodiment, the deltaEGFR dsRNA and the
non-dsRNA therapeutic agent are administered at the same time. In
another embodiment, the deltaEGFR dsRNA is administered with an IL6
dsRNA, such as for the treatment of cancer.
[0015] In certain embodiments, the patient has a cancer, e.g., a
tumor, such as an astrocytic tumor, or a glioma.
[0016] In one aspect, the invention provides a method for
inhibiting the expression of a deltaEGFR gene in a cell by
performing the following steps: [0017] (a) introducing into the
cell a double-stranded ribonucleic acid (dsRNA), wherein the dsRNA
includes at least two sequences that are complementary to each
other. The dsRNA has a sense strand having a first sequence and an
antisense strand having a second sequence; the antisense strand has
a region of complementarity that is substantially complementary to
at least a part of a mRNA encoding deltaEGFR, and where the region
of complementarity is less than 30 nucleotides in length, generally
19-24 nucleotides in length, and where the dsRNA, upon contact with
a cell expressing the deltaEGFR, inhibits expression of the
deltaEGFR gene by at least 40%; [0018] and [0019] (b) maintaining
the cell produced in step (a) for a time sufficient to obtain
degradation of the mRNA transcript of deltaEGFR gene, thereby
inhibiting expression of the deltaEGFR gene in the cell.
[0020] In one embodiment, the dsRNA that inhibits expression of the
deltaEGFR gene also inhibits expression of a wildtype EGFR gene in
the cell. Typically, such a dsRNA can inhibit both deltaEGFR
expression and wildtype EGFR expression, because the antisense
strand has a region of complementarity that is substantially
complementary to at least a part of an mRNA encoding deltaEGFR and
at least part of an mRNA encoding wildtype EGFR.
[0021] In another embodiment, the method is for inhibiting gene
expression in a tumor cell.
[0022] In another aspect, the invention provides methods for
treating, preventing or managing pathological processes mediated by
deltaEGFR expression, e.g., a cancer, such as a glioma, e.g., a
glial tumor of the central nervous system, such as a grade I, II,
III, or IV glioma. For example, a dsRNA targeting deltaEGFR is used
to treat a grade III glioma, such as anaplastic astrocytoma, or a
grade IV glioma, such as a glioblastoma multiforme. In other
embodiments, a dsRNA targeting deltaEGFR is used to treat a
carcinoma of the breast, ovary, cervix, kidney, or squamous cell.
In one embodiment, the deltaEGFR dsRNA is administered with a
second dsRNA, such as an IL6 dsRNA, for treatment of a disorder
associated with deltaEGFR expression. The IL6 and deltaEGFR dsRNAs
can be administered in combination or sequentially. In yet another
embodiment, an IL6 dsRNA alone is administered to treat a disorder
associated with deltaEGFR expression.
[0023] A method featured in the invention can include administering
to a patient in need of such treatment, prevention or management a
therapeutically or prophylactically effective amount of one or more
of the dsRNAs featured in the invention, e.g., one or both of a
dsRNA targeting deltaEGFR or IL6. In one embodiment the patient has
cancer. In another embodiment, administration of the dsRNA
targeting deltaEGFR and/or the dsRNA targeting IL6, alleviates or
relieves the severity of at least one symptom of the
deltaEGFR-mediated disorder in the patient.
[0024] In another aspect, the invention provides a vector for
inhibiting the expression of a deltaEGFR gene in a cell. In one
embodiment, the vector includes at least one regulatory sequence
operably linked to a nucleotide sequence that encodes at least one
strand of one of a dsRNA featured in the invention.
[0025] In another aspect, the invention provides a vector for
inhibiting the expression of an IL6 gene in a cell. In one
embodiment, the vector includes at least one regulatory sequence
operably linked to a nucleotide sequence that encodes at least one
strand of one of a dsRNA featured in the invention.
[0026] In yet another aspect, the invention provides a cell
containing a vector for inhibiting the expression of a deltaEGFR
gene in a cell. The vector includes a regulatory sequence operably
linked to a nucleotide sequence that encodes at least one strand of
one of the deltaEGFR dsRNA featured in the invention. In one
embodiment, the cell also contains a vector for inhibiting
expression of an IL6 gene in a cell. This vector also has a
regulatory sequence operably linked to a nucleotide sequence that
encodes at least one strand of an IL6 dsRNA featured in the
invention.
[0027] In yet another aspect, the invention provides a composition
containing a deltaEGFR dsRNA, in combination with a second dsRNA
targeting a second gene involved in a pathological disease, and
useful for treating the disease, e.g., cancer. In one embodiment,
the second dsRNA is a dsRNA targeting IL6.
[0028] The details of one or more embodiments of the invention are
set forth in the description below. Other features, objects, and
advantages of the invention will be apparent from the description
and the drawings, and from the claims.
BRIEF DESCRIPTION OF THE FIGURES
[0029] FIGS. 1A and 1B are graphs showing tumor growth kinetics
(FIG. 1A) and tumor volume (FIG. 1B) following injection of cells
subcutaneously into the right flank of 4 to 5 weeks-old female
athymic nude mice.
[0030] FIGS. 2A and 2B show hematoxylin and eosin (H&E)
staining of brain cryo-sections from mice. FIG. 2A is H&E
staining of brain cryo-sections from nude mice injected
intracranially with cells, and FIG. 2B shows H&E staining of
brain cryo-sections of mice injected intracranially with cells
either alone or mixed with deltaEGFR over-expressing astrocytes
(upper panel). The lower panel shows the presence of wtEGFR
astrocytes within the tumor by immunofluorescence (GFP IF).
[0031] FIG. 3A is a graph showing the composition of various tumor
samples in 4 to 5 week old mice injected with the indicated cell
types, as assayed by X-Gal staining. FIG. 3B is a graph showing the
composition of tumor samples as assayed by flow cytometry using
Ab-1 (FITC) and Ab-5 (APC) antibodies to stain tumors formed after
infection of U87wt mixed with U87delta at the indicated ratios.
[0032] FIG. 4 is a panel of Western blots showing analysis of EGFR
activation and known downstream signaling molecules in U87wt cells
stimulated with serial dilutions of U87delta CM or negative control
U87Par CM, or positive control EGF ligand.
[0033] FIGS. 5A-5C are Western blots showing the activity of siRNAs
targeting deltaEGFR (FIG. 5A), wildtype EGFR (FIG. 5B), or both
mutant and wildtype receptors (FIG. 5C). C1, C2, C3, and CA
indicate negative controls (untransfected cells).
[0034] FIGS. 6A-6C are Western blots showing the activity of siRNAs
targeting deltaEGFR
[0035] (FIG. 6A) or wildtype EGFR (FIG. 6C). FIG. 6B shows the
activity of siRNAs specific for deltaEGFR (AD-13375) or wtEGFR
(AD-13377) in U87-deltaEGFR cells or U87-wtEGFR cells,
respectively. Luc and (-) indicate negative controls (cells
transfected with an irrelevant gene siRNA (targeting luciferase)
and untransfected cells, respectively).
[0036] FIGS. 7A and 7B are Western blots showing dose response
activity of siRNA activity in U87-deltaEGFR cells (FIG. 7A) and in
U87-wtEGFR cells (FIG. 7B). U87-deltaEGFR cells were transfected
with siRNAs specific for deltaEGFR, and for both mutant and
wildtype receptors. U87-wtEGFR cells were transfected with siRNAs
specific for wtEGFR, and for both mutant and wildtype receptors.
C1, C2, C3, C4, C5, and C6 indicate negative controls
(untransfected cells).
[0037] FIGS. 8A-8D are Western blots showing durability of the
effect of unstabilized (FIGS. 8A and 8B) and stabilized (FIGS. 8C
and 8D) siRNAs. In FIGS. 8A and 8C, U87-deltaEGFR cells were
transfected with non-stabilized (FIG. 8A) or stabilized (FIG. 8C)
siRNAs. In FIGS. 8B and 8D, U87-wtEGFR cells were transfected with
unstabilized (FIG. 8B) or stabilized (FIG. 8D) siRNAs. Lysates were
prepared and Western blots were performed at the indicated day
post-transfection. Luc and (-) indicate negative controls (cells
transfected with an irrelevant gene siRNA (targeting luciferase)
and untransfected cells, respectively).
[0038] FIGS. 9A and 9B are graphs showing the effects of siRNAs on
tumorigenicity in mice injected with U87-wtEGFR cells (FIG. 9A) and
in mice injected with U87-deltaEGFR cells (FIG. 9B). Lysates were
prepared and Western blots were performed at the indicated day
post-transfection. Data are shown as mean.+-.standard deviation
(SD).
[0039] FIGS. 10A and 10B are graphs showing tumor kinetics (FIG.
10A) and volume (FIG. 10B) in nude mice injected with U87delta
cells and then injected intratumorally with 5 mg of deltaEGFR
siRNA#1 or irrelevant siRNA (siRNA luc).
[0040] FIG. 11 is the mRNA sequence of IL-6 reported at GenBank
Accession No. NM_000600.2 (record dated Jan. 4, 2009, GI No.
155369258; SEQ ID NO:274).
[0041] FIGS. 12A and 12B are graphs showing screening analysis of
stabilized siRNAs designed for IL-6 by ELISA. FIG. 12A demonstrates
the effect of the siRNAs on their target cytokines. Specificity of
the siRNAs for IL-6 was assessed by quantifying IL-8 levels (FIG.
12B). Values are mean.+-.SE of 2 independent samples. ("Neg": siRNA
targeting an irrelevant sequence).
[0042] FIGS. 13A and 13B are graphs showing dose-response analysis
of stabilized IL-6 siRNAs. Quantification of secreted IL-6 (FIG.
13A) and IL-8 (FIG. 13B) in supernatants from U87-.DELTA.EGFR cells
transfected with 100, 20, 4, and 0.8 nM of different IL-6 siRNAs
was performed by ELISA. Values are mean.+-.SE of 2 independent
samples. ("Neg": siRNA targeting an irrelevant sequence).
[0043] FIG. 14 is a graph illustrating durability analysis of
stabilized IL-6 siRNAs. Quantification of secreted IL-6 in
supernatants of U87-.DELTA.EGFR cells at different days after
transfection with the siRNAs was performed by ELISA. ("Neg2": siRNA
targeting an irrelevant sequence).
[0044] FIGS. 15A and 15B are two graphs illustrating ex vivo
tumorigenicity test of IL-6-specific stabilized siRNAs. The graphs
show tumor growth kinetics (top) and tumor volume at the end of the
experiment (bottom) after injection into nude mice of U87-deltaEGFR
cells transfected with stabilized siRNAs against luciferase or GFP,
IL-6 (AD-15644 and AD-15660). The experimental group included 6
animals. Data are shown as mean.+-.SE.
[0045] FIGS. 16A and 16B are graphs illustrating the efficacy of in
vivo delivery of IL-6 siRNAs (AD-15644 and AD-15660). Tumor growth
kinetics (FIG. 11A) and tumor volume at the end of the experiment
(FIG. 11B) was reduced in tumors treated with IL-6 specific siRNA
AD-15660, but not in tumors treated with a siRNA against
luciferase. Each group included 4 animals. Data are shown as
mean.+-.SE.
[0046] FIGS. 17A and 17B are graphs illustrating tumor volume (A)
and tumor growth kinetics (B) after subcutaneous injection of the
indicated cell types into nude mice. Tumor volume (FIG. 17A) was
assayed at day 32 after injection.
[0047] FIG. 18 is a graph illustrating quantification of soft agar
colony formation of U87wt colonies formed after treatment with
normal media (Neg), or U87.DELTA. (.DELTA.CM (conditioned medium)),
U87Par (parental CM), or U87Par-IL6 (parental-IL6) CM (*:
p<0.05; ** p<0.001).
[0048] FIG. 19 is a graph illustrating quantification of soft agar
colony formation of U87wt colonies formed after treatment with
normal media (negative control, Neg), or with U87.DELTA. cell CM
untreated (.DELTA.CM) or pretreated with IL-6 neutralizing antibody
(.DELTA.CM+IL-6 Ab) (**: p<0.001).
[0049] FIGS. 20A and 20 B are graphs illustrating tumor growth
kinetics (A) and tumor volume at day 20 (B) after subcutaneous
injection of U87wt, U87.DELTA.-Luc siRNA or U87.DELTA.-IL6 siRNA
cells only, or U87wt cells mixed with U87.DELTA.-Luc siRNA or
U87.DELTA.-IL6 siRNA cells at a ratio of 90:10.
[0050] FIGS. 21A and 21B represent the mRNA sequence of wtEGFR (SEQ
ID NO:1) (GenBank Accession No. NM_005228; record dated Aug. 24,
2008, GI No. 41327737). The underlined nucleotides are deleted in
deltaEGFR mRNA sequence.
DETAILED DESCRIPTION OF THE INVENTION
[0051] The invention provides dsRNAs and methods of using the
dsRNAs for inhibiting the expression of a deltaEGFR gene in a cell
or a mammal where the dsRNA targets the deltaEGFR gene. In some
embodiments, the dsRNAs featured in the invention target both a
deltaEGFR gene and a wildtype EGFR (wtEGFR) gene. The invention
also provides dsRNAs and methods of using the dsRNAs for inhibiting
the expression of an IL6 gene in a cell or a mammal where the dsRNA
targets the IL6 gene. The invention provides compositions and
methods for treating pathological conditions and diseases, such as
a cancer, in a mammal caused by the expression of the deltaEGFR or
IL6 genes. dsRNA directs the sequence-specific degradation of mRNA
through a process known as RNA interference (RNAi).
[0052] The dsRNAs of the compositions featured herein include an
RNA strand (the antisense strand) having a region which is less
than 30 nucleotides in length, generally 19-24 nucleotides in
length, and is substantially complementary to at least part of an
mRNA transcript of the deltaEGFR gene. The use of these dsRNAs
enables the targeted degradation of mRNAs of genes that are
implicated in replication or maintenance of cancer cells in
mammals. Very low dosages of deltaEGFR or IL6 dsRNAs in particular
can specifically and efficiently mediate RNAi, resulting in
significant inhibition of expression of the deltaEGFR and IL6
genes. Using cell-based and animal assays, the present inventors
have demonstrated that dsRNAs targeting deltaEGFR alone, or
targeting both deltaEGFR and wtEGFR, can specifically and
efficiently mediate RNAi, resulting in significant inhibition of
expression of one or both of the deltaEGFR or EGFR genes. Thus,
methods and compositions including these dsRNAs are useful for
treating pathological processes that can be mediated by down
regulating deltaEGFR and EGFR, such as in the treatment of
cancer.
[0053] Using cell-based and animal assays, the present inventors
have also demonstrated that dsRNAs targeting IL6 can also
specifically and efficiently mediate RNAi, resulting in significant
inhibition of expression of an IL6 gene. Thus, methods and
compositions including these dsRNAs are useful for treating
pathological processes that can be mediated by down regulating IL6,
such as in the treatment of cancer.
[0054] The methods and compositions containing the deltaEGFR or IL6
dsRNA featured in the invention are useful for treating
pathological processes mediated by deltaEGFR or IL6 expression,
e.g., cancer, such as glioma.
[0055] The following detailed description discloses how to make and
use the compositions containing dsRNAs to inhibit the expression of
the deltaEGFR or IL6 genes, as well as compositions and methods for
treating diseases and disorders caused by the expression of these
genes, such as leukemia. The pharmaceutical compositions featured
in the invention include a dsRNA having an antisense strand
comprising a region of complementarity which is less than 30
nucleotides in length, generally 19-24 nucleotides in length, and
is substantially complementary to at least part of an RNA
transcript of the deltaEGFR or IL6 gene, together with a
pharmaceutically acceptable carrier. The compositions featured in
the invention also include a dsRNA having an antisense strand
having a region of complementarity which is less than 30
nucleotides in length, generally 19-24 nucleotides in length, and
which is substantially complementary to at least part of an RNA
transcript of the deltaEGFR or IL6 gene.
[0056] Accordingly, in some aspects, pharmaceutical compositions
containing the deltaEGFR dsRNA and a pharmaceutically acceptable
carrier, methods of using the compositions to inhibit expression of
the deltaEGFR gene, and methods of using the pharmaceutical
compositions to treat diseases caused by expression of the
deltaEGFR gene are featured in the invention.
[0057] In other aspects, pharmaceutical compositions containing the
IL6 dsRNA and a pharmaceutically acceptable carrier, methods of
using the compositions to inhibit expression of an IL6 gene, and
methods of using the pharmaceutical compositions to treat diseases
caused by expression of the IL6 gene are featured in the
invention.
I. Definitions
[0058] For convenience, the meaning of certain terms and phrases
used in the specification, examples, and appended claims, are
provided below. If there is an apparent discrepancy between the
usage of a term in other parts of this specification and its
definition provided in this section, the definition in this section
shall prevail.
[0059] "G," "C," "A," "T" and "U" each generally stand for a
nucleotide that contains guanine, cytosine, adenine, thymidine and
uracil as a base, respectively. However, it will be understood that
the term "ribonucleotide" or "nucleotide" can also refer to a
modified nucleotide, as further detailed below, or a surrogate
replacement moiety. The skilled person is well aware that guanine,
cytosine, adenine, thymidine, and uracil may be replaced by other
moieties without substantially altering the base pairing properties
of an oligonucleotide comprising a nucleotide bearing such
replacement moiety. For example, without limitation, a nucleotide
comprising inosine as its base may base pair with nucleotides
containing adenine, cytosine, or uracil. Hence, nucleotides
containing uracil, guanine, or adenine may be replaced in the
nucleotide sequences of dsRNA featured in the invention by a
nucleotide containing, for example, inosine. In another example,
adenine and cytosine anywhere in the oligonucleotide can be
replaced with guanine and uracil, respectively to form G-U Wobble
base pairing with the target mRNA. Sequences containing such
replacement moieties are suitable for the compositions and methods
featured in the invention.
[0060] As used herein, "deltaEGFR" refers to an in-frame deletion
of exons 2-7 from the EGFR gene. deltaEGFR is also known as "de 2-7
EGFR" (Nishikawa et al. "A mutant epidermal growth factor receptor
common in human glioma confers enhanced tumorigenicity" Proc. Natl.
Acad. Sci. USA 91:7727-7731, 1994), "EGFR-de2-7", "EGFR*",
".DELTA.EGFR", and "EGFRvIII." The sequence of deltaEGFR is
equivalent to the sequence shown at FIGS. 21A and 21B carrying a
deletion of nucleotides 335 through 1135.
[0061] As used herein, "wild-type EGFR" ("wtEGFR") refers to a
non-mutant EGFR gene (e.g., an endogenous EGFR gene) in a cell,
such as in a non-transformed, or non-cancerous cell in a human.
EGFR is also known as EC2.7.10.1 (Epidermal Growth Factor Receptor
Precursor), ERBB (Receptor Protein Tyrosine Kinase ErbB1), ERBB1,
HER1, PIG61 (cell proliferation-inducing protein 61), mENA, avian
erythroblastic leukemia viral (v-erb-b) oncogene homolog, and cell
growth inhibiting protein 40. The sequence of four alternative
wildtype EGFR mRNA transcripts can be found at Genbank Accession
Numbers NM_005228.3 (record dated Aug. 24, 2008, GI No. 41327737;
see FIGS. 21A and 21B), NM_201282.1 (record dated Aug. 24, 2008, GI
No. 41327731), NM_201283.1 (record dated Aug. 24, 2008, GI No.
41327733), and NM_201284.1 (record dated Aug. 24, 2008, GI No.
41327735).
[0062] As used herein "Interleukin-6" ("IL-6") refers to an IL-6
gene (e.g., an endogenous IL-6 gene) in a cell, such as in a
non-transformed, or non-cancerous cell in a human.
[0063] IL-6 is also known as Interleukin 6; IFNB2 (Interferon beta
2, or interferon, beta 2); BSF-2 (B-cell stimulatory factor 2);
BSF2; CDF (CTL differentiation factor); HGF (hybridoma growth
factor); HSF. The mRNA sequence of IL-6 is at GenBank Accession No.
NM_000600.2 (FIG. 11) (record dated Jan. 4, 2009, GI No.
155369258).
[0064] As used herein, "target sequence" of a dsRNA refers to a
contiguous portion of the nucleotide sequence of an mRNA molecule
formed during the transcription of the target gene, e.g., a
deltaEGFR gene or an IL-6 gene, including mRNA that is a product of
RNA processing of a primary transcription product.
[0065] As used herein, the term "strand comprising a sequence"
refers to an oligonucleotide comprising a chain of nucleotides that
is described by the sequence referred to using the standard
nucleotide nomenclature.
[0066] As used herein, and unless otherwise indicated, the term
"complementary," when used to describe a first nucleotide sequence
in relation to a second nucleotide sequence, refers to the ability
of an oligonucleotide or polynucleotide comprising the first
nucleotide sequence to hybridize and form a duplex structure under
certain conditions with an oligonucleotide or polynucleotide
comprising the second nucleotide sequence, as will be understood by
the skilled person. Such conditions can, for example, be stringent
conditions, where stringent conditions may include: 400 mM NaCl, 40
mM PIPES pH 6.4, 1 mM EDTA, 50.degree. C. or 70.degree. C. for
12-16 hours followed by washing. Other conditions, such as
physiologically relevant conditions as may be encountered inside an
organism, can apply. The skilled person will be able to determine
the set of conditions most appropriate for a test of
complementarity of two sequences in accordance with the ultimate
application of the hybridized nucleotides.
[0067] This includes base-pairing of the oligonucleotide or
polynucleotide comprising the first nucleotide sequence to the
oligonucleotide or polynucleotide comprising the second nucleotide
sequence over the entire length of the first and second nucleotide
sequence. Such sequences can be referred to as "fully
complementary" with respect to each other herein. However, where a
first sequence is referred to as "substantially complementary" with
respect to a second sequence herein, the two sequences can be fully
complementary, or they may form one or more, but generally not more
than 4, 3 or 2 mismatched base pairs upon hybridization, while
retaining the ability to hybridize under the conditions most
relevant to their ultimate application. However, where two
oligonucleotides are designed to form, upon hybridization, one or
more single stranded overhangs, such overhangs shall not be
regarded as mismatches with regard to the determination of
complementarity. For example, a dsRNA comprising one
oligonucleotide 21 nucleotides in length and another
oligonucleotide 23 nucleotides in length, wherein the longer
oligonucleotide comprises a sequence of 21 nucleotides that is
fully complementary to the shorter oligonucleotide, may yet be
referred to as "fully complementary" for the purposes described
herein.
[0068] "Complementary" sequences, as used herein, may also include,
or be formed entirely from, non-Watson-Crick base pairs and/or base
pairs formed from non-natural and modified nucleotides, insofar as
the above requirements with respect to their ability to hybridize
are fulfilled. Such non-Watson-Crick base pairs includes, but not
limited to, G:U Wobble or Hoogstein base pairing.
[0069] The terms "complementary," "fully complementary" and
"substantially complementary" herein may be used with respect to
the base matching between the sense strand and the antisense strand
of a dsRNA, or between the antisense strand of a dsRNA and a target
sequence, as will be understood from the context of their use.
[0070] As used herein, a polynucleotide that is "substantially
complementary to at least part of" a messenger RNA (mRNA) refers to
a polynucleotide that is substantially complementary to a
contiguous portion of an mRNA of interest (e.g., encoding deltaEGFR
or IL6). For example, a polynucleotide is complementary to at least
a part of a deltaEGFR mRNA if the sequence is substantially
complementary to a non-interrupted portion of an mRNA encoding
deltaEGFR. Similarly, a polynucleotide is complementary to at least
a part of a wtEGFR mRNA if the sequence is substantially
complementary to a non-interrupted portion of an mRNA encoding
wtEGFR.
[0071] The term "double-stranded RNA" or "dsRNA," as used herein,
refers to a complex of ribonucleic acid molecules, having a duplex
structure comprising two anti-parallel and substantially
complementary, as defined above, nucleic acid strands. The two
strands forming the duplex structure may be different portions of
one larger RNA molecule, or they may be separate RNA molecules.
Where the two strands are part of one larger molecule, and
therefore are connected by an uninterrupted chain of nucleotides
between the 3'-end of one strand and the 5'end of the respective
other strand forming the duplex structure, the connecting RNA chain
is referred to as a "hairpin loop." Where the two strands are
connected covalently by means other than an uninterrupted chain of
nucleotides between the 3'-end of one strand and the 5' end of the
respective other strand forming the duplex structure, the
connecting structure is referred to as a "linker." The RNA strands
may have the same or a different number of nucleotides. The maximum
number of base pairs is the number of nucleotides in the shortest
strand of the dsRNA minus any overhangs that are present in the
duplex. In addition to the duplex structure, a dsRNA may comprise
one or more nucleotide overhangs.
[0072] As used herein, a "nucleotide overhang" refers to the
unpaired nucleotide or nucleotides that protrude from the duplex
structure of a dsRNA when a 3'-end of one strand of the dsRNA
extends beyond the 5'-end of the other strand, or vice versa.
"Blunt" or "blunt end" means that there are no unpaired nucleotides
at that end of the dsRNA, i.e., no nucleotide overhang. A "blunt
ended" dsRNA is a dsRNA that is double-stranded over its entire
length, i.e., no nucleotide overhang at either end of the
molecule.
[0073] The term "antisense strand" refers to the strand of a dsRNA
which includes a region that is substantially complementary to a
target sequence. As used herein, the term "region of
complementarity" refers to the region on the antisense strand that
is substantially complementary to a sequence, for example a target
sequence, as defined herein. Where the region of complementarity is
not fully complementary to the target sequence, the mismatches may
be in the internal or terminal regions of the molecule. Generally,
the most tolerated mismatches are in the terminal regions, e.g.,
within 6, 5, 4, 3, or 2 nucleotides of the 5' and/or 3'
terminus.
[0074] The term "sense strand," as used herein, refers to the
strand of a dsRNA that includes a region that is substantially
complementary to a region of the antisense strand.
[0075] The term "identity" is the relationship between two or more
polynucleotide sequences, as determined by comparing the sequences.
Identity also means the degree of sequence relatedness between
polynucleotide sequences, as determined by the match between
strings of such sequences. While there exist a number of methods to
measure identity between two polynucleotide sequences, the term is
well known to skilled artisans (see, e.g., Sequence Analysis in
Molecular Biology, von Heinje, G., Academic Press (1987); and
Sequence Analysis Primer, Gribskov., M. and Devereux, J., eds., M.
Stockton Press, New York (1991)). "Substantially identical," as
used herein, means there is a very high degree of homology (e.g.,
100% sequence identity) between the sense strand of the dsRNA and
the corresponding part of the target gene. However, dsRNA having
greater than 90%, or 95% sequence identity may be used in the
present invention, and thus sequence variations that might be
expected due to genetic mutation, strain polymorphism, or
evolutionary divergence can be tolerated. The dsRNA is typically
100% complementary to the target RNA, but in some embodiments, the
dsRNA may contain single or multiple base-pair random mismatches
between the RNA and the target gene.
[0076] As used herein, the term "SNALP" refers to a stable nucleic
acid-lipid particle. A SNALP represents a vesicle of lipids coating
a reduced aqueous interior comprising a nucleic acid such as an
iRNA agent or a plasmid from which an iRNA agent is transcribed.
SNALPs are described, e.g., in U.S. Patent Application Publication
Nos. 20060240093, 20070135372, and U.S. Ser. No. 61/045,228 filed
Apr. 15, 2008. These applications are hereby incorporated by
reference.
[0077] "Introducing into a cell," when referring to a dsRNA, means
facilitating uptake or absorption into the cell, as is understood
by those skilled in the art. Absorption or uptake of dsRNA can
occur through unaided diffusive or active cellular processes, or by
auxiliary agents or devices. The meaning of this term is not
limited to cells in vitro; a dsRNA may also be "introduced into a
cell," wherein the cell is part of a living organism. In such
instance, introduction into the cell will include the delivery to
the organism. For example, for in vivo delivery, dsRNA can be
injected into a tissue site or administered systemically. In vivo
delivery can also be by a beta-glucan delivery system, such as
those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S.
Publication No. 2005/0281781. U.S. Pat. Nos. 5,032,401 and
5,607,677, and U.S. Publication No. 2005/0281781 are hereby
incorporated by reference in their entirety. In vitro introduction
into a cell includes methods known in the art such as
electroporation and lipofection.
