U.S. patent number RE47,320 [Application Number 15/386,791] was granted by the patent office on 2019-03-26 for modulation of cd40 expression.
This patent grant is currently assigned to Ionis Pharmaceuticals, Inc.. The grantee listed for this patent is Ionis Pharmaceuticals, Inc.. Invention is credited to C. Frank Bennett, Lex M. Cowsert, Susan M. Freier.
United States Patent |
RE47,320 |
Bennett , et al. |
March 26, 2019 |
Modulation of CD40 expression
Abstract
Disclosed herein are antisense compounds and methods for
decreasing CD40. Examples of disease conditions that can be
ameliorated with the administration of antisense compounds targeted
to CD40 include hyperproliferative disorders, graft versus host
disease (GVHD), graft rejection, asthma, airway
hyperresponsiveness, chronic obstructive pulmonary disease (COPD),
multiple sclerosis (MS), systemic lupus erythematosus (SLE), and
certain forms of arthritis.
Inventors: |
Bennett; C. Frank (Carlsbad,
CA), Cowsert; Lex M. (New Braunfels, TX), Freier; Susan
M. (San Diego, CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Ionis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
Ionis Pharmaceuticals, Inc.
(Carlsbad, CA)
|
Family
ID: |
40419509 |
Appl.
No.: |
15/386,791 |
Filed: |
December 21, 2016 |
PCT
Filed: |
November 20, 2008 |
PCT No.: |
PCT/US2008/012998 |
371(c)(1),(2),(4) Date: |
February 15, 2011 |
PCT
Pub. No.: |
WO2009/067243 |
PCT
Pub. Date: |
May 28, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
|
60989421 |
Nov 20, 2007 |
|
|
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Reissue of: |
12743797 |
Nov 20, 2008 |
8916531 |
Dec 23, 2014 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P
29/00 (20180101); C12N 15/113 (20130101); C12N
15/1138 (20130101); C12N 15/1138 (20130101); C12N
15/113 (20130101); A61P 35/00 (20180101); C12N
2310/11 (20130101); C12N 2310/3231 (20130101); C12N
2310/346 (20130101); C12N 2310/346 (20130101); C12N
2310/321 (20130101); C12N 2310/3341 (20130101); C12N
2310/3231 (20130101); C12N 2310/341 (20130101); C12N
2310/315 (20130101); C12N 2310/11 (20130101); C12N
2310/315 (20130101); C12N 2310/3341 (20130101); C12N
2310/341 (20130101); C12N 2310/321 (20130101); C12N
2310/321 (20130101); C12N 2310/321 (20130101); C12N
2310/3525 (20130101); C12N 2310/3525 (20130101) |
Current International
Class: |
C12N
15/113 (20100101) |
Field of
Search: |
;536/24.5,23.1,25.3 |
References Cited
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|
Primary Examiner: Ponnaluri; Padmashri
Attorney, Agent or Firm: Knobbe, Martens, Olson & Bear,
LLP
Parent Case Text
RELATED APPLICATIONS
This application is a 35 U.S.C .sctn.371 national phase application
of international application serial no. PCT/US2008/012998, filed on
Nov. 20, 2008, which is a non-provisional of and claims priority to
U.S. patent application Ser. No. 60/989421, filed on Nov. 20, 2007,
the disclosure of each of which is incorporated herein by reference
in its entirety.
Claims
The invention claimed is:
1. A modified antisense compound 12 to 30 nucleobases in length and
having a nucleobase sequence that is at least 90% complementary to
an equal length portion of the human CD40 gene .[.but not to other
sequences throughout the human genome.]., selected from the
following regions of SEQ ID NO: 4: (a) positions .[.11250-12685,
corresponding to intron 6.]. .Iadd.11801-12591.Iaddend.; (b)
positions 2943-6367, corresponding to intron 1; (c) positions
6447-6780, corresponding to intron 2; (d) positions 6907-7157,
corresponding to intron 3; (e) positions 7305-7673, corresponding
to intron 4; (f) positions 7768-11187, corresponding to intron 5;
(g) positions 12773-12877, corresponding to intron 7; (h) positions
12907-13429, corresponding to intron 8; and (i) positions
13662-16001, which forms part of exon 9 or a region 3' to exon
9.
.[.2. The antisense compound of claim 1, wherein the nucleobase
sequence is at least 90% complementary to an equal length portion
of positions 12527-12685 of SEQ ID NO: 4..].
3. The antisense compound of claim .[.2.]. .Iadd.1.Iaddend., having
a nucleobase sequence comprising at least 8 contiguous nucleobases
of the nucleobase sequence of SEQ ID NO: 208, wherein the
nucleobase sequence of the compound is at least 95% complementary
to the sequence shown in SEQ ID NO: 4.
4. A modified antisense compound 20 nucleobases in length and
consisting of the nucleobase sequence of SEQ ID NO: 208.
5. The antisense compound of claim 1, wherein said antisense
compound is an antisense oligonucleotide.
6. The antisense compound of claim 5, wherein at least one
internucleoside linkage is a modified internucleoside linkage.
7. The antisense compound of claim 6, wherein each internucleoside
linkage is a phosphorothioate internucleoside linkage.
8. The antisense compound of claim 5, wherein at least one
nucleoside comprises a modified sugar.
9. The antisense compound of claim 8, wherein at least one modified
sugar is a bicyclic sugar.
10. The antisense compound of claim 9, wherein the at least one
bicyclic sugar comprises a 4'-CH(CH.sub.3)--O-2' bridge.
11. The antisense compound of claim 8, wherein at least one
modified sugar comprises a 2'-O-methoxyethyl.
12. The antisense compound of claim 1, wherein at least one said
nucleobase is a modified nucleobase.
13. The antisense compound of claim 12, wherein the modified
nucleobase is a 5-methylcytosine.
14. The antisense compound of claim 1, wherein the compound is an
oligonucleotide comprising: a gap segment consisting of linked
deoxynucleosides; a 5' wing segment consisting of linked
nucleosides; a 3' wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment
and the 3' wing segment and wherein each nucleoside of each wing
segment comprises a modified sugar.
15. The antisense compound of claim 14, wherein the oligonucleotide
comprises: a gap segment consisting of ten linked deoxynucleosides;
a 5' wing segment consisting of five linked nucleosides; a 3' wing
segment consisting of five linked nucleosides; wherein the gap
segment is positioned between the 5' wing segment and the 3' wing
segment, wherein each nucleoside of each wing segment comprises a
2'-O-methoxyethyl sugar; and wherein each internucleoside linkage
of said antisense compound is a phosphorothioate linkage.
16. The antisense compound of claim 14, wherein the oligonucleotide
comprises: a gap segment consisting of fifteen linked
deoxynucleosides; a 5' wing segment consisting of two linked
nucleosides; a 3' wing segment consisting of three linked
nucleosides; wherein the gap segment is positioned between the 5'
wing segment and the 3' wing segment, wherein each nucleoside of
each wing segment comprises a 2'-O-methoxyethyl sugar; and wherein
each internucleoside linkage of said antisense compound is a
phosphorothioate linkage.
17. The antisense compound of claim 15 or 16, wherein every
cytosine is a 5-methylcytosine.
18. An antisense oligonucleotide 20 nucleobases in length having
the sequence of nucleobases as set forth in SEQ ID NO:208, wherein
each cytosine is a 5-methylcytosine, each internucleoside linkage
is a phosphorothioate linkage, nucleotides 1-5 and 16-20 are
2'-O-methoxyethyl nucleotides, and nucleotides 6-15 are
2'-deoxynucleotides.
19. A composition comprising an antisense compound of claim 1 or 18
or a salt thereof and a pharmaceutically acceptable carrier or
diluent.
20. A method comprising administering to an animal an antisense
compound of claim 1 or an oligonucleotide of claim 18.
.Iadd.21. The antisense compound of claim 1, wherein said antisense
compound is 15, 16, 17, 18, 19, 20 or 21 nucleobases in length.
.Iaddend.
.Iadd.22. The antisense compound of claim 21, wherein the
nucleobase sequence of the compound is at least 95% complementary
to the sequence shown in SEQ ID NO: 4. .Iaddend.
.Iadd.23. The antisense compound of claim 21, wherein the
nucleobase sequence of the compound is 100% complementary to the
sequence shown in SEQ ID NO: 4. .Iaddend.
Description
SEQUENCE LISTING
The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled 33841-513SEQLIST.txt, created Nov. 19, 2008, which is
67 Kb in size. The information in the electronic format of the
sequence listing is incorporated herein by reference in its
entirety. This sequence listing is identical to the sequence
listing filed on Nov. 20, 2007, with the exception of the addition
of SEQ ID NO: 237.
FIELD OF THE INVENTION
The present invention provides methods and compositions for lowing
levels of CD40 in an animal. Such methods and compositions are
useful as anti-inflammatory compounds and anti-tumor compounds.
BACKGROUND OF THE INVENTION
The immune system serves a vital role in protecting the body
against infectious agents. It is well established, however, that a
number of disease states and/or disorders are a result of either
abnormal or undesirable activation of immune responses. Common
examples include graft versus host disease (GVHD) and graft
rejection, and autoimmune linked diseases such as multiple
sclerosis (MS), systemic lupus erythematosus (SLE), and certain
forms of arthritis.
In general, an immune response is activated as a result of either
tissue injury or infection. Both cases involve the recruitment and
activation of a number of immune system effector cells (e.g., B-
and T-lymphocytes, macrophages, eosinophils, neutrophils) in a
process coordinated through a series of complex cell-cell
interactions. A typical scenario by which an immune response is
mounted against a foreign protein is as follows: foreign proteins
captured by antigen presenting cells (APC's) such as macrophages or
dendritic cells are processed and displayed on the cell surface of
the APC. Circulating T-helper cells which express an immunoglobulin
that recognizes (i.e. binds) the displayed antigen undergo
activation by the APC. These activated T-helpers in turn activate
appropriate B-cell clones to proliferate and differentiate into
plasma cells that produce and secrete humoral antibodies targeted
against the foreign antigen. The secreted humoral antibodies are
free to circulate and bind to any cells expressing the foreign
protein on their cell surface, in effect marking the cell for
destruction by other immune effector cells. In each of the stages
described above, direct cell-cell contact between the involved cell
types is required in order for activation to occur. (Gruss et al.,
Leuk. Lymphoma 1989, 24:393). In recent years, a number of cell
surface receptors that mediate these cell-cell contact dependent
activation events have been identified. Among these cell surface
receptors is CD40 and its physiological ligand, CD40 Ligand (CD40L)
which is also known as CD154.
CD40 was first characterized as a receptor expressed on
B-lymphocytes. It was later found that engagement of B-cell CD40
with CD40L expressed on activated T-cells is essential for T-cell
dependent B-cell activation (i.e. proliferation, immunoglobulin
secretion, and class switching). It was subsequently revealed that
functional CD40 is expressed on a variety of cell types other than
B-cells, including macrophages, dendritic cells, thymic epithelial
cells, Langerhans cells, and endothelial cells. These studies have
led to the current belief that CD40 plays a broad role in immune
regulation by mediating interactions of T-cells with B-cells as
well as other cell types. In support of this notion, it has been
shown that stimulation of CD40 in macrophages and dendritic results
is required for T-cell activation during antigen presentation.
(Gruss et al., Leuk. Lymphoma, 1997, 24:393). Recent evidence
points to a role for CD40 in tissue inflammation as well.
Production of the inflammatory mediators IL-12 and nitric oxide by
macrophages have been shown to be CD40 dependent. (Buhlmann and
Noelle, J. Clin. Immunol., 1996, 16:83). In endothelial cells,
stimulation of CD40 by CD40L has been found to induce surface
expression of E-selectin, ICAM-1, and VCAM-1, promoting adhesion of
leukocytes to sites of inflammation (Buhlmann and Noelle, J. Clin.
Immunol., 1996, 16:83); Gruss et al., Leuk. Lymphoma, 1997,
24:393). Finally, a number of reports have documented
overexpression of CD40 in epithelial and hematopoietic tumors as
well as tumor infiltrating endothelial cells, indicating that CD40
may play a role in tumor growth and/or angiogenesis as well (Gruss
et al., Leuk. Lymphoma, 1997, 24:393; Kluth et al., Cancer Res.,
1997, 57:891).
Due to the pivotal role that CD40 plays in humoral immunity, the
potential exists that therapeutic strategies aimed at
downregulating CD40 or interfering with CD40 signaling may provide
a novel class of agents useful in treating a number of immune
associated disorders, including but not limited to
graft-versus-host disease (GVHD), graft rejection, and autoimmune
diseases such as multiple sclerosis (MS), systemic lupus
erythematosus (SLE), and certain forms of arthritis. Inhibitors of
CD40 may also prove useful as anti-inflammatory compounds, and
could therefore be useful as treatment for a variety of
inflammatory and allergic conditions such as asthma, rheumatoid
arthritis, allograft rejections, inflammatory bowel disease,
autoimmune encephalomyelitis, thyroiditis, various dermatological
conditions, and psoriasis. Recently, both CD40 and CD154 have been
shown to be expressed on vascular endothelial cells, vascular
smooth muscle cells and macrophages present in atherosclerotic
plaques, suggesting that inflammation and immunity contribute to
the atherogenic process. That this process involves CD40 signaling
is suggested by several studies in mouse models in which disruption
of CD154 (by knockout or by monoclonal antibody) reduced the
progression or size of atherosclerotic lesions. (Mach et al.,
Nature, 1998, 394:200-3; Lutgens et al., 1999, Nat. Med.
5:1313-6).
Finally, as more is learned of the association between CD40
overexpression and tumor growth, inhibitors of CD40 may prove
useful as anti-tumor agents and inhibitors of other
hyperproliferative conditions as well.
Currently, there are no known therapeutic agents which effectively
inhibit the synthesis of CD40. To date, strategies aimed at
inhibiting CD40 function have involved the use of a variety of
agents that disrupt CD40/CD40L binding. These include monoclonal
antibodies directed against either CD40 or CD40L, soluble forms of
CD40, and synthetic peptides derived from a second CD40 binding
protein, A20. The use of neutralizing antibodies against CD40
and/or CD40L in animal models has provided evidence that inhibition
of CD40 signaling would have therapeutic benefit for GVHD,
allograft rejection, rheumatoid arthritis, SLE, MS, and B-cell
lymphoma. (Buhlmann and Noelle, J. Clin. Immunol, 1996, 16:83).
Clinical investigations were initiated using anti-CD154 monoclonal
antibody in patients with lupus nephritis. However, studies were
terminated due to the development of thrombotic events. (Boumpas et
al., 2003, Arthritis Rheum. 2003, 48:719-27).
Due to the problems associated with the use of large proteins as
therapeutic agents, there is a long-felt need for additional agents
capable of effectively inhibiting CD40 function. Antisense
oligonucleotides avoid many of the pitfalls of current agents used
to block CD40/CD40L interactions and may therefore prove to be
uniquely useful in a number of therapeutic, diagnostic and research
applications. U.S. Pat. No. 6,197,584 (Bennett and Cowsert)
discloses antisense compounds targeted to CD40.
SUMMARY OF THE INVENTION
Provided herein are antisense compounds, compositions, and methods
for the treatment and prevention of inflammatory conditions and
cancer.
Antisense compounds described herein may be 12 to 30 nucleobases in
length targeted to a CD40 nucleic acid. In certain embodiments, the
CD40 nucleic acid may be any of the sequences as set forth in
GENBANK.RTM. Accession No. X60592.1, incorporated herein as SEQ ID
NO: 1; GENBANK.RTM. Accession No. H50598.1, incorporated herein as
SEQ ID NO: 2; GENBANK.RTM. Accession No. AA203290.1, incorporated
herein as SEQ ID NO: 3; and nucleotides 9797000 to nucleotide
9813000 of GENBANK Accession No. NT_011362.9, incorporated herein
as SEQ ID NO: 4, or GENBANK.RTM. Accession No. BC064518.1,
incorporated herein as SEQ ID NO: 237.
The antisense compound may be 12 to 30 nucleobases in length and
may have a nucleobase sequence comprising at least 8 contiguous
nucleobases complementary to an equal length portion of an intron
region of the CD40 gene, selected from the following regions of SEQ
ID NO: 4:
(a) positions 11250-12685, corresponding to intron 6;
(b) positions 2943-6367, corresponding to intron 1,
(c) positions 6447-6780, corresponding to intron 2,
(d) positions 6907-7157, corresponding to intron 3,
(e) positions 7305-7673, corresponding to intron 4,
(f) positions 7768-11187, corresponding to intron 5,
(g) positions 12773-12877, corresponding to intron 7, or
(h) positions 12907-13429, corresponding to intron 8,
wherein the remaining part or parts of the antisense compound are
at least 70% complementary to the sequence shown in SEQ ID NO: 4.
Preferably, the remaining parts of the antisense compound are at
least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or, most preferably, 100%
complementary to the sequence shown in SEQ ID NO: 4.
Preferably, the antisense compound may comprise at least 8
contiguous nucleobases complementary to an equal length portion of
positions 12527 to 12685 of SEQ ID NO: 4, which is a region that
can be either part of intron 6, or can be part of an alternative
version of exon 7 when a different splice acceptor site is
selected. Preferably, the antisense compound has a nucleobase
sequence comprising at least 8 contiguous nucleobases of the
nucleobase sequence of SEQ ID NO: 208, wherein the nucleobase
sequence of the compound is at least 70% complementary to the
sequence shown in SEQ ID NO: 4. Preferably, the antisense compound
is at least 75%, 80%, 85%, 90%, 95%, 98%, 99%, or, most preferably,
100% complementary to the sequence shown in SEQ ID NO: 4. More
preferably, the antisense compound has the sequence of SEQ ID NO:
208. Even more preferably, the antisense compound is 20 nucleobases
in length and consists of the nucleobase sequence of SEQ ID NO:
208. Most preferably, the antisense compound is an antisense
oligonucleotide 20 nucleotides in length having the sequence of
nucleotides as set forth in SEQ ID NO:208, wherein each cytosine is
a 5-methylcytosine, each internucleoside linkage is a
phosphorothioate linkage, nucleotides 1-5 and 16-20 are
2'-O-methoxyethyl nucleotides, and nucleotides 6-15 are
2'-deoxynucleotides; most preferably the antisense compound is ISIS
396236.
In an alternative embodiment, the antisense compound may be 12 to
30 nucleobases in length and have a nucleobase sequence comprising
at least 8 contiguous nucleobases complementary to an equal length
portion of a region of the CD40 gene, corresponding to positions
13662-16001 of SEQ ID NO: 4, which forms part of exon 9 or a region
3' to exon 9, wherein the remaining parts of the antisense compound
are at least 70% complementary to the sequence shown in SEQ ID NO:
4. Preferably, the target region of the CD40 gene corresponds to
positions 13877-14084, even more preferably to positions
13937-13996, of SEQ ID NO: 4. Preferably, the remaining parts of
the antisense compound are at least 75%, 80%, 85%, 90%, 95%, 98%,
99%, or, most preferably, 100% complementary to the sequence shown
in SEQ ID NO: 4.
In yet another alternative embodiment, the antisense compound is 12
to 30 nucleobases in length and has a nucleobase sequence
complementary to the sequence shown in SEQ ID NO: 1, starting at
position 69 or 70 of SEQ ID NO: 1, wherein the nucleobase sequence
is at least 95% complementary to the sequence shown in SEQ ID NO:
1. Preferably, the nucleobase sequence is essentially complementary
to the sequence shown in SEQ ID NO: 1. More preferably, the
nucleobase sequence is selected from the sequences of SEQ ID Nos:
90 and 163. Even more preferably, the antisense compound has a
nucleobase sequence of SEQ ID NO: 90. Even more preferably, the
antisense compound is 18 or 20 nucleobases in length and consists
of the nucleobase sequence of SEQ ID NO: 90 or SEQ ID NO: 163. The
antisense compound may be ISIS26163, ISIS396201 or ISIS396278.
Preferably, the antisense compound is an antisense oligonucleotide
18 nucleotides in length having the sequence of nucleotides as set
forth in SEQ ID NO: 90, wherein each cytosine is a
5-methylcytosine, each internucleoside linkage is a
phosphorothioate linkage, nucleotides 1-4 and 15-18 are
2'-O-methoxyethyl nucleotides, and nucleotides 5 to 14 are
2'-deoxynucleotides. Most preferably, the antisense compound is
ISIS26163.
An antisense compound according to the invention may comprise a
modified oligonucleotide consisting of 12 to 30 linked nucleosides
and having a nucleobase sequence comprising at least 12 contiguous
nucleobases of a nucleobase sequence selected from among the
nucleobase sequences recited in SEQ ID NOs: 5 to 236. Preferably,
the compound consists of a single-stranded modified
oligonucleotide. Preferably, the nucleobase sequence of the
modified oligonucleotide is 100% complementary to a nucleobase
sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4,
or SEQ ID NO: 237.
The antisense compound may comprise linked nucleosides. Preferably,
the antisense compound is an antisense oligonucleotide.
The antisense compound may be a single-stranded or double-stranded
oligonucleotide. Preferably, the antisense compound is a
single-stranded oligonucleotide.
The antisense oligonucleotide may be modified, wherein at least one
internucleoside linkage is a modified internucleoside linkage. The
internucleoside linkage may be a phosphorothioate internucleoside
linkage.
The antisense oligonucleotide may be modified, wherein at least one
nucleoside comprises a modified sugar. The modified sugar may be a
bicyclic sugar. Preferably, the at least one bicyclic sugar
comprises a 4'-CH(CH3)--O-2' bridge. The modified sugar may
comprise a 2'-O-methoxyethyl. The antisense compound may comprise
at least one tetrahydropyran modified nucleoside, wherein a
tetrahydropyran ring replaces the furanose ring. Preferably, each
of the at least one tetrahydropyran modified nucleoside has the
structure
##STR00001## wherein Bx is an optionally protected heterocyclic
base moiety.
The antisense compound may comprise a modified nucleobase. The
modified nucleobase may be a 5-methylcytosine. Preferably, every
cytosine is a 5-methylcytosine.
The antisense compound may be a gapmer, for example an
oligonucleotide comprising:
a gap segment consisting of linked deoxynucleosides;
a 5' wing segment consisting of linked nucleosides;
a 3' wing segment consisting of linked nucleosides;
wherein the gap segment is positioned between the 5' wing segment
and the 3' wing segment and wherein each nucleoside of each wing
segment comprises a modified sugar. Preferably, each nucleoside of
each wing segment comprises a 2'-O-methoxyethyl sugar; and
preferably each internucleoside linkage is a phosphorothioate
linkage.
The antisense oligonucleotide may be a 5-10-5 MOE gapmer or a
2-15-3 MOE gapmer. The antisense oligonucleotide may consist of 20
linked nucleosides.
The antisense oligonucleotide may be a 4-10-4 MOE gapmer. The
antisense oligonucleotide may consist of 18 linked nucleosides.
Compositions described herein may comprise an oligonucleotide
consisting of 12 to 30 linked nucleosides, targeted to a CD40
nucleic acid or a salt thereof and a pharmaceutically acceptable
carrier or diluent.
The composition may comprise a single-stranded or double-stranded
oligonucleotide.
Another embodiment of the invention is a pharmaceutical composition
comprising an antisense compound as described above and a liposome
or a lipid based delivery system. Preferably, said liposome is an
amphoteric liposome. Preferably, said amphoteric liposome is formed
from a lipid phase comprising an amphoteric lipid or a mixture of
lipid components with amphoteric properties. Said amphoteric
liposome may further comprise one or more neutral or zwitterionic
lipids. More preferably, said amphoteric liposome is formed from a
lipid phase comprising (a) about 15 mol % POPC, about 45 mol %
DOPE, about 20 mol % MoChol, about 20 mol % Chems (b) about 60 mol
% POPC, about 10 mol % DOTAP, about 30 mol % Chems (c) about 30 mol
% POPC, about 10 mol % DOTAP, about 20 mol % Chems, about 40 mol %
Chol (d) about 60 mol % POPC, about 20 mol % HistChol, about 20 mol
% Chol.
A further embodiment of the invention is an antisense compound or
composition as described above for medical use. Yet a further
embodiment of the invention is an antisense compound or a
composition as described above for the treatment of cancer or an
inflammatory or immune associated condition. The treatment may
further comprise administering a second drug, which may be
administered separately or concomitantly with the antisense
compound of the invention.
Methods described herein may comprise administering to an animal an
antisense compound as described above, preferably an antisense
compound comprising an oligonucleotide consisting of 12 to 30
linked nucleosides targeted to a CD40 nucleic acid, or a
composition comprising said antisense compound. Preferably, the
animal is a human.
Administration of the antisense compound and/or the second drug may
be by parenteral administration, topical administration, oral
administration or aerosol administration. Parenteral administration
may be any of subcutaneous or intravenous administration.
DETAILED DESCRIPTION OF THE INVENTION
It is to be understood that both the foregoing general description
and the following detailed description are exemplary and
explanatory only and are not restrictive of the invention, as
claimed. Herein, the use of the singular includes the plural unless
specifically stated otherwise. As used herein, the use of "or"
means "and/or" unless stated otherwise. Furthermore, the use of the
term "including" as well as other forms, such as "includes" and
"included", is not limiting. Also, terms such as "element" or
"component" encompass both elements and components comprising one
unit and elements and components that comprise more than one
subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes
only and are not to be construed as limiting the subject matter
described. All documents, or portions of documents, cited in this
application, including, but not limited to, patents, patent
applications, articles, books, and treatises, are hereby expressly
incorporated by reference in their entirety for any purpose.
Definitions
Unless specific definitions are provided, the nomenclature utilized
in connection with, and the procedures and techniques of,
analytical chemistry, synthetic organic chemistry, and medicinal
and pharmaceutical chemistry described herein are those well known
and commonly used in the art. Standard techniques may be used for
chemical synthesis, and chemical analysis. Where permitted, all
patents, applications, published applications and other
publications, GENBANK Accession Numbers and associated sequence
information obtainable through databases such as National Center
for Biotechnology Information (NCBI) and other data referred to
throughout in the disclosure herein are incorporated by reference
in their entirety.
Unless otherwise indicated, the following terms have the following
meanings:
"2'-O-methoxyethyl" (also 2'-MOE and
2'-O(CH.sub.2).sub.2--OCH.sub.3) refers to an O-methoxy-ethyl
modification of the 2' position of a furosyl ring. A
2'-O-methoxyethyl modified sugar is a modified sugar.
"2'-O-methoxyethyl nucleotide" means a nucleotide comprising a
2'-O-methoxyethyl modified sugar moiety.
"5-methylcytosine" means a cytosine modified with a methyl group
attached to the 5' position. A 5-methylcytosine is a modified
nucleobase.
"Acceptable safety profile" means a pattern of side effects that is
within clinically acceptable limits.
"Active pharmaceutical ingredient" means the substance or
substances in a pharmaceutical composition that provides a desired
effect.
"Active target region" means a target region to which one or more
active antisense compounds is targeted. "Active antisense
compounds" means antisense compounds that reduce target nucleic
acid levels.
"Administered concomitantly" refers to the co-administration of two
agents in any manner in which the pharmacological effects of both
are manifest in the patient at the same time. Concomitant
administration does not require that both agents be administered in
a single pharmaceutical composition, in the same dosage form, or by
the same route of administration.
"Administering" means providing a pharmaceutical agent to an
individual, and includes, but is not limited to administering by a
medical professional and self-administering.
"Antisense compound" means an oligomeric compound that is capable
of undergoing hybridization to a target nucleic acid through
hydrogen bonding.
"Antisense inhibition" means reduction of target nucleic acid
levels in the presence of an antisense compound complementary to a
target nucleic acid compared to target nucleic acid levels in the
absence of the antisense compound.
"Antisense oligonucleotide" means a single-stranded oligonucleotide
having a nucleobase sequence that permits hybridization to a
corresponding region or segment of a target nucleic acid.
"Bicyclic sugar" means a furosyl ring modified by the bridging of
two non-geminal ring atoms. A bicyclic sugar is a modified
sugar.
"Bicyclic nucleic acid" or "BNA" or "bicyclic nucleoside" or
"bicyclic nucleotide" refers to a nucleoside or nucleotide wherein
the furanose portion of the nucleoside includes a bridge connecting
two carbon atoms on the furanose ring, thereby forming a bicyclic
ring system. As used herein, unless otherwise indicated, the term
"methyleneoxy BNA" alone refers to .beta.-D-methyleneoxy BNA.
"Cap structure" or "terminal cap moiety" means chemical
modifications, which have been incorporated at either terminus of
an antisense compound.
"Chimeric antisense compounds" means antisense compounds that have
at least 2 chemically distinct regions, each position having a
plurality of subunits. A "gapmer" means an antisense compound in
which an internal position having a plurality of nucleotides that
supports RNaseH cleavage is positioned between external regions
having one or more nucleotides that are chemically distinct from
the nucleosides of the internal region. A "gap segment" means the
plurality of nucleotides that make up the internal region of a
gapmer. A "wing segment" means the external region of a gapmer.
"Co-administration" means administration of two or more
pharmaceutical agents to an individual. The two or more
pharmaceutical agents may be in a single pharmaceutical
composition, or may be in separate pharmaceutical compositions.
Each of the two or more pharmaceutical agents may be administered
through the same or different routes of administration.
Co-administration encompasses administration in parallel or
sequentially.
"Complementarity" means the capacity for pairing between
nucleobases of a first nucleic acid and a second nucleic acid.
"Comply" means the adherence with a recommended therapy by a
individual.
"Contiguous nucleobases" means nucleobases immediately adjacent to
each other.
"Diluent" means an ingredient in a composition that lacks
pharmacological activity, but is pharmaceutically necessary or
desirable. For example, in drugs that are injected the diluent may
be a liquid, e.g. saline solution.
"Dose" means a specified quantity of a pharmaceutical agent
provided in a single administration, or in a specified time period.
In certain embodiments, a dose may be administered in two or more
boluses, tablets, or injections. For example, in certain
embodiments, where subcutaneous administration is desired, the
desired dose requires a volume not easily accommodated by a single
injection. In such embodiments, two or more injections may be used
to achieve the desired dose. In certain embodiments, a dose may be
administered in two or more injections to minimize injection site
reaction in a individual. In other embodiments, the pharmaceutical
agent is administered by infusion over an extended period of time
or continuously. Doses may be stated as the amount of
pharmaceutical agent per hour, day, week or month.
"Dosage unit" means a form in which a pharmaceutical agent is
provided, e.g. pill, tablet, or other dosage unit known in the art.
In certain embodiments, a dosage unit is a vial containing
lyophilized antisense oligonucleotide. In certain embodiments, a
dosage unit is a vial containing reconstituted antisense
oligonucleotide.
"Duration" means the period of time during which an activity or
event continues. In certain embodiments, the duration of treatment
is the period of time during which doses of a pharmaceutical agent
are administered.
"Efficacy" means the ability to produce a desired effect.
"CD40 nucleic acid" means any nucleic acid encoding CD40. For
example, in certain embodiments, a CD40 nucleic acid includes,
without limitation, a DNA sequence encoding CD40, an RNA sequence
transcribed from DNA encoding CD40, and an mRNA sequence encoding
CD40. "CD40 mRNA" means an mRNA encoding a CD40 protein.
"Fully complementary" means each nucleobase of a first nucleic acid
has a complementary nucleobase in a second nucleic acid. In certain
embodiments, a first nucleic acid is an antisense compound and a
target nucleic acid is a second nucleic acid. In certain such
embodiments, an antisense oligonucleotide is a first nucleic acid
and a target nucleic acid is a second nucleic acid.
"Gap-widened" means an antisense compound has a gap segment of 12
or more contiguous 2'-deoxyribonucleotides positioned between and
immediately adjacent to 5' and 3' wing segments having from one to
six nucleotides having modified sugar moieties. "Immediately
adjacent" means there are no intervening nucleotides between the
immediately adjacent elements.
"Hybridization" means the annealing of complementary nucleic acid
molecules. In certain embodiments, complementary nucleic acid
molecules include, but are not limited to, an antisense compound
and a nucleic acid target. In certain such embodiments,
complementary nucleic acid molecules include, but are not limited
to, an antisense oligonucleotide and a nucleic acid target
"Individual" means a human or non-human animal selected for
treatment or therapy.
"Individual compliance" means adherence to a recommended or
prescribed therapy by a individual.
"Injection site reaction" means inflammation or abnormal redness of
skin at a site of injection in a individual.
"Internucleoside linkage" refers to the chemical bond between
nucleosides.
"Linked nucleosides" means adjacent nucleosides which are bonded
together.
"Modified internucleoside linkage" refers to a substitution and/or
any change from a naturally occurring internucleoside bond (i.e. a
phosphodiester internucleoside bond).
"Modified oligonucleotide" means an oligonucleotide comprising a
modified internucleoside linkage, a modified sugar, and/or a
modified nucleobase.
"Modified sugar" refers to a substitution and/or any change from a
natural sugar.
"Modified nucleobase" means any nucleobase other than adenine,
cytosine, guanine, thymidine, or uracil. An "unmodified nucleobase"
means the purine bases adenine (A) and guanine (G), and the
pyrimidine bases thymine (T), cytosine (C) and uracil (U).
"Modified nucleotide" means a nucleotide having, independently, a
modified sugar moiety, modified internucleoside linkage, or
modified nucleobase. A "modified nucleoside" means a nucleotide
having, independently, a modified sugar moiety or modified
nucleobase.