[0078] The terms "silence," "inhibit the expression of,"
"down-regulate the expression of," "suppress the expression of,"
and the like, insofar as they refer to a deltaEGFR or IL6 gene,
refer to the at least partial suppression of expression of the
deltaEGFR or IL6 gene, as manifested by a reduction of the amount
of deltaEGFR or IL6 mRNA which may be isolated or detected from a
first cell or group of cells in which the deltaEGFR or IL6 gene is
transcribed and which has or have been treated such that the
expression of the deltaEGFR or IL6 gene is inhibited, as compared
to a second cell or group of cells substantially identical to the
first cell or group of cells but which has or have not been so
treated (control cells). The degree of inhibition is usually
expressed in terms of
( mRNA in control cells ) - ( mRNA in treated cells ) ( mRNA in
control cells ) 100 % ##EQU00001##
[0079] Alternatively, the degree of inhibition may be given in
terms of a reduction of a parameter that is functionally linked to
gene expression, e.g., the amount of protein encoded by the
deltaEGFR or IL6 gene which is secreted by a cell, or the number of
cells displaying a certain phenotype, e.g., apoptosis. In
principle, gene silencing may be determined in any cell expressing
the target gene, either constitutively or by genomic engineering,
and by any appropriate assay. However, when a reference is needed
in order to determine whether a given dsRNA inhibits the expression
of a deltaEGFR gene or an IL6 gene by a certain degree and
therefore is encompassed by the instant invention, the assays
provided in the Examples below shall serve as such reference. For
example, deltaEGFR gene silencing may be determined in
U87-deltaEGFR (Nishikawa et al., PNAS 91:7727-7731, 1994) or
U87-wtEGFR (Nagana et al., Cancer Research 56:5079-5086, 1996)
cells.
[0080] In some embodiments, expression of the deltaEGFR gene or IL6
gene is suppressed by at least about 20%, 25%, 30%, 35%, 40%, 45%,
or 50% by administration of a double-stranded oligonucleotide
featured in the invention. In some embodiments, the deltaEGFR or
IL6 gene is suppressed by at least about 60%, 70%, or 80% by
administration of the double-stranded oligonucleotide featured in
the invention. In some embodiments, the deltaEGFR gene is
suppressed by at least about 85%, 90%, or 95% by administration of
the double-stranded oligonucleotide featured in the invention.
Table 4, for example, and FIGS. 7-9 indicate a range of inhibition
of expression obtained in in vitro and ex vivo assays using various
deltaEGFR dsRNA molecules at various concentrations.
[0081] As used herein in the context of deltaEGFR or IL6
expression, the terms "treat," "treatment," and the like, refer to
relief from or alleviation of pathological processes mediated by
deltaEGFR or IL6 gene expression. Insofar as they relate to any of
the other conditions recited herein below (other than pathological
processes mediated by deltaEGFR expression), the terms "treat,"
"treatment," and the like mean to relieve or alleviate at least one
symptom associated with such condition, or to slow or reverse the
progression of such condition, such as the slowing and progression
of glioma.
[0082] As used herein, the phrases "therapeutically effective
amount" and "prophylactically effective amount" refer to an amount
that provides a therapeutic benefit in the treatment, prevention,
or management of pathological processes mediated by deltaEGFR or
IL6 expression or an overt symptom of pathological processes
mediated by deltaEGFR or IL6 expression. The specific amount that
is therapeutically effective can be readily determined by an
ordinary medical practitioner, and may vary depending on factors
known in the art, such as, for example, the type of pathological
processes mediated by deltaEGFR or IL6 expression, the patient's
history and age, the stage of pathological processes mediated by
deltaEGFR or IL6 expression, and the administration of other
anti-pathological processes mediated by deltaEGFR or IL6 expression
agents.
[0083] As used herein, a "pharmaceutical composition" comprises a
pharmacologically effective amount of a dsRNA and a
pharmaceutically acceptable carrier. As used herein,
"pharmacologically effective amount," "therapeutically effective
amount" or simply "effective amount" refers to that amount of an
RNA effective to produce the intended pharmacological, therapeutic
or preventive result. For example, if a given clinical treatment is
considered effective when there is at least a 25% reduction in a
measurable parameter associated with a disease or disorder, a
therapeutically effective amount of a drug for the treatment of
that disease or disorder is the amount necessary to effect at least
a 25% reduction in that parameter.
[0084] The term "pharmaceutically acceptable carrier" refers to a
carrier for administration of a therapeutic agent. Such carriers
include, but are not limited to, saline, buffered saline, dextrose,
water, glycerol, ethanol, and combinations thereof. The term
specifically excludes cell culture medium. For drugs administered
orally, pharmaceutically acceptable carriers include, but are not
limited to pharmaceutically acceptable excipients such as inert
diluents, disintegrating agents, binding agents, lubricating
agents, sweetening agents, flavoring agents, coloring agents and
preservatives. Suitable inert diluents include sodium and calcium
carbonate, sodium and calcium phosphate, and lactose, while corn
starch and alginic acid are suitable disintegrating agents. Binding
agents may include starch and gelatin, while the lubricating agent,
if present, will generally be magnesium stearate, stearic acid or
talc. If desired, the tablets may be coated with a material such as
glyceryl monostearate or glyceryl distearate, to delay absorption
in the gastrointestinal tract.
[0085] As used herein, a "transformed cell" is a cell into which a
vector has been introduced from which a dsRNA molecule may be
expressed.
II. Double-Stranded Ribonucleic Acid (dsRNA)
[0086] In one embodiment, the invention provides double-stranded
ribonucleic acid (dsRNA) molecule for inhibiting expression of a
deltaEGFR gene in a cell or mammal, e.g., in a human having a
cancer, such as a glioma, where the dsRNA includes an antisense
strand having a region of complementarity which is complementary to
at least a part of an mRNA formed in the expression of the
deltaEGFR gene, and where the region of complementarity is less
than 30 nucleotides in length, generally 19-24 nucleotides in
length, and where said dsRNA, upon contact with a cell expressing
said deltaEGFR gene, inhibits the expression of said deltaEGFR gene
by at least 30% as assayed by, for example, a PCR or branched DNA
(bDNA)-based method, or by a protein-based method, such as by
Western blot. Expression of the deltaEGFR gene can be reduced by at
least 30% when measured by an assay as described in the Examples
below. The expression of wtEGFR may also be reduced by at least
30%, e.g., as assayed by a method described herein, and the level
of reduced expression of deltaEGFR and wtEGFR may be different. The
reduction in deltaEGFR or wtEGFR expression can also be assayed by
measuring protein levels, such as by Western blot analysis.
[0087] In one embodiment, the invention provides a double-stranded
ribonucleic acid (dsRNA) molecule for inhibiting expression of an
IL6 gene in a cell or mammal, e.g., in a human having a cancer,
such as a glioma, where the dsRNA includes an antisense strand
having a region of complementarity which is complementary to at
least a part of an mRNA formed in the expression of the IL6 gene,
and where the region of complementarity is less than 30 nucleotides
in length, generally 19-24 nucleotides in length, and where the
dsRNA, upon contact with a cell expressing the IL6 gene, inhibits
the expression of the gene by at least 30% as assayed by, for
example, a PCR or branched DNA (bDNA)-based method, or by a
protein-based method, such as by Western blot.
[0088] A dsRNA featured in the invention, e.g., a dsRNA targeting
deltaEGFR or IL6 mRNA, includes two RNA strands that are
sufficiently complementary to hybridize to form a duplex structure.
One strand of the dsRNA (the antisense strand) includes a region of
complementarity that is substantially complementary, and generally
fully complementary, to a target sequence, derived from the
sequence of an mRNA formed during the expression of the target
gene, the other strand (the sense strand) includes a region that is
complementary to the antisense strand, such that the two strands
hybridize and form a duplex structure when combined under suitable
conditions. Optionally, the region of the antisense strand that is
substantially complementary to a sequence of a deltaEGFR mRNA is
also substantially complementary to a wtEGFR mRNA.
[0089] Generally, the duplex structure of a dsRNA featured herein
is between 15 and 30, more generally between 18 and 25, yet more
generally between 19 and 24, and most generally between 19 and 21
base pairs in length. Similarly, the region of complementarity to
the target sequence is between 15 and 30, more generally between 18
and 25, yet more generally between 19 and 24, and most generally
between 19 and 21 nucleotides in length. In some embodiments, the
dsRNA is between 10 and 15 nucleotides in length, and in other
embodiments, the dsRNA is between 25 and 30 nucleotides in length.
The dsRNA featured in the invention may further include one or more
single-stranded nucleotide overhangs. The dsRNA can be synthesized
by standard methods known in the art as further discussed below,
e.g., by use of an automated DNA synthesizer, such as are
commercially available from, for example, Biosearch, Applied
Biosystems, Inc. In one embodiment, the deltaEGFR gene is a human
deltaEGFR gene, and the wtEGFR gene is a human wtEGFR gene. In
specific embodiments, the first sequence is a sense strand of the
dsRNA that includes a sense sequence from Tables 2 or 3, and the
second sequence is an antisense strand that includes an antisense
sequence from Tables 2 or 3. Alternative antisense agents that
target elsewhere in the target sequence provided in Tables 2 or 3
can readily be determined using the target sequence and the
flanking deltaEGFR sequence.
[0090] The dsRNA targeting deltaEGFR will include at least two
nucleotide sequences selected from the groups of sequences provided
in Tables 2 or 3. One of the two sequences is complementary to the
other of the two sequences, with one of the sequences being
substantially complementary to a sequence of an mRNA generated in
the expression of the deltaEGFR gene. As such, the dsRNA will
include two oligonucleotides, where one oligonucleotide is
described as the sense strand in Tables 2 or 3 and the second
oligonucleotide is described as the antisense strand in Tables 2 or
3.
[0091] In some embodiments, the dsRNA will target an IL-6 gene,
e.g., a human IL-6 gene. In certain embodiments, the first sequence
of the dsRNA is a sense strand that includes a sense sequence from
Tables 5-8, and the second sequence is an antisense strand that
includes an antisense sequence from Tables 5-8. Alternative
antisense agents that target elsewhere in the target sequence
provided in Tables 5-8 can readily be determined using the target
sequence and the flanking IL-6 sequence.
[0092] A dsRNA targeting IL-6 will include at least two nucleotide
sequences selected from the groups of sequences provided in Tables
5-8. One of the two sequences is complementary to the other of the
two sequences, with one of the sequences being substantially
complementary to a sequence of an mRNA generated in the expression
of the IL-6 gene. As such, the dsRNA will include two
oligonucleotides, where one oligonucleotide is described as the
sense strand in Tables 6-10 and the second oligonucleotide is
described as the antisense strand in Tables 6-10.
[0093] In certain embodiments, the IL-6 dsRNA does not have a sense
or antisense strand consisting of the sequences shown in Tables 6A
or 6B of WO 2007/064846. In other embodiments, the dsRNA does not
consist of the sequence of SEQ ID NO:1 of US2008/0234218, and its
complementary sequence of SEQ NO:2; the sequence of SEQ ID NO:3 of
US2008/0234218, and its complementary sequence of SEQ NO:4; or the
sequence of SEQ ID NO:5 of US2008/0234218, and its complementary
sequence of SEQ NO:6.
[0094] The skilled person is well aware that dsRNAs having a duplex
structure of between 20 and 23, but specifically 21, base pairs
have been hailed as particularly effective in inducing RNA
interference (Elbashir et al., EMBO 2001, 20:6877-6888). However,
others have found that shorter or longer dsRNAs can be effective as
well. In the embodiments described above, by virtue of the nature
of the oligonucleotide sequences provided in Tables 2 and 3, and
5-8, the dsRNAs featured in the invention can include at least one
strand of a length of minimally 21 nt. It can be reasonably
expected that shorter dsRNAs having one of the sequences of Tables
2 or 3, or 5-8 minus only a few nucleotides on one or both ends may
be similarly effective as compared to the dsRNAs described above.
Hence, dsRNAs having a partial sequence of at least 15, 16, 17, 18,
19, 20, or more contiguous nucleotides from one of the sequences of
Tables 2, 3 and 5-8, and differing in their ability to inhibit the
expression of the respective target genes, e.g., as measured by a
FACS assay as described herein below by not more than 5, 10, 15,
20, 25, or 30% inhibition from a dsRNA comprising the full
sequence, are contemplated by the invention. Further, dsRNAs that
cleave within the desired target sequence can readily be made using
the corresponding deltaEGFR or IL6 antisense sequence and a
complementary sense sequence.
[0095] In addition, the dsRNAs provided in Tables 2 and 3 identify
a site in a deltaEGFR mRNA and the wtEGFR sequence that is
susceptible to RNAi based cleavage, and the dsRNAs provided in
Tables 5-8 identify a site in an IL6 mRNA susceptible to RNAi based
cleavage As such, the present invention further provides dsRNAs
that target within the sequence targeted by one of the other agents
featured in the invention. As used herein, a second dsRNA is said
to target within the sequence of a first dsRNA if the second dsRNA
cleaves the message anywhere within the mRNA that is complementary
to the antisense strand of the first dsRNA. Such a second dsRNA
will generally consist of at least 15 contiguous nucleotides from
one of the sequences provided in Tables 2, 3 or 5-8, coupled to an
additional nucleotide sequence taken from the region contiguous to
the selected sequence in the target gene, e.g., the deltaEGFR gene,
the wtEGFR gene, or the IL6 gene. For example, the last 15
nucleotides of SEQ ID NO:2 combined with the next six nucleotides
from the target deltaEGFR gene produces a single strand agent of 21
nucleotides that is based on one of the sequences provided in
Tables 2 and 3.
[0096] The dsRNA featured in the invention can contain one or more
mismatches to the target sequence. In one embodiment, the dsRNA
contains no more than 3 mismatches. If the antisense strand of the
dsRNA contains mismatches to a target sequence, it is preferable
that the area of mismatch not be located in the center of the
region of complementarity. If the antisense strand of the dsRNA
contains mismatches to the target sequence, it is preferable that
the mismatch be restricted to 5 nucleotides from either end, for
example 5, 4, 3, 2, or 1 nucleotide from either the 5' or 3' end of
the region of complementarity. For example, for a 23 nucleotide
dsRNA strand which is complementary to a region of the deltaEGFR
gene, the dsRNA generally does not contain any mismatch within the
central 13 nucleotides. The methods described within the invention
can be used to determine whether a dsRNA containing a mismatch to a
target sequence is effective in inhibiting the expression of the
target gene, e.g., a deltaEGFR gene or an IL6 gene. Consideration
of the efficacy of dsRNAs with mismatches in inhibiting expression
of the target gene is important, especially if the particular
region of complementarity in the target gene is known to have
polymorphic sequence variation within the population.
[0097] In one embodiment, at least one end of the dsRNA has a
single-stranded nucleotide overhang of 1 to 4, generally 1 or 2
nucleotides. dsRNAs having at least one nucleotide overhang have
unexpectedly superior inhibitory properties than their blunt-ended
counterparts. Moreover, the present inventors have discovered that
the presence of only one nucleotide overhang strengthens the
interference activity of the dsRNA, without affecting its overall
stability. dsRNA having only one overhang has proven particularly
stable and effective in vivo, as well as in a variety of cells,
cell culture mediums, blood, and serum. Generally, the
single-stranded overhang is located at the 3'-terminal end of the
antisense strand or, alternatively, at the 3'-terminal end of the
sense strand. The dsRNA may also have a blunt end, generally
located at the 5'-end of the antisense strand. Such dsRNAs have
improved stability and inhibitory activity, thus allowing
administration at low dosages, i.e., less than 5 mg/kg body weight
of the recipient per day. In one embodiment, the antisense strand
of the dsRNA has a 1-10 nucleotide overhang at the 3'-end and/or
the 5' end. In another embodiment, the sense strand of the dsRNA
has a 1-10 nucleotide overhang at the 3' end and/or the 5' end. In
another embodiment, one or more of the nucleotides in the overhang
is replaced with a nucleoside thiophosphate.
[0098] In yet another embodiment, the dsRNA is chemically modified
to enhance stability. The nucleic acids featured in the invention
may be synthesized and/or modified by methods well established in
the art, such as those described in "Current protocols in nucleic
acid chemistry," Beaucage, S. L. et al. (Edrs.), John Wiley &
Sons, Inc., New York, N.Y., USA, which is hereby incorporated
herein by reference. Specific examples of dsRNA compounds useful in
this invention include dsRNAs containing modified backbones or no
natural internucleoside linkages. As defined in this specification,
dsRNAs having modified backbones include those that retain a
phosphorus atom in the backbone and those that do not have a
phosphorus atom in the backbone. For the purposes of this
specification, and as sometimes referenced in the art, modified
dsRNAs that do not have a phosphorus atom in their internucleoside
backbone can also be considered to be oligonucleosides.
[0099] Modified dsRNA backbones include, for example,
phosphorothioates, chiral phosphorothioates, phosphorodithioates,
phosphotriesters, aminoalkylphosphotriesters, methyl and other
alkyl phosphonates including 3'-alkylene phosphonates and chiral
phosphonates, phosphinates, phosphoramidates including 3'-amino
phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, and boranophosphates having normal
3'-5' linkages, 2'-5' linked analogs of these, and those) having
inverted polarity wherein the adjacent pairs of nucleoside units
are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed
salts and free acid forms are also included.
[0100] Representative U.S. patents that teach the preparation of
the above phosphorus-containing linkages include, but are not
limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111;
5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is
incorporated herein by reference
[0101] Modified dsRNA backbones that do not include a phosphorus
atom have backbones that are formed by short chain alkyl or
cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or
cycloalkyl internucleoside linkages, or ore or more short chain
heteroatomic or heterocyclic internucleoside linkages. These
include those having morpholino linkages (formed in part from the
sugar portion of a nucleoside); siloxane backbones; sulfide,
sulfoxide and sulfone backbones; formacetyl and thioformacetyl
backbones; methylene formacetyl and thioformacetyl backbones;
alkene containing backbones; sulfamate backbones; methyleneimino
and methylenehydrazino backbones; sulfonate and sulfonamide
backbones; amide backbones; and others having mixed N, O, S and
CH.sub.2 component parts.
[0102] Representative U.S. patents that teach the preparation of
the above oligonucleosides include, but are not limited to, U.S.
Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360;
5,677,437; and, 5,677,439, each of which is incorporated herein by
reference.
[0103] In other suitable dsRNA mimetics, both the sugar and the
internucleoside linkage, i.e., the backbone, of the nucleotide
units are replaced with novel groups. The base units are maintained
for hybridization with an appropriate nucleic acid target compound.
One such oligomeric compound, a dsRNA mimetic that has been shown
to have excellent hybridization properties, is referred to as a
peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of
a dsRNA is replaced with an amide containing backbone, in
particular an aminoethylglycine backbone. The nucleobases are
retained and are bound directly or indirectly to aza nitrogen atoms
of the amide portion of the backbone. Representative U.S. patents
that teach the preparation of PNA compounds include, but are not
limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262,
each of which is incorporated herein by reference. Further teaching
of PNA compounds can be found in Nielsen et al., Science, 1991,
254, 1497-1500.
[0104] Most embodiments featured in the invention include dsRNAs
with phosphorothioate backbones and oligonucleosides with
heteroatom backbones, and in particular --CH.sub.2--NH--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--O--CH.sub.2-- [known as a methylene
(methylimino) or MMI backbone],
--CH.sub.2--O--N(CH.sub.3)--CH.sub.2--,
--CH.sub.2--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2-- and
--N(CH.sub.3)--CH.sub.2--CH.sub.2-- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2--] of
the above-referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above-referenced U.S. Pat. No. 5,602,240. In some
embodiments, the dsRNAs featured herein have morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
[0105] Modified dsRNAs may also contain one or more substituted
sugar moieties. The dsRNAs featured herein can have one of the
following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or
N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the
alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl. Exemplary suitable modifications include
O[(CH.sub.2).sub.nO].sub.m--CH.sub.3, O(CH.sub.2).sub.nOCH.sub.3,
O(CH.sub.2).sub.nNH.sub.2, O(CH.sub.2).sub.nCH.sub.3,
O(CH.sub.2).sub.nONH.sub.2, and
O(CH.sub.2).sub.n[(CH.sub.2).sub.nCH.sub.3)].sub.2, where n and m
are from 1 to about 10. In other embodiments, dsRNAs include one of
the following at the 2' position: C.sub.1 to C.sub.10 lower alkyl,
substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl,
SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3, SOCH.sub.3,
SO.sub.2CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3, NH.sub.2,
heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of a dsRNA, or a group for improving the
pharmacodynamic properties of a dsRNA, and other substituents
having similar properties. In some embodiments, the modification
includes a 2'-methoxyethoxy (2'-O--CH.sub.2CH.sub.2OCH.sub.3, also
known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv.
Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another
exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a
O(CH.sub.2).sub.2ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE,
as described in examples hereinbelow, and
2'-dimethylaminoethoxyethoxy (also known in the art as
2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2--O--CH.sub.2--N(CH.sub.2).sub.2, also described in
examples hereinbelow.
[0106] Other modifications include 2'-methoxy (2'-OCH.sub.3),
2'-aminopropoxy (2'-OCH.sub.2CH.sub.2CH.sub.2NH.sub.2) and
2'-fluoro (2'-F). Similar modifications may also be made at other
positions on the dsRNA, particularly the 3' position of the sugar
on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5'
position of 5' terminal nucleotide. DsRNAs may also have sugar
mimetics such as cyclobutyl moieties in place of the pentofuranosyl
sugar. Representative U.S. patents that teach the preparation of
such modified sugar structures include, but are not limited to,
U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;
5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;
5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which
are commonly owned with the instant application, and each of which
is incorporated herein by reference in its entirety.
[0107] DsRNAs may also include nucleobase (often referred to in the
art simply as "base") modifications or substitutions. As used
herein, "unmodified" or "natural" nucleobases include the purine
bases adenine (A) and guanine (G), and the pyrimidine bases thymine
(T), cytosine (C) and uracil (U). Modified nucleobases include
other synthetic and natural nucleobases such as 5-methylcytosine
(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,
5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo
uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl
anal other 8-substituted adenines and guanines, 5-halo,
particularly 5-bromo, 5-trifluoromethyl and other 5-substituted
uracils and cytosines, 7-methylguanine and 7-methyladenine,
8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine
and 3-deazaguanine and 3-deazaadenine. Further nucleobases include
those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The
Concise Encyclopedia Of Polymer Science And Engineering, pages
858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these
disclosed by Englisch et al., Angewandte Chemie, International
Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y S.,
Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,
S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these
nucleobases are particularly useful for increasing the binding
affinity of the oligomeric compounds featured in the invention.
These include 5-substituted pyrimidines, 6-azapyrimidines and N-2,
N-6 and 0-6 substituted purines, including 2-aminopropyladenine,
5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
substitutions have been shown to increase nucleic acid duplex
stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and
Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, Boca
Raton, 1993, pp. 276-278) and are exemplary base substitutions,
even more particularly when combined with 2'-O-methoxyethyl sugar
modifications.
[0108] Representative U.S. patents that teach the preparation of
certain of the above noted modified nucleobases as well as other
modified nucleobases include, but are not limited to, the above
noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205;
5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;
5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;
5,594,121, 5,596,091; 5,614,617; and 5,681,941, each of which is
herein incorporated by reference, and U.S. Pat. No. 5,750,692, also
incorporated herein by reference.
[0109] Another modification of the dsRNAs featured in the invention
involves chemically linking to the dsRNA one or more moieties or
conjugates which enhance the activity, cellular distribution or
cellular uptake of the dsRNA. Such moieties include but are not
limited to lipid moieties such as a cholesterol moiety (Letsinger
et al., Proc. Natl. Acid. Sci. USA, 1989, 86: 6553-6556), cholic
acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060),
a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., Ann. N.Y.
Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem.
Let., 1993, 3:2765-2770), a thiocholesterol (Oberhauser et al.,
Nucl. Acids Res., 1992, 20:533-538), an aliphatic chain, e.g.,
dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J,
1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330;
Svinarchuk et al., Biochimie, 1993, 75:49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium
1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18:3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995,
14:969-973), or adamantane acetic acid (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651-3654), a palmityl moiety (Mishra
et al., Biochim Biophys. Acta, 1995, 1264:229-237), or an
octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke
et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).
[0110] Representative U.S. patents that teach the preparation of
such dsRNA conjugates include, but are not limited to, U.S. Pat.
Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;
5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124;
5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718;
5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;
4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830;
5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;
5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098;
5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667;
5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371;
5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941, each of
which is herein incorporated by reference.
[0111] It is not necessary for all positions in a given compound to
be uniformly modified, and in fact more than one of the
aforementioned modifications may be incorporated in a single
compound or even at a single nucleoside within a dsRNA. The present
invention also includes dsRNA compounds which are chimeric
compounds. "Chimeric" dsRNA compounds or "chimeras," in the context
of this invention, are dsRNA compounds, particularly dsRNAs, which
contain two or more chemically distinct regions, each made up of at
least one monomer unit, i.e., a nucleotide in the case of a dsRNA
compound. These dsRNAs typically contain at least one region
wherein the dsRNA is modified so as to confer upon the dsRNA
increased resistance to nuclease degradation, increased cellular
uptake, and/or increased binding affinity for the target nucleic
acid. An additional region of the dsRNA may serve as a substrate
for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way
of example, RNase H is a cellular endonuclease which cleaves the
RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore,
results in cleavage of the RNA target, thereby greatly enhancing
the efficiency of dsRNA inhibition of gene expression.
Consequently, comparable results can often be obtained with shorter
dsRNAs when chimeric dsRNAs are used, compared to phosphorothioate
deoxydsRNAs hybridizing to the same target region. Cleavage of the
RNA target can be routinely detected by gel electrophoresis and, if
necessary, associated nucleic acid hybridization techniques known
in the art.
[0112] In certain instances, the dsRNA may be modified by a
non-ligand group. A number of non-ligand molecules have been
conjugated to dsRNAs in order to enhance the activity, cellular
distribution or cellular uptake of the dsRNA, and procedures for
performing such conjugations are available in the scientific
literature. Such non-ligand moieties have included lipid moieties,
such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.
Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol
(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan
et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol
(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic
chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et
al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990,
259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a
phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res.,
1990, 18:3777), a polyamine or a polyethylene glycol chain
(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or
adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,
36:3651), a palmityl moiety (Mishra et al., Biochim Biophys. Acta,
1995, 1264:229), or an octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277:923). Representative United States
patents that teach the preparation of such dsRNA conjugates have
been listed above. Typical conjugation protocols involve the
synthesis of dsRNAs bearing an aminolinker at one or more positions
of the sequence. The amino group is then reacted with the molecule
being conjugated using appropriate coupling or activating reagents.
The conjugation reaction may be performed either with the dsRNA
still bound to the solid support or following cleavage of the dsRNA
in solution phase. Purification of the dsRNA conjugate by HPLC
typically affords the pure conjugate.
[0113] Vector Encoded dsRNAs
[0114] In another aspect, dsRNA molecules featured in the
invention, e.g., deltaEGFR and IL6 dsRNAs, are expressed from
transcription units inserted into DNA or RNA vectors (see, e.g.,
Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al.,
International PCT Publication No. WO 00/22113, Conrad,
International PCT Publication No. WO 00/22114, and Conrad, U.S.
Pat. No. 6,054,299). These transgenes can be introduced as a linear
construct, a circular plasmid, or a viral vector, which can be
incorporated and inherited as a transgene integrated into the host
genome. The transgene can also be constructed to permit it to be
inherited as an extrachromosomal plasmid (Gassmann, et al., Proc.
Natl. Acad. Sci. USA (1995) 92:1292).
[0115] The individual strands of a dsRNA can be transcribed by
promoters on two separate expression vectors and co-transfected
into a target cell. Alternatively each individual strand of the
dsRNA can be transcribed by promoters both of which are located on
the same expression plasmid. In one embodiment, a dsRNA is
expressed as an inverted repeat joined by a linker polynucleotide
sequence such that the dsRNA has a stem and loop structure.
[0116] The recombinant dsRNA expression vectors are generally DNA
plasmids or viral vectors. dsRNA expressing viral vectors can be
constructed based on, but not limited to, adeno-associated virus
(for a review, see Muzyczka, et al., Curr. Topics Micro. Immunol.
(1992) 158:97-129)); adenovirus (see, for example, Berkner, et al.,
BioTechniques (1998) 6:616), Rosenfeld et al. (1991, Science
252:431-434), and Rosenfeld et al. (1992), Cell 68:143-155)); or
alphavirus as well as others known in the art. Retroviruses have
been used to introduce a variety of genes into many different cell
types, including epithelial cells, in vitro and/or in vivo (see,
e.g., Eglitis, et al., Science (1985) 230:1395-1398; Danos and
Mulligan, Proc. Natl. Acad. Sci. USA (1998) 85:6460-6464; Wilson et
al., 1988, Proc. NatI. Acad. Sci. USA 85:3014-3018; Armentano et
al., 1990, Proc. Natl. Acad. Sci. USA 87:61416145; Huber et al.,
1991, Proc. NatI. Acad. Sci. USA 88:8039-8043; Ferry et al., 1991,
Proc. NatI. Acad. Sci. USA 88:8377-8381; Chowdhury et al., 1991,
Science 254:1802-1805; van Beusechem. et al., 1992, Proc. Nad.