"Modified sugar moiety" means a sugar moiety having any
substitution and/or change from a natural sugar moiety.
"Motif" means the pattern of unmodified and modified nucleosides in
an antisense compound.
"Naturally occurring internucleoside linkage" means a 3' to 5'
phosphodiester linkage.
"Natural sugar moiety" means a sugar moiety found in DNA (2'-H) or
RNA (2'-OH).
"Non-complementary nucleobase" or "mismatch" means a nucleobase of
a first nucleic acid that is not capable of pairing with the
corresponding nucleobase of a second or target nucleic acid.
"Nucleoside" means a nucleobase linked to a sugar.
As used herein the term "nucleoside mimetic" is intended to include
those structures used to replace the sugar or the sugar and the
base and not necessarily the linkage at one or more positions of an
oligomeric compound such as for example nucleoside mimetics having
morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or
tricyclo sugar mimetics e.g. non furanose sugar units.
"Nucleobase" means a heterocyclic moiety capable of pairing with a
base of another nucleic acid.
"Nucleobase sequence" means the order of contiguous nucleobases
independent of any sugar, linkage, and/or nucleobase
modification.
"Nucleotide" means a nucleoside having a phosphate group covalently
linked to the sugar portion of the nucleoside.
The term "nucleotide mimetic" is intended to include those
structures used to replace the nucleoside and the linkage at one or
more positions of an oligomeric compound such as for example
peptide nucleic acids or morpholinos (morpholinos linked by
--N(H)--C(.dbd.O)--O-- or other non-phosphodiester linkage).
"Oligomeric compound" means a polymer or oligomer of linked
monomeric subunits which is capable of hybridizing to at least a
region of a nucleic acid molecule.
"Oligonucleotide" means an oligonucleotide in which the
internucleoside linkages do not contain a phosphorus atom.
"Oligonucleotide" means a polymer or oligomer of linked nucleosides
each of which can be modified or unmodified, independent one from
another.
"Parenteral administration," means administration through injection
or infusion. Parenteral administration includes, but is not limited
to, subcutaneous administration, intravenous administration, or
intramuscular administration.
"Pharmaceutical agent" means a substance that provides a
therapeutic benefit when administered to a individual. For example,
in certain embodiments, an antisense oligonucleotide targeted to
CD40 is pharmaceutical agent.
"Pharmaceutically acceptable salts" means physiologically and
pharmaceutically acceptable salts of antisense compounds, i.e.,
salts that retain the desired biological activity of the parent
oligonucleotide and do not impart undesired toxicological effects
thereto.
"Pharmaceutical composition" means a mixture of substances suitable
for administering to an individual. For example, a pharmaceutical
composition may comprise one or more antisense oligonucleotides or
a combination of antisense oligonucleotides and non-antisense
active agents and a sterile aqueous solution or other
pharmaceutically acceptable additive.
"Phosphorothioate linkage" means a linkage between nucleosides
where the phosphodiester bond is modified by replacing one of the
non-bridging oxygen atoms with a sulfur atom. A phosphorothioate
linkage is a modified internucleoside linkage.
"Portion" means a defined number of contiguous (i.e. linked)
nucleobases of a nucleic acid. In certain embodiments, a portion is
a defined number of contiguous nucleobases of a target nucleic
acid. In certain embodiments, a portion is a defined number of
contiguous nucleobases of an antisense compound.
"Prodrug" means a therapeutic agent that is prepared in an inactive
form that is converted to an active form (i.e., drug) within the
body or cells thereof by the action of endogenous enzymes or other
chemicals and/or conditions.
"Recommended therapy" means a therapeutic regimen recommended by a
medical professional for the treatment, amelioration, or prevention
of a disease.
"Side effects" means physiological responses attributable to a
treatment other than desired effects. In certain embodiments, side
effects include, without limitation, injection site reactions,
liver function test abnormalities, renal function abnormalities,
liver toxicity, renal toxicity, central nervous system
abnormalities, and myopathies. For example, increased
aminotransferase levels in serum may indicate liver toxicity or
liver function abnormality. For example, increased bilirubin may
indicate liver toxicity or liver function abnormality.
"Single-stranded modified oligonucleotide" means a modified
oligonucleotide which is not hybridized to a complementary
strand.
"Specifically hybridizable" means an antisense compound that
hybridizes to a target nucleic acid to induce a desired effect,
while exhibiting minimal or no effects on non-target nucleic
acids.
The term "sugar surrogate" overlaps with the slightly broader term
"nucleoside mimetic" but is intended to indicate replacement of the
sugar unit (furanose ring) only. The tetrahydropyranyl rings
provided herein are illustrative of an example of a sugar surrogate
wherein the furanose sugar group has been replaced with a
tetrahydropyranyl ring system.
"Stringent hybridization conditions" means conditions under which a
nucleic acid molecule, such as an antisense compound, will
hybridize to a target nucleic acid sequence, but to a minimal
number of other sequences. Stringent conditions are
sequence-dependent and will vary in different circumstances. In the
context of this invention, "stringent conditions" under which
oligomeric compounds hybridize to a target sequence are determined
by the nature and composition of the oligomeric compounds and the
assays in which they are being investigated.
"Subcutaneous administration" means administration just below the
skin. "Intravenous administration" means administration into a
vein.
"Targeted" or "targeted to" means having a nucleobase sequence that
will allow specific hybridization of an antisense compound to a
target nucleic acid to induce a desired effect. In certain
embodiments, a desired effect is reduction of a target nucleic
acid. In certain such embodiments, a desired effect is reduction of
a CD40 mRNA.
"Targeting" means the process of design and selection of an
antisense compound that will specifically hybridize to a target
nucleic acid and induce a desired effect.
"Target nucleic acid," "target RNA," "target RNA transcript" and
"nucleic acid target" all mean a nucleic acid capable of being
targeted by antisense compounds. Target nucleic acids may include,
but are not limited to, DNA, RNA (including, but not limited to
pre-mRNA and mRNA or portions thereof) transcribed from DNA
encoding a target, and also miRNA.
"Target region" means a portion of a target nucleic acid to which
one or more antisense compounds is targeted.
"Target segment" means the sequence of nucleotides of a target
nucleic acid to which an antisense compound is targeted. "5' target
site" refers to the 5'-most nucleotide of a target segment. "3'
target site" refers to the 3'-most nucleotide of a target
segment.
"Therapeutically effective amount" means an amount of a
pharmaceutical agent that provides a therapeutic benefit to an
individual.
"Unmodified nucleotide" means a nucleotide composed of naturally
occurring nucleobases, sugar moieties and internucleoside linkages.
In certain embodiments, an unmodified nucleotide is an RNA
nucleotide (i.e., .beta.-D-ribonucleosides) or a DNA nucleotide
(i.e., .beta.-D-deoxyribonucleoside).
Antisense Compounds
Antisense compounds include, but are not limited to,
oligonucleotides, oligonucleosides, oligonucleotide analogs,
oligonucleotide mimetics, antisense oligonucleotides, and siRNAs.
An oligomeric compound may be "antisense" to a target nucleic acid,
meaning that is capable of undergoing hybridization to a target
nucleic acid through hydrogen bonding.
In certain embodiments, an antisense compound has a nucleobase
sequence that, when written in the 5' to 3' direction, comprises
the reverse complement of the target segment of a target nucleic
acid to which it is targeted. In certain such embodiments, an
antisense oligonucleotide has a nucleobase sequence that, when
written in the 5' to 3' direction, comprises the reverse complement
of the target segment of a target nucleic acid to which it is
targeted.
In certain embodiments, an antisense compound targeted to a CD40
nucleic acid is 12 to 30 subunits in length. In other words,
antisense compounds are from 12 to 30 linked subunits. In other
embodiments, the antisense compound is 8 to 80, 12 to 50, 15 to 30,
18 to 24, 19 to 22, or 20 linked subunits. In certain such
embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,
32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48,
49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80
linked subunits in length, or a range defined by any two of the
above values. In some embodiments the antisense compound is an
antisense oligonucleotide, and the linked subunits are
nucleotides.
In certain embodiments, a shortened or truncated antisense compound
targeted to a CD40 nucleic acid has a single subunit deleted from
the 5' end (5' truncation), or alternatively from the 3' end (3'
truncation). A shortened or truncated antisense compound targeted
to a CD40 nucleic acid may have two subunits deleted from the 5'
end, or alternatively may have two subunits deleted from the 3'
end, of the antisense compound. Alternatively, the deleted
nucleosides may be dispersed throughout the antisense compound, for
example, in an antisense compound having one nucleoside deleted
from the 5' end and one nucleoside deleted from the 3' end.
When a single additional subunit is present in a lengthened
antisense compound, the additional subunit may be located at the 5'
or 3' end of the antisense compound. When two are more additional
subunits are present, the added subunits may be adjacent to each
other, for example, in an antisense compound having two subunits
added to the 5' end (5' addition), or alternatively to the 3' end
(3' addition), of the antisense compound. Alternatively, the added
subunits may be dispersed throughout the antisense compound, for
example, in an antisense compound having one subunit added to the
5' end and one subunit added to the 3' end.
It is possible to increase or decrease the length of an antisense
compound, such as an antisense oligonucleotide, and/or introduce
mismatch bases without eliminating activity. For example, in Woolf
et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of
antisense oligonucleotides 13-25 nucleobases in length were tested
for their ability to induce cleavage of a target RNA in an oocyte
injection model. Antisense oligonucleotides 25 nucleobases in
length with 8 or 11 mismatch bases near the ends of the antisense
oligonucleotides were able to direct specific cleavage of the
target mRNA, albeit to a lesser extent than the antisense
oligonucleotides that contained no mismatches. Similarly, target
specific cleavage was achieved using 13 nucleobase antisense
oligonucleotides, including those with 1 or 3 mismatches.
Gautschi et al (J. Natl. Cancer Inst. 93:463-471, March 2001)
demonstrated the ability of an oligonucleotide having 100%
complementarity to the bcl-2 mRNA and having 3 mismatches to the
bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in
vitro and in vivo. Furthermore, this oligonucleotide demonstrated
potent anti-tumor activity in vivo.
Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a
series of tandem 14 nucleobase antisense oligonucleotides, and a 28
and 42 nucleobase antisense oligonucleotides comprised of the
sequence of two or three of the tandem antisense oligonucleotides,
respectively, for their ability to arrest translation of human DHFR
in a rabbit reticulocyte assay. Each of the three 14 nucleobase
antisense oligonucleotides alone was able to inhibit translation,
albeit at a more modest level than the 28 or 42 nucleobase
antisense oligonucleotides.
Bhanot et al. (PCT/US2007/068401) provided short antisense
compounds, including compounds comprising chemically-modified
high-affinity monomers 8 to 16 monomers in length. These short
antisense compounds were shown to be useful for reducing target
nucleic acids and/or proteins in cells, tissues, and animals with
increased potency and improved therapeutic index. Short antisense
compounds were effective at lower doses than previously described
antisense compounds, allowing for a reduction in toxicity and cost
of treatment. In addition, the described short antisense compounds
have greater potential for oral dosing.
Antisense Compound Motifs
In certain embodiments, antisense compounds targeted to a CD40
nucleic acid have chemically modified subunits arranged in
patterns, or motifs, to confer to the antisense compounds
properties such as enhanced the inhibitory activity, increased
binding affinity for a target nucleic acid, or resistance to
degradation by in vivo nucleases.
Chimeric antisense compounds typically contain at least one region
modified so as to confer increased resistance to nuclease
degradation, increased cellular uptake, increased binding affinity
for the target nucleic acid, and/or increased inhibitory activity.
A second region of a chimeric antisense compound may optionally
serve as a substrate for the cellular endonuclease RNase H, which
cleaves the RNA strand of an RNA:DNA duplex.
Antisense compounds having a gapmer motif are considered chimeric
antisense compounds. In a gapmer an internal region having a
plurality of nucleotides that supports RNaseH cleavage is
positioned between external regions having a plurality of
nucleotides that are chemically distinct from the nucleosides of
the internal region. In the case of an antisense oligonucleotide
having a gapmer motif, the gap segment generally serves as the
substrate for endonuclease cleavage, while the wing segments
comprise modified nucleosides. In a preferred embodiment, the
regions of a gapmer are differentiated by the types of sugar
moieties comprising each distinct region. The types of sugar
moieties that are used to differentiate the regions of a gapmer may
in some embodiments include .beta.-D-ribonucleosides,
.beta.-D-deoxyribonucleosides, 2'-modified nucleosides (such
2'-modified nucleosides may include 2'-MOE, and 2'-O--CH.sub.3,
among others), and bicyclic sugar modified nucleosides (such
bicyclic sugar modified nucleosides may include those having a
4'-(CH.sub.2)n-O-2' bridge, where n=1 or n=2). Preferably, each
distinct region comprises uniform sugar moieties. The wing-gap-wing
motif is frequently described as "X-Y-Z", where "X" represents the
length of the 5' wing region, "Y" represents the length of the gap
region, and "Z" represents the length of the 3' wing region. Any of
the antisense compounds described herein can have a gapmer motif.
In some embodiments, X and Z are the same, in other embodiments
they are different. In a preferred embodiment, Y is between 8 and
15 nucleotides. X, Y or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more
nucleotides. Thus, gapmers of the present invention include, but
are not limited to, for example 5-10-5, 4-8-4, 4-10-4, 2-15-3,
4-12-3, 4-12-4, 3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1 or
2-8-2.
In some embodiments, the antisense compound as a "wingmer" motif,
having a wing-gap or gap-wing configuration, i.e. an X-Y or Y-Z
configuration as described above for the gapmer configuration.
Thus, wingmer configurations of the present invention include, but
are not limited to, for example 5-10, 8-4, 4-12, 12-4, 3-14, 16-2,
18-1, 10-3, 2-10, 1-10 or 8-2.
In one embodiment, antisense compounds targeted to a CD40 nucleic
acid possess a 5-10-5 gapmer motif.
In some embodiments, an antisense compound targeted to a CD40
nucleic acid has a gap-widened motif. In other embodiments, an
antisense oligonucleotide targeted to a CD40 nucleic acid has a
gap-widened motif.
In one embodiment, a gap-widened antisense oligonucleotide targeted
to a CD40 nucleic acid has a gap segment of fourteen
2'-deoxyribonucleotides positioned between wing segments of three
chemically modified nucleosides. In one embodiment, the chemical
modification comprises a 2'-sugar modification. In another
embodiment, the chemical modification comprises a 2'-MOE sugar
modification.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
Nucleotide sequences that encode CD40 include, without limitation,
the following: GENBANK Accession No. X60592.1, first deposited with
GENBANK.RTM. on Apr. 21, 1993 and incorporated herein as SEQ ID NO:
1; GENBANK.RTM. Accession No. H50598.1, first deposited with
GENBANK.RTM. on Sep. 19, 1995, and incorporated herein as SEQ ID
NO: 2; GENBANK Accession No. AA203290.1, first deposited with
GENBANK.RTM. on Jan. 25, 1997, and incorporated herein as SEQ ID
NO: 3; and nucleotides 9797000 to 9813000 of GENBANK Accession No.
NT_011362.9, first deposited with GENBANK.RTM. on Nov. 29, 2000,
and incorporated herein as SEQ ID NO: 4, and GENBANK.RTM. Accession
No. BC064518.1, incorporated herein as SEQ ID NO: 237.
It is understood that the sequence set forth in each SEQ ID NO in
the Examples contained herein is independent of any modification to
a sugar moiety, an internucleoside linkage, or a nucleobase. As
such, antisense compounds defined by a SEQ ID NO may comprise,
independently, one or more modifications to a sugar moiety, an
internucleoside linkage, or a nucleobase. Antisense compounds
described by Isis Number (Isis No) indicate a combination of
nucleobase sequence and motif.
In one embodiment, a target region is a structurally defined region
of the nucleic acid. For example, a target region may encompass a
3' UTR, a 5' UTR, an exon, an intron, a coding region, a
translation initiation region, translation termination region, or
other defined nucleic acid region. The structurally defined regions
for CD40 can be obtained by accession number from sequence
databases such as NCBI and such information is incorporated herein
by reference. In other embodiments, a target region may encompass
the sequence from a 5' target site of one target segment within the
target region to a 3' target site of another target segment within
the target region.
Targeting includes determination of at least one target segment to
which an antisense compound hybridizes, such that a desired effect
occurs. In certain embodiments, the desired effect is a reduction
in mRNA target nucleic acid levels. In other embodiments, the
desired effect is reduction of levels of protein encoded by the
target nucleic acid or a phenotypic change associated with the
target nucleic acid.
A target region may contain one or more target segments. Multiple
target segments within a target region may be overlapping.
Alternatively, they may be non-overlapping. In one embodiment,
target segments within a target region are separated by no more
than about 300 nucleotides. In other embodiments, target segments
within a target region are separated by no more than about, 250,
200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on
the target nucleic acid. In another embodiment, target segments
within a target region are separated by no more than about 5
nucleotides on the target nucleic acid. In additional embodiments,
target segments are contiguous.
Suitable target segments may be found within a 5' UTR, a coding
region, a 3' UTR, an intron, or an exon. Target segments containing
a start codon or a stop codon are also suitable target segments. A
suitable target segment may specifically exclude a certain
structurally defined region such as the start codon or stop
codon.
The determination of suitable target segments may include a
comparison of the sequence of a target nucleic acid to other
sequences throughout the genome. For example, the BLAST algorithm
may be used to identify regions of similarity amongst different
nucleic acids. This comparison can prevent the selection of
antisense compound sequences that may hybridize in a non-specific
manner to sequences other than a selected target nucleic acid
(i.e., non-target or off-target sequences).
There may be variation in activity (e.g., as defined by percent
reduction of target nucleic acid levels) of the antisense compounds
within an active target region. In one embodiment, reductions in
CD40 mRNA levels are indicative of inhibition of CD40 expression.
Reductions in levels of a CD40 protein are also indicative of
inhibition of target mRNA expression. Further, phenotypic changes
are indicative of inhibition of CD40 expression. For example,
changes in cell morphology over time or treatment dose as well as
changes in levels of cellular components such as proteins, lipids,
nucleic acids, hormones, saccharides, or metals is indicative of
inhibition of CD40 expression. Reduction of eosinophils is
indicative of inhibition of CD40 expression. Measurements of
cellular status which include pH, stage of cell cycle, intake or
excretion of biological indicators by the cell are also endpoints
of interest.
Analysis of the genotype of the cell (measurement of the expression
of one or more of the genes of the cell) after treatment is also
used as an indicator of the efficacy or potency of the CD40
inhibitors. Hallmark genes, or those genes suspected to be
associated with a specific disease state, condition, or phenotype,
are measured in both treated and untreated cells.
Genomic Structure, Exons and Introns
Although some eukaryotic mRNA transcripts are directly translated,
many contain one or more regions, known as "introns," which are
excised from a transcript before it is translated. The remaining
(and therefore translated) regions are known as "exons" and are
spliced together to form a continuous mRNA sequence. Targeting
splice sites, i.e., intron-exon junctions or exon-intron junctions,
may also be particularly useful in situations where aberrant
splicing is implicated in disease, or where an overproduction of a
particular splice product is implicated in disease. Aberrant fusion
junctions due to rearrangements or deletions are also preferred
target sites. mRNA transcripts produced via the process of splicing
of two (or more) mRNAs from different gene sources are known as
"fusion transcripts". It is also known that introns can be
effectively targeted using antisense compounds targeted to, for
example, DNA or pre-mRNA.
It is also known in the art that alternative RNA transcripts can be
produced from the same genomic region of DNA. These alternative
transcripts are generally known as "variants". More specifically,
"pre-mRNA variants" are transcripts produced from the same genomic
DNA that differ from other transcripts produced from the same
genomic DNA in either their start or stop position and contain both
intronic and exonic sequence.
Upon excision of one or more exon or intron regions, or portions
thereof during splicing, pre-mRNA variants produce smaller "mRNA
variants". Consequently, mRNA variants are processed pre-mRNA
variants and each unique pre-mRNA variant must always produce a
unique mRNA variant as a result of splicing. These mRNA variants
are also known as "alternative splice variants". If no splicing of
the pre-mRNA variant occurs then the pre-mRNA variant is identical
to the mRNA variant.
Hybridization
In some embodiments, hybridization occurs between an antisense
compound disclosed herein and a CD40 nucleic acid. The most common
mechanism of hybridization involves hydrogen bonding (e.g.,
Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding)
between complementary nucleobases of the nucleic acid
molecules.
Hybridization can occur under varying conditions. Stringent
conditions are sequence-dependent and are determined by the nature
and composition of the nucleic acid molecules to be hybridized.
Methods of determining whether a sequence is specifically
hybridizable to a target nucleic acid are well known in the art. In
one embodiment, the antisense compounds provided herein are
specifically hybridizable with a CD40 nucleic acid.
Complementarity
An antisense compound and a target nucleic acid are complementary
to each other when a sufficient number of nucleobases of the
antisense compound can hydrogen bond with the corresponding
nucleobases of the target nucleic acid, such that a desired effect
will occur (e.g., antisense inhibition of a target nucleic acid,
such as a CD40 nucleic acid).
Non-complementary nucleobases between an antisense compound and a
CD40 nucleic acid may be tolerated provided that the antisense
compound remains able to specifically hybridize to a target nucleic
acid. Moreover, an antisense compound may hybridize over one or
more segments of a CD40 nucleic acid such that intervening or
adjacent segments are not involved in the hybridization event
(e.g., a loop structure, mismatch or hairpin structure).
In some embodiments, the antisense compounds provided herein are at
least 70%, at least 75%, at least 80%, at least 85%, at least 90%,
at least 95%, at least 96%, at least 97%, at least 98% or at least
99% complementary to a CD40 nucleic acid. Percent complementarity
of an antisense compound with a target nucleic acid can be
determined using routine methods. For example, an antisense
compound in which 18 of 20 nucleobases of the antisense compound
are complementary to a target region, and would therefore
specifically hybridize, would represent 90 percent complementarity.
In this example, the remaining noncomplementary nucleobases may be
clustered or interspersed with complementary nucleobases and need
not be contiguous to each other or to complementary nucleobases. As
such, an antisense compound which is 18 nucleobases in length
having 4 (four) noncomplementary nucleobases which are flanked by
two regions of complete complementarity with the target nucleic
acid would have 77.8% overall complementarity with the target
nucleic acid and would thus fall within the scope of the present
invention. Percent complementarity of an antisense compound with a
region of a target nucleic acid can be determined routinely using
BLAST programs (basic local alignment search tools) and PowerBLAST
programs known in the art (Altschul et al., J. Mol. Biol., 1990,
215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656).
Percent homology, sequence identity or complementarity, can be
determined by, for example, the Gap program (Wisconsin Sequence
Analysis Package, Version 8 for Unix, Genetics Computer Group,
University Research Park, Madison Wis.), using default settings,
which uses the algorithm of Smith and Waterman (Adv. Appl. Math.,
1981, 2, 482 489).
In other embodiments, the antisense compounds provided herein are
fully complementary (i.e, 100% complementary) to a target nucleic
acid. For example, an antisense compound may be fully complementary
to a CD40 nucleic acid, or a target region, or a target segment or
target sequence thereof. As used herein, "fully complementary"
means each nucleobase of an antisense compound is capable of
precise base pairing with the corresponding nucleobases of a target
nucleic acid.
The location of a non-complementary nucleobase may be at the 5' end
or 3' end of the antisense compound. Alternatively, the
non-complementary nucleobase or nucleobases may be at an internal
position of the antisense compound. When two or more
non-complementary nucleobases are present, they may be contiguous
(i.e. linked) or non-contiguous. In one embodiment, a
non-complementary nucleobase is located in the wing segment of a
gapmer antisense oligonucleotide.
In one embodiment, antisense compounds up to 20 nucleobases in
length comprise no more than 4, no more than 3, no more than 2 or
no more than 1 non-complementary nucleobase(s) relative to a target
nucleic acid, such as a CD40 nucleic acid.
In another embodiment, antisense compounds up to 30 nucleobases in
length comprise no more than 6, no more than 5, no more than 4, no
more than 3, no more than 2 or no more than 1 non-complementary
nucleobase(s) relative to a target nucleic acid, such as a CD40
nucleic acid.
The antisense compounds provided herein also include those which
are complementary to a portion of a target nucleic acid. As used
herein, "portion" refers to a defined number of contiguous (i.e.
linked) nucleobases within a region or segment of a target nucleic
acid. A "portion" can also refer to a defined number of contiguous
nucleobases of an antisense compound. In one embodiment, the
antisense compounds are complementary to at least an 8 nucleobase
portion of a target segment. In another embodiment, the antisense
compounds are complementary to at least a 12 nucleobase portion of
a target segment. In yet another embodiment, the antisense
compounds are complementary to at least a 15 nucleobase portion of
a target segment. Also contemplated are antisense compounds that
are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17,
18, 19, 20, or more nucleobase portion of a target segment, or a
range defined by any two of these values.
Identity
The antisense compounds provided herein may also have a defined
percent identity to a particular nucleotide sequence, SEQ ID NO, or
compound represented by a specific Isis number. As used herein, an
antisense compound is identical to the sequence disclosed herein if
it has the same nucleobase pairing ability. For example, a RNA
which contains uracil in place of thymidine in a disclosed DNA
sequence would be considered identical to the DNA sequence since
both uracil and thymidine pair with adenine. Shortened and
lengthened versions of the antisense compounds described herein as
well as compounds having non-identical bases relative to the
antisense compounds provided herein also are contemplated. The
non-identical bases may be adjacent to each other or dispersed
throughout the antisense compound. Percent identity of an antisense
compound is calculated according to the number of bases that have
identical base pairing relative to the sequence to which it is
being compared.
In one embodiment, the antisense compounds are at least 70%, 75%,
80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or
more of the antisense compounds or SEQ ID NOs, or a portion
thereof, disclosed herein.
Modifications
A nucleoside is a base-sugar combination. The nucleobase (also
known as base) portion of the nucleoside is normally a heterocyclic
base moiety. Nucleotides are nucleosides that further include a
phosphate group covalently linked to the sugar portion of the
nucleoside. For those nucleosides that include a pentofuranosyl
sugar, the phosphate group can be linked to the 2', 3' or 5'
hydroxyl moiety of the sugar. Oligonucleotides are formed through
the covalent linkage of adjacent nucleosides to one another, to
form a linear polymeric oligonucleotide. Within the oligonucleotide
structure, the phosphate groups are commonly referred to as forming
the internucleoside linkages of the oligonucleotide.
Modifications to antisense compounds encompass substitutions or
changes to internucleoside linkages, sugar moieties, or
nucleobases. Modified antisense compounds are often preferred over
native forms because of desirable properties such as, for example,
enhanced cellular uptake, enhanced affinity for nucleic acid
target, increased stability in the presence of nucleases, or
increased inhibitory activity.
Chemically modified nucleosides may also be employed to increase
the binding affinity of a shortened or truncated antisense
oligonucleotide for its target nucleic acid. Consequently,
comparable results can often be obtained with shorter antisense
compounds that have such chemically modified nucleosides.
Modified Internucleoside Linkages
The naturally occurring internucleoside linkage of RNA and DNA is a
3' to 5' phosphodiester linkage. Antisense compounds having one or
more modified, i.e. non-naturally occurring, internucleoside
linkages are often selected over antisense compounds having
naturally occurring internucleoside linkages because of desirable
properties such as, for example, enhanced cellular uptake, enhanced
affinity for target nucleic acids, and increased stability in the
presence of nucleases.
Oligonucleotides having modified internucleoside linkages include
internucleoside linkages that retain a phosphorus atom as well as
internucleoside linkages that do not have a phosphorus atom.
Representative phosphorus containing internucleoside linkages
include, but are not limited to, phosphodiesters, phosphotriesters,
methylphosphonates, phosphonoacetates, phosphoramidate, and
phosphorothioates or phosphorodithioates. Internucleoside linkages
that do not have a phosphorus atom include, amongst others,
methylene(methylimino) or MMI linkages, morpholino linkages or
amide linkages. In peptide nucleic acids (PNA) the sugar backbone
is replaced with an amide containing backbone.
Methods of preparation of phosphorous-containing and
non-phosphorous-containing linkages are well known.
Representative United States patents that teach the preparation of
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;
5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717;
5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677;
5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;
5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218;
5,672,697; 5,625,050 and U.S. Pat. No. 6,693,187, each of which is
herein incorporated by reference.
Representative United States patents that teach the preparation of
non-phosphorous-containing linkages include, but are not limited
to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134;
5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;
5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086;
5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704;
5,623,070; 5,663,312; 5,633,360; 5,677,437; 5,792,608; 5,646,269
and 5,677,439, each of which is herein incorporated by
reference.
In one embodiment, antisense compounds targeted to a CD40 nucleic
acid comprise one or more modified internucleoside linkages. In
some embodiments, the modified internucleoside linkages are
phosphorothioate linkages. In other embodiments, each
internucleoside linkage of an antisense compound is a
phosphorothioate internucleoside linkage.
Modified Sugar Moieties
Antisense compounds of the invention can optionally contain one or
more nucleosides wherein the sugar group has been modified. Such
sugar modified nucleosides may impart enhanced nuclease stability,
increased binding affinity or some other beneficial biological
property to the antisense compounds. In certain embodiments,
nucleosides are modified by modification of the ribofuranose ring.
Such modifications include without limitation, addition of
substituent groups, bridging of non-geminal ring atoms to form a
bicyclic nucleic acid (BNA), as in locked nucleic acids (LNA),
replacement of the ribosyl ring oxygen atom with S, N(R), or
C(R1)(R)2 (R.dbd.H, C1-C12 alkyl or a protecting group) and
combinations thereof. Examples of chemically modified sugars
include 2'-F-5'-methyl substituted nucleoside (see PCT
International Application WO 2008/101157 published on Aug. 21, 2008
for other disclosed 5',2'-bis substituted nucleosides) or
replacement of the ribosyl ring oxygen atom with S with further
substitution at the 2'-position (see published U.S. Patent
Application US2005-0130923, published on Jun. 16, 2005) or
alternatively 5'-substitution of a BNA (see PCT International
Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA
is substituted with for example a 5'-methyl or a 5'-vinyl
group).
Examples of nucleosides having modified sugar moieties include
without limitation nucleosides comprising 5'-vinyl, 5'-methyl (R or
S), 4'-S, 2'-F, 2'-OCH.sub.3 (known as 2'-OMe) and
2'-O(CH.sub.2).sub.2OCH.sub.3 (known as 2'MOE) substituent groups.
The substituent at the 2' position can also be selected from allyl,
amino, azido, thio, O-allyl, O--C1-C10 alkyl, OCF.sub.3,
O--CH.sub.2CH.sub.2CH.sub.2NH.sub.2, O(CH.sub.2).sub.2SCH.sub.3,
O(CH.sub.2).sub.2--O--N(Rm)(Rn) such as 2'-dimethylaminooxyethoxy
(2'-O--(CH.sub.2).sub.2ON(CH.sub.3).sub.2 or 2'-DMAOE),
O(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--N(Rm)(Rn) such as
2'-dimethylaminoethoxyethoxy
(2'-O--(CH.sub.2).sub.2--O--(CH.sub.2).sub.2--N(CH.sub.3).sub.2 or
2'-DMAEOE and O--CH.sub.2--C(.dbd.O)--N(Rm)(Rn), where each Rm and
Rn is, independently, H or substituted or unsubstituted C1-C10
alkyl.
Examples of bicyclic nucleic acids (BNAs) include without
limitation nucleosides comprising a bridge between the 4' and the
2' ribosyl ring atoms, e.g. a 4'-(CH.sub.2).sub.n--O-2' bridge,
where n=1 or n=2. In certain embodiments, antisense compounds
provided herein include one or more BNA nucleosides wherein the
bridge comprises one of the formulas: 4'-(CH.sub.2)--O-2' (LNA);
4'-(CH.sub.2)--S-2'; 4'-(CH.sub.2)--O-2' (LNA);
4'-(CH.sub.2).sub.2--O-2' (ENA); 4'-C(CH.sub.3).sub.2--O-2' (see
PCT/US2008/068922); 4'-CH(CH.sub.3)--O-2' and
4'CH(CH.sub.2OCH.sub.3)--O-2' (see U.S. Pat. No. 7,399,845, issued
on Jul. 15, 2008); 4'-CH.sub.2--N(OCH.sub.3)-2' (see
PCT/US2008/064591); 4'-CH.sub.2--O--N(CH.sub.3)-2' (see published
U.S. Patent Application US2004-0171570, published Sep. 2, 2004);
4'-CH.sub.2--N(R)--O-2' (see U.S. Pat. No. 7,427,672, issued on
Sep. 23, 2008); 4'-CH.sub.2--C(CH.sub.3)-2' and
4'-CH.sub.2--C(.dbd.CH.sub.2)-2' (see PCT/US2008/066154); and
wherein R is, independently, H, C1-C12 alkyl, or a protecting
group. Each of the foregoing BNAs include various stereochemical
sugar configurations including for example .alpha.-L-ribofuranose
and .beta.-D-ribofuranose (See PCT international application
PCT/DK98/00393, published on Mar. 25, 1999 as WO99/14226).