Acad. Sci. USA 89:7640-19; Kay et al., 1992, Human Gene Therapy
3:641-647; Dai et al., 1992, Proc. Natl. Acad. Sci. USA
89:10892-10895; Hwu et al., 1993, J. Immunol. 150:4104-4115; U.S.
Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO
89/07136; PCT Application WO 89/02468; PCT Application WO 89/05345;
and PCT Application WO 92/07573). Recombinant retroviral vectors
capable of transducing and expressing genes inserted into the
genome of a cell can be produced by transfecting the recombinant
retroviral genome into suitable packaging cell lines such as PA317
and Psi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone
et al., 1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant
adenoviral vectors can be used to infect a wide variety of cells
and tissues in susceptible hosts (e.g., rat, hamster, dog, and
chimpanzee) (Hsu et al., 1992, J. Infectious Disease, 166:769), and
also have the advantage of not requiring mitotically active cells
for infection.
[0117] Any viral vector capable of accepting the coding sequences
for the dsRNA molecule(s) to be expressed can be used, for example
vectors derived from adenovirus (AV); adeno-associated virus (AAV);
retroviruses (e.g, lentiviruses (LV), Rhabdoviruses, murine
leukemia virus); herpes virus, and the like. The tropism of viral
vectors can be modified by pseudotyping the vectors with envelope
proteins or other surface antigens from other viruses, or by
substituting different viral capsid proteins, as appropriate.
[0118] For example, lentiviral vectors featured in the invention
can be pseudotyped with surface proteins from vesicular stomatitis
virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors
featured in the invention can be made to target different cells by
engineering the vectors to express different capsid protein
serotypes. For example, an AAV vector expressing a serotype 2
capsid on a serotype 2 genome is called AAV 2/2. This serotype 2
capsid gene in the AAV 2/2 vector can be replaced by a serotype 5
capsid gene to produce an AAV 2/5 vector. Techniques for
constructing AAV vectors which express different capsid protein
serotypes are within the skill in the art; see, e.g., Rabinowitz J
E et al. (2002), J Virol 76:791-801, the entire disclosure of which
is herein incorporated by reference.
[0119] Selection of recombinant viral vectors suitable for use in
the invention, methods for inserting nucleic acid sequences for
expressing the dsRNA into the vector, and methods of delivering the
viral vector to the cells of interest are within the skill in the
art. See, for example, Domburg R (1995), Gene Therap. 2: 301-310;
Eglitis M A (1988), Biotechniques 6: 608-614; Miller A D (1990),
Hum Gene Therap. 1: 5-14; Anderson W F (1998), Nature 392: 25-30;
and Rubinson D A et al., Nat. Genet. 33: 401-406, the entire
disclosures of which are herein incorporated by reference.
[0120] Viral vectors can be derived from AV and AAV. In one
embodiment, the dsRNA featured in the invention is expressed as two
separate, complementary single-stranded RNA molecules from a
recombinant AAV vector having, for example, either the U6 or H1 RNA
promoters, or the cytomegalovirus (CMV) promoter.
[0121] A suitable AV vector for expressing the dsRNA featured in
the invention, a method for constructing the recombinant AV vector,
and a method for delivering the vector into target cells, are
described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.
[0122] Suitable AAV vectors for expressing the dsRNA featured in
the invention, methods for constructing the recombinant AV vector,
and methods for delivering the vectors into target cells are
described in Samulski R et al. (1987), J. Virol. 61: 3096-3101;
Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al.
(1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S. Pat.
No. 5,139,941; International Patent Application No. WO 94/13788;
and International Patent Application No. WO 93/24641, the entire
disclosures of which are herein incorporated by reference.
[0123] The promoter driving dsRNA expression in either a DNA
plasmid or viral vector featured in the invention may be a
eukaryotic RNA polymerase I (e.g., ribosomal RNA promoter), RNA
polymerase II (e.g., CMV early promoter or actin promoter or U1
snRNA promoter) or generally RNA polymerase III promoter (e.g., U6
snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example
the T7 promoter, provided the expression plasmid also encodes T7
RNA polymerase required for transcription from a T7 promoter. The
promoter can also direct transgene expression to the pancreas (see,
e.g., the insulin regulatory sequence for pancreas (Bucchini et
al., 1986, Proc. Natl. Acad. Sci. USA 83:2511-2515)).
[0124] In addition, expression of the transgene can be precisely
regulated, for example, by using an inducible regulatory sequence
and expression systems such as a regulatory sequence that is
sensitive to certain physiological regulators, e.g., circulating
glucose levels, or hormones (Docherty et al., 1994, FASEB J.
8:20-24). Such inducible expression systems, suitable for the
control of transgene expression in cells or in mammals include
regulation by ecdysone, by estrogen, progesterone, tetracycline,
chemical inducers of dimerization, and
isopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in
the art would be able to choose the appropriate regulatory/promoter
sequence based on the intended use of the dsRNA transgene.
[0125] Generally, recombinant vectors capable of expressing dsRNA
molecules are delivered as described below, and persist in target
cells. Alternatively, viral vectors can be used that provide for
transient expression of dsRNA molecules. Such vectors can be
repeatedly administered as necessary. Once expressed, the dsRNAs
bind to target RNA and modulate its function or expression.
Delivery of dsRNA expressing vectors can be systemic, such as by
intravenous or intramuscular administration, by administration to
target cells ex-planted from the patient followed by reintroduction
into the patient, or by any other means that allows for
introduction into a desired target cell.
[0126] dsRNA expression DNA plasmids are typically transfected into
target cells as a complex with cationic lipid carriers (e.g.,
Oligofectamine) or non-cationic lipid-based carriers (e.g.,
Transit-TKO.TM.). Multiple lipid transfections for dsRNA-mediated
knockdowns targeting different regions of a single target gene or
multiple target genes over a period of a week or more are also
contemplated by the invention. Successful introduction of vectors
into host cells can be monitored using various known methods. For
example, transient transfection can be signaled with a reporter,
such as a fluorescent marker, such as Green Fluorescent Protein
(GFP). Stable transfection of cells ex vivo can be ensured using
markers that provide the transfected cell with resistance to
specific environmental factors (e.g., antibiotics and drugs), such
as hygromycin B resistance.
[0127] The deltaEGFR- and IL6-specific dsRNA molecules can also be
inserted into vectors and used as gene therapy vectors for human
patients. Gene therapy vectors can be delivered to a subject by,
for example, intravenous injection, local administration (see U.S.
Pat. No. 5,328,470) or by stereotactic injection (see e.g., Chen et
al. (1994) Proc. Natl. Acad. Sci. USA 91:3054-3057). The
pharmaceutical preparation of the gene therapy vector can include
the gene therapy vector in an acceptable diluent, or can include a
slow release matrix in which the gene delivery vehicle is imbedded.
Alternatively, where the complete gene delivery vector can be
produced intact from recombinant cells, e.g., retroviral vectors,
the pharmaceutical preparation can include one or more cells which
produce the gene delivery system.
III. Pharmaceutical Compositions Containing dsRNA
[0128] In one embodiment, the invention provides pharmaceutical
compositions containing a dsRNA, as described herein, and a
pharmaceutically acceptable carrier. The pharmaceutical composition
containing the dsRNA is useful for treating a disease or disorder
associated with the expression or activity of the deltaEGFR gene
and/or the IL6 gene, such as pathological processes mediated by
deltaEGFR or IL6 expression. Such pharmaceutical compositions are
formulated based on the mode of delivery. One example is a
composition formulated for direct delivery into the brain
parenchyma, e.g., by infusion into the brain, such as by continuous
pump infusion. Another example is a composition formulated for
intraventricular or intrathecal delivery into the cerebrospinal
fluid, e.g., by bolus or continuous pump infusion. Another example
is a compositions formulated for systemic administration via
parenteral delivery, e.g., by intravenous (IV) delivery.
[0129] The pharmaceutical compositions featured herein are
administered in dosages sufficient to inhibit expression of the
target gene, e.g, the deltaEGFR or IL6 gene. In general, a suitable
dose of dsRNA will be in the range of 0.01 to 200.0 milligrams per
kilogram body weight of the recipient per day, generally in the
range of 0.02 to 50 mg per kilogram body weight per day. For
example, the dsRNA can be administered at 0.01 mg/kg, 0.05 mg/kg,
0.1 mg/kg, 0.5 mg/kg, 1 mg/kg, 2 mg/kg, 3 mg/kg, 10 mg/kg, 20
mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kg per single dose. The
pharmaceutical composition may be administered once daily, or the
dsRNA may be administered as two, three, or more sub-doses at
appropriate intervals throughout the day or even using continuous
infusion or delivery through a controlled release formulation. In
that case, the dsRNA contained in each sub-dose must be
correspondingly smaller in order to achieve the total daily dosage.
The dosage unit can also be compounded for delivery over several
days, e.g., using a conventional sustained release formulation
which provides sustained release of the dsRNA over a several day
period. Sustained release formulations are well known in the art
and are particularly useful for delivery of agents at a particular
site, such as could be used with the agents featured in the
invention. In this embodiment, the dosage unit contains a
corresponding multiple of the daily dose.
[0130] The effect of a single dose of dsRNA on target RNA levels,
e.g., deltaEGFR levels (or both deltaEGFR and wtEGFR levels) or IL6
levels, is long lasting, such that subsequent doses are
administered at not more than 3, 4, or 5 day intervals, or at not
more than 1, 2, 3, or 4 week intervals.
[0131] The present invention includes pharmaceutical compositions
that can be delivered by injection directly into the brain. The
injection can be by stereotactic injection into the brain tumor
directly, or into a particular region of the brain (e.g., into
white matter, such as the corona radiata, or the substantia nigra,
cortex, hippocampus, striatum, or globus pallidus), or the dsRNA
can be delivered into multiple regions of the central nervous
system (e.g., into multiple regions of the brain, and/or into the
spinal cord). The dsRNA can also be delivered into diffuse regions
of the brain (e.g., diffuse delivery to the cortex of the
brain).
[0132] In one embodiment, a dsRNA targeting deltaEGFR or IL-6 can
be delivered by way of a cannula or other delivery device having
one end implanted in a tissue, e.g., the brain, e.g., the white
matter, such as the corona radiata, or the substantia nigra,
cortex, hippocampus, striatum, corpus callosum or globus pallidus
of the brain. In one embodiment, the cannula or other delivery
device has one end implanted into a tumor in the brain. The cannula
can be connected to a reservoir of the dsRNA composition. The flow
or delivery can be mediated by a pump. In one embodiment, a pump
and reservoir are implanted in an area distant from the tissue,
e.g., in the abdomen, and delivery is effected by a conduit leading
from the pump or reservoir to the site of release. Infusion of the
dsRNA composition into the brain can be over several hours or for
several days, e.g., for 1, 2, 3, 5, or 7 days or more. Devices for
delivery to the brain are described, for example, in U.S. Pat. Nos.
6,093,180, and 5,814,014. In another embodiment, the pump is
externalized (not implanted). Infusion of the dsRNA composition
into the brain can be over several hours or for several days up to
approximately 7 days, e.g., for 1, 2, 3, 5, or 7 days.
[0133] The skilled artisan will appreciate that certain factors may
influence the dosage and timing required to effectively treat a
subject, including but not limited to the severity of the disease
or disorder, previous treatments, the general health and/or age of
the subject, and other diseases present. Moreover, treatment of a
subject with a therapeutically effective amount of a composition
can include a single treatment or a series of treatments. Estimates
of effective dosages and in vivo half-lives for the individual
dsRNAs encompassed by the invention can be made using conventional
methodologies or on the basis of in vivo testing using an
appropriate animal model, as described elsewhere herein.
[0134] Advances in mouse genetics have generated a number of mouse
models for the study of various human diseases, such as
pathological processes mediated by deltaEGFR or IL6 expression.
Such models are used for in vivo testing of dsRNA, as well as for
determining a therapeutically effective dose.
[0135] The present invention also includes pharmaceutical
compositions and formulations which include the dsRNA compounds
featured in the invention. The pharmaceutical compositions may be
administered in a number of ways depending upon whether local or
systemic treatment is desired and upon the area to be treated.
Administration may be topical (e.g., by transdermal patch),
pulmonary, e.g., by inhalation or insufflation of powders or
aerosols, including by nebulizer; intratracheal, intranasal,
epidermal and transdermal, oral or parenteral. Parenteral
administration includes intravenous, intraarterial, subcutaneous,
intraperitoneal or intramuscular injection or infusion; subdermal,
e.g., via an implanted device; or intracranial, e.g., by
intraparenchymal, intrathecal or intraventricular,
administration.
[0136] Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful. Suitable
topical formulations include those in which the dsRNAs featured in
the invention are in admixture with a topical delivery agent such
as lipids, liposomes, fatty acids, fatty acid esters, steroids,
chelating agents and surfactants. Suitable lipids and liposomes
include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine,
dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl
choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and
cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and
dioleoylphosphatidyl ethanolamine DOTMA). DsRNAs featured in the
invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, dsRNAs may be complexed to lipids, in particular to
cationic lipids. Suitable fatty acids and esters include but are
not limited to arachidonic acid, oleic acid, eicosanoic acid,
lauric acid, caprylic acid, capric acid, myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,
1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or
a C.sub.1-10 alkyl ester (e.g., isopropylmyristate IPM),
monoglyceride, diglyceride or pharmaceutically acceptable salt
thereof. Topical formulations are described in detail in U.S. Pat.
No. 6,747,014, which is incorporated herein by reference in its
entirety.
[0137] In one embodiment, a deltaEGFR or an IL-6 dsRNA featured in
the invention is fully encapsulated in the lipid formulation (e.g.,
to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid
particle). As used herein, the term "SNALP" refers to a stable
nucleic acid-lipid particle, including SPLP. As used herein, the
term "SPLP" refers to a nucleic acid-lipid particle comprising
plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs
typically contain a cationic lipid, a non-cationic lipid, and a
lipid that prevents aggregation of the particle (e.g., a PEG-lipid
conjugate). SNALPs and SPLPs are extremely useful for systemic
applications, as they exhibit extended circulation lifetimes
following intravenous (i.v.) injection and accumulate at distal
sites (e.g., sites physically separated from the administration
site). SPLPs include "pSPLP," which include an encapsulated
condensing agent-nucleic acid complex as set forth in PCT
Publication No. WO 00/03683. The particles typically have a mean
diameter of about 50 nm to about 150 nm, more typically about 60 nm
to about 130 nm, more typically about 70 nm to about 110 nm, most
typically about 70 to about 90 nm, and are substantially nontoxic.
In addition, the nucleic acids when present in the nucleic
acid-lipid particles are resistant in aqueous solution to
degradation with a nuclease. Nucleic acid-lipid particles and their
method of preparation are disclosed in, e.g., U.S. Pat. Nos.
5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; and PCT
Publication No. WO 96/40964.
[0138] In one embodiment, the lipid to drug ratio (mass/mass ratio)
(e.g., lipid to dsRNA ratio) will be in the range of from about 1:1
to about 50:1, from about 1:1 to about 25:1, from about 3:1 to
about 15:1, from about 4:1 to about 10:1, from about 5:1 to about
9:1, or about 6:1 to about 9:1.
[0139] The cationic lipid may be, for example,
N,N-dioleyl-N,N-dimethylammonium chloride (DODAC),
N,N-distearyl-N,N-dimethylammonium bromide (DDAB),
N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride
(DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium
chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),
1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),
1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),
1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),
1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),
1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),
1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),
1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),
1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),
1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt
(DLin-TMA.C1), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride
salt (DLin-TAP.C1), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane
(DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),
3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),
1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane
(DLin-EG-DMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane
(DLin-K-DMA), or a mixture thereof. The cationic lipid may comprise
from about 20 mol % to about 50 mol % or about 40 mol % of the
total lipid present in the particle.
[0140] The non-cationic lipid may be an anionic lipid or a neutral
lipid including, but not limited to, distearoylphosphatidylcholine
(DSPC), dioleoylphosphatidylcholine (DOPC),
dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol
(DOPG), dipalmitoylphosphatidylglycerol (DPPG),
dioleoyl-phosphatidylethanolamine (DOPE),
palmitoyloleoylphosphatidylcholine (POPC),
palmitoyloleoyl-phosphatidylethanolamine (POPE),
dioleoyl-phosphatidylethanolamine
4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal),
dipalmitoyl phosphatidyl ethanolamine (DPPE),
dimyristoylphosphoethanolamine (DMPE),
distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,
16-O-dimethyl PE, 18-1-trans PE,
1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or
a mixture thereof. The non-cationic lipid may be from about 5 mol %
to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol
is included, of the total lipid present in the particle.
[0141] The conjugated lipid that inhibits aggregation of particles
may be, for example, a polyethyleneglycol (PEG)-lipid including,
without limitation, a PEG-diacylglycerol (DAG), a
PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide
(Cer), or a mixture thereof. The PEG-DAA conjugate may be, for
example, a PEG-dilauryloxypropyl (Ci.sub.2), a
PEG-dimyristyloxypropyl (Ci.sub.4), a PEG-dipalmityloxypropyl
(Ci.sub.6), or a PEG-distearyloxypropyl (Ci.sub.8). The conjugated
lipid that prevents aggregation of particles may be from 0 mol % to
about 20 mol % or about 2 mol % of the total lipid present in the
particle.
[0142] In some embodiments, the nucleic acid-lipid particle further
includes cholesterol at, e.g., about 10 mol % to about 60 mol % or
about 48 mol % of the total lipid present in the particle.
[0143] In one embodiment, the lipidoid ND98.4HCl (MW 1487) (Formula
1), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar
Lipids) can be used to prepare lipid-siRNA nanoparticles (i.e.,
LNP01 particles). Stock solutions of each in ethanol can be
prepared as follows: ND98, 133 mg/mL; Cholesterol, 25 mg/mL,
PEG-Ceramide C16, 100 mg/mL. The ND98, Cholesterol, and
PEG-Ceramide C16 stock solutions can then be combined in a, e.g.,
42:48:10 molar ratio. The combined lipid solution can be mixed with
aqueous siRNA (e.g., in sodium acetate pH 5) such that the final
ethanol concentration is about 35-45% and the final sodium acetate
concentration is about 100-300 mM. Lipid-siRNA nanoparticles
typically form spontaneously upon mixing. Depending on the desired
particle size distribution, the resultant nanoparticle mixture can
be extruded through a polycarbonate membrane (e.g., 100 nm cut-off)
using, for example, a thermobarrel extruder, such as Lipex Extruder
(Northern Lipids, Inc). In some cases, the extrusion step can be
omitted. Ethanol removal and simultaneous buffer exchange can be
accomplished by, for example, dialysis or tangential flow
filtration. Buffer can be exchanged with, for example, phosphate
buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH
7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.
##STR00001##
[0144] LNP01 formulations are described, e.g., in International
Application Publication No. WO 2008/042973, which is hereby
incorporated by reference.
[0145] Formulations prepared by either the standard or
extrusion-free method can be characterized in similar manners. For
example, formulations are typically characterized by visual
inspection. They should be whitish translucent solutions free from
aggregates or sediment. Particle size and particle size
distribution of lipid-nanoparticles can be measured by light
scattering using, for example, a Malvern Zetasizer Nano ZS
(Malvern, USA). Particles should be about 20-300 nm, such as 40-100
nm in size. The particle size distribution should be unimodal. The
total siRNA concentration in the formulation, as well as the
entrapped fraction, is estimated using a dye exclusion assay. A
sample of the formulated siRNA can be incubated with an RNA-binding
dye, such as Ribogreen (Molecular Probes) in the presence or
absence of a formulation disrupting surfactant, e.g., 0.5%
Triton-X100. The total siRNA in the formulation can be determined
by the signal from the sample containing the surfactant, relative
to a standard curve. The entrapped fraction is determined by
subtracting the "free" siRNA content (as measured by the signal in
the absence of surfactant) from the total siRNA content. Percent
entrapped siRNA is typically >85%. For SNALP formulation, the
particle size is at least 30 nm, at least 40 nm, at least 50 nm, at
least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at
least 100 nm, at least 110 nm, and at least 120 nm. The suitable
range is typically about at least 50 nm to about at least 110 nm,
about at least 60 nm to about at least 100 nm, or about at least 80
nm to about at least 90 nm.
[0146] Compositions and formulations for oral administration
include powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. In some embodiments, oral formulations are those
in which dsRNAs featured in the invention are administered in
conjunction with one or more penetration enhancers surfactants and
chelators. Suitable surfactants include fatty acids and/or esters
or salts thereof, bile acids and/or salts thereof. Suitable bile
acids/salts include chenodeoxycholic acid (CDCA) and
ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic
acid, deoxycholic acid, glucholic acid, glycholic acid,
glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid,
sodium tauro-24,25-dihydro-fusidate and sodium
glycodihydrofusidate. Suitable fatty acids include arachidonic
acid, undecanoic acid, oleic acid, lauric acid, caprylic acid,
capric acid, myristic acid, palmitic acid, stearic acid, linoleic
acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,
glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an
acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or
a pharmaceutically acceptable salt thereof (e.g., sodium). In some
embodiments, combinations of penetration enhancers are used, for
example, fatty acids/salts in combination with bile acids/salts.
One exemplary combination is the sodium salt of lauric acid, capric
acid and UDCA. Further penetration enhancers include
polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether.
DsRNAs featured in the invention may be delivered orally, in
granular form including sprayed dried particles, or complexed to
form micro or nanoparticles. DsRNA complexing agents include
poly-amino acids; polyimines; polyacrylates; polyalkylacrylates,
polyoxethanes, polyalkylcyanoacrylates; cationized gelatins,
albumins, starches, acrylates, polyethyleneglycols (PEG) and
starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines,
pollulans, celluloses and starches. Suitable complexing agents
include chitosan, N-trimethylchitosan, poly-L-lysine,
polyhistidine, polyornithine, polyspermines, protamine,
polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE),
polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate),
poly(ethylcyanoacrylate), poly(butylcyanoacrylate),
poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate),
DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide,
DEAE-albumin and DEAE-dextran, polymethylacrylate,
polyhexylacrylate, poly(D,L-lactic acid),
poly(DL-lactic-co-glycolic acid (PLGA), alginate, and
polyethyleneglycol (PEG). Oral formulations for dsRNAs and their
preparation are described in detail in U.S. Pat. No. 6,887,906, US
Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which
is incorporated herein by reference in their entirety.
[0147] Compositions and formulations for parenteral,
intraparenchymal (into the brain), intrathecal, intraventricular or
intrahepatic administration may include sterile aqueous solutions
which may also contain buffers, diluents and other suitable
additives such as, but not limited to, penetration enhancers,
carrier compounds and other pharmaceutically acceptable carriers or
excipients.
[0148] Pharmaceutical compositions featured in the invention
include, but are not limited to, solutions, emulsions, and
liposome-containing formulations. These compositions may be
generated from a variety of components that include, but are not
limited to, preformed liquids, self-emulsifying solids and
self-emulsifying semisolids. Particularly preferred are
formulations that target the liver when treating hepatic disorders
such as hepatic carcinoma.
[0149] The pharmaceutical formulations, which may conveniently be
presented in unit dosage form, may be prepared according to
conventional techniques well known in the pharmaceutical industry.
Such techniques include the step of bringing into association the
active ingredients with the pharmaceutical carrier(s) or
excipient(s). In general, the formulations are prepared by
uniformly and intimately bringing into association the active
ingredients with liquid carriers or finely divided solid carriers
or both, and then, if necessary, shaping the product.
[0150] The compositions may be formulated into any of many possible
dosage forms such as, but not limited to, tablets, capsules, gel
capsules, liquid syrups, soft gels, suppositories, and enemas. The
compositions may also be formulated as suspensions in aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain
substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
[0151] Emulsions
[0152] The compositions may be prepared and formulated as
emulsions. Emulsions are typically heterogenous systems of one
liquid dispersed in another in the form of droplets usually
exceeding 0.1.mu.m in diameter (Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;
Higuchi et al., in Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often
biphasic systems comprising two immiscible liquid phases intimately
mixed and dispersed with each other. In general, emulsions may be
of either the water-in-oil (w/o) or the oil-in-water (o/w) variety.
When an aqueous phase is finely divided into and dispersed as
minute droplets into a bulk oily phase, the resulting composition
is called a water-in-oil (w/o) emulsion. Alternatively, when an
oily phase is finely divided into and dispersed as minute droplets
into a bulk aqueous phase, the resulting composition is called an
oil-in-water (o/w) emulsion. Emulsions may contain additional
components in addition to the dispersed phases, and the active drug
which may be present as a solution in either the aqueous phase,
oily phase or itself as a separate phase. Pharmaceutical excipients
such as emulsifiers, stabilizers, dyes, and anti-oxidants may also
be present in emulsions as needed. Pharmaceutical emulsions may
also be multiple emulsions that are comprised of more than two
phases such as, for example, in the case of oil-in-water-in-oil
(o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex
formulations often provide certain advantages that simple binary
emulsions do not. Multiple emulsions in which individual oil
droplets of an o/w emulsion enclose small water droplets constitute
a w/o/w emulsion. Likewise a system of oil droplets enclosed in
globules of water stabilized in an oily continuous phase provides
an o/w/o emulsion.
[0153] Emulsions are characterized by little or no thermodynamic
stability. Often, the dispersed or discontinuous phase of the
emulsion is well dispersed into the external or continuous phase
and maintained in this form through the means of emulsifiers or the
viscosity of the formulation. Either of the phases of the emulsion
may be a semisolid or a solid, as is the case of emulsion-style
ointment bases and creams. Other means of stabilizing emulsions
entail the use of emulsifiers that may be incorporated into either
phase of the emulsion. Emulsifiers may broadly be classified into
four categories: synthetic surfactants, naturally occurring
emulsifiers, absorption bases, and finely dispersed solids (Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
[0154] Synthetic surfactants, also known as surface active agents,
have found wide applicability in the formulation of emulsions and
have been reviewed in the literature (Rieger, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).
Surfactants are typically amphiphilic and comprise a hydrophilic
and a hydrophobic portion. The ratio of the hydrophilic to the
hydrophobic nature of the surfactant has been termed the
hydrophile/lipophile balance (HLB) and is a valuable tool in
categorizing and selecting surfactants in the preparation of
formulations. Surfactants may be classified into different classes
based on the nature of the hydrophilic group: nonionic, anionic,
cationic and amphoteric (Rieger, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 285).
[0155] Naturally occurring emulsifiers used in emulsion
formulations include lanolin, beeswax, phosphatides, lecithin and
acacia. Absorption bases possess hydrophilic properties such that
they can soak up water to form w/o emulsions yet retain their
semisolid consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, nonswelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
[0156] A large variety of non-emulsifying materials are also
included in emulsion formulations and contribute to the properties
of emulsions. These include fats, oils, waxes, fatty acids, fatty
alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and antioxidants (Block, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199).
[0157] Hydrophilic colloids or hydrocolloids include naturally
occurring gums and synthetic polymers such as polysaccharides (for
example, acacia, agar, alginic acid, carrageenan, guar gum, karaya
gum, and tragacanth), cellulose derivatives (for example,
carboxymethylcellulose and carboxypropylcellulose), and synthetic
polymers (for example, carbomers, cellulose ethers, and
carboxyvinyl polymers). These disperse or swell in water to form
colloidal solutions that stabilize emulsions by forming strong
interfacial films around the dispersed-phase droplets and by
increasing the viscosity of the external phase.
[0158] Since emulsions often contain a number of ingredients such
as carbohydrates, proteins, sterols and phosphatides that may
readily support the growth of microbes, these formulations often
incorporate preservatives. Commonly used preservatives included in
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Antioxidants are also
commonly added to emulsion formulations to prevent deterioration of
the formulation. Antioxidants used may be free radical scavengers
such as tocopherols, alkyl gallates, butylated hydroxyanisole,
butylated hydroxytoluene, or reducing agents such as ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric
acid, tartaric acid, and lecithin.
[0159] The application of emulsion formulations via dermatological,
oral and parenteral routes and methods for their manufacture have
been reviewed in the literature (Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for
oral delivery have been very widely used because of ease of
formulation, as well as efficacy from an absorption and
bioavailability standpoint (Rosoff, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base
laxatives, oil-soluble vitamins and high fat nutritive preparations
are among the materials that have commonly been administered orally
as o/w emulsions.
[0160] In one embodiment, the compositions of dsRNAs and nucleic
acids are formulated as microemulsions. A microemulsion may be
defined as a system of water, oil and amphiphile which is a single
optically isotropic and thermodynamically stable liquid solution
(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245). Typically microemulsions are systems that are prepared by
first dispersing an oil in an aqueous surfactant solution and then
adding a sufficient amount of a fourth component, generally an
intermediate chain-length alcohol to form a transparent system.