In certain embodiments, nucleosides are modified by replacement of
the ribosyl ring with a sugar surrogate. Such modification includes
without limitation, replacement of the ribosyl ring with a
surrogate ring system (sometimes referred to as DNA analogs) such
as a morpholino ring, a cyclohexenyl ring, a cyclohexyl ring or a
tetrahydropyranyl ring such as one having one of the formula:
##STR00002## wherein Bx is an optionally protected heterocyclic
base moiety.
Many other bicyclo and tricyclo sugar surrogate ring systems are
also know in the art that can be used to modify nucleosides for
incorporation into antisense compounds (see for example Leumann, C.
J., Bioorg. Med. Chem. 10, (2002), 841-854). Such ring systems can
undergo various additional substitutions to enhance activity.
Methods for the preparations of modified sugars are well known to
those skilled in the art. Representative United States patents that
teach the preparation of modified sugars include, but are not
limited to U.S. Pat. No. 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, each of
which is herein incorporated by reference.
In certain embodiments, a 2'-modified nucleoside has a bicyclic
sugar moiety. In certain such embodiments, the bicyclic sugar
moiety is a D sugar in the alpha configuration. In certain such
embodiments, the bicyclic sugar moiety is a D sugar in the beta
configuration. In certain such embodiments, the bicyclic sugar
moiety is an L sugar in the alpha configuration. In certain such
embodiments, the bicyclic sugar moiety is an L sugar in the beta
configuration.
In certain embodiments, the bicyclic sugar moiety comprises a
bridge group between the 2' and the 4'-carbon atoms. In certain
such embodiments, the bridge group comprises from 1 to 8 linked
biradical groups. In certain embodiments, the bicyclic sugar moiety
comprises from 1 to 4 linked biradical groups. In certain
embodiments, the bicyclic sugar moiety comprises 2 or 3 linked
biradical groups. In certain embodiments, the bicyclic sugar moiety
comprises 2 linked biradical groups. In certain embodiments, a
linked biradical group is selected from --O--, --S--, --N(R1)-,
--C(R1)(R2)-, --C(R1)=C(R1)-, --C(R1)=N--, --C(.dbd.NR1)-,
--Si(R1)(R2)-, --S(.dbd.O).sub.2--, --S(.dbd.O)--, --C(.dbd.O)--
and --C(.dbd.S)--; where each R1 and R2 is, independently, H,
hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl,
substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12
alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocycle
radical, a substituted hetero-cycle radical, heteroaryl,
substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7
alicyclic radical, halogen, substituted oxy (--O--), amino,
substituted amino, azido, carboxyl, substituted carboxyl, acyl,
substituted acyl, CN, thiol, substituted thiol, sulfonyl
(S(.dbd.O).sub.2--H), substituted sulfonyl, sulfoxyl (S(.dbd.O)--H)
or substituted sulfoxyl; and each substituent group is,
independently, halogen, C1-C12 alkyl, substituted C1-C12 alkyl,
C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl,
substituted C2-C12 alkynyl, amino, substituted amino, acyl,
substituted acyl, C1-C12 aminoalkyl, C1-C12 aminoalkoxy,
substituted C1-C12 aminoalkyl, substituted C1-C12 aminoalkoxy or a
protecting group.
In some embodiments, the bicyclic sugar moiety is bridged between
the 2' and 4' carbon atoms with a biradical group selected from
--O--(CH.sub.2)p-, --O--CH.sub.2--, --O--CH.sub.2CH2,
--O--CH(alkyl)-, --NH--(CH.sub.2)p-, --N(alkyl)-(CH.sub.2)p-,
--O--CH(alkyl)-, --(CH(alkyl))(CH.sub.2)p-, --NH--O--(CH.sub.2)p-,
--N(alkyl)-O--(CH.sub.2)p-, or --O--N(alkyl)-(CH.sub.2)p-, wherein
p is 1, 2, 3, 4 or 5 and each alkyl group can be further
substituted. In certain embodiments, p is 1, 2 or 3.
In one aspect, each of said bridges is, independently,
--[C(R1)(R2)]n-, --[C(R1)(R2)]n-O--, --C(R1R2)-N(R1)-O-- or
--C(R1R2)-O--N(R1)-. In another aspect, each of said bridges is,
independently,
4'-(CH.sub.2).sub.3-2',4'-(CH.sub.2).sub.2-2',4'-CH.sub.2--O-2',4'-(CH.su-
b.2).sub.2--O-2',4'-CH.sub.2--O--N(R1)-2' and
4'-CH.sub.2--N(R1)-O-2'- wherein each R1 is, independently, H, a
protecting group or C1-C12 alkyl.
In nucleotides having modified sugar moieties, the nucleobase
moieties (natural, modified or a combination thereof) are
maintained for hybridization with an appropriate nucleic acid
target.
In one embodiment, antisense compounds targeted to a CD40 nucleic
acid comprise one or more nucleotides having modified sugar
moieties. In a preferred embodiment, the modified sugar moiety is
2'-MOE. In other embodiments, the 2'-MOE modified nucleotides are
arranged in a gapmer motif.
Modified Nucleobases
Nucleobase (or base) modifications or substitutions are
structurally distinguishable from, yet functionally interchangeable
with, naturally occurring or synthetic unmodified nucleobases. Both
natural and modified nucleobases are capable of participating in
hydrogen bonding. Such nucleobase modifications may impart nuclease
stability, binding affinity or some other beneficial biological
property to antisense compounds. Modified nucleobases include
synthetic and natural nucleobases such as, for example,
5-methylcytosine (5-me-C). Certain nucleobase substitutions,
including 5-methylcytosine substitutions, are particularly useful
for increasing the binding affinity of an antisense compound for a
target nucleic acid. For example, 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., Antisense Research and Applications, CRC Press, Boca Raton,
1993, pp. 276-278).
Additional unmodified nucleobases include 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
(--C.ident.C--CH.sub.3) uracil and cytosine and other alkynyl
derivatives of pyrimidine bases, 6-azo uracil, cytosine and
thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines
and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and
other 5-substituted uracils and cytosines, 7-methylguanine and
7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine.
Heterocyclic base moieties may also include those in which the
purine or pyrimidine base is replaced with other heterocycles, for
example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and
2-pyridone. Nucleobases that are particularly useful for increasing
the binding affinity of antisense compounds include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted
purines, including 2 aminopropyladenine, 5-propynyluracil and
5-propynylcytosine.
Methods for the preparations of modified nucleobases are well known
to those skilled in the art. Representative United States patents
that teach the preparation of modified nucleobases include, but are
not limited to U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos.
4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272;
5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540;
5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,645,985; 5,830,653;
5,763,588; 6,005,096; 5,750,692 and 5,681,941 each of which is
herein incorporated by reference.
In one embodiment, antisense compounds targeted to a CD40 nucleic
acid comprise one or more modified nucleobases. In an additional
embodiment, gap-widened antisense oligonucleotides targeted to a
CD40 nucleic acid comprise one or more modified nucleobases. In
some embodiments, the modified nucleobase is 5-methylcytosine. In
further embodiments, each cytosine is a 5-methylcytosine.
Conjugated Antisense Compounds
Antisense compounds may be covalently linked to one or more
moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the resulting antisense
oligonucleotides. Typical conjugate groups include cholesterol
moieties and lipid moieties. Additional conjugate groups include
carbohydrates, phospholipids, biotin, phenazine, folate,
phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines,
coumarins, and dyes.
Antisense compounds can also be modified to have one or more
stabilizing groups that are generally attached to one or both
termini of antisense compounds to enhance properties such as, for
example, nuclease stability. Included in stabilizing groups are cap
structures. These terminal modifications protect the antisense
compound having terminal nucleic acid from exonuclease degradation,
and can help in delivery and/or localization within a cell. The cap
can be present at the 5'-terminus (5'-cap), or at the 3'-terminus
(3'-cap), or can be present on both termini. Cap structures are
well known in the art and include, for example, inverted deoxy
abasic caps. Further 3' and 5'-stabilizing groups that can be used
to cap one or both ends of an antisense compound to impart nuclease
stability include those disclosed in WO 03/004602 published on Jan.
16, 2003.
Compositions and Methods for Formulating Pharmaceutical
Compositions
Antisense oligonucleotides may be admixed with pharmaceutically
acceptable active and/or inert substances for the preparation of
pharmaceutical compositions or formulations. Compositions and
methods for the formulation of pharmaceutical compositions are
dependent upon a number of criteria, including, but not limited to,
route of administration, extent of disease, or dose to be
administered.
Antisense compound targeted to a CD40 nucleic acid can be utilized
in pharmaceutical compositions by combining the antisense compound
with a suitable pharmaceutically acceptable diluent or carrier. A
pharmaceutically acceptable diluent includes for example
phosphate-buffered saline (PBS). PBS is a diluent suitable for use
in compositions to be delivered parenterally. Accordingly, in one
embodiment, employed in the methods described herein is a
pharmaceutical composition comprising an antisense compound
targeted to a CD40 nucleic acid and a pharmaceutically acceptable
diluent. In one embodiment, the pharmaceutically acceptable diluent
is PBS. In other embodiments, the antisense compound is an
antisense oligonucleotide.
Pharmaceutical compositions comprising antisense compounds
encompass any pharmaceutically acceptable salts, esters, or salts
of such esters, or any other oligonucleotide which, upon
administration to an animal, including a human, is capable of
providing (directly or indirectly) the biologically active
metabolite or residue thereof. Accordingly, for example, the
disclosure is also drawn to pharmaceutically acceptable salts of
antisense compounds, prodrugs, pharmaceutically acceptable salts of
such prodrugs, and other bioequivalents. Suitable pharmaceutically
acceptable salts include, but are not limited to, sodium and
potassium salts.
A prodrug can include the incorporation of additional nucleosides
at one or both ends of an antisense compound which are cleaved by
endogenous nucleases within the body, to form the active antisense
compound.
The present invention also includes pharmaceutical compositions and
formulations which include the antisense compounds of the
invention. The pharmaceutical compositions of the present invention
may be administered in a number of ways depending upon whether
local or systemic treatment is desired and upon the area to be
treated. Administration may be topical (including ophthalmic and to
mucous membranes including vaginal and rectal delivery), pulmonary,
e.g., by inhalation or insufflation of powders or aerosols,
including by nebulizer; intratracheal, intranasal, epidermal and
transdermal), oral or parenteral. Parenteral administration
includes intravenous, intraarterial, subcutaneous, intraperitoneal
or intramuscular injection or infusion; or intracranial, e.g.,
intrathecal or intraventricular, administration. Oligonucleotides
with at least one 2'-O-methoxyethyl modification are believed to be
particularly useful for oral administration.
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.
The pharmaceutical formulations of the present invention, which may
conveniently be presented in unit dosage form, may be prepared
according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general, the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
The compositions of the present invention may be formulated into
any of many possible dosage forms such as, but not limited to,
tablets, capsules, gel capsules, liquid syrups, soft gels,
suppositories, and enemas. The compositions of the present
invention may also be formulated as suspensions in aqueous,
non-aqueous or mixed media. Aqueous suspensions may further contain
substances which increase the viscosity of the suspension
including, for example, sodium carboxymethylcellulose, sorbitol
and/or dextran. The suspension may also contain stabilizers.
Pharmaceutical compositions of the present invention include, but
are not limited to, solutions, emulsions, foams and
liposome-containing formulations. The pharmaceutical compositions
and formulations of the present invention may comprise one or more
penetration enhancers, carriers, excipients or other active or
inactive ingredients.
Emulsions are typically heterogenous systems of one liquid
dispersed in another in the form of droplets usually exceeding 0.1
.mu.m in diameter. 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. Microemulsions are included as an
embodiment of the present invention. Emulsions and their uses are
well known in the art and are further described in U.S. Pat. No.
6,287,860, which is incorporated herein in its entirety.
Formulations of the present invention include liposomal
formulations. As used in the present invention, the term "liposome"
means a vesicle composed of amphiphilic lipids arranged in a
spherical bilayer or bilayers. Liposomes are unilamellar or
multilamellar vesicles which have a membrane formed from a
lipophilic material and an aqueous interior that contains the
composition to be delivered. Cationic liposomes are positively
charged liposomes which are believed to interact with negatively
charged DNA molecules to form a stable complex. Liposomes that are
pH-sensitive or negatively-charged are believed to entrap DNA
rather than complex with it. Both cationic and noncationic
liposomes have been used to deliver DNA to cells.
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 comprises one or more glycolipids or is
derivatized with one or more hydrophilic polymers, such as a
polyethylene glycol (PEG) moiety. Liposomes and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety.
Other liposomes or lipid based delivery systems known in the art
are described for example in WO 05/105152; WO 06/069782; Morrissey
et al., Nature Biotechnology, 23 (8), 1002-1007, 2005; WO
05/007196; Wheeler et al., Gene Therapy, 6 (2), 271-281, 1999; WO
02/34236; Budker et al., Nature Biotechnology, 14 (6), 760-764,
1996; U.S. Pat. No. 5,965,434; U.S. Pat. No. 5,635,487; Spagnou et
al., Biochemistry, 43 (42), 13348-13356, 2004; U.S. Pat. No.
6,756,054; WO 06/016097 and U.S. Pat. No. 5,785,992; WO 04/035523,
each of which is herein incorporated by reference.
In a preferred embodiment of the invention amphoteric liposomes may
be used as formulations which include the inventive antisense
compounds. Amphoteric liposomes are a class of liposomes having an
anionic or neutral charge at pH 7.5 and a cationic charge at pH 4.
Reference is made to WO 02/066012 by Panzner et al. which is
incorporated herein by reference. The use, selection and
manufacturing of amphoteric liposomes for the transfection of cells
is further described in WO 05/094783 of Ended et al., WO 07/031,333
of Panzner et al., WO 07/107,304 of Panzner et al. and WO
08/043,575 of Panzner et al. Amphoteric liposomes have an excellent
bio-distribution and are well tolerated in animals. They can
encapsulate nucleic acid molecules with high efficiency. WO
06/048329 of Panzner et al., which is incorporated herein by
reference in its entirety, describes pharmaceutical compositions
comprising amphoteric liposomes and oligonucleotides which are
adapted to target nucleic acids encoding CD40.
By "amphoteric" is meant herein that the liposomes comprise charged
groups of both anionic and cationic character wherein:
(i) at least one of the charged groups has a pKa between 4 and
7.4,
(ii) the cationic charge prevails at pH 4 and
(iii) the anionic charge prevails at pH 7.4;
whereby the liposomes have an isoelectric point of zero net charge
between pH 4 and pH 7.4. Amphoteric character, by this definition,
is different from "zwitterionic character", because zwitterions do
not have a pK in the range mentioned above. In consequence,
zwitterions are essentially neutral over a range of pH values.
Phosphatidylcholine or phosphatidylethanolamines, for example, are
neutral lipids with zwitterionic character.
Amphoteric liposomes may be formed from a lipid phase comprising an
amphoteric lipid. In some embodiments said lipid phase may comprise
5 to 30 mol. % of said amphoteric lipid, preferably 10 to 25 mol.
%.
Suitable amphoteric lipids are disclosed in WO 02/066489 and WO
03/070735 by Panzner et al. Preferably, said amphoteric lipid is
selected from the group consisting of HistChol, HistDG,
isoHistSuccDG, Acylcarnosin and HCChol. (A glossary of such
abbreviated forms of the names of the lipids referred to herein is
included below for ease of reference. A number of such
abbreviations are those that are commonly used by those skilled in
the art.)
Alternatively, said amphoteric liposomes may be formed from a lipid
phase comprising a mixture of lipid components with amphoteric
properties. Such amphoteric liposomes may be formed from
pH-responsive anionic and/or cationic components, as disclosed for
example in WO 02/066012. Cationic lipids sensitive to pH are
disclosed in WO 02/066489 and WO 03/070220 and in the references
made therein, in particular in Budker, et al. 1996, Nat Biotechnol.
14 (6):760-4, and can be used in combination with constitutively
charged anionic lipids or with anionic lipids that are sensitive to
pH.
Alternatively, the cationic charge may be introduced from
constitutively charged lipids that are known to those skilled in
the art in combination with a pH sensitive anionic lipid.
Combinations of constitutively charged anionic and cationic lipids,
e.g. DOTAP and DPPG, are not preferred. Thus, in some presently
preferred embodiments of the invention, said mixture of lipid
components may comprise (i) a stable cationic lipid and a
chargeable anionic lipid, (ii) a chargeable cationic lipid and
chargeable anionic lipid or (iii) a stable anionic lipid and a
chargeable cationic lipid.
Preferred cationic components include DMTAP, DPTAP, DOTAP, DC-Chol,
MoChol, HisChol, DPIM, CHIM, DOME, DDAB, DAC-Chol, TC-Chol, DOTMA,
DOGS, (C18).sub.2Gly.sup.+ N,N-dioctadecylamido-glycin, CTAB, CPyC,
DODAP and DOEPC.
Preferred anionic lipids for use with the invention include
DOGSucc, POGSucc, DMGSucc, DPGSucc, DMPS, DPPS, DOPS, POPS, DMPG,
DPPG, DOPG, POPG, DMPA, DPPA, DOPA, POPA, CHEMS and CetylP.
Preferably, such an amphoteric mixture of lipids does not
constitute more than about 70 mol. % of the lipid phase. In some
embodiments, said mixture may constitute not more than 50 mol. % of
the lipid phase; preferably said lipid phase comprises about 20 to
about 40 mol. % of such a mixture.
In some embodiments, said lipid phase may further comprise a
neutral lipid, preferably a neutral phospholipid, such as a
phosphatidylcholine. Presently preferred phosphatidylcholines
include POPC, natural or hydrogenated soy bean PC, natural or
hydrogenated egg PC, DMPC, DPPC, DSPC and DOPC. More preferably,
said phosphatidylcholine comprises POPC, non-hydrogenated soy bean
PC or non-hydrogenated egg PC.
The lipid phase may comprise at least 15 mol. % of said
phosphatidylcholine, preferably at least 20 mol. %. In some
embodiments, said lipid phase may comprise no less than about 25
mol. % phosphatidylcholine. Alternatively, said lipid phase may
comprise no less than about 40 mol. % phosphatidylcholine.
A presently preferred formulation in accordance with the present
invention comprises a liposome having about 60 mol. % POPC, about
10 mol. % DOTAP and about 30 mol. % CHEMS.
In some embodiments said neutral lipid may comprise a
phosphatidylethanolamine or a mixture of phosphatidylcholine and
phosphatidylethanolamine. Said neutral phosphatidylcholines or
phosphatidylethanolamines or mixtures of the two may be present in
the lipid phase in the molar amount (mol. %) not constituted by the
other components of the lipid phase, but to at least 20 mol. % (the
total for the lipid phase being 100 mol. %).
Preferred phosphatidylethanolamines include DOPE, DMPE and
DPPE.
In some embodiments said neutral lipid may comprise POPC and
DOPE.
Advantageously, said lipid phase may comprise a mixture of anionic
and cationic lipids with amphoteric properties, phosphatidylcholine
and phosphatidylethanolamine. Amphoteric liposomes formed from such
a lipid phase may be serum-stable and therefore suitable for
systemic delivery. Preferably said lipid phase comprises MoChol as
a cationic lipid and CHEMS or DMG-Succ as an anionic lipid.
Further presently preferred amphoteric liposomes for use as
formulations which include antisense compounds of the present
invention may be selected from the group consisting of: (a) about
15 mol. % POPC, about 45 mol. % DOPE, about 20 mol. % MoChol and
about 20 mol. % CHEMS; (b) about 10 mol. % POPC, about 30 mol. %
DOPE, about 30 mol. % MoChol and about 30 mol. % CHEMS; (c) about
10 mol. % POPC, about 30 mol. % DOPE, about 20 mol. % MoChol and
about 40 mol. % CHEMS; (d) about 6 mol. % POPC, about 24 mol. %
DOPE, about 47 mol. % MoChol and about 23 mol. % CHEMS.
Alternatively, said lipid phase may comprise a mixture of anionic
and cationic lipids with amphoteric properties a neutral
phosphatidylcholine and cholesterol. Such liposomes may also be
serum-stable. In some embodiments, said lipid phase may comprise
from 30 mol. % to 50 mol. % cholesterol, preferably from about 35
mol. % to about 45 mol. %. Alternatively, said lipid phase may
comprise phosphatidylcholine and from 10 mol. % to 25 mol. %
cholesterol, preferably from about 15 mol. % to about 25 mol.
%.
A presently preferred formulation comprises 10 to 25 mol. %
amphoteric lipid, e.g. HistChol, HistDG or Acylcarnosin, 15 to 25
mol. % cholesterol and the remainder being POPC, soy bean PC, egg
PC, DMPC, DPPC or DOPC, preferably POPC; for example about 60 mol.
% POPC, about 20 mol. % HistChol and about 20 mol. % Chol.
Another presently preferred formulation in accordance with the
present invention comprises a liposome including a mix of lipid
components with amphoteric properties and having about 30 mol. %
POPC, about 10 mol. % DOTAP, about 20 mol. % CHEMS and about 40
mol. % Chol.
The amphoteric liposomes may have a size in the range 50 to 500 nm,
preferably 100 to 500 nm, more preferably 150 and 300 nm.
The amphoteric liposome formulations of the present invention may
be formulated for use as a colloid in a suitable pharmacologically
acceptable vehicle. Vehicles such as water, saline, phosphate
buffered saline and the like are well known to those skilled in the
art for this purpose.
In some embodiments, the amphoteric liposome formulations of the
present invention may be administered at a physiological pH of
between about 7 and about 8. To this end, the formulation
comprising the antisense compound, excipient and vehicle may be
formulated to have a pH in this range.
The amphoteric liposome formulations of the invention may be
manufactured using suitable methods that are known to those skilled
in the art. Such methods include, but are not limited to, extrusion
through membranes of defined pore size, injection of lipid
solutions in ethanol into a water phase containing the cargo to be
encapsulated, or high pressure homogenisation.
A solution of the oligonucleotide may be contacted with said
excipient at a neutral pH, thereby resulting in volume inclusion of
a certain percentage of the solution. An high concentrations of the
excipient, ranging from about 50 mM to about 150 mM, is preferred
to achieve substantial encapsulation of the active agent.
Amphoteric liposomes used as formulations in accordance with the
present invention offer the distinct advantage of binding
oligonucleotides at or below their isoelectric point, thereby
concentrating said active agent at the liposome surface. This
process is described in more detail in WO 02/066012.
Irrespective of the actual production process used to make the
amphoteric liposome formulations, in some embodiments,
non-encapsulated oligonucleotide may be removed from the liposomes
after the initial production step in which the liposomes are formed
as tight containers. Again, the technical literature and the
references included herein describe such methodology in detail and
suitable process steps may include, but are not limited to, size
exclusion chromatography, sedimentation, dialysis, ultrafiltration
and diafiltration.
However, the removal of any non-encapsulated oligonucleotide is not
required for performance of the invention, and in some embodiments
the composition may comprise free as well as entrapped drug.
In some aspects of the invention the amphoteric liposome
formulations which include the inventive antisense compounds may be
used as pharmaceutical compositions for the prevention or treatment
of an inflammatory, immune or autoimmune disorder of a human or
non-human animal such as graft rejection, graft-versus-host
disease, multiple sclerosis, systemic lupus erythematosous,
rheumatoid arthritis, asthma, inflammatory bowel disease, psoriasis
or thyroiditis, Morbus Crohn and Colitis ulcerosa.
Glossary Of Common Abbreviated Lipid Names
DMPC Dimyristoylphosphatidylcholine DPPC
Dipalmitoylphosphatidylcholine DSPC Distearoylphosphatidylcholine
POPC Palmitoyl-oleoylphosphatidylcholine DOPC
Dioleoylphosphatidylcholine DOPE Dioleoylphosphatidylethanolamine
DMPE Dimyristoylphosphatidylethanolamine DPPE
Dipalmitoylphosphatidylethanolamine DOPG
Dioleoylphosphatidylglycerol POPG
Palmitoyl-oleoylphosphatidylglycerol DMPG
Dimyristoylphosphatidylglycerol DPPG
Dipalmitoylphosphatidylglycerol DMPS Dimyristoylphosphatidylserine
DPPS Dipalmitoylphosphatidylserine DOPS Dioleoylphosphatidylserine
POPS Palmitoyl-oleoylphosphatidylserine DMPA
Dimyristoylphosphatidic acid DPPA Dipalmitoylphosphatidic acid DOPA
Dioleoylphosphatidic acid POPA Palmitoyl-oleoylphosphatidic acid
CHEMS Cholesterolhemisuccinate DC-Chol
3-.beta.-[N--(N',N'-dimethylethane) carbamoyl]cholesterol CetylP
Cetylphosphate DODAP (1,2)-dioleoyloxypropyl)-N,N-dimethylammonium
chloride DOEPC 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine
DAC-Chol 3-.beta.-[N--(N,N'-dimethylethane) carbamoyl]cholesterol
TC-Chol
3-.beta.-[N--(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol
DOTMA (1,2-dioleyloxypropyl)-N,N,N-trimethylammoniumchlorid)
(Lipofectin.RTM.) DOGS ((C18).sub.2GlySper3.sup.+)
N,N-dioctadecylamido-glycylspermine (Transfectam.RTM.) CTAB
Cetyl-trimethylammoniumbromide, CPyC Cetyl-pyridiniumchloride DOTAP
(1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium salt DMTAP
(1,2-dimyristoyloxypropyl)-N,N,N-trimethylammonium salt DPTAP
(1,2-dipalmitoyloxypropyl)-N,N,N-trimethylammonium salt DOTMA
(1,2-dioleyloxypropyl)-N,N,N-trimethylammonium chloride) DORIE
(1,2-dioleyloxypropyl)-3 dimethylhydroxyethyl ammoniumbromide) DDAB
Dimethyldioctadecylammonium bromide DPIM
4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole CHIM
Histaminyl-Cholesterolcarbamate MoChol
4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate His Chol
Histaminyl-Cholesterolhemisuccinate. HCChol
N.alpha.-Histidinyl-Cholesterolcarbamate HistChol
N.alpha.-Histidinyl-Cholesterol-hemisuccinate. AC Acylcarnosine,
Stearyl- & Palmitoylcarnosine HistDG
1,2-Dipalmitoylglycerol-hemisuccinate-N.alpha.-Histidinyl-hemisuccinate,
& Distearoyl-, Dimyristoyl-, Dioleoyl- or
palmitoyl-oleoylderivatives IsoHistSuccDG
1,2-Dipalmitoylglycerol-O.alpha.-Histidinyl-N.alpha.-hemisuccinat,
& Distearoyl-, Dimyristoyl-, Dioleoyl- or
palmitoyl-oleoylderivatives DGSucc
1,2-Dipalmitoyglycerol-3-hemisuccinate & Distearoyl-,
Dimyristoyl-Dioleoyl- or palmitoyl-oleoyl-derivatives
The pharmaceutical formulations and compositions of the present
invention may also include surfactants. The use of surfactants in
drug products, formulations and in emulsions is well known in the
art. Surfactants and their uses are further described in U.S. Pat.
No. 6,287,860, which is incorporated herein in its entirety. In one
embodiment, the present invention employs various penetration
enhancers to effect the efficient delivery of nucleic acids,
particularly oligonucleotides. In addition to aiding the diffusion
of non-lipophilic drugs across cell membranes, penetration
enhancers also enhance the permeability of lipophilic drugs.
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. Penetration
enhancers and their uses are further described in U.S. Pat. No.
6,287,860, which is incorporated herein in its entirety.
One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
Preferred formulations for topical administration include those in
which the oligonucleotides of the invention are in admixture with a
topical delivery agent such as lipids, liposomes, fatty acids,
fatty acid esters, steroids, chelating agents and surfactants.
Preferred lipids and liposomes include neutral (e.g.
dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl
choline DMPC, distearolyphosphatidyl choline) negative (e.g.
dimyristoylphosphatidyl glycerol DMPG); cationic (e.g.
dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl
ethanolamine DOTMA) or amphoteric lipids or lipid mixtures wherein
a mixture of cationic and anionic lipids displays amphoteric
properties. For topical or other administration, oligonucleotides
of the invention may be encapsulated within liposomes or may form
complexes thereto, in particular to cationic liposomes.
Alternatively, oligonucleotides may be complexed to lipids, in
particular to cationic lipids. Preferred fatty acids and esters,
pharmaceutically acceptable salts thereof, and their uses are
further described in U.S. Pat. No. 6,287,860, which is incorporated
herein in its entirety. Topical formulations are described in
detail in U.S. patent application Ser. No. 09/315,298 filed on May
20, 1999, which is incorporated herein by reference in its
entirety.
Compositions and formulations for oral administration include
powders or granules, microparticulates, nanoparticulates,
suspensions or solutions in water or non-aqueous media, capsules,
gel capsules, sachets, tablets or minitablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable. Preferred oral formulations are those in which
oligonucleotides of the invention are administered in conjunction
with one or more penetration enhancers surfactants and chelators.
Preferred surfactants include fatty acids and/or esters or salts
thereof, bile acids and/or salts thereof. Preferred bile
acids/salts and fatty acids and their uses are further described in
U.S. Pat. No. 6,287,860, which is incorporated herein in its
entirety. Also preferred are combinations of penetration enhancers,
for example, fatty acids/salts in combination with bile
acids/salts. A particularly preferred combination is the sodium
salt of lauric acid, capric acid and UDCA. Further penetration
enhancers include polyoxyethylene-9-lauryl ether,
polyoxyethylene-20-cetyl ether. Oligonucleotides of the invention
may be delivered orally, in granular form including sprayed dried
particles, or complexed to form micro or nanoparticles.
Oligonucleotide complexing agents and their uses are further
described in U.S. Pat. No. 6,287,860, which is incorporated herein
in its entirety. Oral formulations for oligonucleotides and their
preparation are described in detail in U.S. application Ser. Nos.
09/108,673 (filed Jul. 1, 1998), 09/315,298 (filed May 20, 1999)
and 10/071,822, filed Feb. 8, 2002, each of which is incorporated
herein by reference in their entirety.
Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
Certain embodiments of the invention provide pharmaceutical
compositions containing one or more oligomeric compounds and one or
more other active agents which function by a non-antisense
mechanism, such as for example chemotherapeutic agents or
antiinflammatory drugs. Examples of such chemotherapeutic agents
include but are not limited to cancer chemotherapeutic drugs such
as daunorubicin, daunomycin, dactinomycin, doxorubicin, epirubicin,
idarubicin, esorubicin, bleomycin, mafosfamide, ifosfamide,
cytosine arabinoside, bis-chloroethylnitrosurea, busulfan,
mitomycin C, actinomycin D, mithramycin, prednisone,
hydroxyprogesterone, testosterone, tamoxifen, dacarbazine,
procarbazine, hexamethylmelamine, pentamethylmelamine,
mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,
nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,
6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea,
deoxycoformycin, 4-hydroxyperoxycyclophosphoramide, 5-fluorouracil
(5-FU), 5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX),
colchicine, taxol, vincristine, vinblastine, etoposide (VP-16),
trimetrexate, irinotecan, topotecan, gemcitabine, teniposide,
cisplatin and diethylstilbestrol (DES). When used with the
compounds of the invention, such chemotherapeutic agents may be
used individually (e.g., 5-FU and oligonucleotide), sequentially
(e.g., 5-FU and oligonucleotide for a period of time followed by
MTX and oligonucleotide), or in combination with one or more other
such chemotherapeutic agents (e.g., 5-FU, MTX and oligonucleotide,
or 5-FU, radiotherapy and oligonucleotide).
Anti-inflammatory drugs, including but not limited to non-steroidal
anti-inflammatory drugs and corticosteroids, and antiviral drugs,
including but not limited to ribivirin, vidarabine, acyclovir and
ganciclovir, may also be combined in compositions of the invention.
Combinations of antisense compounds and other non-antisense drugs
are also within the scope of this invention. Two or more combined
compounds may be used together or sequentially.
In another related embodiment, compositions of the invention may
contain one or more antisense compounds, particularly
oligonucleotides, targeted to a first nucleic acid and one or more
additional antisense compounds targeted to a second nucleic acid
target. Alternatively, compositions of the invention may contain
two or more antisense compounds targeted to different regions of
the same nucleic acid target. Numerous examples of antisense
compounds are known in the art. Two or more combined compounds may
be used together or sequentially.
Cell Culture and Antisense Compounds Treatment
The effects of antisense compounds on the level, activity or
expression of CD40 nucleic acids can be tested in vitro in a
variety of cell types. Cell types used for such analyses are
available from commercial vendors (e.g. American Type Culture
Collection, Manassas, Va.; Zen-Bio, Inc., Research Triangle Park,
N.C.; Clonetics Corporation, Walkersville, Md.) and cells are
cultured according to the vendor's instructions using commercially
available reagents (e.g. Invitrogen Life Technologies, Carlsbad,
Calif.). Illustrative cell types include, but are not limited to,
HepG2 cells, Hep3B cells, HuVEC cells, T24, A549, and primary
hepatocytes.
In vitro Testing of Antisense Oligonucleotides
Described herein are methods for treatment of cells with antisense
oligonucleotides, which can be modified appropriately for treatment
with other antisense compounds.
In general, cells are treated with antisense oligonucleotides when
the cells reach approximately 60-80% confluency in culture.