Therefore, microemulsions have also been described as
thermodynamically stable, isotropically clear dispersions of two
immiscible liquids that are stabilized by interfacial films of
surface-active molecules (Leung and Shah, in: Controlled Release of
Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH
Publishers, New York, pages 185-215). Microemulsions commonly are
prepared via a combination of three to five components that include
oil, water, surfactant, cosurfactant and electrolyte. Whether the
microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w)
type is dependent on the properties of the oil and surfactant used
and on the structure and geometric packing of the polar heads and
hydrocarbon tails of the surfactant molecules (Schott, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 1985, p. 271).
[0161] The phenomenological approach utilizing phase diagrams has
been extensively studied and has yielded a comprehensive knowledge,
to one skilled in the art, of how to formulate microemulsions
(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger
and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,
volume 1, p. 335). Compared to conventional emulsions,
microemulsions offer the advantage of solubilizing water-insoluble
drugs in a formulation of thermodynamically stable droplets that
are formed spontaneously.
[0162] Surfactants used in the preparation of microemulsions
include, but are not limited to, ionic surfactants, non-ionic
surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol
fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol
monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol
pentaoleate (PO500), decaglycerol monocaprate (MCA750),
decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750),
decaglycerol decaoleate (DAO750), alone or in combination with
cosurfactants. The cosurfactant, usually a short-chain alcohol such
as ethanol, 1-propanol, and 1-butanol, serves to increase the
interfacial fluidity by penetrating into the surfactant film and
consequently creating a disordered film because of the void space
generated among surfactant molecules. Microemulsions may, however,
be prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase may typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase may include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C8-C12) mono, di, and tri-glycerides,
polyoxyethylated glyceryl fatty acid esters, fatty alcohols,
polyglycolized glycerides, saturated polyglycolized C8-C10
glycerides, vegetable oils and silicone oil.
[0163] Microemulsions are particularly of interest from the
standpoint of drug solubilization and the enhanced absorption of
drugs. Lipid based microemulsions (both o/w and w/o) have been
proposed to enhance the oral bioavailability of drugs, including
peptides (Constantinides et al., Pharmaceutical Research, 1994, 11,
1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13,
205). Microemulsions afford advantages of improved drug
solubilization, protection of drug from enzymatic hydrolysis,
possible enhancement of drug absorption due to surfactant-induced
alterations in membrane fluidity and permeability, ease of
preparation, ease of oral administration over solid dosage forms,
improved clinical potency, and decreased toxicity (Constantinides
et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.
Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form
spontaneously when their components are brought together at ambient
temperature. This may be particularly advantageous when formulating
thermolabile drugs, peptides or dsRNAs. Microemulsions have also
been effective in the transdermal delivery of active components in
both cosmetic and pharmaceutical applications. It is expected that
the microemulsion compositions and formulations will facilitate the
increased systemic absorption of dsRNAs and nucleic acids from the
gastrointestinal tract, as well as improve the local cellular
uptake of dsRNAs and nucleic acids.
[0164] Microemulsions may also contain additional components and
additives such as sorbitan monostearate (Grill 3), Labrasol, and
penetration enhancers to improve the properties of the formulation
and to enhance the absorption of the dsRNAs and nucleic acids
featured herein. Penetration enhancers used in the microemulsions
may be classified as belonging to one of five broad
categories--surfactants, fatty acids, bile salts, chelating agents,
and non-chelating non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these
classes has been discussed above.
[0165] Liposomes
[0166] There are many organized surfactant structures besides
microemulsions that have been studied and used for the formulation
of drugs. These include monolayers, micelles, bilayers and
vesicles. Vesicles, such as liposomes, have attracted great
interest because of their specificity and the duration of action
they offer from the standpoint of drug delivery. As used in the
present invention, the term "liposome" means a vesicle composed of
amphiphilic lipids arranged in a spherical bilayer or bilayers.
[0167] Liposomes are unilamellar or multilamellar vesicles which
have a membrane formed from a lipophilic material and an aqueous
interior. The aqueous portion contains the composition to be
delivered. Cationic liposomes possess the advantage of being able
to fuse to the cell wall. Non-cationic liposomes, although not able
to fuse as efficiently with the cell wall, are taken up by
macrophages in vivo.
[0168] In order to cross intact mammalian skin, lipid vesicles must
pass through a series of fine pores, each with a diameter less than
50 nm, under the influence of a suitable transdermal gradient.
Therefore, it is desirable to use a liposome which is highly
deformable and able to pass through such fine pores.
[0169] Further advantages of liposomes include; liposomes obtained
from natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; liposomes can protect encapsulated drugs in their internal
compartments from metabolism and degradation (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
Important considerations in the preparation of liposome
formulations are the lipid surface charge, vesicle size and the
aqueous volume of the liposomes.
[0170] Liposomes are useful for the transfer and delivery of active
ingredients to the site of action. Because the liposomal membrane
is structurally similar to biological membranes, when liposomes are
applied to a tissue, the liposomes start to merge with the cellular
membranes and as the merging of the liposome and cell progresses,
the liposomal contents are emptied into the cell where the active
agent may act.
[0171] Liposomal formulations have been the focus of extensive
investigation as the mode of delivery for many drugs. There is
growing evidence that for topical administration, liposomes present
several advantages over other formulations. Such advantages include
reduced side-effects related to high systemic absorption of the
administered drug, increased accumulation of the administered drug
at the desired target, and the ability to administer a wide variety
of drugs, both hydrophilic and hydrophobic, into the skin.
[0172] Several reports have detailed the ability of liposomes to
deliver agents including high-molecular weight DNA into the skin.
Compounds including analgesics, antibodies, hormones and
high-molecular weight DNAs have been administered to the skin. The
majority of applications resulted in the targeting of the upper
epidermis
[0173] Liposomes fall into two broad classes. Cationic liposomes
are positively charged liposomes which interact with the negatively
charged DNA molecules to form a stable complex. The positively
charged DNA/liposome complex binds to the negatively charged cell
surface and is internalized in an endosome. Due to the acidic pH
within the endosome, the liposomes are ruptured, releasing their
contents into the cell cytoplasm (Wang et al., Biochem. Biophys.
Res. Commun., 1987, 147, 980-985).
[0174] Liposomes which are pH-sensitive or negatively-charged,
entrap DNA rather than complex with it. Since both the DNA and the
lipid are similarly charged, repulsion rather than complex
formation occurs. Nevertheless, some DNA is entrapped within the
aqueous interior of these liposomes. pH-sensitive liposomes have
been used to deliver DNA encoding the thymidine kinase gene to cell
monolayers in culture. Expression of the exogenous gene was
detected in the target cells (Zhou et al., Journal of Controlled
Release, 1992, 19, 269-274).
[0175] One major type of liposomal composition includes
phospholipids other than naturally-derived phosphatidylcholine.
Neutral liposome compositions, for example, can be formed from
dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl
phosphatidylcholine (DPPC). Anionic liposome compositions generally
are formed from dimyristoyl phosphatidylglycerol, while anionic
fusogenic liposomes are formed primarily from dioleoyl
phosphatidylethanolamine (DOPE). Another type of liposomal
composition is formed from phosphatidylcholine (PC) such as, for
example, soybean PC, and egg PC. Another type is formed from
mixtures of phospholipid and/or phosphatidylcholine and/or
cholesterol.
[0176] Several studies have assessed the topical delivery of
liposomal drug formulations to the skin. Application of liposomes
containing interferon to guinea pig skin resulted in a reduction of
skin herpes sores while delivery of interferon via other means
(e.g., as a solution or as an emulsion) were ineffective (Weiner et
al., Journal of Drug Targeting, 1992, 2, 405-410). Further, an
additional study tested the efficacy of interferon administered as
part of a liposomal formulation to the administration of interferon
using an aqueous system, and concluded that the liposomal
formulation was superior to aqueous administration (du Plessis et
al., Antiviral Research, 1992, 18, 259-265).
[0177] Non-ionic liposomal systems have also been examined to
determine their utility in the delivery of drugs to the skin, in
particular systems comprising non-ionic surfactant and cholesterol.
Non-ionic liposomal formulations comprising Novasome.TM. I
(glyceryl dilaurate/cholesterol/po-lyoxyethylene-10-stearyl ether)
and Novasome.TM. II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).
[0178] Liposomes also include "sterically stabilized" liposomes, a
term which, as used herein, refers to liposomes comprising one or
more specialized lipids that, when incorporated into liposomes,
result in enhanced circulation lifetimes relative to liposomes
lacking such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome (A) comprises one or more glycolipids, such
as monosialoganglioside G.sub.M1, or (B) is derivatized with one or
more hydrophilic polymers, such as a polyethylene glycol (PEG)
moiety. While not wishing to be bound by any particular theory, it
is thought in the art that, at least for sterically stabilized
liposomes containing gangliosides, sphingomyelin, or
PEG-derivatized lipids, the enhanced circulation half-life of these
sterically stabilized liposomes derives from a reduced uptake into
cells of the reticuloendothelial system (RES) (Allen et al., FEBS
Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53,
3765).
[0179] Various liposomes comprising one or more glycolipids are
known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci.,
1987, 507, 64) reported the ability of monosialoganglioside
G.sub.M1, galactocerebroside sulfate and phosphatidylinositol to
improve blood half-lives of liposomes. These findings were
expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A.,
1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to
Allen et al., disclose liposomes comprising (1) sphingomyelin and
(2) the ganglioside G.sub.M1 or a galactocerebroside sulfate ester.
U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes
comprising sphingomyelin. Liposomes comprising
1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499
(Lim et al).
[0180] Many liposomes comprising lipids derivatized with one or
more hydrophilic polymers, and methods of preparation thereof, are
known in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53,
2778) described liposomes comprising a nonionic detergent,
2C.sub.1215 G, that contains a PEG moiety. Illum et al. (FEBS
Lett., 1984, 167, 79) noted that hydrophilic coating of polystyrene
particles with polymeric glycols results in significantly enhanced
blood half-lives. Synthetic phospholipids modified by the
attachment of carboxylic groups of polyalkylene glycols (e.g., PEG)
are described by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899).
Klibanov et al. (FEBS Lett., 1990, 268, 235) described experiments
demonstrating that liposomes comprising phosphatidylethanolamine
(PE) derivatized with PEG or PEG stearate have significant
increases in blood circulation half-lives. Blume et al. (Biochimica
et Biophysica Acta, 1990, 1029, 91) extended such observations to
other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from
the combination of distearoylphosphatidylethanolamine (DSPE) and
PEG. Liposomes having covalently bound PEG moieties on their
external surface are described in European Patent No. EP 0 445 131
B1 and WO 90/04384 to Fisher. Liposome compositions containing 1-20
mole percent of PE derivatized with PEG, and methods of use
thereof, are described by Woodle et al. (U.S. Pat. Nos. 5,013,556
and 5,356,633) and Martin et al. (U.S. Pat. No. 5,213,804 and
European Patent No. EP 0 496 813 B1). Liposomes comprising a number
of other lipid-polymer conjugates are disclosed in WO 91/05545 and
U.S. Pat. No. 5,225,212 (both to Martin et al.) and in WO 94/20073
(Zalipsky et al.) Liposomes comprising PEG-modified ceramide lipids
are described in WO 96/10391 (Choi et al). U.S. Pat. No. 5,540,935
(Miyazaki et al.) and U.S. Pat. No. 5,556,948 (Tagawa et al.)
describe PEG-containing liposomes that can be further derivatized
with functional moieties on their surfaces.
[0181] A limited number of liposomes comprising nucleic acids are
known in the art. WO 96/40062 to Thierry et al. discloses methods
for encapsulating high molecular weight nucleic acids in liposomes.
U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded
liposomes and asserts that the contents of such liposomes may
include a dsRNA. U.S. Pat. No. 5,665,710 to Rahman et al. describes
certain methods of encapsulating oligodeoxynucleotides in
liposomes. WO 97/04787 to Love et al. discloses liposomes
comprising dsRNAs targeted to the raf gene.
[0182] Transfersomes are yet another type of liposomes, and are
highly deformable lipid aggregates which are attractive candidates
for drug delivery vehicles. Transfersomes may be described as lipid
droplets which are so highly deformable that they are easily able
to penetrate through pores which are smaller than the droplet.
Transfersomes are adaptable to the environment in which they are
used, e.g., they are self-optimizing (adaptive to the shape of
pores in the skin), self-repairing, frequently reach their targets
without fragmenting, and often self-loading. To make transfersomes
it is possible to add surface edge-activators, usually surfactants,
to a standard liposomal composition. Transfersomes have been used
to deliver serum albumin to the skin. The transfersome-mediated
delivery of serum albumin has been shown to be as effective as
subcutaneous injection of a solution containing serum albumin.
[0183] Surfactants find wide application in formulations such as
emulsions (including microemulsions) and liposomes. The most common
way of classifying and ranking the properties of the many different
types of surfactants, both natural and synthetic, is by the use of
the hydrophile/lipophile balance (HLB). The nature of the
hydrophilic group (also known as the "head") provides the most
useful means for categorizing the different surfactants used in
formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel
Dekker, Inc., New York, N.Y., 1988, p. 285).
[0184] If the surfactant molecule is not ionized, it is classified
as a nonionic surfactant. Nonionic surfactants find wide
application in pharmaceutical and cosmetic products and are usable
over a wide range of pH values. In general their HLB values range
from 2 to about 18 depending on their structure. Nonionic
surfactants include nonionic esters such as ethylene glycol esters,
propylene glycol esters, glyceryl esters, polyglyceryl esters,
sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic
alkanolamides and ethers such as fatty alcohol ethoxylates,
propoxylated alcohols, and ethoxylated/propoxylated block polymers
are also included in this class. The polyoxyethylene surfactants
are the most popular members of the nonionic surfactant class.
[0185] If the surfactant molecule carries a negative charge when it
is dissolved or dispersed in water, the surfactant is classified as
anionic. Anionic surfactants include carboxylates such as soaps,
acyl lactylates, acyl amides of amino acids, esters of sulfuric
acid such as alkyl sulfates and ethoxylated alkyl sulfates,
sulfonates such as alkyl benzene sulfonates, acyl isethionates,
acyl taurates and sulfosuccinates, and phosphates. The most
important members of the anionic surfactant class are the alkyl
sulfates and the soaps.
[0186] If the surfactant molecule carries a positive charge when it
is dissolved or dispersed in water, the surfactant is classified as
cationic. Cationic surfactants include quaternary ammonium salts
and ethoxylated amines. The quaternary ammonium salts are the most
used members of this class.
[0187] If the surfactant molecule has the ability to carry either a
positive or negative charge, the surfactant is classified as
amphoteric. Amphoteric surfactants include acrylic acid
derivatives, substituted alkylamides, N-alkylbetaines and
phosphatides.
[0188] The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).
[0189] Penetration Enhancers
[0190] In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly dsRNAs, to the skin of animals. Most drugs are
present in solution in both ionized and nonionized forms. However,
usually only lipid soluble or lipophilic drugs readily cross cell
membranes. It has been discovered that even non-lipophilic drugs
may cross cell membranes if the membrane to be crossed is treated
with a penetration enhancer. In addition to aiding the diffusion of
non-lipophilic drugs across cell membranes, penetration enhancers
also enhance the permeability of lipophilic drugs.
[0191] Penetration enhancers may be classified as belonging to one
of five broad categories, i.e., surfactants, fatty acids, bile
salts, chelating agents, and non-chelating non-surfactants (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
p.92). Each of the above mentioned classes of penetration enhancers
are described below in greater detail.
[0192] Surfactants: In connection with the present invention,
surfactants (or "surface-active agents") are chemical entities
which, when dissolved in an aqueous solution, reduce the surface
tension of the solution or the interfacial tension between the
aqueous solution and another liquid, with the result that
absorption of dsRNAs through the mucosa is enhanced. In addition to
bile salts and fatty acids, these penetration enhancers include,
for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether
and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews
in Therapeutic Drug Carrier Systems, 1991, p.92); and
perfluorochemical emulsions, such as FC-43. Takahashi et al., J.
Pharm. Pharmacol., 1988, 40, 252).
[0193] Fatty acids: Various fatty acids and their derivatives which
act as penetration enhancers include, for example, oleic acid,
lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic
acid, stearic acid, linoleic acid, linolenic acid, dicaprate,
tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin,
caprylic acid, arachidonic acid, glycerol 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines,
C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and
t-butyl), and mono- and di-glycerides thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carryier Systems,
1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,
1992, 44, 651-654).
[0194] Bile salts: The physiological role of bile includes the
facilitation of dispersion and absorption of lipids and fat-soluble
vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.
Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural
bile salts, and their synthetic derivatives, act as penetration
enhancers. Thus the term "bile salts" includes any of the naturally
occurring components of bile as well as any of their synthetic
derivatives. Suitable bile salts include, for example, cholic acid
(or its pharmaceutically acceptable sodium salt, sodium cholate),
dehydrocholic acid (sodium dehydrocholate), deoxycholic acid
(sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic
acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm.
Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990,
79, 579-583).
[0195] Chelating Agents: Chelating agents, as used in connection
with the present invention, can be defined as compounds that remove
metallic ions from solution by forming complexes therewith, with
the result that absorption of dsRNAs through the mucosa is
enhanced. With regards to their use as penetration enhancers in the
present invention, chelating agents have the added advantage of
also serving as DNase inhibitors, as most characterized DNA
nucleases require a divalent metal ion for catalysis and are thus
inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,
315-339). Suitable chelating agents include but are not limited to
disodium ethylenediaminetetraacetate (EDTA), citric acid,
salicylates (e.g., sodium salicylate, 5-methoxysalicylate and
homovanilate), N-acyl derivatives of collagen, laureth-9 and
N-amino acyl derivatives of beta-diketones (enamines)(Lee et al.,
Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page
92; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14,
43-51).
[0196] Non-chelating non-surfactants: As used herein, non-chelating
non-surfactant penetration enhancing compounds can be defined as
compounds that demonstrate insignificant activity as chelating
agents or as surfactants but that nonetheless enhance absorption of
dsRNAs through the alimentary mucosa (Muranishi, Critical Reviews
in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of
penetration enhancers include, for example, unsaturated cyclic
ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et
al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621-626).
[0197] Agents that enhance uptake of dsRNAs at the cellular level
may also be added to the pharmaceutical and other compositions
featured in the invention. For example, cationic lipids, such as
lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic
glycerol derivatives, and polycationic molecules, such as
polylysine (Lollo et al., PCT Application WO 97/30731), are also
known to enhance the cellular uptake of dsRNAs.
[0198] Other agents may be utilized to enhance the penetration of
the administered nucleic acids, including glycols such as ethylene
glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and
terpenes such as limonene and menthone.
[0199] Carriers
[0200] Certain compositions also incorporate carrier compounds in
the formulation. As used herein, "carrier compound" or "carrier"
can refer to a nucleic acid, or analog thereof, which is inert
(i.e., does not possess biological activity per se) but is
recognized as a nucleic acid by in vivo processes that reduce the
bioavailability of a nucleic acid having biological activity by,
for example, degrading the biologically active nucleic acid or
promoting its removal from circulation. The coadministration of a
nucleic acid and a carrier compound, typically with an excess of
the latter substance, can result in a substantial reduction of the
amount of nucleic acid recovered in the liver, kidney or other
extracirculatory reservoirs, presumably due to competition between
the carrier compound and the nucleic acid for a common receptor.
For example, the recovery of a partially phosphorothioate dsRNA in
hepatic tissue can be reduced when it is coadministered with
polyinosinic acid, dextran sulfate, polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et
al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA
& Nucl. Acid Drug Dev., 1996, 6, 177-183.
[0201] Excipients
[0202] In contrast to a carrier compound, a "pharmaceutical
carrier" or "excipient" is a pharmaceutically acceptable solvent,
suspending agent or any other pharmacologically inert vehicle for
delivering one or more nucleic acids to an animal. The excipient
may be liquid or solid and is selected, with the planned manner of
administration in mind, so as to provide for the desired bulk,
consistency, etc., when combined with a nucleic acid and the other
components of a given pharmaceutical composition. Typical
pharmaceutical carriers include, but are not limited to, binding
agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or
hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and
other sugars, microcrystalline cellulose, pectin, gelatin, calcium
sulfate, ethyl cellulose, polyacrylates or calcium hydrogen
phosphate, etc.); lubricants (e.g., magnesium stearate, talc,
silica, colloidal silicon dioxide, stearic acid, metallic
stearates, hydrogenated vegetable oils, corn starch, polyethylene
glycols, sodium benzoate, sodium acetate, etc.); disintegrants
(e.g., starch, sodium starch glycolate, etc.); and wetting agents
(e.g., sodium lauryl sulphate, etc).
[0203] Pharmaceutically acceptable organic or inorganic excipient
suitable for non-parenteral administration, which do not
deleteriously react with nucleic acids, can also be used to
formulate the compositions. Suitable pharmaceutically acceptable
carriers include, but are not limited to, water, salt solutions,
alcohols, polyethylene glycols, gelatin, lactose, amylose,
magnesium stearate, talc, silicic acid, viscous paraffin,
hydroxymethylcellulose, polyvinylpyrrolidone and the like.
[0204] Formulations for topical administration of nucleic acids may
include sterile and non-sterile aqueous solutions, non-aqueous
solutions in common solvents such as alcohols, or solutions of the
nucleic acids in liquid or solid oil bases. The solutions may also
contain buffers, diluents and other suitable additives.
Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can be used.
[0205] Suitable pharmaceutically acceptable excipients include, but
are not limited to, water, salt solutions, alcohol, polyethylene
glycols, gelatin, lactose, amylose, magnesium stearate, talc,
silicic acid, viscous paraffin, hydroxymethylcellulose,
polyvinylpyrrolidone and the like.
[0206] Other Components
[0207] The compositions featured in the invention may additionally
contain other adjunct components conventionally found in
pharmaceutical compositions, at their art-established usage levels.
Thus, for example, the compositions may contain additional,
compatible, pharmaceutically-active materials such as, for example,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the compositions, such as dyes,
flavoring agents, preservatives, antioxidants, opacifiers,
thickening agents and stabilizers. However, such materials, when
added, should not unduly interfere with the biological activities
of the components of the compositions. The formulations can be
sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the nucleic acid(s) of the
formulation.
[0208] Aqueous suspensions may contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
[0209] In some embodiments, pharmaceutical compositions featured in
the invention include (a) one or more dsRNA compounds and (b) one
or more other chemotherapeutic agents which function by a non-RNAi
mechanism. Examples of such chemotherapeutic agents include but are
not limited to temozolomide, daunorubicin, daunomycin,
dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,
bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,
bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,
mithramycin, prednisone, hydroxyprogesterone, testosterone,
tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,
pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,
methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphor-amide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,
taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate,
irinotecan, topotecan, gemcitabine, teniposide, cisplatin and
diethylstilbestrol (DES). See, generally, The Merck Manual of
Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al.,
eds., Rahway, N.J. When used with the dsRNAs featured in the
invention, such chemotherapeutic agents may be used individually
(e.g., 5-FU and oligonucleotide), sequentially (e.g., 5-FU and
oligonucleotide for a period of time followed by MTX and
oligonucleotide), or in combination with one or more other such
chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide, or
5-FU, radiotherapy and oligonucleotide). Anti-inflammatory drugs,
including but not limited to nonsteroidal anti-inflammatory drugs
and corticosteroids, and antiviral drugs, including but not limited
to ribivirin, vidarabine, acyclovir and ganciclovir, may also be
combined in compositions featured in the invention. See, generally,
The Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al.,
eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively).
Other non-RNAi chemotherapeutic agents are also within the scope of
this invention. Two or more combined compounds may be used together
or sequentially.
[0210] Toxicity and therapeutic efficacy of such compounds can be
determined by standard pharmaceutical procedures in cell cultures
or experimental animals, e.g., for determining the LD50 (the dose
lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds that exhibit
high therapeutic indices are generally preferred.
[0211] The data obtained from cell culture assays and animal
studies can be used in formulation a range of dosage for use in
humans. The dosage of compositions featured in the invention lies
generally within a range of circulating concentrations that include
the ED50 with little or no toxicity. The dosage may vary within
this range depending upon the dosage form employed and the route of
administration utilized. For any compound used in the methods
featured in the invention, the therapeutically effective dose can
be estimated initially from cell culture assays. A dose may be
formulated in animal models to achieve a circulating plasma
concentration range of the compound or, when appropriate, of the
polypeptide product of a target sequence (e.g., achieving a
decreased concentration of the polypeptide) that includes the IC50
(i.e., the concentration of the test compound which achieves a
half-maximal inhibition of symptoms) as determined in cell culture.
Such information can be used to more accurately determine useful
doses in humans. Levels in plasma may be measured, for example, by
high performance liquid chromatography.
[0212] In addition to their administration, as discussed above, the
dsRNAs featured in the invention can be administered in combination
with other known agents effective in treatment of pathological
processes mediated by deltaEGFR or IL6 expression. In any event,
the administering physician can adjust the amount and timing of
dsRNA administration on the basis of results observed using
standard measures of efficacy known in the art or described
herein.
[0213] Methods for Treating Diseases Caused by Expression of
deltaEGFR or IL6 Genes
[0214] The invention relates in particular to the use of a dsRNA
targeting deltaEGFR or IL6 and compositions containing at least one
such dsRNA, for the treatment of a deltaEGFR-mediated disorder or
disease. For example, a dsRNA targeting a deltaEGFR gene can be
useful for the treatment of a proliferative disorder, such as
cancer, such as glioma, e.g., a glial tumor of the central nervous
system, such as a grade I, II, III, or IV glioma. For example, a
composition containing a dsRNA targeting deltaEGFR can be used to
treat a grade III glioma, such as anaplastic astrocytoma, or a
grade IV glioma, such as a glioblastoma multiforme. The glioma can
be an ependymoma, astrocytoma, oligodendroglioma, or a mixed
glioma, such as an oligoastrocytoma. A composition containing a
dsRNA targeting a mutant EGFR, e.g., deltaEGFR or an IL6, is used
to treat a carcinoma of the breast, ovary, cervix, kidney, or a
squamous cell. The dsRNA targeting deltaEGFR can also target
wtEGFR.
[0215] A composition containing a dsRNA targeting a mutant EGFR,
e.g., a deltaEGFR or an IL6, may also be used to treat other tumors
and cancers, such as breast cancer, lung cancer, head and neck
cancer, brain cancer, abdominal cancer, colon cancer, colorectal
cancer, esophagus cancer, gastrointestinal cancer, tongue cancer,
neuroblastoma, osteosarcoma, ovarian cancer, pancreatic cancer,
prostate cancer, cervical cancer (e.g., squamous carcinoma of the
cervix), lymphoid tumor, retinoblastoma, Wilm's tumor, multiple
myeloma and for the treatment of skin cancer, like melanoma, for
the treatment of lymphomas and blood cancer. The compositions
featured herein can be used to treat a tumor of the brain or
spine.
[0216] A dsRNA targeting deltaEGFR or IL6 may be used to treat a
proliferative disorder or differentiative disorder. Examples of
cellular proliferative and/or differentiative disorders include
cancer, e.g., carcinoma, sarcoma, metastatic disorders or
hematopoietic neoplastic disorders, e.g., leukemias. A metastatic
tumor can arise from a multitude of primary tumor types, including
those of prostate, colon, lung, breast and liver origin. As used
herein, the terms "cancer," "hyperproliferative," and "neoplastic"
refer to cells having the capacity for autonomous growth, i.e., an
abnormal state or condition characterized by rapidly proliferating
cell growth. These terms are meant to include all types of
cancerous growths or oncogenic processes, metastatic tissues or
malignantly transformed cells, tissues, or organs, irrespective of
histopathologic type or stage of invasiveness. Proliferative
disorders also include hematopoietic neoplastic disorders,
including diseases involving hyperplastic/neoplastic cells of
hematopoictic origin, e.g., arising from myeloid, lymphoid or
erythroid lineages, or precursor cells thereof.
[0217] The invention further relates to the use of a dsRNA or a
pharmaceutical composition thereof, e.g., for treating a cancer, in
combination with other pharmaceuticals and/or other therapeutic
methods, e.g., with known pharmaceuticals and/or known therapeutic
methods, such as, for example, those which are currently employed
for treating these disorders. In one example, administration of a
dsRNA targeting deltaEGFR can be administered in combination with a
chemotherapeutic agent, such as temozolomide, deoxycoformycin,
cisplatin, cyclophosphamide, 5-fluorouracil, adriamycin,
daunorubicin, tamoxifen aunorubicin, daunomycin, dactinomycin,
doxorubicin, epirubicin, idarubicin, esorubicin, bleomycin,
mafosfamide, ifosfamide, cytosine arabinoside,
bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,
mithramycin, prednisone, hydroxyprogesterone, testosterone,
tamoxifen, dacarbazine, procarbazine, hexamethylmelamine,
pentamethylmelamine, mitoxantrone, amsacrine, chlorambucil,
methylcyclohexylnitrosurea, nitrogen mustards, melphalan,
cyclophosphamide, 6-mercaptopurine, 6-thioguanine, cytarabine,
5-azacytidine, hydroxyurea, deoxycoformycin,
4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),
5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine,
taxol, vincristine, vinblastine, etoposide (VP-16), trimetrexate,
irinotecan, topotecan, gemcitabine, teniposide, cisplatin and
diethylstilbestrol (DES). See, generally, The Merck Manual of
Diagnosis and Therapy, 15th Ed. 1987, pp. 1206-1228, Berkow et al.,
eds., Rahway, N.J. When used with the dsRNAs featured in the
invention, such chemotherapeutic agents may be used individually,
sequentially (e.g., dsRNA for a period of time, followed by
chemotherapy), or in combination with one or more other such agents
(e.g., chemotherapy and dsRNA). Two or more combined compounds may
be used together or sequentially.
[0218] In one embodiment, a deltaEGFR dsRNA is administered in
combination with at least one additional therapeutic agent, such as
a second dsRNA targeting a different nucleic acid, e.g., an IL6
dsRNA, for the treatment of a condition or a symptom of a
condition, such as for the treatment of a cancer. In some
embodiments, the second therapeutic agent is a chemotherapeutic
agent.
[0219] The dsRNA and an additional therapeutic agent can be
administered in the same combination, e.g., intracranially or
parenterally, or the additional therapeutic agent can be
administered as part of a separate composition, e.g.,
intracranially or parenterally, or by another method described
herein.
[0220] Treatment with a dsRNA targeting deltaEGFR can also be
performed in combination with radiation therapy, including external
beam radiation, such as for treatment of tumors of the brain. A
dsRNA featured herein may be administered before or after a
surgical procedure to treat a cancer (e.g., to remove a tumor),
such as resection of a brain tumor.
[0221] The invention also relates to the use of a dsRNA targeting
IL6 and compositions containing at least one such dsRNA, for the
treatment of a IL6 or a deltaEGFR-mediated disorder or disease. For
example, an IL-6 dsRNA featured in the invention may be used to
treat a hematological disorder, such as plasma cell dyscrasia,
leukemia or lymphoma; proliferative glomerulonephritis; an
inflammatory disease, such as rheumatoid arthritis, or an
inflammatory bowel disease, such as Crohn's disease or ulcerative
colitis; diabetes; septic shock; bacterial infections; viral
infections, including HIV-1 infections; osteoporosis; autoimmune
disorders, such as chronic immune deficiency syndrome or autoimmune
deficiency syndrome (AIDS); neural disorders, such as multiple
sclerosis, HTLV1-associated myelopathy or bacterial meningitis,
systemic lupus erythematosus and vasculitis-associated central
nervous system diseases; or other disorders of the central nervous
system, including Alzheimer's disease, hypochondria, epilepsy,
migraine, pain, Parkin's disease or schizophrenia.
[0222] An IL-6 dsRNA featured in the invention may also be used to
prevent allograft rejection or xenograft rejection and
ischemia/reperfusion injury in solid organ or tissue
transplantation. For example, an IL-6 dsRNA can be administered to
prevent rejection of a transplanted organ, such as a transplanted
kidney, liver, lung, pancrease, heart, small bowel, cornea,
epithelial cells, vascular endothelium, vascular smooth muscle
cells, myocardium and passenger leukocytes resident in the organ at
the time of transplantation.
[0223] Treatment with a dsRNA targeting IL-6 can be performed in
combination with a second dsRNA also targeting IL-6, and which
targets a different sequence than a first dsRNA targeting IL-6. A
dsRNA targeting IL-6 can also be administered in combination with
one or more dsRNAs targeting other cytokines, immunomodulatory or
immunoeffector genes, such as the C3 (complement component 3) gene,
ICAM1 (intercellular adhesion molecule 1), VCAM-1 (vascular cell
adhesion molecule 1), IFN-gamma (interferon gamma), IL-1
(interleukin-1), IL-8 (interleukin-8), TNF-alpha (tumor necrosis
factor-alpha), CD80, CD86, MHC-II (major histocombatibility
complex-II), MHC-I (major histocombatibility complex-I), CD28,
CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) or PV-B19
(parvovirus B19). As stated above, the IL6 dsRNA can also be
administered in combination with a dsRNA targeting deltaEGFR, and
optionally, also targeting wtEGFR, such as for the treatment of a
deltaEGFR mediated disease, such as a cancer.
[0224] Patients can be administered a therapeutic amount of dsRNA,
such as 0.01 mg/kg, 0.02 mg/kg, 0.1 mg/kg, 0.5 mg/kg, 1.0 mg/kg,
1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The dsRNA can be
administered by intracranial infusion over a period of time, such
as over a 30 minute, 1 hour, 2 hour, 3 hour or 4 hour period. The
administration is repeated, for example, on a regular basis, such
as biweekly (i.e., every two weeks) for one month, two months,
three months, four months or longer. After an initial treatment
regimen, the treatments can be administered on a less frequent
basis. For example, after administration biweekly for three months,
administration can be repeated once per month, for six months or a
year or longer. Intracranial infusion can be continuos.
Administration of the dsRNA can reduce target RNA and protein
levels, e.g., deltaEGFR or IL-6 levels, in the cerebrospinal fluid
of the patient by at least 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%
or 90% or more. Alternatively, the dsRNA can be administered by
intravenous infusion over a period of time, such as over a 5
minute, 10 minute, 15 minute, 20 minute, or 25 minute period. The
administration is repeated, for example, on a regular basis, such
as biweekly (i.e., every two weeks) for one month, two months,
three months, four months or longer. After an initial treatment
regimen, the treatments can be administered on a less frequent
basis. For example, after administration biweekly for three months,
administration can be repeated once per month, for six months or a
year or longer. Administration of the dsRNA can reduce deltaEGFR
levels in the blood or urine of the patient by at least 20%, 25%,
30%, 40%, 50%, 60%, 70%, 80% or 90% or more.
[0225] Before administration of a full dose of the dsRNA, patients
can be administered a smaller dose, such as 5% of the total dose,
and monitored for adverse effects, such as an allergic reaction.
Patients can be monitored for adverse effects depending on the
formulation. For example, if the dsRNA is formulated in a lipid,
the patient can be administered a smaller dose, and then monitored
for elevated lipid levels or blood pressure. In another example,
the patient can be monitored for unwanted immunostimulatory
effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)
levels.
[0226] Many EGFR- and IL6-associated diseases and disorders are
hereditary. Therefore, a patient in need of a deltaEGFR dsRNA can
be identified by taking a family history. A healthcare provider,
such as a doctor, nurse, or family member, can take a family
history before prescribing or administering a deltaEGFR dsRNA.
[0227] Owing to the inhibitory effects on deltaEGFR expression, and
of the inhibitory effects of IL6 overexpression, a composition
according to the invention or a pharmaceutical composition prepared
therefrom can enhance the quality of life of a subject.
[0228] Methods for Inhibiting Expression of a deltaEGFR or IL6
Gene
[0229] In yet another aspect, the invention provides a method for
inhibiting expression of a deltaEGFR gene in a mammal. The method
includes administering a composition featured in the invention to
the mammal such that expression of the target deltaEGFR gene and,
optionally, a wtEGFR gene is decreased or silenced. In one aspect,
the invention provides a method for inhibiting expression of an IL6
gene in a mammal. The method includes administering a composition
featured in the invention to the mammal such that expression of the
target IL6 gene is decreased or silenced.
[0230] When the organism to be treated is a mammal such as a human,
the composition may be administered by any means known in the art
including, but not limited to oral or parenteral routes, including
intracranial (e.g., intraventricular, intraparenchymal and
intrathecal), intravenous, intramuscular, subcutaneous,
transdermal, airway (aerosol), nasal, rectal, and topical
(including buccal and sublingual) administration. In certain
embodiments, the compositions are administered by intravenous
infusion or injection. In other embodiments, the compositions are
administered by intracranial infusion or injection.
[0231] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the dsRNAs and
methods featured in the invention, suitable methods and materials
are described below. All publications, patent applications,
patents, and other references mentioned herein are incorporated by
reference in their entirety. In case of conflict, the present
specification, including definitions, will control. In addition,
the materials, methods, and examples are illustrative only and not
intended to be limiting.
EXAMPLES
Example 1. dsRNA Synthesis
[0232] Source of Reagents
[0233] Where the source of a reagent is not specifically given
herein, such reagent may be obtained from any supplier of reagents
for molecular biology at a quality/purity standard for application
in molecular biology.
[0234] siRNA Synthesis
[0235] Single-stranded RNAs were produced by solid phase synthesis
on a scale of 1 .mu.mole using an Expedite 8909 synthesizer
(Applied Biosystems, Applera Deutschland GmbH, Darmstadt, Germany)
and controlled pore glass (CPG, 500 .ANG., Proligo Biochemie GmbH,
Hamburg, Germany) as solid support. RNA and RNA containing
2'-O-methyl nucleotides were generated by solid phase synthesis
employing the corresponding phosphoramidites and 2'-O-methyl
phosphoramidites, respectively (Proligo Biochemie GmbH, Hamburg,
Germany). These building blocks were incorporated at selected sites
within the sequence of the oligoribonucleotide chain using standard
nucleoside phosphoramidite chemistry such as described in Current
protocols in nucleic acid chemistry, Beaucage, S. L. et al.
(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA.
Phosphorothioate linkages were introduced by replacement of the
iodine oxidizer solution with a solution of the Beaucage reagent
(Chruachem Ltd, Glasgow, UK) in acetonitrile (1%). Further
ancillary reagents were obtained from Mallinckrodt Baker
(Griesheim, Germany).
[0236] Deprotection and purification of the crude
oligoribonucleotides by anion exchange HPLC were carried out
according to established procedures. Yields and concentrations were
determined by UV absorption of a solution of the respective RNA at
a wavelength of 260 nm using a spectral photometer (DU 640B,
Beckman Coulter GmbH, UnterschleiBheim, Germany). Double stranded
RNA was generated by mixing an equimolar solution of complementary
strands in annealing buffer (20 mM sodium phosphate, pH 6.8; 100 mM
sodium chloride), heated in a water bath at 85-90.degree. C. for 3
minutes and cooled to room temperature over a period of 3-4 hours.
The annealed RNA solution was stored at -20.degree. C. until
use.
[0237] For the synthesis of 3'-cholesterol-conjugated siRNAs
(herein referred to as --Chol-3'), an appropriately modified solid
support is used for RNA synthesis. The modified solid support is
prepared as follows:
Diethyl-2-azabutane-1,4-dicarboxylate AA
##STR00002##
[0239] A 4.7 M aqueous solution of sodium hydroxide (50 mL) is
added into a stirred, ice-cooled solution of ethyl glycinate
hydrochloride (32.19 g, 0.23 mole) in water (50 mL). Then, ethyl
acrylate (23.1 g, 0.23 mole) is added and the mixture is stirred at
room temperature until completion of the reaction is ascertained by
TLC. After 19 h the solution is partitioned with dichloromethane
(3.times.100 mL). The organic layer is dried with anhydrous sodium
sulfate, filtered and evaporated. The residue is distilled to
afford AA (28.8 g, 61%).
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl-
]-amino}-propionic Acid Ethyl Ester AB
##STR00003##
[0241] Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) is dissolved
in dichloromethane (50 mL) and cooled with ice.
Diisopropylcarbodiimde (3.25 g, 3.99 mL, 25.83 mmol) is added to
the solution at 0.degree. C. It is then followed by the addition of
Diethyl-azabutane-1,4-dicarboxylate (5 g, 24.6 mmol) and
dimethylamino pyridine (0.305 g, 2.5 mmol). The solution is brought
to room temperature and stirred further for 6 h. Completion of the
reaction is ascertained by TLC. The reaction mixture is
concentrated under vacuum and ethyl acetate is added to precipitate
diisopropyl urea. The suspension is filtered. The filtrate is
washed with 5% aqueous hydrochloric acid, 5% sodium bicarbonate and
water. The combined organic layer is dried over sodium sulfate and
concentrated to give the crude product which is purified by column
chromatography (50% EtOAC/Hexanes) to yield 11.87 g (88%) of
AB.
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic Acid
Ethyl Ester AC
##STR00004##
[0243]
3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-he-
xanoyl]-amino}-propionic acid ethyl ester AB (11.5 g, 21.3 mmol) is
dissolved in 20% piperidine in dimethylformamide at 0.degree. C.
The solution is continued stirring for 1 h. The reaction mixture is
concentrated under vacuum, water is added to the residue, and the
product is extracted with ethyl acetate. The crude product is
purified by conversion into its hydrochloride salt.
3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,1-
5,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-h-
exanoyl}ethoxycarbonylmethyl-amino)-propionic Acid Ethyl Ester
AD
##STR00005##
[0245] The hydrochloride salt of
3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid
ethyl ester AC (4.7 g, 14.8 mmol) is taken up in dichloromethane.
The suspension is cooled to 0.degree. C. on ice. To the suspension
diisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) is added. To the
resulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol)
is added. The reaction mixture is stirred overnight. The reaction
mixture is diluted with dichloromethane and ished with 10%
hydrochloric acid. The product is purified by flash chromatography
(10.3 g, 92%).
1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15-
,16,17-tetradecahydro-1H-cyclopenta[a]
phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxyli-
c Acid Ethyl Ester AE
##STR00006##
[0247] Potassium t-butoxide (1.1 g, 9.8 mmol) is slurried in 30 mL
of dry toluene. The mixture is cooled to 0.degree. C. on ice and 5
g (6.6 mmol) of diester AD is added slowly with stirring within 20
mins. The temperature is kept below 5.degree. C. during the
addition. The stirring is continued for 30 mins at 0.degree. C. and
1 mL of glacial acetic acid is added, immediately followed by 4 g
of NaH.sub.2PO.sub.4.H.sub.2O in 40 mL of water The resultant
mixture is extracted twice with 100 mL of dichloromethane each and
the combined organic extracts are washed twice with 10 mL of
phosphate buffer each, dried, and evaporated to dryness. The
residue is dissolved in 60 mL of toluene, cooled to 0.degree. C.
and extracted with three 50 mL portions of cold pH 9.5 carbonate
buffer. The aqueous extracts are adjusted to pH 3 with phosphoric
acid, and extracted with five 40 mL portions of chloroform which
are combined, dried and evaporated to dryness. The residue is
purified by column chromatography using 25% ethylacetate/hexane to
afford 1.9 g of b-ketoester (39%).
[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamic
Acid
17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1-
7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl Ester AF
##STR00007##
[0249] Methanol (2 mL) is added dropwise over a period of 1 h to a
refluxing mixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium
borohydride (0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring
is continued at reflux temperature for 1 h. After cooling to room
temperature, 1 N HCl (12.5 mL) is added, the mixture is extracted
with ethylacetate (3.times.40 mL). The combined ethylacetate layer
is dried over anhydrous sodium sulfate and concentrated under
vacuum to yield the product which is purified by column
chromatography (10% MeOH/CHCl.sub.3) (89%).
(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-
-yl}-6-oxo-hexyl)-carbamic Acid
17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,1-
7-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl Ester AG
##STR00008##
[0251] Diol AF (1.25 gm 1.994 mmol) is dried by evaporating with
pyridine (2.times.5 mL) in vacuo. Anhydrous pyridine (10 mL) and
4,4'-dimethoxytritylchloride (0.724 g, 2.13 mmol) are added with
stirring. The reaction is carried out at room temperature
overnight. The reaction is quenched by the addition of methanol.
The reaction mixture is concentrated under vacuum and to the
residue dichloromethane (50 mL) is added. The organic layer is
washed with 1M aqueous sodium bicarbonate. The organic layer is
dried over anhydrous sodium sulfate, filtered and concentrated. The
residual pyridine is removed by evaporating with toluene. The crude
product is purified by column chromatography (2% MeOH/Chloroform,
Rf=0.5 in 5% MeOH/CHCl.sub.3) (1.75 g, 95%).
Succinic Acid
Mono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimet-
hyl-hexyl)-10,13-dimethyl
2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H
cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)
Ester AH
##STR00009##
[0253] Compound AG (1.0 g, 1.05 mmol) is mixed with succinic
anhydride (0.150 g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and
dried in a vacuum at 40.degree. C. overnight. The mixture is
dissolved in anhydrous dichloroethane (3 mL), triethylamine (0.318
g, 0.440 mL, 3.15 mmol) is added and the solution is stirred at
room temperature under argon atmosphere for 16 h. It is then
diluted with dichloromethane (40 mL) and washed with ice cold
aqueous citric acid (5 wt %, 30 mL) and water (2.times.20 mL). The
organic phase is dried over anhydrous sodium sulfate and
concentrated to dryness. The residue is used as such for the next
step.
[0254] Cholesterol Derivatised CPG AI
##STR00010##
[0255] Succinate AH (0.254 g, 0.242 mmol) is dissolved in a mixture
of dichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP
(0.0296 g, 0.242 mmol) in acetonitrile (1.25 mL),
2,2'-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) in
acetonitrile/dichloroethane (3:1, 1.25 mL) are added successively.
To the resulting solution triphenylphosphine (0.064 g, 0.242 mmol)
in acetonitrile (0.6 ml) is added. The reaction mixture turned
bright orange in color. The solution is agitated briefly using a
wrist-action shaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG)
(1.5 g, 61 mM) is added. The suspension is agitated for 2 h. The
CPG is filtered through a sintered funnel and washed with
acetonitrile, dichloromethane and ether successively. Unreacted
amino groups are masked using acetic anhydride/pyridine. The
achieved loading of the CPG is measured by taking UV measurement
(37 mM/g).
[0256] The synthesis of siRNAs bearing a 5'-12-dodecanoic acid
bisdecylamide group (herein referred to as "5'-C32-") or a
5'-cholesteryl derivative group (herein referred to as "5'-Chol-")
is performed as described in WO 2004/065601, except that, for the
cholesteryl derivative, the oxidation step is performed using the
Beaucage reagent in order to introduce a phosphorothioate linkage
at the 5'-end of the nucleic acid oligomer.
[0257] Nucleic acid sequences are represented below using standard
nomenclature, and specifically the abbreviations of Table 1.
TABLE-US-00001 TABLE 1 Abbreviations of nucleotide monomers used in
nucleic acid sequence representation. It will be understood that
these monomers, when present in an oligonucleotide, are mutually
linked by 5'-3'-phosphodiester bonds. Abbreviation Nucleotide(s) A
adenosine C cytidine G guanosine T thymidine U uridine N any
nucleotide (G, A, C, or T) a 2'-O-methyladenosine c
2'-O-methylcytidine g 2'-O-methylguanosine u 2'-O-methyluridine sT
phosphorothioate linkage
Example 2. .DELTA.EGFR Enhances Tumorigenicity of Wt EGFR
Over-Expressing Cells after Subcutaneous Injection into Nude
Mice
[0258] U87MG (U87Par) cells were engineered to over-express wtEGFR
(U87wt), mutant EGFR (de 2-7EGFR, deltaEGFR, .DELTA.EGFR or
EGFRvIII) (U87.DELTA.) or the kinase-deficient .DELTA.EGFR
(U87.DELTA.K). Cell lines that were mixed with U87wt were
engineered to express the LacZ gene and so could be distinguished
by X-Gal staining. Cell populations expressing equivalent levels of
over-expressed receptors were selected by FACS. 1.times.10.sup.6
U87wt cells (U87wt 100%), or these cells mixed with U87Par,
U87.DELTA.K or U87.DELTA. in ratios 90-10% or 99-1%, were injected
subcutaneously into the right flank of 4 to 5 weeks-old female
athymic nude mice and tumor volume was measured periodically. Tumor
growth kinetics (FIG. 1A) and tumor volume at the end of the
experiment (FIG. 1B) were measured.
[0259] A strong tumor growth enhancement was observed when U87wt
cells were co-injected with U87.DELTA. at either 90-10% or 99-1%
ratios. This tumor potentiation was not observed when U87wt cells
were injected together with U87Par or U87.DELTA.K, corroborating
our hypothesis that deltaEGFR induces enhanced proliferation, and
that the enhanced proliferation is dependent on the kinase
activation of deltaEGFR. Tumor volumes at day 21 after injection of
U87wt+U87delta cells were significantly bigger than the theoretical
volume of the sum of two different population volumes.
Example 3. deltaEGFR Enhanced Tumorigenicity of wtEGFR
Over-Expressing U87 (A) and Ink4/Arf.sup.-/- Murine Astrocyte Cells
(B)
[0260] 0.5.times.10.sup.6 cells (100%) were injected intracranially
into nude mice using a guide-screw system as described by Lal S. et
al. (J Neurosurg 92(2):326-333, 2000). Time matched mice were
sacrificed and brains were removed, fixed in 4% PFA and embedded in
OCT. H&E stain was performed on 6 um cryo-sections in order to
compare tumor size between U87wt, U87.DELTA. and mixtures 90-10% or
99-1% of U87wt and U87.DELTA., respectively. Similar to the results
in FIGS. 1A and 1B, a strong tumor growth potentiation was observed
when U87wt cells were mixed with U87.DELTA.. This effect was more
evident when injections were performed with 99% U87wt with 1%
U87.DELTA. (FIG. 2A). No tumor enhancement was observed when U87wt
cells were mixed with U87Par or U87.DELTA.K, confirming the role of
catalytically active .DELTA.EGFR in the tumorigenic growth
promotion of glioma cells expressing wtEGFR. These results
indicated that U87.DELTA. not only can enhance heterogeneous tumor
growth subcutaneously, but also intracranially.
[0261] To confirm that these results were not cell line specific,
the trans-proliferation model was further tested with murine
Ink4/Arf.sup.-/- astrocytes engineered to over-express wt and
deltaEGFR. Previously it had been shown that Ink4/Arf.sup.-/-
deltaEGFR astrocytes are tumorigenic upon intracranial injection in
nude mice. However Ink4/Arf.sup.-/-, wtEGFR astrocytes require the
introduction of EGF to elicit this effect (Bachoo et al., Cancer
Cell 1:269-77, 2002). It was therefore hypothesized that deltaEGFR
over-expressing astrocytes might be able to promote the
tumorigenicity of wtEGFR over-expressing astrocytes if the cells
were co-injected. In order to demonstrate the presence of wtEGFR
astrocytes within the tumor, these cells were tagged with nuclear
GFP and injected either alone or mixed with deltaEGFR
over-expressing astrocytes (FIG. 2B). As shown in the upper panel
of FIG. 2B, tumor size at day 22 after injection was significantly
bigger in mice co-injected with 90% wtEGFR and 10% deltaEGFR
Ink4/Arf.sup.-/- murine astrocytes than with 10% of deltaEGFR
Ink4/Arf.sup.-/- astrocytes alone. It was also confirmed that
wtEGFR astrocytes did not form tumors. These results not only
demonstrated that there was tumor growth potentiation when wtEGFR
astrocytes were mixed with deltaEGFR astrocytes, but also showed
the presence of wtEGFR astrocytes within the tumor by
immunofluorescence (GFP IF) (FIG. 2B, lower panel). Interestingly,
a small number of GFP positive cells were detected in mice injected
with wtEGFR astrocytes only, which may represent dormant cells that
could be activated upon activation of the receptor.
Example 4: Analysis of Tumor Composition by X-Gal Staining and Flow
Cytometry
[0262] To determine the relative cell type composition of the mixed
U87 xenografts, X-Gal staining was performed to detect LacZ-tagged
U87delta cells. Representative images of X-Gal stained tumors
obtained after injection of admixed U87wt and U87delta cells show a
significant number of U87delta cells (LacZ positive) among U87wt
cells (LacZ negative). Specifically, the large tumors that resulted
from an initial inoculum ratio of 90% U87wt combined with 10%
U87delta cells resulted in a final composition of 51.5% U87wt and
48.5% U87delta cells, while tumors from mice injected with 99%
U87wt plus 1% U87delta were composed of 58.7% U87wt and a 41.3%
U87delta cells. In contrast, the small tumors formed by the mixture
of U87wt with U87.DELTA.K or U87Parental cells were predominantly
composed of U87wt cells (FIG. 3A). Notably, the absolute tumor
volume attributable to U87 wt cells was approximately 3-10-fold
greater in tumors resulting from an injection of U87wt plus
U87delta cells than in tumors resulting from an injection of U87wt
cells alone, or U87wt with U87.DELTA.K or U87Parental cells,
demonstrating the dramatic growth effect of U87.DELTA. cells on
U87wt cells within the same tumor.
[0263] To more accurately quantify tumor composition, differential
fluorescence activated cell sorting analysis was performed on
single cell suspensions of subcutaneous xenografted mixed tumors
stained with two different antibodies against EGFR, one antibody
that recognizes the wt and mutant receptor (Ab-1) and another
antibody that only recognizes the wt receptor (Ab-5). Using this
approach, tumors generated 24 days post injection of 90% U87wt plus
10% U87delta cells were shown to consist of 52.8.+-.17.3%
(mean.+-.SD) wt and 47.2.+-.17.3% deltaEGF receptors (FIG. 3B),
confirming the LacZ staining results. This further illustrates that
DEGFR-expressing cells do not exert an overwhelmingly dominant
growth advantage in heterogeneous tumors containing amplified
levels of wtEGFR, but rather stimulate the latter to grow more
robustly.
Example 5: Treatment of U87wt Cells with U87delta Conditioned Media
(CM) Activates EGFR
[0264] To analyze the effect of deltaEGFR cells on wtEGFR
activation, conditioned media was collected from 48 h-starved
deltaEGFR cells and used to stimulate U87wt cells, also starved 48
h. Western blot analysis of EGFR activation and known signaling
molecules downstream of the receptor was performed on lysates of
U87wt cells stimulated for 15 minutes with serial dilutions of
U87delta CM, negative control U87Par CM or positive control EGF
ligand. Membranes were interrogated with anti-pTyr monoclonal
antibody (4G10) to check the activation of the EGFR, and with
phospho-specific antibodies directed to the major known
transduction proteins involved in tumorigenesis in GBMs
(gliobastoma multiformes): Akt, ERK1/2 (a.k.a., MAPK) and
STAT3.
[0265] FIG. 4 shows that these pathways were activated in response
to the U87.DELTA. CM stimulation, as shown by the increase of the
phosphorylated forms of those proteins in a dose-dependent manner.
In contrast, CM from U87Par cells failed to activate EGFR or any of
these pathways. Notably the level of phosphorylation of these
proteins, except EGFR, is at the same extent for the undiluted
conditioned medium and the high dose of EGF that was used,
indicating that activation of EGFR downstream signaling is
efficiently mimicked by factors secreted from U87.DELTA..
Activation of these pathways was also elicited in part by abundant
IL-6 produced and secreted by the deltaEGFR-expressing cells.
[0266] Similarly, wtEGFR phosphorylation was observed to be
significantly higher (p<0.05) in mixed tumors than in tumors
obtained after injection of U87wt alone (0.576.+-.0.166 vs
0.19.+-.0.007), while no differences in phosphorylation were
detected for .DELTA.EGFR in the mixed tumors compared to the tumors
generated from injection of U87.DELTA. alone (1.215.+-.0.225 vs
1.179.+-.0.260).
[0267] To determine whether the activation of these intracellular
pathways is dependent on the kinase activity of EGFR, the effect of
.DELTA. CM in the presence of the EGFR inhibitor AG1478 was
analyzed. Pre-treating U87wt cells with this inhibitor for 30
minutes prior to CM stimulation, completely abolished the
activation of EGFR. Moreover, it was also observed that activation
of Akt and ERK was maintained at the level of untreated cells.
Conversely, the activation of STAT3 caused by exposure to the
.DELTA. CM was not affected by blocking the activity of EGFR with
this inhibitor. These results suggested that at least two (or more)
soluble factors exist in the .DELTA. CM, producing different
effects on target U87wt cells: (i) activation of EGFR and its
intracellular signaling surrogates (Akt, ERK), via receptor kinase
activity, and (ii) STAT3 activation independent of EGFR
stimulation.
Example 6. wtEGFR Activation is not Caused by any of its Natural
Ligands
[0268] The results obtained with .DELTA. CM would suggested that
U87.DELTA. cells secrete one or more factors responsible for EGFR
activation in wtEGFR cells. In order to test whether these were
known ligands for the EGFR, neutralization using an EGFR ligand
trap was performed. This recombinant ligand-binding protein
consisted of the extracellular portion of the EGFR bound to a human
Fc fragment and bound the known EGFR ligands with high affinity.