One reagent commonly used to introduce antisense oligonucleotides
into cultured cells includes the cationic lipid transfection
reagent LIPOFECTIN.RTM. (Invitrogen, Carlsbad, Calif.). Antisense
oligonucleotides are mixed with LIPOFECTIN.RTM. in OPTI-MEM.RTM. 1
(Invitrogen, Carlsbad, Calif.) to achieve the desired final
concentration of antisense oligonucleotide and a LIPOFECTIN.RTM.
concentration that typically ranges 2 to 12 ug/mL per 100 nM
antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into
cultured cells includes LIPOFECTAMINE.RTM. (Invitrogen, Carlsbad,
Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE.RTM.
in OPTI-MEM.RTM. 1 reduced serum medium (Invitrogen, Carlsbad,
Calif.) to achieve the desired concentration of antisense
oligonucleotide and a LIPOFECTAMINE.RTM. concentration that
typically ranges 2 to 12 ug/mL per 100 nM antisense
oligonucleotide.
Cells are treated with antisense oligonucleotides by routine
methods. Cells are typically harvested 16-24 hours after antisense
oligonucleotide treatment, at which time RNA or protein levels of
target nucleic acids are measured by methods known in the art and
described herein. In general, when treatments are performed in
multiple replicates, the data are presented as the average of the
replicate treatments.
The concentration of antisense oligonucleotide used varies from
cell line to cell line. Methods to determine the optimal antisense
oligonucleotide concentration for a particular cell line are well
known in the art. Antisense oligonucleotides are typically used at
concentrations ranging from 1 nM to 300 nM.
RNA Isolation
RNA analysis can be performed on total cellular RNA or poly(A)+
mRNA. Methods of RNA isolation are well known in the art. RNA is
prepared using methods well known in the art, for example, using
the TRIZOL.RTM. Reagent (Invitrogen, Carlsbad, Calif.) according to
the manufacturer's recommended protocols.
Analysis of Inhibition of Target Levels or Expression
Inhibition of levels or expression of a CD40 nucleic acid can be
assayed in a variety of ways known in the art. For example, target
nucleic acid levels can be quantitated by, e.g., Northern blot
analysis, competitive polymerase chain reaction (PCR), or
quantitative real-time PCR. RNA analysis can be performed on total
cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well
known in the art. Northern blot analysis is also routine in the
art. Quantitative real-time PCR can be conveniently accomplished
using the commercially available ABI PRISM.RTM. 7600, 7700, or 7900
Sequence Detection System, available from PE-Applied Biosystems,
Foster City, Calif. and used according to manufacturer's
instructions.
Quantitative Real-Time PCR Analysis of Target RNA Levels
Quantitation of target RNA levels may be accomplished by
quantitative real-time PCR using the ABI PRISM.RTM. 7600, 7700, or
7900 Sequence Detection System (PE-Applied Biosystems, Foster City,
Calif.) according to manufacturer's instructions. Methods of
quantitative real-time PCR are well known in the art.
Prior to real-time PCR, the isolated RNA is subjected to a reverse
transcriptase (RT) reaction, which produces complementary DNA
(cDNA) that is then used as the substrate for the real-time PCR
amplification. The RT and real-time PCR reactions are performed
sequentially in the same sample well. RT and real-time PCR reagents
are obtained from Invitrogen (Carlsbad, Calif.). RT, real-time-PCR
reactions are carried out by methods well known to those skilled in
the art.
Gene (or RNA) target quantities obtained by real time PCR are
normalized using either the expression level of a gene whose
expression is constant, such as GAPDH or by quantifying total RNA
using RIBOGREEN.RTM. (Invitrogen, Inc. Carlsbad, Calif.). GAPDH
expression is quantified by real time PCR, by being run
simultaneously with the target, multiplexing, or separately. Total
RNA is quantified using RIBOGREEN.RTM. RNA quantification reagent
(Invetrogen, Inc. Eugene, Oreg.). Methods of RNA quantification by
RIBOGREEN.RTM. are taught in Jones, L. J., et al, (Analytical
Biochemistry, 1998, 265, 368-374). A CYTOFLUOR.RTM. 4000 instrument
(PE Applied Biosystems) is used to measure RIBOGREEN.RTM.
fluorescence.
Probes and primers are designed to hybridize to a CD40 nucleic
acid. Methods for designing real-time PCR probes and primers are
well known in the art, and may include the use of software such as
PRIMER EXPRESS.RTM. Software (Applied Biosystems, Foster City,
Calif.).
Analysis of Protein Levels
Antisense inhibition of CD40 nucleic acids can be assessed by
measuring CD40 protein levels. Protein levels of CD40 can be
evaluated or quantitated in a variety of ways well known in the
art, such as immunoprecipitation, Western blot analysis
(immunoblotting), enzyme-linked immunosorbent assay (ELISA),
quantitative protein assays, protein activity assays (for example,
caspase activity assays), immunohistochemistry, immunocytochemistry
or fluorescence-activated cell sorting (FACS). Antibodies directed
to a target can be identified and obtained from a variety of
sources, such as the MSRS catalog of antibodies (Aerie Corporation,
Birmingham, Mich.), or can be prepared via conventional monoclonal
or polyclonal antibody generation methods well known in the art.
Antibodies useful for the detection of human and rat CD40 are
commercially available.
In vivo Testing of Antisense Compounds
Antisense compounds, for example, antisense oligonucleotides, are
tested in animals to assess their ability to inhibit expression of
CD40 and produce phenotypic changes, such as changes in cell
morphology over time or treatment dose as well as changes in levels
of cellular components such as proteins, nucleic acids, hormones,
cytokines, and eosinophils. Testing may be performed in normal
animals, or in experimental disease models. For administration to
animals, antisense oligonucleotides are formulated in a
pharmaceutically acceptable diluent, such as phosphate-buffered
saline. Administration includes pulmonary administration, aerosol
administration, topical administration, and parenteral routes of
administration, such as intraperitoneal, intravenous, and
subcutaneous. Calculation of antisense oligonucleotide dosage and
dosing frequency is within the abilities of those skilled in the
art, and depends upon factors such as route of administration and
animal body weight. Following a period of treatment with antisense
oligonucleotides, RNA is isolated from liver tissue and changes in
CD40 nucleic acid expression are measured.
Certain Indications
In certain embodiments, the invention provides methods of treating
an individual comprising administering one or more pharmaceutical
compositions of the present invention. In certain embodiments, the
individual has an inflammatory or hyperproliferative disorder. In
certain embodiments the invention provides methods for
prophylactically reducing CD40 expression in an individual. Certain
embodiments include treating an individual in need thereof by
administering to an individual a therapeutically effective amount
of an antisense compound targeted to a CD40 nucleic acid.
In one embodiment, administration of a therapeutically effective
amount of an antisense compound targeted to a CD40 nucleic acid is
accompanied by monitoring of eosinophils in an individual, to
determine an individual's response to administration of the
antisense compound. An individual's response to administration of
the antisense compound is used by a physician to determine the
amount and duration of therapeutic intervention.
In one embodiment, administration of an antisense compound targeted
to a CD40 nucleic acid results in reduction of CD40 expression by
at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80,
85, 90, 95 or 99%, or a range defined by any two of these values.
In some embodiments, administration of a CD40 antisense compound
increases the measure by at least 15, 20, 25, 30, 35, 40, 45, 50,
55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by
any two of these values. In some embodiments, administration of a
CD40 antisense compound decreases the measure by at least 15, 20,
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%,
or a range defined by any two of these values.
In certain embodiments pharmaceutical composition comprising an
antisense compound targeted to CD40 is used for the preparation of
a medicament for treating a patient suffering or susceptible to an
inflammatory condition or a hyperproliferative disorder.
The formulation of therapeutic compositions and their subsequent
administration (dosing) is believed to be within the skill of those
in the art. Dosing is dependent on severity and responsiveness of
the disease state to be treated, with the course of treatment
lasting from several days to several months, or until a cure is
effected or a diminution of the disease state is achieved. Optimal
dosing schedules can be calculated from measurements of drug
accumulation in the body of the patient. Persons of ordinary skill
can easily determine optimum dosages, dosing methodologies and
repetition rates. Optimum dosages may vary depending on the
relative potency of individual oligonucleotides, and can generally
be estimated based on EC50s found to be effective in in vitro and
in vivo animal models. In general, dosage is from 0.01 ug to 100 g
per kg of body weight, and may be given once or more daily, weekly,
monthly or yearly, or even once every 2 to 20 years. Persons of
ordinary skill in the art can easily estimate repetition rates for
dosing based on measured residence times and concentrations of the
drug in bodily fluids or tissues. Following successful treatment,
it may be desirable to have the patient undergo maintenance therapy
to prevent the recurrence of the disease state, wherein the
oligonucleotide is administered in maintenance doses, ranging from
0.01 ug to 100 g per kg of body weight, once or more daily, to once
every 20 years.
Certain Combination Therapies
In certain embodiments, one or more pharmaceutical compositions of
the present invention are co-administered with one or more other
pharmaceutical agents. In certain embodiments, such one or more
other pharmaceutical agents are designed to treat the same disease
or condition as the one or more pharmaceutical compositions of the
present invention. In certain embodiments, such one or more other
pharmaceutical agents are designed to treat a different disease or
condition as the one or more pharmaceutical compositions of the
present invention. In certain embodiments, such one or more other
pharmaceutical agents are designed to treat an undesired effect of
one or more pharmaceutical compositions of the present invention.
In certain embodiments, one or more pharmaceutical compositions of
the present invention are co-administered with another
pharmaceutical agent to treat an undesired effect of that other
pharmaceutical agent. In certain embodiments, one or more
pharmaceutical compositions of the present invention and one or
more other pharmaceutical agents are administered at the same time.
In certain embodiments, one or more pharmaceutical compositions of
the present invention and one or more other pharmaceutical agents
are administered at different times. In certain embodiments, one or
more pharmaceutical compositions of the present invention and one
or more other pharmaceutical agents are prepared together in a
single formulation. In certain embodiments, one or more
pharmaceutical compositions of the present invention and one or
more other pharmaceutical agents are prepared separately.
In certain embodiments, pharmaceutical agents that may be
co-administered with a pharmaceutical composition of the present
invention include steroids and/or chemotherapeutic agents. In
certain such embodiments, pharmaceutical agents that may be
co-administered with a pharmaceutical composition of the present
invention include, but are not limited to prednisone,
corticosteroids, and paclitaxel. In certain such embodiments, the
agent is administered prior to administration of a pharmaceutical
composition of the present invention. In certain such embodiments,
the agent is administered following administration of a
pharmaceutical composition of the present invention. In certain
such embodiments the agent is administered at the same time as a
pharmaceutical composition of the present invention. In certain
such embodiments the dose of a co-administered agent is the same as
the dose that would be administered if the agent was administered
alone. In certain such embodiments the dose of a co-administered
agent is lower than the dose that would be administered if the
agent was administered alone. In certain such embodiments the dose
of a co-administered agent is greater than the dose that would be
administered if the agent was administered alone.
In certain embodiments, the co-administration of a second compound
enhances the effect of a first compound, such that
co-administration of the compounds results in an effect that is
greater than the effect of administering the first compound alone.
In other embodiments, the co-administration results in effects that
are additive of the effects of the compounds when administered
alone. In other embodiments, the co-administration results in
effects that are supra-additive of the effects of the compounds
when administered alone. In some embodiments, the first compound is
an antisense compound. In some embodiments, the second compound is
an antisense compound.
EXAMPLES
Nonlimiting Disclosure and Incorporation by Reference
While certain compounds, compositions and methods described herein
have been described with specificity in accordance with certain
embodiments, the following examples serve only to illustrate the
compounds described herein and are not intended to limit the same.
Each of the references recited in the present application is
incorporated herein by reference in its entirety.
Example 1
Antisense Inhibition of Human CD40 In Vitro
Antisense oligonucleotides targeted to a CD40 nucleic acid were
tested for their effects on CD40 mRNA in vitro. When cultured
cells, grown in a 96-well plate, reached 80% confluency, they were
treated with 150 nM antisense oligonucleotide. After a treatment
period of approximately 24 hours, RNA was isolated from the cells
and CD40 mRNA levels were measured by quantitative real-time PCR,
as described herein. CD40 mRNA levels were adjusted according to
total RNA content as measured by normalization to RIBOGREEN.RTM..
Results are presented as percent inhibition of CD40, relative to
untreated control cells in Table 1.
The antisense oligonucleotides were designed as 18-mers with
phosphorothioate backbones (internucleoside linkages) throughout.
"5' target site" indicates the 5'-most nucleotide which the
antisense oligonucleotide is targeted to SEQ ID NO: 1 (GENBANK.RTM.
Accession No X60592.1). Data are averages from three
experiments.
TABLE-US-00001 TABLE 1 Inhibition of human CD40 mRNA levels by
fully phosphorothioate oligodeoxynucleotides Target SEQ Target
Target SEQ Oligo ID Start Stop Target % ID ID NO Site Site Region
Sequence (5' to 3') Inhibition NO 18623 1 18 35 5' UTR
CCAGGCGGCAGGACCACT 31 5 18624 1 20 37 5' UTR GACCAGGCGGCAGGACCA 28
6 18625 1 26 43 5' UTR AGGTGAGACCAGGCGGCA 22 7 18626 1 48 65 AUG
CAGAGGCAGACGAACCAT 0 8 18627 1 49 66 Coding GCAGAGGCAGACGAACCA 0 9
18628 1 73 90 Coding GCAAGCAGCCCCAGAGGA 0 10 18629 1 78 95 Coding
GGTCAGCAAGCAGCCCCA 30 11 18630 1 84 101 Coding GACAGCGGTCAGCAAGCA 0
12 18631 1 88 105 Coding GATGGACAGCGGTCAGCA 0 13 18632 1 92 109
Coding TCTGGATGGACAGCGGTC 0 14 18633 1 98 115 Coding
GGTGGTTCTGGATGGACA 0 15 18634 1 101 118 Coding GTGGGTGGTTCTGGATGG 0
16 18635 1 104 121 Coding GCAGTGGGTGGTTCTGGA 0 17 18636 1 152 169
Coding CACAAAGAACAGCACTGA 0 18 18637 1 156 173 Coding
CTGGCACAAAGAACAGCA 0 19 18638 1 162 179 Coding TCCTGGCTGGCACAAAGA 0
20 18639 1 165 182 Coding CTGTCCTGGCTGGCACAA 5 21 18640 1 176 193
Coding CTCACCAGTTTCTGTCCT 0 22 18641 1 179 196 Coding
TCACTCACCAGTTTCTGT 0 23 18642 1 185 202 Coding GTGCAGTCACTCACCAGT 0
24 18643 1 190 207 Coding ACTCTGTGCAGTCACTCA 0 25 18644 1 196 213
Coding CAGTGAACTCTGTGCAGT 5 26 18645 1 205 222 Coding
ATTCCGTTTCAGTGAACT 0 27 18646 1 211 228 Coding GAAGGCATTCCGTTTCAG 9
28 18647 1 222 239 Coding TTCACCGCAAGGAAGGCA 0 29 18648 1 250 267
Coding CTCTGTTCCAGGTGTCTA 0 30 18649 1 267 284 Coding
CTGGTGGCAGTGTGTCTC 0 31 18650 1 286 303 Coding TGGGGTCGCAGTATTTGT 0
32 18651 1 289 306 Coding GGTTGGGGTCGCAGTATT 0 33 18652 1 292 309
Coding CTAGGTTGGGGTCGCAGT 0 34 18653 1 318 335 Coding
GGTGCCCTTCTGCTGGAC 20 35 18654 1 322 339 Coding CTGAGGTGCCCTTCTGCT
16 36 18655 1 332 349 Coding GTGTCTGTTTCTGAGGTG 0 37 18656 1 334
351 Coding TGGTGTCTGTTTCTGAGG 0 38 18657 1 345 362 Coding
ACAGGTGCAGATGGTGTC 0 39 18658 1 348 365 Coding TTCACAGGTGCAGATGGT 0
40 18659 1 360 377 Coding GTGCCAGCCTTCTTCACA 6 41 18660 1 364 381
Coding TACAGTGCCAGCCTTCTT 8 42 18661 1 391 408 Coding
GGACACAGCTCTCACAGG 0 43 18662 1 395 412 Coding TGCAGGACACAGCTCTCA 0
44 18663 1 401 418 Coding GAGCGGTGCAGGACACAG 0 45 18664 1 416 433
Coding AAGCCGGGCGAGCATGAG 0 46 18665 1 432 449 Coding
AATCTGCTTGACCCCAAA 6 47 18666 1 446 463 Coding GAAACCCCTGTAGCAATC 0
48 18667 1 452 469 Coding GTATCAGAAACCCCTGTA 0 49 18668 1 463 480
Coding GCTCGCAGATGGTATCAG 0 50 18669 1 468 485 Coding
GCAGGGCTCGCAGATGGT 34 51 18670 1 471 488 Coding TGGGCAGGGCTCGCAGAT
0 52 18671 1 474 491 Coding GACTGGGCAGGGCTCGCA 3 53 18672 1 490 507
Coding CATTGGAGAAGAAGCCGA 0 54 18673 1 497 514 Coding
GATGACACATTGGAGAAG 0 55 18674 1 500 517 Coding GCAGATGACACATTGGAG 0
56 18675 1 506 523 Coding TCGAAAGCAGATGACACA 0 57 18676 1 524 541
Coding GTCCAAGGGTGACATTTT 8 58 18677 1 532 549 Coding
CACAGCTTGTCCAAGGGT 0 59 18678 1 539 556 Coding TTGGTCTCACAGCTTGTC 0
60 18679 1 546 563 Coding CAGGTCTTTGGTCTCACA 7 61 18680 1 558 575
Coding CTGTTGCACAACCAGGTC 19 62 18681 1 570 587 Coding
GTTTGTGCCTGCCTGTTG 2 63 18682 1 575 592 Coding GTCTTGTTTGTGCCTGCC 0
64 18683 1 590 607 Coding CCACAGACAACATCAGTC 0 65 18684 1 597 614
Coding CTGGGGACCACAGACAAC 0 66 18685 1 607 624 Coding
TCAGCCGATCCTGGGGAC 0 67 18686 1 621 638 Coding CACCACCAGGGCTCTCAG
23 68 18687 1 626 643 Coding GGGATCACCACCAGGGCT 0 69 18688 1 657
674 Coding GAGGATGGCAAACAGGAT 0 70 18689 1 668 685 Coding
ACCAGCACCAAGAGGATG 0 71 18690 1 679 696 Coding TTTTGATAAAGACCAGCA 0
72 18691 1 703 720 Coding TATTGGTTGGCTTCTTGG 0 73 18692 1 729 746
Coding GGGTTCCTGCTTGGGGTG 0 74 18693 1 750 767 Coding
GTCGGGAAAATTGATCTC 0 75 18694 1 754 771 Coding GATCGTCGGGAAAATTGA 0
76 18695 1 765 782 Coding GGAGCCAGGAAGATCGTC 0 77 18696 1 766 783
Coding TGGAGCCAGGAAGATCGT 0 78 18697 1 780 797 Coding
TGGAGCAGCAGTGTTGGA 0 79 18698 1 796 813 Coding GTAAAGTCTCCTGCACTG 0
80 18699 1 806 823 Coding TGGCATCCATGTAAAGTC 0 81 18700 1 810 827
Coding CGGTTGGCATCCATGTAA 0 82 18701 1 834 851 Coding
CTCTTTGCCATCCTCCTG 4 83 18702 1 861 878 Coding CTGTCTCTCCTGCACTGA 0
84 18703 1 873 890 Stop GGTGCAGCCTCACTGTCT 0 85 18704 1 910 927 3'
UTR AACTGCCTGTTTGCCCAC 34 86 18705 1 954 971 3' UTR
CTTCTGCCTGCACCCCTG 0 87 18706 1 976 993 3' UTR ACTGACTGGGCATAGCTC 0
88
Example 2
Antisense Inhibition of Human CD40 In Vitro
Antisense oligonucleotides targeted to a CD40 nucleic acid were
tested for their effects on CD40 mRNA in vitro. T24 cells at a
density of 7000 cells per well in a 96-well plate were treated with
150 nM antisense oligonucleotide. After a treatment period of
approximately 24 hours, RNA was isolated from the cells and CD40
mRNA levels were measured by quantitative real-time PCR, as
described herein. CD40 mRNA levels were adjusted according to GAPDH
content, a housekeeping gene. Results are presented as percent
inhibition of CD40, relative to untreated control cells in Table
2.