Pre-incubation of recombinant EGF with 10 .mu.g/ml of the ligand
trap reduced the activation of EGFR as well as Akt and ERK,
however, no effect on either EGFR or these two downstream pathways
was observed when .DELTA. CM was pretreated with the ligand trap.
As expected, STAT3 activation was not affected in the presence of
the ligand trap. To further demonstrate that EGFR ligands were not
involved in the transactivation of U87wt by .DELTA. CM, ELISA was
used to quantify the concentrations of EGF, TGF-.alpha.,
amphiregulin, HB-EGF and betacellulin in U87.DELTA. CM as well as
in control U87Parental, U87wt and U87.DELTA.K CM. All of these
tested EGFR ligands were either undetectable or showed no
significant increase in expression in .DELTA. CM when compared to
the conditioned media from the other cell lines. Given the
possibility that active soluble EGFR ligands may be released from
the surface of the target cells by proteolytic cleavage of
membrane-anchored precursors (Sanderson et al., 2006), .DELTA. CM
was tested for the ability to induce the release of EGFR ligands
expressed on the surface of U87wt cells. U87wt cells were
stimulated with serum-free medium, recombinant EGF and U87.DELTA.
CM, and then these media were collected to analyze by ELISA changes
in EGFR ligand concentration after the stimulation. None of the
tested ligands showed any significant change in concentration,
indicating that EGFR is not stimulated by soluble factors shed from
the membrane of wtEGFR cells. Confirmation of these results was
achieved by incubation of .DELTA. CM with neutralizing antibodies
against each EGFR ligand; these also failed to block the ability of
the CM to activate EGFR, while each antibody tested was able to
block the activity of the respective recombinant ligand. In
summary, U87.DELTA. cells do not produce a detectable EGF family
ligand activity, pointing to other factors driving inter-cellular
activation of wtEGFR.
Example 7. IL-6 is Over-Expressed in U87delta Cells and in
deltaEGFR-Positive GBM Clinical Samples
[0269] To identify soluble factors expressed by U87.DELTA. cells
that could potentially mediate intercellular communication with and
promote the proliferation of U87wt cells, a cytokine array was used
to qualitatively detect 79 human cytokines and growth factors in
supernatants of cultured cells. With this approach, IL-6 was found
to be significantly upregulated in U87.DELTA. cells compared to the
other U87 cell lines. To further quantify IL-6 upregulation in
U87.DELTA. CM, an ELISA assay was performed on supernatants from
the different U87 cell lines collected after 48 hours starvation.
The values obtained (pg/ml/4.times.105 cells) illustrate a 13-fold
increase of IL-6 secretion for U87.DELTA. (3813.+-.2) compared to
U87Parental cells (299.+-.25), while no significant increase was
detected with U87wt (567.+-.85) or U87.DELTA.K (355.+-.75) CM.
[0270] Nineteen GBM tumor samples, U87 cell lines and one normal
brain sample were analyzed for .DELTA.EGFR and IL-6 RNA expression
by real time PCR. As expected, U87.DELTA. demonstrated a
significant higher (p<0.001) expression of IL-6, while no
significant differences in IL-6 expression were observed between
the rest of U87 cell lines. Notably, we observed a very significant
correlation between .DELTA.EGFR and IL-6 expression (p=0.0034) in
the GBM tumor samples. All tumor samples that presented .DELTA.EGFR
expression also showed high IL-6 expression (8/8), while only three
tumors that did not show .DELTA.EGFR expression over-expressed IL-6
(3/11).
Example 8. siRNAs Specific for deltaEGFR or wtEGFR Reduced Tumor
Growth after Subcutaneous Injection of Ex Vivo Transfected
Cells
[0271] The present inventors observed that in vitro treatment of
cells expressing wtEGFR with conditioned media from cells
overexpressing deltaEGFR resulted in activation of STATS, Akt,
Erk1/2 and wtEGFR. In vivo tumor growth potentiation was also
observed when wtEGFR overexpressing cells were mixed with deltaEGFR
expressing cells, but not when those cells were mixed with cells
with normal levels of wtEGFR or overexpressing a dead kinase
version of deltaEGFR. Based on these observations, siRNA technology
was used to knock-down either wt or deltaEGFR to assess the effect
of specific receptor ablation on tumorigenicity and contribution to
heterogeneity. As shown below, siRNAs specific for deltaEGFR or
wtEGFR were able to reduce tumor growth after subcutaneous
injection of ex vivo transfected cells.
[0272] In Vitro siRNA Screening:
[0273] siRNAs non-stabilized (Table 2) and stabilized (Table 3),
designed to be specific (i) to wtEGFR, (ii) to deltaEGFR, and (iii)
to both receptors, were synthesized. U87-wtEGFR (Nagane et al,
Cancer Research, 56: 5079-5086, 1996) and U87-deltaEGFR (Nishikawa
et al, PNAS, 91: 7727-7731, 1994) cells were used as test cell
lines to assess the specificity of these siRNA molecules. The term
"deltaEGFR," as used in this example, refers to an EGFR gene
construct deleted for exons 2-7. U87-deltaEGFR cells are
recombinant cells expressing an EGFR gene deleted for exons 2-7 as
described in Nishikawa et al. (1994). U87-wtEGFR cells are
recombinant cells expressing a wtEGFR gene as described in
Nishikawa et al. (1994).
[0274] Cells were seeded in 24 well plates at 48,000 cells per well
in DMEM medium (Cellgro) supplemented with 10% fetal bovine serum
and L-Glutamine. The following day, siRNAs were transfected at 100
nM, 10 nM and 1 nM concentrations using Lipofectamine.TM. 2000
(Invitrogen) and OptiMEM (Gibco). Twenty four hours after
transfection, the medium was changed to DMEM supplemented with 10%
fetal bovine serum, penicillin/streptomycin and L-Glutamine.
[0275] Cells non-transfected as well as transfected with a siRNA
specific for GFP or Luciferase protein were included as negative
controls.
[0276] Three days after transfection, protein lysates were prepared
using RIPA buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.5, 1 mM EDTA,
1% NP-40, 0.1% SDS and 0.5% sodium deoxycholate) supplemented with
protease inhibitors (Roche) and 5 .mu.g of protein were resolved on
12% NuPAGE Bis-Tris acrylamide gels (Invitrogen). Gels were blotted
onto nitrocellulose membranes, blocked with 5% milk in TBS-Tween
and probed with anti-EGFR monoclonal antibody, C13, which
recognizes both wt and mutant EGFR (BD). Membrane bound C13
antibody was detected with HRP-conjugated anti-mouse IgG (Dako)
followed by chemiluminiscence. Nitrocellulose membranes were also
probed with anti-actin antibody as a positive control for protein
loading.
[0277] In Vitro Specificity Test:
[0278] Specificity for the receptor for which each siRNA candidate
was designed was assessed by transfecting siRNAs at 100 nM dose in
the U87 cell line that expresses the other receptor. Expression
knock-down was assessed by western blot as previously
described.
[0279] To exclude that any reduction in the cytokine synthesis was
caused by off-target effects of the siRNAs, the concentration of
both IL-6 and IL-8 was measured in the samples where an siRNA was
found to have a strong effect on the expression of IL-6, and
compared to non-transfected cells as well as cells transfected with
an siRNA against GFP or Luciferase
[0280] Cells non-transfected as well as transfected with an siRNA
against GFP or Luciferase protein were included as negative
controls.
[0281] In Vitro Dose-Response Analysis:
[0282] siRNAs determined to be specific and able to knock-down the
expression of the receptor for which they were designed were tested
again in U87-deltaEGFR and U87-wtEGFR to determine the minimal
effective dose to achieve receptor expression knock-down. Cells
were seeded in 24 well plates at 48,000 cells per well and siRNAs
were transfected the following day at 100, 25, 5 and 1 nM
concentrations using Lipofectamine.TM. 2000. Twenty four hours
after transfection, medium was changed and two days after protein
lysates were obtained as described previously. Cells
non-transfected as well as transfected with a siRNA against GFP or
Luciferase protein were included as negative controls. Receptor
expression was analyzed by western blot as described
previously.
[0283] In Vitro Durability Test:
[0284] siRNAs determined to be robust in the ablation of deltaEGFR
or wtEGFR expression, when transfected at low concentration, were
tested for suppression durability using U87-deltaEGFR and
U87-wtEGFR cell lines.
[0285] Cells were seeded in 24 well plates at 48,000 cells per well
and siRNAs were transfected the following day at 25 nM
concentration using Lipofectamine.TM. 2000. Protein lysates were
obtained using RIPA buffer supplemented with protease inhibitors at
days 3, 5, 7, 10, 14 and 17 post-transfection. Cells
non-transfected as well as transfected with a siRNA against GFP or
Luciferase protein were included as negative controls. Receptor
expression was analyzed by western blot as described
previously.
[0286] Ex Vivo Experiments:
[0287] To determine the effect of specific receptor knock-down on
tumorigenicity, cells were transfected and then injected
subcutaneously into nude mice.
[0288] Briefly, 25 cm.sup.2 plates were seeded with
1.3.times.10.sup.6 cells and one day after they were transfected
with 25 nM or 100 nM siRNA using Lipofectamine.TM. 2000. The
following day, the medium was changed and cells were split into
larger dishes when they were almost confluent.
[0289] Cells non-transfected as well as transfected with a siRNA
against GFP or Luciferase protein were included as negative
controls.
[0290] One million U87-wtEGFR or 0.5.times.10.sup.6 U87-deltaEGFR
transfected cells were resuspended in 100 .mu.l PBS. Cells were
injected subcutaneously into the right flank of Nu/Nu mice using a
1 ml syringe with a 26G needle. Tumor volume was measured starting
at day 5 after injection and was calculated using the formula
0.5.times.L.times.W.sup.2. Mice were euthanized when tumor volume
reached 1500 mm.sup.3.
[0291] Protein lysates were prepared from the remaining cells of
the injection using RIPA buffer supplemented with protease
inhibitors. Receptor expression was analyzed by western blot as
described previously.
[0292] Results
[0293] In Vitro siRNA Screening (Table 4):
[0294] Non-Stabilized siRNAs:
[0295] DeltaEGFR-Specific siRNAs:
[0296] Western blot analysis demonstrated that 7 of 8
deltaEGFR-specific siRNAs were capable of complete elimination of
the mutant receptor expressed in U87-deltaEGFR cells when tested at
100 nM (AL-DP-6901-6907) while three (AL-DP-6902, -6903, -6906)
were able to effect a modest receptor reduction as low as 1 nM
concentration (Table 4 and FIG. 4A). One siRNA, AL-DP-6908, had no
effect. To show specificity for the mutant receptor, each
deltaEGFR-specific siRNA was tested against U87-wtEGFR cells. Only
one of the siRNAs (AL-DP-6906) that reduced deltaEGFR expression
was also able to reduce wtEGFR expression.
[0297] wtEGFR-Specific siRNAs:
[0298] As above, western blot analysis was used to demonstrate that
5 of 8 wtEGFR-specific siRNAs were capable of complete elimination
of the wt receptor expressed in U87-wtEGFR cells when tested at 100
nM (AL-DP-6918-6921 and -6923), while two (AL-DP-6919 and -6920)
were able to effect a modest receptor reduction as low as 1 nM
concentration (Table 4 and FIG. 5B). Three siRNAs (AL-DP-6917,
-6922, and -6924) had little or no effect. To show specificity for
the wt receptor, each active wtEGFR-specific siRNA (AL-DP-6918-6921
and -6923) was tested against U87-deltaEGFR cells with none showing
reduction of the mutant receptor.
[0299] Wt and deltaEGFR-Specific siRNAs:
[0300] As above, western blot analysis was used to demonstrate the
specificity of 8 siRNAs designed to knock-down the expression of
both wt and deltaEGFR. Of this series, three siRNAs were able to
simultaneously knock-down both receptors (AL-DP-6913, -6915, and
-6916) albeit the effect was stronger for suppressing deltaEGFR
(Table 4 and FIG. 5C).
[0301] In FIGS. 5A-5C, two samples were run for each treatment
condition. Membranes were blotted with c13 antibody to detect EGFR
and beta-actin antibody to confirm equivalent loading between
lanes.
[0302] Stabilized siRNAs:
[0303] DeltaEGFR-Specific siRNAs:
[0304] Western blot analysis demonstrated that 8 of 8
deltaEGFR-specific siRNAs were capable of reducing mutant receptor
expression in U87-deltaEGFR cells when tested at 25 nM (Table 4 and
FIGS. 6A-B). To show specificity for the mutant receptor,
deltaEGFR-specific siRNAs (AD-15416, AD-15417, AD-13375, AD-15418)
were tested against U87-wtEGFR cells. None of siRNAs tested
resulted in reduced wtEGFR expression.
[0305] wtEGFR-Specific siRNAs:
[0306] Western blot analysis demonstrated that 4 of 8
wtEGFR-specific siRNAs strongly suppress wtEGFR protein expression
(AD-16177, AD-16178, AD-16179 and AD-13377), while 2 of these 8
siRNAs were able to moderately reduce wtEGFR protein expression
(AD-13376 and AD-13378) (Table 4 and FIG. 6C). Two of the 8 siRNAs
had little or no effect on wtEGFR protein levels (AD-16180 and
AD-16181).
[0307] In FIGS. 6A-6C, two samples were run for each treatment
condition. Membranes were blotted with c13 antibody to detect EGFR
and beta-actin antibody to confirm equivalent loading between
lanes.
[0308] In Vitro Dose-Response Analysis:
[0309] Non-Stabilized siRNAs:
[0310] DeltaEGFR-Specific siRNAs:
[0311] Western blot analysis demonstrate that the four
deltaEGFR-specific siRNAs tested (AL-DP-6901-6903, and -6905) were
capable of complete elimination of the mutant receptor expressed in
U87-deltaEGFR cells when transfected at 25 nM, nearly complete
elimination at 5 nM and partial elimination at 1 nM (FIG. 7A).
[0312] wtEGFR-Specific siRNAs:
[0313] As above, western blot analysis was used to demonstrate that
4 of 4 wtEGFR-specific siRNAs (AL-DP-6918-6921) were capable to
varying degrees of wt receptor elimination in U87-wtEGFR cells
(FIG. 7B).
[0314] Wt and deltaEGFR-Specific siRNAs:
[0315] AL-DP-6913 and AL-DP-6916 were able to suppress expression
of both wt and deltaEGFR protein in a dose-dependent manner (FIGS.
7A-B).
[0316] In FIGS. 7A and 7B, two samples were run for each treatment
condition. Membranes were blotted with c13 antibody to detect
EGFR.
[0317] In Vitro Durability Test:
[0318] Non-Stabilized siRNAs:
[0319] DeltaEGFR-Specific siRNAs:
[0320] Western blot analysis demonstrated that 4 of 4
deltaEGFR-specific siRNAs analyzed (AL-DP-6901, AL-DP-6902,
AL-DP-6903, AL-DP-6905) were capable of complete elimination of
mutant receptor expression in U87-deltaEGFR cells at 7 days after
siRNA transfection, while siRNA AL-DP-6905 was capable of durable
suppression as far as 10 days after siRNA transfection (FIG.
8A).
[0321] wtEGFR-Specific siRNAs:
[0322] As above, western blot analysis was used to demonstrate that
2 of 2 wtEGFR-specific siRNAs analyzed (AL-DP-6920, AL-DP-6921)
were capable of complete wt receptor expression elimination in
U87-wtEGFR cells out to 7 days after siRNA transfection; however,
for both siRNAs, receptor levels were restored to control levels by
10 days post transfection (FIG. 8B).
[0323] Stabilized siRNAs:
[0324] DeltaEGFR-Specific siRNAs:
[0325] Western blot analysis demonstrated that 3 of 5
deltaEGFR-specific siRNAs analyzed (AD-15416, AD15417, AD-13374,
AD-13375, AD-15418) were capable of complete elimination of mutant
receptor expression in U87-deltaEGFR cells 5 days post
transfection, while siRNA AD-15416 was capable of durable and
complete suppression as far as 7 days post transfection (FIG. 8C).
Receptor expression levels were completely restored by day 12 post
transfection for all stabilized siRNAs tested.
[0326] wtEGFR-Specific siRNAs:
[0327] As above, western blot analysis was used to demonstrate that
AD-13377 wtEGFR-specific siRNA was capable of complete wt receptor
protein elimination in U87-wtEGFR cells out to 7 days after
transfection (FIG. 8D). However, receptor levels were restored to
control levels by 10 days post transfection.
[0328] Ex Vivo Experiments:
[0329] Non-Stabilized siRNAs:
[0330] DeltaEGFR-Specific siRNAs:
[0331] U87-deltaEGFR cells were transfected with 100 nM siRNA and
injected subcutaneously into nude mice. Treatment with siRNAs
AL-DP-6901 and AL-DP-6902 (FIG. 9B) resulted in a substantial
reduction of U87-deltaEGFR tumor growth. As a negative control,
cells non transfected or transfected with an irrelevant GFP siRNA
were included in the study (FIG. 9B). In both of these groups,
substantial tumor growth occurred.
[0332] wtEGFR-Specific siRNAs:
[0333] U87-wtEGFR cells were transfected with 25 nM siRNA
concentration and injected subcutaneously into nude mice. Treatment
with siRNAs AL-DP-6920 and AL-DP-6921 (FIG. 9A) resulted in
ablation of U87-wtEGFR tumor growth. As a negative control, cells
non transfected or transfected with an irrelevant GFP siRNA were
included in the study (FIG. 9A). In both of these groups,
substantial tumor growth occurred.
[0334] In FIGS. 9A and 9B, each treatment group included six
animals.
[0335] U87delta cells were injected into nude mice and at days 13
(approximately 160 mm.sup.3) and 16, 5 mg of deltaEGFR siRNA#1 or
irrelevant siRNA (siRNA luc) were administrated intratumorally
using JetPei (Polyplus) following the manufacturer's instructions
followed by the monitoring of tumor growth as above. These initial
results illustrate modest tumor reduction can be achieved in vivo
with siRNAs targeting deltaEGFR FIGS. 10A and 10B.
TABLE-US-00002 TABLE 2 Sequences of dsRNAs tested for deltaEGFR and
wtEGFR gene expression inhibiting activity Target position of 5'
base of sense strand (see FIGS. 21A SEQ SEQ Duplex and ID Sense
strand sequence ID Antisense strand name 21B) Specificity NO:
(5'-3') NO: sequence (5'-3') AL-DP-6901 320 deltaEGFR 2
UGGAGGAAAAGAAAGGUAATT 3 UUACCUUUCUUUUCCUCCATT AL-DP-6902 321
deltaEGFR 4 GGAGGAAAAGAAAGGUAAUTT 5 AUUACCUUUCUUUUCCUCCTT
AL-DP-6903 322 deltaEGFR 6 GAGGAAAAGAAAGGUAAUUTT 7
AAUUACCUUUCUUUUCCUCTT AL-DP-6904 319 deltaEGFR 8
CUGGAGGAAAAGAAAGGUATT 9 UACCUUUCUUUUCCUCCAGTT AL-DP-6905 323
deltaEGFR 10 AGGAAAAGAAAGGUAAUUATT 11 UAAUUACCUUUCUUUUCCUTT
AL-DP-6906 324 deltaEGFR 12 GGAAAAGAAAGGUAAUUAUTT 13
AUAAUUACCUUUCUUUUCCTT AL-DP-6907 330 deltaEGFR 14
GAAAGGUAAUUAUGUGGUGTT 15 CACCACAUAAUUACCUUUCTT AL-DP-6908 329
deltaEGFR 16 AGAAAGGUAAUUAUGUGGUTT 17 ACCACAUAAUUACCUUUCUTT
AL-DP-6909 40 deltaEGFR 18 ACGGUGUGAGCGCCCGACGTT 19
CGUCGGGCGCUCACACCGUTT and wtEGFR AL-DP-6910 1150 deltaEGFR 20
ACAGAUCACGGCUCGUGCGTT 21 CGCACGAGCCGUGAUCUGUTT and wtEGFR
AL-DP-6911 1156 deltaEGFR 22 CACGGCUCGUGCGUCCGAGTT 23
CUCGGACGCACGAGCCGUGTT and wtEGFR AL-DP-6912 117 deltaEGFR 24
CGACAGGCCACCUCGUCGGTT 25 CCGACGAGGUGGCCUGUCGTT and wtEGFR
AL-DP-6913 1147 deltaEGFR 26 GUGACAGAUCACGGCUCGUTT 27
ACGAGCCGUGAUCUGUCACTT and wtEGFR AL-DP-6914 129 deltaEGFR 28
UCGUCGGCGUCCGCCCGAGTT 29 CUCGGGCGGACGCCGACGATT and wtEGFR
AL-DP-6915 197 deltaEGFR 30 CCGUCCAGUAUUGAUCGGGTT 31
CCCGAUCAAUACUGGACGGTT and wtEGFR AL-DP-6916 1146 deltaEGFR 32
GGUGACAGAUCACGGCUCGTT 33 CGAGCCGUGAUCUGUCACCTT and wtEGFR
AL-DP-6917 997 wtEGFR 34 UGCCGCAAAUUCCGAGACGTT 35
CGUCUCGGAAUUUGCGGCATT AL-DP-6918 683 wtEGFR 36
GCGCCGUGCGGUUCAGCAATT 37 UUGCUGAACCGCACGGCGCTT AL-DP-6919 999
wtEGFR 38 CCGCAAAUUCCGAGACGAATT 39 UUCGUCUCGGAAUUUGCGGTT AL-DP-6920
337 wtEGFR 40 UGCCAAGGCACGAGUAACATT 41 UGUUACUCGUGCCUUGGCATT
AL-DP-6921 569 wtEGFR 42 GAGGAAAUAUGUACUACGATT 43
UCGUAGUACAUAUUUCCUCTT AL-DP-6922 668 wtEGFR 44
AGGAAAUCCUGCAUGGCGCTT 45 GCGCCAUGCAGGAUUUCCUTT AL-DP-6923 677
wtEGFR 46 UGCAUGGCGCCGUGCGGUUTT 47 AACCGCACGGCGCCAUGCATT AL-DP-6924
732 wtEGFR 48 CCAGUGGCGGGACAUAGUCTT 49 GACUAUGUCCCGCCACUGGTT
TABLE-US-00003 TABLE 3 Sequences of dsRNAs with stabilizing
modifications tested for deltaEGFR and wtEGFR gene expression
inhibiting activity Target position of 5' base of sense strand (see
FIGS. SEQ SEQ Duplex 21A and ID Sense strand sequence ID Antisense
strand name 21B) NO: (5'-3') NO: sequence (5'-3') AD- 320 50
uGGAGGAAAAGAAAGGuAATsT 51 UuACCUUUCUUUUCCUCcATsT 15416 AD- 321 52
GGAGGAAAAGAAAGGuAAuTsT 53 AUuACCUUUCUUUUCCUCCTsT 15417 AD- 322 54
GAGGAAAAGAAAGGuAAuuTsT 55 AAUuACCUUUCUUUUCCUCTsT 13373 AD- 319 56
cuGGAGGAAAAGAAAGGuATsT 57 uACCUUUCUUUUCCUCcAGTsT 13374 AD- 323 58
AGGAAAAGAAAGGuAAuuATsT 59 uAAUuACCUUUCUUUUCCUTsT 13375 AD- 324 60
GGAAAAGAAAGGuAAuuAuTsT 61 AuAAUuACCUUUCUUUUCCTsT 15418 AD- 325 62
GAAAGGuAAuuAuGuGGuGTsT 63 cACcAcAuAAUuACCUUUCTsT 15419 AD- 329 64
AGAAAGGuAAuuAuGuGGuTsT 65 ACcAcAuAAUuACCUUUCUTsT 15420 AD- 997 66
uGccGcAAAuuccGAGAcGTsT 67 CGUCUCGGAAUUUGCGGcATsT 16177 AD- 683 68
GcGccGuGcGGuucAGcAATsT 69 UUGCUGAACCGcACGGCGCTsT 13376 AD- 999 70
ccGcAAAuuccGAGAcGAATsT 71 UUCGUCUCGGAAUUuGCGGTsT 16178 AD- 337 72
uGccAAGGcAcGAGuAAcATsT 73 UGUuACUCGUGCCUUGGcATsT 16179 AD- 569 74
GAGGAAAuAuGuAcuAcGATsT 75 UCGuAGuAcAuAUUUCCUCTsT 13377 AD- 668 76
AGGAAAuccuGcAuGGcGcTsT 77 GCGCcAUGcAGGAUUUCCUTsT 16180 AD- 677 78
uGcAuGGcGccGuGcGGuuTsT 79 AACCGcACGGCGCcAUGcATsT 13378 AD- 732 80
ccAGuGGcGGGAcAuAGucTsT 81 GACuAUGUCCCGCcACUGGTsT 16181
TABLE-US-00004 TABLE 4 Summary of the western blot results of the
knock-down of delta or wtEGFR expression after transfection of
non-stabilized or stabilized siRNAs. Unmodified U87- U87-
Durability Modified U87- U87- Durability siRNA Specificity
deltaEGFR wtEGFR (days) siRNA deltaEGFR wtEGFR (days) AL-DP-6901
deltaEGFR + - 7-10 AD-15416 + - 7-12 AL-DP-6902 deltaEGFR + - 7-10
AD-15417 + - 7-12 AL-DP-6903 deltaEGFR + - 7-10 AD-13373 + x x
AL-DP-6904 deltaEGFR + - x AD-13374 + x 5-7 AL-DP-6905 deltaEGFR +
- 10-14 AD-13375 + - 5-7 AL-DP-6906 deltaEGFR + + x AD-15418 + -
7-12 AL-DP-6907 deltaEGFR + x x AD-15419 + x x AL-DP-6908 deltaEGFR
- x x AD-15420 + x x AL-DP-6909 both +/- + x none na na na
AL-DP-6910 both + - x none na na na AL-DP-6911 both - - x none na
na na AL-DP-6912 both - - x none na na na AL-DP-6913 both + + 7-10
none na na na AL-DP-6914 both - - x none na na na AL-DP-6915 both +
+ x none na na na AL-DP-6916 both + + 7-10 none na na na AL-DP-6917
wtEGFR x +/- x AD-16177 - +/- x AL-DP-6918 wtEGFR - + x AD-13376 x
+/- x AL-DP-6919 wtEGFR - + x AD-16178 - + x AL-DP-6920 wtEGFR - +
7-10 AD-16179 - + x AL-DP-6921 wtEGFR - + 7-10 AD-13377 - +
.gtoreq.7 AL-DP-6922 wtEGFR x - x AD-16180 x - x AL-DP-6923 wtEGFR
- + x AD-13378 x - x AL-DP-6924 wtEGFR x - x AD-16181 x - x - = no
reduction of expression + = reduction of expression +/- = small
reduction of expression x = not determined na = not available
Example 9. siRNAs Specific for IL-6 Reduced Tumor Growth In Vivo
after Subcutaneous Injection of U87.DELTA. Cells
[0336] Methods
[0337] In vitro siRNA screening: U87-.DELTA.EGFR (Nishikawa et al,
PNAS 91: 7727-7731, 1994) cells over-express the IL-6 cytokine, and
the importance of IL-6 secretion was underscored by demonstrating
that the in vivo growth of wtEGFR-expressing cells could be
enhanced when mixed with parental glioma cells engineered to
overexpress IL-6 (see Example 10 below).
[0338] To further test the role of IL-6 in enhanced cell
proliferation, 24 stabilized siRNAs were designed to be specific to
IL-6 (AD-15637 to AD-15660) and were synthesized. The sequences of
the siRNAs and their target position on the IL-6 mRNA (GenBank
Accession No. NM_000600.2, version Jan. 4, 2009) (FIG. 11) are
provided in Table 5.
[0339] Cells were seeded in 24 well plates at 48,000 cells per well
in DMEM medium (Cellgro) supplemented with 10% fetal bovine serum
and L-Glutamine. The following day, siRNAs were transfected at 100
nM using Lipofectamine.TM. 2000 (Invitrogen.TM.) and Opti-MEM.RTM.
(Gibco.TM.). Cells non-transfected as well as transfected with an
siRNA specific for GFP or Luciferase were included as negative
controls. Forty-eight hours after transfection, the medium was
changed to DMEM-serum-free supplemented with
penicillin/streptomycin and L-Glutamine after washing the cells
with serum-free medium. After twenty-four hours of
serum-starvation, supernatants were collected, centrifuged to
remove cell debris, and either analyzed immediately or frozen at
-80.degree. C. Quantification of IL-6/IL-8 in supernatants was
assessed by ELISA. Briefly, 96-well plates (MaxiSorp, Nunc) were
coated overnight at room temperature with the capture antibody
diluted in PBS. The following day, the plates were blocked in
blocking buffer composed of PBS containing 1% BSA and 5% sucrose.
The standards (recombinant human IL-6 and IL-8) and the samples
were diluted in diluent buffer (1.lamda.TBS, 0.5% BSA, 0.05%
Tween-20) and incubated 2 hours at room temperature. The plates
were then washed with PBS 0.05% Tween-20 and incubated with the
biotinylated detection antibody and then with streptavidin-HRP
(Biosource) both diluted in diluent buffer. The HRP activity was
determined by using Tetramethylbenzidine (Sigma) as substrate. The
enzymatic reaction was stopped with 1 N sulfuric acid and the
absorbance was measured at 450 nm with wavelength correction set to
540 nm using a Tecan Genios Pro microplate reader. The absorbance
readings were converted using a four parameter logistic curve.
[0340] In Vitro Specificity Test:
[0341] To exclude that any reduction in the cytokine synthesis was
caused by off-target effects of the siRNAs, the concentration of
both IL-6 and IL-8 was measured in the samples where an siRNA was
found to have a strong effect on the expression of IL-6, and
compared to non-transfected cells as well as cells transfected with
an siRNA against GFP or Luciferase.
[0342] In Vitro Dose-Response Analysis:
[0343] siRNAs determined to be specifically able to knock-down the
secretion of IL-6 were tested again in U87-.DELTA.EGFR to determine
the minimal effective dose to achieve cytokine secretion
knock-down. Cells were seeded in 24 well plates at 48,000 cells per
well and siRNAs were transfected the following day at 100, 20, 4
and 0.8 nM concentrations using Lipofectamine.TM. 2000. Forty-eight
hours after transfection the medium was changed to serum-free
medium and twenty-four hours later the supernatants were collected
and centrifuged as described previously. Cells non-transfected as
well as transfected with an siRNA against GFP or Luciferase were
included as negative controls. IL-6 or IL-8 secretion was analyzed
by ELISA.
[0344] In Vitro Durability Test:
[0345] siRNAs determined to be robust in the ablation of IL-6
secretion, when transfected at low concentration, were tested for
suppression durability using U87-.DELTA.EGFR cells. Cells were
seeded in 24 well plates at 48,000 cells per well and siRNAs were
transfected the following day at 25 nM concentration using
Lipofectamine.TM. 2000. Supernatants were collected at days 3, 7,
and 11 or 14 days post-transfection (in each case after 24 hours
serum-starvation). Cells non-transfected as well as transfected
with an siRNA against GFP or Luciferase were included as negative
controls. IL-6 secretion was analyzed by ELISA.
[0346] In Vitro Proliferation Test:
[0347] Cells were seeded in 24 well plates at 48,000 cells per well
and siRNAs were transfected the following day at 25 nM
concentration using Lipofectamine.TM. 2000. 48 hours after
transfection the cells were trypsinized, counted, and seeded at the
same density in larger dishes to allow them to grow. The cell
proliferation was evaluated by counting the cells at day 4 and at
day 6-8 as indicated. Three independent samples were counted for
each treatment/time point.
[0348] Ex Vivo Experiments:
[0349] To determine the effect of specific IL-6 knock-down on
tumorigenicity, U87-.DELTA.EGFR cells were transfected with the
different siRNAs (AD-15644 and AD-15660) and then injected
subcutaneously into nude mice. Briefly, 1.3.times.10.sup.6 cells
were seeded in 10 cm plates and one day after they were transfected
either with a control siRNA or with a specific siRNA at the
concentration of 25 nM with Lipofectamine.TM. 2000. The following
day, the medium was changed and cells were split into larger
dishes. Transfected U87-.DELTA.EGFR cells were injected
subcutaneously into the right flank of Nu/Nu mice using a 1 ml
syringe with a 26G needle. 5.times.10.sup.5 cells resuspended in
100 .mu.l of PBS were injected into each mouse. Tumor volume was
measured starting at day 5 after injection and was calculated using
the formula 0.5.times.L.times.W.sup.2. Mice were euthanized
according to our animal protocol when tumor volume reached 1500
mm.sup.3.
[0350] In Vivo siRNA Delivery:
[0351] One million cells of U87-wtEGFR mixed with U87-.DELTA.EGFR
in a ratio 90:10% respectively were injected subcutaneously into 4
to 5 weeks-old female nude mice. Treatment of tumors was started
after 13 days when the tumor volume reached approximately 80 mm3.
JetPEI/siRNA complexes were prepared following manufacturer
instructions (Polyplus Transfection, Illkirch, France) in 5%
Glucose at N/P ratio of 15 and 70 .mu.l of the complex was injected
intratumorally at a dose of 10 .mu.g siRNA/mouse every two days.
Tumor volumes were measured every second day from the commencement
of siRNA delivery through day 21 of treatment.
[0352] Results
[0353] In Vitro siRNA Screening (Table 9):
[0354] ELISA analysis demonstrated that 20 of 24 siRNAs designed
against IL-6 were capable of reducing IL-6 secretion in
U87-.DELTA.EGFR cells when tested at 100 nM (Table 9, and FIGS. 12A
and 12B). Only siRNAs AD-15647, AD-15651, AD-15654, and AD-15656
were unable to reduce IL-6 secretion in U87-.DELTA.EGFR cells (FIG.
12A). The strongest effect was obtained with AD-15658 (99.09%), and
the weakest with AD-15642 (34.1%), with an average 82.4% reduction
compared with cells transfected with the control siRNA. IL-8
concentration was tested in the 15 samples (FIG. 12B) where
strongest reduction of IL-6 secretion was observed. IL-8 production
was significantly reduced in 11 of the analyzed samples. In one
case (AD-15641) there was a significant increase in IL-8 secretion.
Two siRNAs designed against IL-6 (AD-15644, AD-15650), showed no
significant effect on the IL-8 secretion in U87-.DELTA.EGFR cells
demonstrating specificity for IL-6. AD-15660 caused a moderate
increase in IL-8 production.
[0355] In Vitro Dose-Response Analysis:
[0356] The five siRNAs that showed the least non-specific effects
were selected, transfected into U87-.DELTA.EGFR cells in serial 1:5
dilutions starting at 100 nM and compared to a control siRNA at 100
nM.
[0357] ELISA analysis demonstrated that the 5 IL-6-specific siRNAs
tested (AD-15644, -15649, -15650, -15657 and -15660) reduced
significantly the secretion of IL-6 when transfected into
U87-.DELTA.EGFR cells at doses as low as 0.8 nM (FIG. 13A). The
concentration of IL-8 was also measured in these samples (FIG. 13B)
showing that siRNAs AD-15650 and AD-15660 caused the least
significant reduction in IL-8 secretion when compared to control
siRNA.
[0358] In Vitro Durability Test:
[0359] ELISA analysis demonstrated that two out of four
IL-6-specific siRNAs analyzed (AD-15644 and AD-15660) were capable
of maximally reducing IL-6 secretion in U87-.DELTA.EGFR cells as
far as day 7 after siRNA transfection. At day 14 these two siRNAs
were still able to suppress IL-6 expression. siRNA AD-15650 was
capable of suppression of IL-6 secretion only until day three, and
AD-15657 only until day 7 (FIG. 14).
[0360] In Vitro Proliferation Test:
[0361] The proliferation of U87-.DELTA.EGFR transfected with siRNAs
AD-15644, -15650, and -15660 was monitored in vitro for 6 days, and
compared with cells non transfected or transfected with a control
siRNA. Only AD-15644 and AD-15660 had no effect on cell
proliferation, while AD-15650 almost completely blocked cell
proliferation in vitro.
[0362] Ex Vivo Experiments:
[0363] U87-.DELTA.EGFR cells were transfected with 25 nM siRNA and
injected subcutaneously into nude mice. Treatment with siRNAs
AD-15644 and AD-15660 resulted in no substantial reduction of
U87-.DELTA.EGFR tumor growth (FIGS. 15A and 15B). As a negative
control, cells non transfected or transfected with an irrelevant
Luciferase siRNA were included in the study. In both groups,
substantial tumor growth occurred.
[0364] In Vivo siRNA Delivery:
[0365] Two siRNAs (AD-15644 and AD-15660) that demonstrated
specificity for IL-6 and showed durable, low dose knock-down were
chosen for in vivo studies. Nude mice were injected subcutaneously
with 1.times.10.sup.6 U87wt or U87.DELTA. cells or with
U87wt+U87.DELTA. (90:10%) (FIGS. 9A and 9B) and monitored until
tumors reached 80 mm.sup.3 whereupon ten micrograms of AD-15644 or
AD-15660 siRNA against IL-6 or an siRNA against Luciferase gene
were injected intratumorally every two days. Tumor growth kinetics
(FIG. 16A) and tumor volume (FIG. 16B) at the end of the experiment
was reduced in tumors treated with IL-6 specific siRNA AD-15660,
but not in tumors treated with an siRNA against luciferase or with
AD-15644 siRNA. These results illustrate that the emergence of the
.DELTA.EGFR oncogene during gliomagenesis not only conveys a cell
intrinsic growth potential but also establishes a cell extrinsic
potentiation loop to neighboring cells expressing the amplified
antecedent genetic lesion. These results also illustrate a
potential therapeutic use of IL-6 siRNA to inhibit the tumor
enhancement conferred by .DELTA.EGFR on cells over-expressing
wtEGFR and demonstrate a role for this cytokine in driving glioma
heterogeneity.
Example 10. IL-6 Enhances Heterogeneous Tumorigenic Growth
[0366] To determine whether the over-expression of IL-6 was a
mediator of U87wt tumor enhancement through its secretion from
U87.DELTA. cells, U87Parental cells, which lack the ability to
enhance U87wt tumor growth (FIG. 1A), were engineered to
over-express IL-6 (U87Par-IL6). U87wt alone or mixed with
U87.DELTA., U87Parental or U87Par-IL6 were injected subcutaneously
into nude mice (1.times.10.sup.6 total cells) at a ratio of 90:10%
(FIG. 17A) and resultant tumor volumes were measured over 32 days.
As controls, mice were injected with 10% of the total cell number
(1.times.10.sup.5 cells) of U87.DELTA., U87Parental and U87Par-IL6
cells. As shown in FIGS. 17A and 17B, U87Par-IL6 did not grow
faster than U87Parental. When they were mixed with U87wt cells,
tumor growth kinetics were much faster for the U87wt+U87Par-IL6
mixture than for U87wt+U87Par demonstrating a paracrine tumor
enhancement effect mediated by IL-6 secretion in these composite
tumors. Even though U87Parental and U87Par-IL6 grew more slowly
than U87.DELTA., we observed a potent tumor enhancement when U87wt
were injected with U87Par-IL6 with tumor volumes being nearly
double that of U87wt tumors by the end of the experiment (FIG.
17B). In accordance with the previous results, CM generated from
U87Parental-IL6 was able to potently enhanced
[0367] U87wt colony formation in the in vitro soft agar colony
formation assays (FIG. 18). U87Parental-IL6 CM was able to enhance
U87wt colony formation with the same efficiency as U87.DELTA. CM
(p>0.05). In contrast, U87Parental CM was unable to enhance
colony formation, and no significant differences were found when
U87wt cells were treated with U87Parental or normal media
(p>0.05).
Example 11. siRNA-Mediated Reduction of U87.DELTA.-Produced IL-6
Inhibits Soft Agar Colony Formation of U87wt Cells
[0368] Two siRNAs targeting IL-6 (AD-15644 and AD-15660) were
studied in an in vitro soft agar colony formation assay (FIG. 19).
5.times.10.sup.5 U87.DELTA. cells, U87.DELTA. cells transfected
with 25 nM luciferase siRNA or U87.DELTA. cells transfected with 25
nM of a mixture of AD-15644 and AD-15660 (12.5 nM each) were plated
in 10 cm.sup.2 dishes with 10 ml DMEM supplemented with 10% FBS,
penicillin/streptomycin and L-Glutamine, and media was collected
after 48 hours. Conditioned medium (CM) or normal medium was
filtered and used in the upper layer of agar as well as on top of
the agar. Briefly, the bottom layer of agar was prepared by mixing
equal volumes of 1.2% agar (USB Corporation) and 2.times.DMEM/20%
FBS solutions. Two ml of the resulting 0.6% agar/1.times.DMEM/10%
FBS solution was added to each well of 6 well/plates and let to
solidify at room temperature. The upper layer containing
2.5.times.10.sup.3 U87wt cells was prepared by mixing equal volumes
of 1.2% agar, 2.times.DMEM/20% FBS and conditioned media or normal
media. Plates were kept at room temperature until top agar
solidified and treatment media was added on top of the agar. Plates
were placed at 37.degree. C./5% CO.sub.2 for three weeks. Once a
week, media on top of the agar was replaced with fresh media. Every
treatment was performed in triplicate. After three weeks, plates
were stained with 0.005% crystal violet solution and pictures were
taken using a digital camera illuminated with white light. Colonies
were counted using Image Pro-Analyzer 6.2 Software.
[0369] CM generated from U87.DELTA. cells transfected with siRNA
against the irrelevant luciferase gene (.DELTA. CM Luc siRNA)
resulted in a significant increase in colony number (** p<0.01).
In contrast, CM generated from U87.DELTA. cells transfected with a
mixture of the IL-6 siRNAs AD-15644 and AD-15660 (.DELTA. CM IL-6
siRNA) resulted in a reduction in soft agar colony number to levels
comparable to normal growth media. These results demonstrate that
IL-6 produced from U87.DELTA. cells has an important role in the
promotion of U87wt cell proliferation, and that siRNAs targeting
IL-6 inhibit this promotion of U87wt cell proliferation.
Example 12. siRNA-Mediated Reduction of U87.DELTA.-Produced IL-6
Inhibits Tumor Growth Enhancement of U87wt Cells
[0370] To study the effect of IL-6 secretion from U87.DELTA. cells
on tumor growth enhancement, U87.DELTA. cells were transfected with
a 25 nM dose of a combination of IL-6 siRNAs AD-15644 and AD-15660,
or an siRNA against the irrelevant luciferase gene as a negative
control. 1.3.times.10.sup.6 U87.DELTA. cells were seeded in 10 cm
plates and the following day were transfected using
Lipofectamine.TM. 2000 (Invitrogen) and a mixture of IL-6 siRNAs
AD-15644 and AD-15660 at a concentration of 12.5 nM each, or an
siRNA against the luciferase gene at a concentration of 25 nM.
After 18 hours of transfection, the media was changed and cells
were partitioned into larger plates. The transfected U87.DELTA.
cells (10.sup.5 cells per mouse, 10%) were injected subcutaneously
into nude mice alone or mixed with U87wt cells (9.times.10.sup.5
cells per mouse, 90%). Tumors were measured starting at day 5 after
injection and volumes were calculated as described above.
[0371] As shown in FIGS. 14A and 14B, there was no significant
difference in tumor volume between U87.DELTA. cells transfected
with IL-6 siRNAs or with luciferase siRNA when injected alone
(p>0.05). However, tumor volume was significantly reduced when
U87wt cells were mixed with U87.DELTA. cells transfected with IL-6
siRNAs compared to U87.DELTA. cells transfected with luciferase
siRNA (p<0.05). Tumor volumes obtained at the end of the
experiment when U87.DELTA. cells were transfected with IL-6 siRNAs
and mixed with U87wt were similar to the sum of the volumes
obtained after injection of U87wt (90%) and U87.DELTA. (10%)
indicating a reduction in the proliferation enhancement of U87wt
cells induced by U87.DELTA. cells (FIGS. 20A and 20B). This
reduction of U87wt contribution to tumor volume was confirmed when
we analyzed the cell composition by flow cytometry. A significant
reduction in the proportion of U87wt cells was detected in mixed
tumors where IL-6 was knocked-down (41.8.+-.2.73% U87wt cells in
U87wt+U87.DELTA.-Luciferase siRNA tumors vs. 32.87.+-.2.28% U87wt
cells in U87wt+U87.DELTA.-IL6 siRNA tumors). These results
illustrate a potential therapeutic use of IL-6 siRNAs to inhibit
the tumor enhancement conferred by .DELTA.EGFR on cells
over-expressing wtEGFR.
TABLE-US-00005 TABLE 5 Modified siRNAs targeting IL-6. Target
position of 5' base of sense strand S SEQ AS SEQ Antisense (AS)
Oligo Duplex (see Oligo ID Sense (S) Oligo Sequence Oligo ID
Sequence Name FIG. 11) Name NO: (5' to 3') Name NO: (5' to 3') AD-
803 25860 82 AAAAGuAuGAGcGuuAGGAdTsdT 25861 83
UCCuAACGCUcAuACUUUUdTsdT 15637 AD- 223 25862 84
uGAcAAAcAAAuucGGuAcdTsdT 25863 85 GuACCGAAUUUGUUUGUcAdTsdT 15638
AD- 802 25864 86 uAAAAGuAuGAGcGuuAGGdTsdT 25865 87
CCuAACGCUcAuACUUUuAdTsdT 15639 AD- 804 25866 88
AAAGuAuGAGcGuuAGGAcdTsdT 25867 89 GUCCuAACGCUcAuACUUUdTsdT 15640
AD- 234 25868 90 uucGGuAcAuccucGAcGGdTsdT 25869 91
CCGUCGAGGAUGuACCGAAdTsdT 15641 AD- 235 25870 92
ucGGuAcAuccucGAcGGcdTsdT 25871 93 GCCGUCGAGGAUGuACCGAdTsdT 15642
AD- 222 25872 94 uuGAcAAAcAAAuucGGuAdTsdT 25873 95
uACCGAAUUUGUUUGUcAAdTsdT 15643 AD- 809 25874 96
AuGAGcGuuAGGAcAcuAudTsdT 25875 97 AuAGUGUCCuAACGCUcAUdTsdT 15644
AD- 231 25876 98 AAAuucGGuAcAuccucGAdTsdT 25877 99
UCGAGGAUGuACCGAAUUUdTsdT 15645 AD- 425 25878 100
GAGuuuGAGGuAuAccuAGdTsdT 25879 101 CuAGGuAuACCUcAAACUCdTsdT 15646
AD- 542 25880 102 AAucuAGAuGcAAuAAccAdTsdT 25881 103
UGGUuAUUGcAUCuAGAUUdTsdT 15647 AD- 805 25882 104
AAGuAuGAGcGuuAGGAcAdTsdT 25883 105 UGUCCuAACGCUcAuACUUdTsdT 15648
AD- 806 25884 106 AGuAuGAGcGuuAGGAcAcdTsdT 25885 107
GUGUCCuAACGCUcAuACUdTsdT 15649 AD- 1009 25886 108
AGuGuAGGcuuAccucAAAdTsdT 25887 109 UUUGAGGuAAGCCuAcACUdTsdT 15650
AD- 422 25888 110 uuGGAGuuuGAGGuAuAccdTsdT 25889 111
GGuAuACCUcAAACUCcAAdTsdT 15651 AD- 225 25890 112
AcAAAcAAAuucGGuAcAudTsdT 25891 113 AUGuACCGAAUUUGUUUGUdTsdT 15652
AD- 808 25892 114 uAuGAGcGuuAGGAcAcuAdTsdT 25893 115
uAGUGUCCuAACGCUcAuAdTsdT 15653 AD- 210 25894 116
cuucAGAAcGAAuuGAcAAdTsdT 25895 117 UUGUcAAUUCGUUCUGAAGdTsdT 15654
AD- 680 25896 118 cuGAGGGcucuucGGcAAAdTsdT 25897 119
UUUGCCGAAGAGCCCUcAGdTsdT 15655 AD- 636 25898 120
cucAucucAuucuGcGcAGdTsdT 25899 121 CUGCGcAGAAUGAGAUGAGdTsdT 15656
AD- 1004 25900 122 uGGAAAGuGuAGGcuuAccdTsdT 25901 123
GGuAAGCCuAcACUUUCcAdTsdT 15657 AD- 691 25902 124
ucGGcAAAuGuAGcAuGGGdTsdT 25903 125 CCcAUGCuAcAUUUGCCGAdTsdT 15658
AD- 543 25904 126 AucuAGAuGcAAuAAccAcdTsdT 25905 127
GUGGUuAUUGcAUCuAGAUdTsdT 15659 AD- 811 25906 128
GAGcGuuAGGAcAcuAuuudTsdT 25907 129 AAAuAGUGUCCuAACGCUCdTsdT
15660
TABLE-US-00006 TABLE 6 Unmodified siRNAs targeting IL-6. Position
of 5' base of sense strand on transcript SEQ SEQ (see ID Sense (S)
Oligo Sequence ID Antisense (AS) Oligo Sequence FIG. 11) NO: (5' to
3') NO: (5' to 3') 803 130 AAAAGUAUGAGCGUUAGGA 131
UCCUAACGCUCAUACUUUU 223 132 UGACAAACAAAUUCGGUAC 133
GUACCGAAUUUGUUUGUCA 802 134 UAAAAGUAUGAGCGUUAGG 135
CCUAACGCUCAUACUUUUA 804 136 AAAGUAUGAGCGUUAGGAC 137
GUCCUAACGCUCAUACUUU 234 138 UUCGGUACAUCCUCGACGG 139
CCGUCGAGGAUGUACCGAA 235 140 UCGGUACAUCCUCGACGGC 141
GCCGUCGAGGAUGUACCGA 222 142 UUGACAAACAAAUUCGGUA 143
UACCGAAUUUGUUUGUCAA 809 144 AUGAGCGUUAGGACACUAU 145
AUAGUGUCCUAACGCUCAU 231 146 AAAUUCGGUACAUCCUCGA 147
UCGAGGAUGUACCGAAUUU 425 148 GAGUUUGAGGUAUACCUAG 149
CUAGGUAUACCUCAAACUC 542 150 AAUCUAGAUGCAAUAACCA 151
UGGUUAUUGCAUCUAGAUU 805 152 AAGUAUGAGCGUUAGGACA 153
UGUCCUAACGCUCAUACUU 806 154 AGUAUGAGCGUUAGGACAC 155
GUGUCCUAACGCUCAUACU 1009 156 AGUGUAGGCUUACCUCAAA 157
UUUGAGGUAAGCCUACACU 422 158 UUGGAGUUUGAGGUAUACC 159
GGUAUACCUCAAACUCCAA 225 160 ACAAACAAAUUCGGUACAU 161
AUGUACCGAAUUUGUUUGU 808 162 UAUGAGCGUUAGGACACUA 163
UAGUGUCCUAACGCUCAUA 210 164 CUUCAGAACGAAUUGACAA 165
UUGUCAAUUCGUUCUGAAG 680 166 CUGAGGGCUCUUCGGCAAA 167
UUUGCCGAAGAGCCCUCAG 636 168 CUCAUCUCAUUCUGCGCAG 169
CUGCGCAGAAUGAGAUGAG 1004 170 UGGAAAGUGUAGGCUUACC 171
GGUAAGCCUACACUUUCCA 691 172 UCGGCAAAUGUAGCAUGGG 173
CCCAUGCUACAUUUGCCGA 543 174 AUCUAGAUGCAAUAACCAC 175
GUGGUUAUUGCAUCUAGAU 811 176 GAGCGUUAGGACACUAUUU 177
AAAUAGUGUCCUAACGCUC
TABLE-US-00007 TABLE 7 siRNAs targeting IL-6 and modified with 3'
dinucleotide (NN) overhang. Position of 5' base of sense strand on
transcript SEQ SEQ (see ID Sense (S) Oligo Sequence ID Antisense
(AS) Oligo Sequence FIG. 11) NO: (5' to 3') NO: (5' to 3') 803 178
AAAAGUAUGAGCGUUAGGANN 179 UCCUAACGCUCAUACUUUUNN 223 180
UGACAAACAAAUUCGGUACNN 181 GUACCGAAUUUGUUUGUCANN 802 182
UAAAAGUAUGAGCGUUAGGNN 183 CCUAACGCUCAUACUUUUANN 804 184
AAAGUAUGAGCGUUAGGACNN 185 GUCCUAACGCUCAUACUUUNN 234 186
UUCGGUACAUCCUCGACGGNN 187 CCGUCGAGGAUGUACCGAANN 235 188
UCGGUACAUCCUCGACGGCNN 189 GCCGUCGAGGAUGUACCGANN 222 190
UUGACAAACAAAUUCGGUANN 191 UACCGAAUUUGUUUGUCAANN 809 192
AUGAGCGUUAGGACACUAUNN 193 AUAGUGUCCUAACGCUCAUNN 231 194
AAAUUCGGUACAUCCUCGANN 195 UCGAGGAUGUACCGAAUUUNN 425 196
GAGUUUGAGGUAUACCUAGNN 197 CUAGGUAUACCUCAAACUCNN 542 198
AAUCUAGAUGCAAUAACCANN 199 UGGUUAUUGCAUCUAGAUUNN 805 200
AAGUAUGAGCGUUAGGACANN 201 UGUCCUAACGCUCAUACUUNN 806 202
AGUAUGAGCGUUAGGACACNN 203 GUGUCCUAACGCUCAUACUNN 1009 204
AGUGUAGGCUUACCUCAAANN 205 UUUGAGGUAAGCCUACACUNN 422 206
UUGGAGUUUGAGGUAUACCNN 207 GGUAUACCUCAAACUCCAANN 225 208
ACAAACAAAUUCGGUACAUNN 209 AUGUACCGAAUUUGUUUGUNN 808 210
UAUGAGCGUUAGGACACUANN 211 UAGUGUCCUAACGCUCAUANN 210 212
CUUCAGAACGAAUUGACAANN 213 UUGUCAAUUCGUUCUGAAGNN 680 214
CUGAGGGCUCUUCGGCAAANN 215 UUUGCCGAAGAGCCCUCAGNN 636 216
CUCAUCUCAUUCUGCGCAGNN 217 CUGCGCAGAAUGAGAUGAGNN 1004 218
UGGAAAGUGUAGGCUUACCNN 219 GGUAAGCCUACACUUUCCANN 691 220
UCGGCAAAUGUAGCAUGGGNN 221 CCCAUGCUACAUUUGCCGANN 543 222
AUCUAGAUGCAAUAACCACNN 223 GUGGUUAUUGCAUCUAGAUNN 811 224
GAGCGUUAGGACACUAUUUNN 225 AAAUAGUGUCCUAACGCUCNN
TABLE-US-00008 TABLE 8 siRNAs targeting IL-6 and modified with 3'
dithymidine (dTdT) overhang. Position of 5' base of sense strand on
transcript SEQ SEQ (see ID Sense (S) Oligo Sequence ID Antisense
(AS) Oligo Sequence FIG. 11) NO: (5' to 3') NO: (5' to 3') 803 226
AAAAGUAUGAGCGUUAGGAdTdT 227 UCCUAACGCUCAUACUUUUdTdT 223 228
UGACAAACAAAUUCGGUACdTdT 229 GUACCGAAUUUGUUUGUCAdTdT 802 230
UAAAAGUAUGAGCGUUAGGdTdT 231 CCUAACGCUCAUACUUUUAdTdT 804 232
AAAGUAUGAGCGUUAGGACdTdT 233 GUCCUAACGCUCAUACUUUdTdT 234 234
UUCGGUACAUCCUCGACGGdTdT 235 CCGUCGAGGAUGUACCGAAdTdT 235 236
UCGGUACAUCCUCGACGGCdTdT 237 GCCGUCGAGGAUGUACCGAdTdT 222 238
UUGACAAACAAAUUCGGUAdTdT 239 UACCGAAUUUGUUUGUCAAdTdT 809 240
AUGAGCGUUAGGACACUAUdTdT 241 AUAGUGUCCUAACGCUCAUdTdT 231 242
AAAUUCGGUACAUCCUCGAdTdT 243 UCGAGGAUGUACCGAAUUUdTdT 425 244
GAGUUUGAGGUAUACCUAGdTdT 245 CUAGGUAUACCUCAAACUCdTdT 542 246
AAUCUAGAUGCAAUAACCAdTdT 247 UGGUUAUUGCAUCUAGAUUdTdT 805 248
AAGUAUGAGCGUUAGGACAdTdT 249 UGUCCUAACGCUCAUACUUdTdT 806 250
AGUAUGAGCGUUAGGACACdTdT 251 GUGUCCUAACGCUCAUACUdTdT 1009 252
AGUGUAGGCUUACCUCAAAdTdT 253 UUUGAGGUAAGCCUACACUdTdT 422 254
UUGGAGUUUGAGGUAUACCdTdT 255 GGUAUACCUCAAACUCCAAdTdT 225 256
ACAAACAAAUUCGGUACAUdTdT 257 AUGUACCGAAUUUGUUUGUdTdT 808 258
UAUGAGCGUUAGGACACUAdTdT 259 UAGUGUCCUAACGCUCAUAdTdT 210 260
CUUCAGAACGAAUUGACAAdTdT 261 UUGUCAAUUCGUUCUGAAGdTdT 680 262
CUGAGGGCUCUUCGGCAAAdTdT 263 UUUGCCGAAGAGCCCUCAGdTdT 636 264
CUCAUCUCAUUCUGCGCAGdTdT 265 CUGCGCAGAAUGAGAUGAGdTdT 1004 266
UGGAAAGUGUAGGCUUACCdTdT 267 GGUAAGCCUACACUUUCCAdTdT 691 268
UCGGCAAAUGUAGCAUGGGdTdT 269 CCCAUGCUACAUUUGCCGAdTdT 543 270
AUCUAGAUGCAAUAACCACdTdT 271 GUGGUUAUUGCAUCUAGAUdTdT 811 272
GAGCGUUAGGACACUAUUUdTdT 273 AAAUAGUGUCCUAACGCUCdTdT
TABLE-US-00009 TABLE 9 Effect of IL-6 siRNAs on IL-6 and IL-8 gene
expression. siRNA IL-6 IL-8 Durability (days) AD-15637 + + .times.
AD-15638 + + .times. AD-15639 + + .times. AD-15640 + + .times.
AD-15641 + - .times. AD-15642 +/- .times. .times. AD-15643 +
.times. .times. AD-15644 + - .gtoreq.14 AD-15645 + .times. .times.
AD-15646 + .times. .times. AD-15647 - .times. .times. AD-15648 + +
.times. AD-15649 + + .times. AD-15650 + - 3-7 AD-15651 - .times.
.times. AD-15652 + + .times. AD-15653 + + .times. AD-15654 -
.times. .times. AD-15655 + .times. .times. AD-15656 - .times.
.times. AD-15657 + + 7-14 AD-15658 + + .times. AD-15659 + + .times.
AD-15660 + - .gtoreq.14 - = no reduction of expression + =
reduction of expression +/- = small reduction of expression .times.
= not determined
[0372] Other embodiments are in the claims.
Sequence CWU 1
1
27415616DNAHomo sapiens 1ccccggcgca gcgcggccgc agcagcctcc
gccccccgca cggtgtgagc gcccgacgcg 60gccgaggcgg ccggagtccc gagctagccc
cggcggccgc cgccgcccag accggacgac 120aggccacctc gtcggcgtcc
gcccgagtcc ccgcctcgcc gccaacgcca caaccaccgc 180gcacggcccc
ctgactccgt ccagtattga tcgggagagc cggagcgagc tcttcgggga
240gcagcgatgc gaccctccgg gacggccggg gcagcgctcc tggcgctgct
ggctgcgctc 300tgcccggcga gtcgggctct ggaggaaaag aaagtttgcc
aaggcacgag taacaagctc 360acgcagttgg gcacttttga agatcatttt
ctcagcctcc agaggatgtt caataactgt 420gaggtggtcc ttgggaattt
ggaaattacc tatgtgcaga ggaattatga tctttccttc 480ttaaagacca
tccaggaggt ggctggttat gtcctcattg ccctcaacac agtggagcga
540attcctttgg aaaacctgca gatcatcaga ggaaatatgt actacgaaaa
ttcctatgcc 600ttagcagtct tatctaacta tgatgcaaat aaaaccggac
tgaaggagct gcccatgaga 660aatttacagg aaatcctgca tggcgccgtg
cggttcagca acaaccctgc cctgtgcaac 720gtggagagca tccagtggcg
ggacatagtc agcagtgact ttctcagcaa catgtcgatg 780gacttccaga
accacctggg cagctgccaa aagtgtgatc caagctgtcc caatgggagc
840tgctggggtg caggagagga gaactgccag aaactgacca aaatcatctg
tgcccagcag 900tgctccgggc gctgccgtgg caagtccccc agtgactgct
gccacaacca gtgtgctgca 960ggctgcacag gcccccggga gagcgactgc
ctggtctgcc gcaaattccg agacgaagcc 1020acgtgcaagg acacctgccc
cccactcatg ctctacaacc ccaccacgta ccagatggat 1080gtgaaccccg
agggcaaata cagctttggt gccacctgcg tgaagaagtg tccccgtaat
1140tatgtggtga cagatcacgg ctcgtgcgtc cgagcctgtg gggccgacag
ctatgagatg 1200gaggaagacg gcgtccgcaa gtgtaagaag tgcgaagggc
cttgccgcaa agtgtgtaac 1260ggaataggta ttggtgaatt taaagactca
ctctccataa atgctacgaa tattaaacac 1320ttcaaaaact gcacctccat
cagtggcgat ctccacatcc tgccggtggc atttaggggt 1380gactccttca
cacatactcc tcctctggat ccacaggaac tggatattct gaaaaccgta
1440aaggaaatca cagggttttt gctgattcag gcttggcctg aaaacaggac
ggacctccat 1500gcctttgaga acctagaaat catacgcggc aggaccaagc
aacatggtca gttttctctt 1560gcagtcgtca gcctgaacat aacatccttg
ggattacgct ccctcaagga gataagtgat 1620ggagatgtga taatttcagg
aaacaaaaat ttgtgctatg caaatacaat aaactggaaa 1680aaactgtttg
ggacctccgg tcagaaaacc aaaattataa gcaacagagg tgaaaacagc
1740tgcaaggcca caggccaggt ctgccatgcc ttgtgctccc ccgagggctg
ctggggcccg 1800gagcccaggg actgcgtctc ttgccggaat gtcagccgag
gcagggaatg cgtggacaag 1860tgcaaccttc tggagggtga gccaagggag
tttgtggaga actctgagtg catacagtgc 1920cacccagagt gcctgcctca
ggccatgaac atcacctgca caggacgggg accagacaac 1980tgtatccagt
gtgcccacta cattgacggc ccccactgcg tcaagacctg cccggcagga
2040gtcatgggag aaaacaacac cctggtctgg aagtacgcag acgccggcca
tgtgtgccac 2100ctgtgccatc caaactgcac ctacggatgc actgggccag
gtcttgaagg ctgtccaacg 2160aatgggccta agatcccgtc catcgccact
gggatggtgg gggccctcct cttgctgctg 2220gtggtggccc tggggatcgg
cctcttcatg cgaaggcgcc acatcgttcg gaagcgcacg 2280ctgcggaggc
tgctgcagga gagggagctt gtggagcctc ttacacccag tggagaagct
2340cccaaccaag ctctcttgag gatcttgaag gaaactgaat tcaaaaagat
caaagtgctg 2400ggctccggtg cgttcggcac ggtgtataag ggactctgga
tcccagaagg tgagaaagtt 2460aaaattcccg tcgctatcaa ggaattaaga
gaagcaacat ctccgaaagc caacaaggaa 2520atcctcgatg aagcctacgt
gatggccagc gtggacaacc cccacgtgtg ccgcctgctg 2580ggcatctgcc
tcacctccac cgtgcagctc atcacgcagc tcatgccctt cggctgcctc
2640ctggactatg tccgggaaca caaagacaat attggctccc agtacctgct
caactggtgt 2700gtgcagatcg caaagggcat gaactacttg gaggaccgtc
gcttggtgca ccgcgacctg 2760gcagccagga acgtactggt gaaaacaccg
cagcatgtca agatcacaga ttttgggctg 2820gccaaactgc tgggtgcgga
agagaaagaa taccatgcag aaggaggcaa agtgcctatc 2880aagtggatgg
cattggaatc aattttacac agaatctata cccaccagag tgatgtctgg
2940agctacgggg tgaccgtttg ggagttgatg acctttggat ccaagccata
tgacggaatc 3000cctgccagcg agatctcctc catcctggag aaaggagaac
gcctccctca gccacccata 3060tgtaccatcg atgtctacat gatcatggtc
aagtgctgga tgatagacgc agatagtcgc 3120ccaaagttcc gtgagttgat
catcgaattc tccaaaatgg cccgagaccc ccagcgctac 3180cttgtcattc
agggggatga aagaatgcat ttgccaagtc ctacagactc caacttctac
3240cgtgccctga tggatgaaga agacatggac gacgtggtgg atgccgacga
gtacctcatc 3300ccacagcagg gcttcttcag cagcccctcc acgtcacgga
ctcccctcct gagctctctg 3360agtgcaacca gcaacaattc caccgtggct
tgcattgata gaaatgggct gcaaagctgt 3420cccatcaagg aagacagctt
cttgcagcga tacagctcag accccacagg cgccttgact 3480gaggacagca
tagacgacac cttcctccca gtgcctgaat acataaacca gtccgttccc
3540aaaaggcccg ctggctctgt gcagaatcct gtctatcaca atcagcctct
gaaccccgcg 3600cccagcagag acccacacta ccaggacccc cacagcactg
cagtgggcaa ccccgagtat 3660ctcaacactg tccagcccac ctgtgtcaac
agcacattcg acagccctgc ccactgggcc 3720cagaaaggca gccaccaaat
tagcctggac aaccctgact accagcagga cttctttccc 3780aaggaagcca
agccaaatgg catctttaag ggctccacag ctgaaaatgc agaataccta
3840agggtcgcgc cacaaagcag tgaatttatt ggagcatgac cacggaggat
agtatgagcc 3900ctaaaaatcc agactctttc gatacccagg accaagccac
agcaggtcct ccatcccaac 3960agccatgccc gcattagctc ttagacccac
agactggttt tgcaacgttt acaccgacta 4020gccaggaagt acttccacct
cgggcacatt ttgggaagtt gcattccttt gtcttcaaac 4080tgtgaagcat
ttacagaaac gcatccagca agaatattgt ccctttgagc agaaatttat
4140ctttcaaaga ggtatatttg aaaaaaaaaa aaagtatatg tgaggatttt
tattgattgg 4200ggatcttgga gtttttcatt gtcgctattg atttttactt
caatgggctc ttccaacaag 4260gaagaagctt gctggtagca cttgctaccc
tgagttcatc caggcccaac tgtgagcaag 4320gagcacaagc cacaagtctt
ccagaggatg cttgattcca gtggttctgc ttcaaggctt 4380ccactgcaaa
acactaaaga tccaagaagg ccttcatggc cccagcaggc cggatcggta
4440ctgtatcaag tcatggcagg tacagtagga taagccactc tgtcccttcc
tgggcaaaga 4500agaaacggag gggatggaat tcttccttag acttactttt
gtaaaaatgt ccccacggta 4560cttactcccc actgatggac cagtggtttc
cagtcatgag cgttagactg acttgtttgt 4620cttccattcc attgttttga
aactcagtat gctgcccctg tcttgctgtc atgaaatcag 4680caagagagga
tgacacatca aataataact cggattccag cccacattgg attcatcagc
4740atttggacca atagcccaca gctgagaatg tggaatacct aaggatagca
ccgcttttgt 4800tctcgcaaaa acgtatctcc taatttgagg ctcagatgaa
atgcatcagg tcctttgggg 4860catagatcag aagactacaa aaatgaagct
gctctgaaat ctcctttagc catcacccca 4920accccccaaa attagtttgt
gttacttatg gaagatagtt ttctcctttt acttcacttc 4980aaaagctttt
tactcaaaga gtatatgttc cctccaggtc agctgccccc aaaccccctc
5040cttacgcttt gtcacacaaa aagtgtctct gccttgagtc atctattcaa
gcacttacag 5100ctctggccac aacagggcat tttacaggtg cgaatgacag
tagcattatg agtagtgtgg 5160aattcaggta gtaaatatga aactagggtt
tgaaattgat aatgctttca caacatttgc 5220agatgtttta gaaggaaaaa
agttccttcc taaaataatt tctctacaat tggaagattg 5280gaagattcag
ctagttagga gcccaccttt tttcctaatc tgtgtgtgcc ctgtaacctg
5340actggttaac agcagtcctt tgtaaacagt gttttaaact ctcctagtca
atatccaccc 5400catccaattt atcaaggaag aaatggttca gaaaatattt
tcagcctaca gttatgttca 5460gtcacacaca catacaaaat gttccttttg
cttttaaagt aatttttgac tcccagatca 5520gtcagagccc ctacagcatt
gttaagaaag tatttgattt ttgtctcaat gaaaataaaa 5580ctatattcat
ttccactcta aaaaaaaaaa aaaaaa 5616221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 2uggaggaaaa gaaagguaat t
21321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 3uuaccuuucu
uuuccuccat t 21421DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 4ggaggaaaag aaagguaaut t
21521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 5auuaccuuuc
uuuuccucct t 21621DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 6gaggaaaaga aagguaauut t
21721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 7aauuaccuuu
cuuuuccuct t 21821DNAArtificial Sequencesource/note="Description of
Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 8cuggaggaaa agaaagguat t
21921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 9uaccuuucuu
uuccuccagt t 211021DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 10aggaaaagaa agguaauuat t
211121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 11uaauuaccuu
ucuuuuccut t 211221DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 12ggaaaagaaa gguaauuaut t
211321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 13auaauuaccu
uucuuuucct t 211421DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 14gaaagguaau uauguggugt t
211521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 15caccacauaa
uuaccuuuct t 211621DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 16agaaagguaa uuauguggut t
211721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 17accacauaau
uaccuuucut t 211821DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 18acggugugag cgcccgacgt t
211921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 19cgucgggcgc
ucacaccgut t 212021DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 20acagaucacg gcucgugcgt t
212121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 21cgcacgagcc
gugaucugut t 212221DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 22cacggcucgu gcguccgagt t
212321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 23cucggacgca
cgagccgugt t 212421DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 24cgacaggcca ccucgucggt t
212521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 25ccgacgaggu
ggccugucgt t 212621DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 26gugacagauc acggcucgut t
212721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 27acgagccgug
aucugucact t 212821DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 28ucgucggcgu ccgcccgagt t
212921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 29cucgggcgga
cgccgacgat t 213021DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 30ccguccagua uugaucgggt t
213121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 31cccgaucaau
acuggacggt t 213221DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 32ggugacagau cacggcucgt t
213321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 33cgagccguga
ucugucacct t 213421DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 34ugccgcaaau uccgagacgt t
213521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 35cgucucggaa
uuugcggcat t 213621DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 36gcgccgugcg guucagcaat t
213721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 37uugcugaacc
gcacggcgct t 213821DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 38ccgcaaauuc cgagacgaat t
213921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 39uucgucucgg
aauuugcggt t 214021DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 40ugccaaggca cgaguaacat t
214121DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 41uguuacucgu gccuuggcat t
214221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 42gaggaaauau
guacuacgat t 214321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 43ucguaguaca uauuuccuct t
214421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 44aggaaauccu
gcauggcgct t 214521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 45gcgccaugca ggauuuccut t
214621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 46ugcauggcgc
cgugcgguut t 214721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 47aaccgcacgg cgccaugcat t
214821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 48ccaguggcgg
gacauaguct t 214921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 49gacuaugucc cgccacuggt t
215021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 50uggaggaaaa
gaaagguaat t 215121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 51uuaccuuucu uuuccuccat t
215221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 52ggaggaaaag
aaagguaaut t 215321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 53auuaccuuuc uuuuccucct t
215421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 54gaggaaaaga
aagguaauut t 215521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 55aauuaccuuu cuuuuccuct t
215621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 56cuggaggaaa
agaaagguat t 215721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 57uaccuuucuu uuccuccagt t
215821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 58aggaaaagaa
agguaauuat t 215921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 59uaauuaccuu ucuuuuccut t
216021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 60ggaaaagaaa
gguaauuaut t 216121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 61auaauuaccu uucuuuucct t
216221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 62gaaagguaau
uauguggugt t 216321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 63caccacauaa uuaccuuuct t
216421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 64agaaagguaa
uuauguggut t 216521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 65accacauaau uaccuuucut t
216621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 66ugccgcaaau
uccgagacgt t 216721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 67cgucucggaa uuugcggcat t
216821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 68gcgccgugcg
guucagcaat t 216921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 69uugcugaacc gcacggcgct t
217021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 70ccgcaaauuc
cgagacgaat t 217121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 71uucgucucgg aauuugcggt t
217221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 72ugccaaggca
cgaguaacat t 217321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 73uguuacucgu gccuuggcat t
217421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 74gaggaaauau
guacuacgat t 217521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 75ucguaguaca uauuuccuct t
217621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 76aggaaauccu
gcauggcgct t 217721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 77gcgccaugca ggauuuccut t
217821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 78ugcauggcgc
cgugcgguut t 217921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 79aaccgcacgg cgccaugcat t
218021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 80ccaguggcgg
gacauaguct t 218121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 81gacuaugucc cgccacuggt t
218221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 82aaaaguauga
gcguuaggat t 218321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 83uccuaacgcu cauacuuuut t
218421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 84ugacaaacaa
auucgguact t 218521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 85guaccgaauu uguuugucat t
218621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 86uaaaaguaug
agcguuaggt t 218721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 87ccuaacgcuc auacuuuuat t
218821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 88aaaguaugag
cguuaggact t 218921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 89guccuaacgc ucauacuuut t
219021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 90uucgguacau
ccucgacggt t 219121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 91ccgucgagga uguaccgaat t
219221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 92ucgguacauc
cucgacggct t 219321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 93gccgucgagg auguaccgat t
219421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 94uugacaaaca
aauucgguat t 219521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 95uaccgaauuu guuugucaat t
219621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 96augagcguua
ggacacuaut t 219721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 97auaguguccu aacgcucaut t
219821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 98aaauucggua
cauccucgat t 219921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 99ucgaggaugu accgaauuut t
2110021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 100gaguuugagg
uauaccuagt t 2110121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 101cuagguauac cucaaacuct t
2110221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 102aaucuagaug
caauaaccat t 2110321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 103ugguuauugc aucuagauut t
2110421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 104aaguaugagc
guuaggacat t 2110521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 105uguccuaacg cucauacuut t
2110621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 106aguaugagcg
uuaggacact t 2110721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 107guguccuaac gcucauacut t
2110821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 108aguguaggcu
uaccucaaat t 2110921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 109uuugagguaa gccuacacut t
2111021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 110uuggaguuug
agguauacct t 2111121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 111gguauaccuc aaacuccaat t
2111221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 112acaaacaaau
ucgguacaut t 2111321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 113auguaccgaa uuuguuugut t
2111421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 114uaugagcguu
aggacacuat t 2111521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 115uaguguccua acgcucauat t
2111621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 116cuucagaacg
aauugacaat t 2111721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 117uugucaauuc guucugaagt t
2111821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 118cugagggcuc
uucggcaaat t 2111921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 119uuugccgaag agcccucagt t
2112021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 120cucaucucau
ucugcgcagt t 2112121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 121cugcgcagaa ugagaugagt t
2112221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 122uggaaagugu
aggcuuacct t 2112321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 123gguaagccua cacuuuccat t
2112421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 124ucggcaaaug
uagcaugggt t 2112521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 125cccaugcuac auuugccgat t
2112621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 126aucuagaugc
aauaaccact t 2112721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 127gugguuauug caucuagaut t
2112821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 128gagcguuagg
acacuauuut t 2112921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 129aaauaguguc cuaacgcuct t
2113019RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 130aaaaguauga gcguuagga
1913119RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 131uccuaacgcu cauacuuuu
1913219RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 132ugacaaacaa auucgguac
1913319RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 133guaccgaauu uguuuguca
1913419RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 134uaaaaguaug agcguuagg
1913519RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 135ccuaacgcuc auacuuuua
1913619RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 136aaaguaugag cguuaggac
1913719RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 137guccuaacgc ucauacuuu
1913819RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 138uucgguacau ccucgacgg
1913919RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 139ccgucgagga uguaccgaa
1914019RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 140ucgguacauc cucgacggc
1914119RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 141gccgucgagg auguaccga
1914219RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 142uugacaaaca aauucggua
1914319RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 143uaccgaauuu guuugucaa
1914419RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 144augagcguua ggacacuau
1914519RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 145auaguguccu aacgcucau
1914619RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 146aaauucggua cauccucga
1914719RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 147ucgaggaugu accgaauuu
1914819RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 148gaguuugagg uauaccuag
1914919RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 149cuagguauac cucaaacuc
1915019RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 150aaucuagaug caauaacca
1915119RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 151ugguuauugc aucuagauu
1915219RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 152aaguaugagc guuaggaca
1915319RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 153uguccuaacg cucauacuu
1915419RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 154aguaugagcg uuaggacac
1915519RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 155guguccuaac gcucauacu
1915619RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 156aguguaggcu uaccucaaa
1915719RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 157uuugagguaa gccuacacu
1915819RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 158uuggaguuug agguauacc
1915919RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 159gguauaccuc aaacuccaa
1916019RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 160acaaacaaau ucgguacau
1916119RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 161auguaccgaa uuuguuugu
1916219RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 162uaugagcguu aggacacua
1916319RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 163uaguguccua acgcucaua
1916419RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 164cuucagaacg aauugacaa
1916519RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 165uugucaauuc guucugaag
1916619RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 166cugagggcuc uucggcaaa
1916719RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 167uuugccgaag agcccucag
1916819RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 168cucaucucau ucugcgcag
1916919RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 169cugcgcagaa ugagaugag
1917019RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 170uggaaagugu aggcuuacc
1917119RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 171gguaagccua cacuuucca
1917219RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 172ucggcaaaug uagcauggg
1917319RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 173cccaugcuac auuugccga
1917419RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 174aucuagaugc aauaaccac
1917519RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 175gugguuauug caucuagau
1917619RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 176gagcguuagg acacuauuu
1917719RNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide" 177aaauaguguc cuaacgcuc
1917821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 178aaaaguauga gcguuaggan n 2117921DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 179uccuaacgcu cauacuuuun n
2118021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 180ugacaaacaa auucgguacn n 2118121DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t,
unknown or other 181guaccgaauu uguuugucan n 2118221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 182uaaaaguaug agcguuaggn n
2118321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 183ccuaacgcuc auacuuuuan n 2118421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 184aaaguaugag cguuaggacn n
2118521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 185guccuaacgc ucauacuuun n 2118621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 186uucgguacau ccucgacggn n
2118721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 187ccgucgagga uguaccgaan n 2118821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 188ucgguacauc cucgacggcn n
2118921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 189gccgucgagg auguaccgan n 2119021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 190uugacaaaca aauucgguan n
2119121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 191uaccgaauuu guuugucaan n 2119221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 192augagcguua ggacacuaun n
2119321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 193auaguguccu aacgcucaun n 2119421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 194aaauucggua cauccucgan n
2119521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 195ucgaggaugu accgaauuun n 2119621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 196gaguuugagg uauaccuagn n
2119721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 197cuagguauac cucaaacucn n 2119821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 198aaucuagaug caauaaccan n
2119921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 199ugguuauugc aucuagauun n 2120021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 200aaguaugagc guuaggacan n
2120121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 201uguccuaacg cucauacuun n 2120221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 202aguaugagcg uuaggacacn n
2120321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 203guguccuaac gcucauacun n 2120421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 204aguguaggcu uaccucaaan n
2120521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 205uuugagguaa gccuacacun n 2120621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 206uuggaguuug agguauaccn n
2120721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 207gguauaccuc aaacuccaan n 2120821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 208acaaacaaau ucgguacaun n
2120921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 209auguaccgaa uuuguuugun n 2121021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 210uaugagcguu aggacacuan n
2121121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 211uaguguccua acgcucauan n 2121221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 212cuucagaacg aauugacaan n
2121321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 213uugucaauuc guucugaagn n 2121421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 214cugagggcuc uucggcaaan n
2121521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 215uuugccgaag agcccucagn n 2121621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 216cucaucucau ucugcgcagn n
2121721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 217cugcgcagaa ugagaugagn n 2121821DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 218uggaaagugu aggcuuaccn n
2121921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 219gguaagccua cacuuuccan n 2122021DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 220ucggcaaaug uagcaugggn n
2122121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 221cccaugcuac auuugccgan n 2122221DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 222aucuagaugc aauaaccacn n
2122321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 223gugguuauug caucuagaun n 2122421DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide"modified_base(20)..(21)a, c, g,
u, t, unknown or other 224gagcguuagg acacuauuun n
2122521DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic
oligonucleotide"modified_base(20)..(21)a, c, g, u, t, unknown or
other 225aaauaguguc cuaacgcucn n 2122621DNAArtificial
Sequencesource/note="Description of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 226aaaaguauga gcguuaggat t
2122721DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 227uccuaacgcu
cauacuuuut t 2122821DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 228ugacaaacaa auucgguact t
2122921DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 229guaccgaauu
uguuugucat t 2123021DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 230uaaaaguaug agcguuaggt t
2123121DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 231ccuaacgcuc
auacuuuuat t 2123221DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 232aaaguaugag cguuaggact t
2123321DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 233guccuaacgc ucauacuuut t
2123421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 234uucgguacau
ccucgacggt t 2123521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 235ccgucgagga uguaccgaat t
2123621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 236ucgguacauc
cucgacggct t 2123721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 237gccgucgagg auguaccgat t
2123821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 238uugacaaaca
aauucgguat t 2123921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 239uaccgaauuu guuugucaat t
2124021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 240augagcguua
ggacacuaut t 2124121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 241auaguguccu aacgcucaut t
2124221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 242aaauucggua
cauccucgat t 2124321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 243ucgaggaugu accgaauuut t
2124421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 244gaguuugagg
uauaccuagt t 2124521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 245cuagguauac cucaaacuct t
2124621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 246aaucuagaug
caauaaccat t 2124721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 247ugguuauugc aucuagauut t
2124821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 248aaguaugagc
guuaggacat t 2124921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 249uguccuaacg cucauacuut t
2125021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 250aguaugagcg
uuaggacact t 2125121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 251guguccuaac gcucauacut t
2125221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 252aguguaggcu
uaccucaaat t 2125321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 253uuugagguaa gccuacacut t
2125421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 254uuggaguuug
agguauacct t 2125521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 255gguauaccuc aaacuccaat t
2125621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 256acaaacaaau
ucgguacaut t 2125721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 257auguaccgaa uuuguuugut t
2125821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 258uaugagcguu
aggacacuat t 2125921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 259uaguguccua acgcucauat t
2126021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 260cuucagaacg
aauugacaat t 2126121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 261uugucaauuc guucugaagt t
2126221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 262cugagggcuc
uucggcaaat t 2126321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 263uuugccgaag agcccucagt t
2126421DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 264cucaucucau
ucugcgcagt t 2126521DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 265cugcgcagaa ugagaugagt t
2126621DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 266uggaaagugu
aggcuuacct t 2126721DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 267gguaagccua cacuuuccat t
2126821DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 268ucggcaaaug
uagcaugggt t 2126921DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 269cccaugcuac auuugccgat t
2127021DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 270aucuagaugc
aauaaccact t 2127121DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 271gugguuauug caucuagaut t
2127221DNAArtificial Sequencesource/note="Description of Artificial
Sequence Synthetic oligonucleotide"source/note="Description of
Combined DNA/RNA Molecule Synthetic oligonucleotide" 272gagcguuagg
acacuauuut t 2127321DNAArtificial Sequencesource/note="Description
of Artificial Sequence Synthetic
oligonucleotide"source/note="Description of Combined DNA/RNA
Molecule Synthetic oligonucleotide" 273aaauaguguc cuaacgcuct t
212741131DNAHomo sapiens 274cattctgccc tcgagcccac cgggaacgaa
agagaagctc tatctcccct ccaggagccc 60agctatgaac tccttctcca caagcgcctt
cggtccagtt gccttctccc tggggctgct 120cctggtgttg cctgctgcct
tccctgcccc agtaccccca ggagaagatt ccaaagatgt 180agccgcccca
cacagacagc cactcacctc ttcagaacga attgacaaac aaattcggta
240catcctcgac ggcatctcag ccctgagaaa ggagacatgt aacaagagta
acatgtgtga 300aagcagcaaa gaggcactgg cagaaaacaa cctgaacctt
ccaaagatgg ctgaaaaaga 360tggatgcttc caatctggat tcaatgagga
gacttgcctg gtgaaaatca tcactggtct 420tttggagttt gaggtatacc
tagagtacct ccagaacaga tttgagagta gtgaggaaca 480agccagagct
gtgcagatga gtacaaaagt cctgatccag ttcctgcaga aaaaggcaaa
540gaatctagat gcaataacca cccctgaccc aaccacaaat gccagcctgc
tgacgaagct 600gcaggcacag aaccagtggc tgcaggacat gacaactcat
ctcattctgc gcagctttaa 660ggagttcctg cagtccagcc tgagggctct
tcggcaaatg tagcatgggc acctcagatt 720gttgttgtta atgggcattc
cttcttctgg tcagaaacct gtccactggg cacagaactt 780atgttgttct
ctatggagaa ctaaaagtat gagcgttagg acactatttt aattattttt
840aatttattaa tatttaaata tgtgaagctg agttaattta tgtaagtcat
atttatattt 900ttaagaagta ccacttgaaa cattttatgt attagttttg
aaataataat ggaaagtggc 960tatgcagttt gaatatcctt tgtttcagag
ccagatcatt tcttggaaag tgtaggctta 1020cctcaaataa atggctaact
tatacatatt tttaaagaaa tatttatatt gtatttatat 1080aatgtataaa
tggtttttat accaataaat ggcattttaa aaaattcagc a 1131
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