The antisense oligonucleotides were designed as 4-10-4 gapmers,
where the gap segment comprises 2'-deoxynucleotides and each wing
segment comprises 2'-MOE nucleotides and 5-methylcytosine
substitutions. The antisense oligonucleotides comprise
phosphorothioate backbones (internucleoside linkages) throughout.
"5' target site" indicates the 5'-most nucleotide which the
antisense oligonucleotide is targeted to SEQ ID NO: 1 (GENBANK.RTM.
Accession No X60592.1). Data are averages from three experiments.
"ND" indicates a value was not determined.
TABLE-US-00002 TABLE 2 Inhibition of human CD40 mRNA levels by
chimeric oligonucleotides having 4-10-4 MOE wings and deoxy gap
Target SEQ Target Target SEQ ID Start Stop Target % ID OligoID NO
Site Site Region Sequence (5' to 3') Inhibition NO 19211 1 18 35 5'
UTR CCAGGCGGCAGGACCACT 76 5 19212 1 20 37 5' UTR GACCAGGCGGCAGGACCA
77 6 19213 1 26 43 5' UTR AGGTGAGACCAGGCGGCA 81 7 19214 1 48 65 AUG
CAGAGGCAGACGAACCAT 24 8 19215 1 49 66 Coding GCAGAGGCAGACGAACCA 46
9 19216 1 73 90 Coding GCAAGCAGCCCCAGAGGA 66 10 19217 1 78 95
Coding GGTCAGCAAGCAGCCCCA 75 11 19218 1 84 101 Coding
GACAGCGGTCAGCAAGCA 67 12 19219 1 88 105 Coding GATGGACAGCGGTCAGCA
65 13 19220 1 92 109 Coding TCTGGATGGACAGCGGTC 79 14 19221 1 98 115
Coding GGTGGTTCTGGATGGACA 81 15 19222 1 101 118 Coding
GTGGGTGGTTCTGGATGG 58 16 19223 1 104 121 Coding GCAGTGGGTGGTTCTGGA
74 17 19224 1 152 169 Coding CACAAAGAACAGCACTGA 40 18 19225 1 156
173 Coding CTGGCACAAAGAACAGCA 60 19 19226 1 162 179 Coding
TCCTGGCTGGCACAAAGA 10 20 19227 1 165 182 Coding CTGTCCTGGCTGGCACAA
24 21 19228 1 176 193 Coding CTCACCAGTTTCTGTCCT 22 22 19229 1 179
196 Coding TCACTCACCAGTTTCTGT 41 23 19230 1 185 202 Coding
GTGCAGTCACTCACCAGT 82 24 19231 1 190 207 Coding ACTCTGTGCAGTCACTCA
38 25 19232 1 196 213 Coding CAGTGAACTCTGTGCAGT 40 26 19233 1 205
222 Coding ATTCCGTTTCAGTGAACT 56 27 19234 1 211 228 Coding
GAAGGCATTCCGTTTCAG 32 28 19235 1 222 239 Coding TTCACCGCAAGGAAGGCA
61 29 19236 1 250 267 Coding CTCTGTTCCAGGTGTCTA 62 30 19237 1 267
284 Coding CTGGTGGCAGTGTGTCTC 70 31 19238 1 286 303 Coding
TGGGGTCGCAGTATTTGT 0 32 19239 1 289 306 Coding GGTTGGGGTCGCAGTATT
19 33 19240 1 292 309 Coding CTAGGTTGGGGTCGCAGT 36 34 19241 1 318
335 Coding GGTGCCCTTCTGCTGGAC 79 35 19242 1 322 339 Coding
CTGAGGTGCCCTTCTGCT 70 36 19243 1 332 349 Coding GTGTCTGTTTCTGAGGTG
63 37 19244 1 334 351 Coding TGGTGTCTGTTTCTGAGG 43 38 19245 1 345
362 Coding ACAGGTGCAGATGGTGTC 73 39 19246 1 348 365 Coding
TTCACAGGTGCAGATGGT 48 40 19247 1 360 377 Coding GTGCCAGCCTTCTTCACA
61 41 19248 1 364 381 Coding TACAGTGCCAGCCTTCTT 47 42 19249 1 391
408 Coding GGACACAGCTCTCACAGG 0 43 19250 1 395 412 Coding
TGCAGGACACAGCTCTCA 52 44 19251 1 401 418 Coding GAGCGGTGCAGGACACAG
50 45 19252 1 416 433 Coding AAGCCGGGCGAGCATGAG 32 46 19253 1 432
449 Coding AATCTGCTTGACCCCAAA 0 47 19254 1 446 463 Coding
GAAACCCCTGTAGCAATC 0 48 19255 1 452 469 Coding GTATCAGAAACCCCTGTA
36 49 19256 1 463 480 Coding GCTCGCAGATGGTATCAG 65 50 19257 1 468
485 Coding GCAGGGCTCGCAGATGGT 75 51 19258 1 471 488 Coding
TGGGCAGGGCTCGCAGAT 0 52 19259 1 474 491 Coding GACTGGGCAGGGCTCGCA
82 53 19260 1 490 507 Coding CATTGGAGAAGAAGCCGA 41 54 19261 1 497
514 Coding GATGACACATTGGAGAAG 14 55 19262 1 500 517 Coding
GCAGATGACACATTGGAG 78 56 19263 1 506 523 Coding TCGAAAGCAGATGACACA
59 57 19264 1 524 541 Coding GTCCAAGGGTGACATTTT 71 58 19265 1 532
549 Coding CACAGCTTGTCCAAGGGT 0 59 19266 1 539 556 Coding
TTGGTCTCACAGCTTGTC 46 60 19267 1 546 563 Coding CAGGTCTTTGGTCTCACA
64 61 19268 1 558 575 Coding CTGTTGCACAACCAGGTC 82 62 19269 1 570
587 Coding GTTTGTGCCTGCCTGTTG 70 63 19270 1 575 592 Coding
GTCTTGTTTGTGCCTGCC 69 64 19271 1 590 607 Coding CCACAGACAACATCAGTC
11 65 19272 1 597 614 Coding CTGGGGACCACAGACAAC 9 66 19273 1 607
624 Coding TCAGCCGATCCTGGGGAC 0 67 19274 1 621 638 Coding
CACCACCAGGGCTCTCAG 23 68 19275 1 626 643 Coding GGGATCACCACCAGGGCT
58 69 19276 1 657 674 Coding GAGGATGGCAAACAGGAT 49 70 19277 1 668
685 Coding ACCAGCACCAAGAGGATG ND 71 19278 1 679 696 Coding
TTTTGATAAAGACCAGCA 31 72 19279 1 703 720 Coding TATTGGTTGGCTTCTTGG
49 73 19280 1 729 746 Coding GGGTTCCTGCTTGGGGTG 14 74 19281 1 750
767 Coding GTCGGGAAAATTGATCTC 55 75 19282 1 754 771 Coding
GATCGTCGGGAAAATTGA 0 76 19283 1 765 782 Coding GGAGCCAGGAAGATCGTC
69 77 19284 1 766 783 Coding TGGAGCCAGGAAGATCGT 54 78 19285 1 780
797 Coding TGGAGCAGCAGTGTTGGA 15 79 19286 1 796 813 Coding
GTAAAGTCTCCTGCACTG 31 80 19287 1 806 823 Coding TGGCATCCATGTAAAGTC
65 81 19288 1 810 827 Coding CGGTTGGCATCCATGTAA 34 82 19289 1 834
851 Coding CTCTTTGCCATCCTCCTG 42 83 19290 1 861 878 Coding
CTGTCTCTCCTGCACTGA 26 84 19291 1 873 890 Stop GGTGCAGCCTCACTGTCT 76
85 19292 1 910 927 3' UTR AACTGCCTGTTTGCCCAC 63 86 19293 1 954 971
3' UTR CTTCTGCCTGCACCCCTG 0 87 19294 1 976 993 3' UTR
ACTGACTGGGCATAGCTC 12 88
Example 3
Antisense Inhibition of Human CD40
Antisense oligonucleotides targeted to a CD40 nucleic acid were
tested for their effects on CD40 mRNA in vitro. T24 cells at a
density of 7000 cells per well in a 96-well plate were treated with
100 nM of antisense oligonucleotide. After a treatment period of
approximately 24 hours, RNA was isolated from the cells and CD40
mRNA levels were measured by quantitative real-time PCR, as
described herein. CD40 mRNA levels were adjusted according to GAPDH
content, a housekeeping gene. Results are presented as percent
inhibition of CD40, relative to untreated control cells in Table
3.
The antisense oligonucleotides were designed as 4-10-4 gapmers,
where the gap segment comprises 2'-deoxynucleotides and each wing
segment comprises 2'-MOE nucleotides. The antisense
oligonucleotides comprise phosphorothioate backbones
(internucleoside linkages) and 5-methylcytosine substitutions
throughout. "5' target site" indicates the 5'-most nucleotide which
the antisense oligonucleotide is targeted to SEQ ID NO: 1 (GENBANK
Accession No. X60592.1), SEQ ID NO: 2 (GENBANK.RTM. Accession No.
H50598.1), and SEQ ID NO: 3 (GENBANK.RTM. Accession No.
AA203290.1).
TABLE-US-00003 TABLE 3 Inhibition of human CD40 mRNA levels by
chimeric oligonucleotides having 4-10-4 MOE wings and deoxy gap
Target SEQ Target Target SEQ Oligo ID Start Stop % ID ID NO Site
Site Sequence (5' to 3') Inhibition NO 26162 1 66 83
GCCCCAGAGGACGCACTG 0 89 26163 1 70 87 AGCAGCCCCAGAGGACGC 98 90
26164 1 74 91 AGCAAGCAGCCCCAGAGG 47 91 26165 1 80 97
GCGGTCAGCAAGCAGCCC 54 92 26167 1 95 112 GGTTCTGGATGGACAGCG 66 93
26168 1 102 119 AGTGGGTGGTTCTGGATG 26 94 26169 1 141 158
GCACTGACTGTTTATTAG 43 95 26170 1 154 171 GGCACAAAGAACAGCACT 53 96
26171 1 164 181 TGTCCTGGCTGGCACAAA 29 97 26172 1 171 188
CAGTTTCTGTCCTGGCTG 48 98 26173 1 180 197 GTCACTCACCAGTTTCTG 47 99
26174 1 210 227 AAGGCATTCCGTTTCAGT 57 100 26175 1 224 241
CTTTCACCGCAAGGAAGG 34 101 26176 1 250 267 CTCTGTTCCAGGTGTCTA 78 30
26177 1 257 274 TGTGTCTCTCTGTTCCAG 57 102 26178 1 264 281
GTGGCAGTGTGTCTCTCT 0 103 26179 1 314 331 CCCTTCTGCTGGACCCGA 58 104
26180 1 321 338 TGAGGTGCCCTTCTGCTG 69 105 26181 1 329 346
TCTGTTTCTGAGGTGCCC 44 106 26182 1 336 353 GATGGTGTCTGTTTCTGA 12 107
26183 1 364 381 TACAGTGCCAGCCTTCTT 14 42 26184 1 445 462
AAACCCCTGTAGCAATCT 15 108 26185 1 460 477 CGCAGATGGTATCAGAAA 53 109
26186 1 469 486 GGCAGGGCTCGCAGATGG 79 110 26202 1 485 502
GAGAAGAAGCCGACTGGG 0 111 26187 1 487 504 TGGAGAAGAAGCCGACTG 23 112
26204 1 489 506 ATTGGAGAAGAAGCCGAC 0 113 26205 1 491 508
ACATTGGAGAAGAAGCCG 4 114 26206 1 493 510 ACACATTGGAGAAGAAGC 0 115
26207 1 495 512 TGACACATTGGAGAAGAA 46 116 26188 1 496 513
ATGACACATTGGAGAAGA 0 117 26208 1 497 514 GATGACACATTGGAGAAG 0 55
26189 1 503 520 AAAGCAGATGACACATTG 6 118 26209 1 524 541
GTCCAAGGGTGACATTTT 53 58 26210 1 545 562 AGGTCTTTGGTCTCACAG 81 119
26211 1 555 572 TTGCACAACCAGGTCTTT 48 120 26212 1 570 587
GTTTGTGCCTGCCTGTTG 76 63 26213 1 572 589 TTGTTTGTGCCTGCCTGT 50 121
26214 1 574 591 TCTTGTTTGTGCCTGCCT 87 122 26215 1 576 593
AGTCTTGTTTGTGCCTGC 83 123 26216 1 577 594 CAGTCTTGTTTGTGCCTG 80 124
26217 1 578 595 TCAGTCTTGTTTGTGCCT 88 125 26218 1 580 597
CATCAGTCTTGTTTGTGC 52 126 26219 1 590 607 CCACAGACAACATCAGTC 16 65
26220 1 592 609 GACCACAGACAACATCAG 11 127 26221 1 594 611
GGGACCACAGACAACATC 40 128 26222 1 622 639 TCACCACCAGGGCTCTCA 37 129
26223 1 624 641 GATCACCACCAGGGCTCT 82 130 26224 1 658 675
AGAGGATGGCAAACAGGA 33 131 26225 1 659 676 AAGAGGATGGCAAACAGG 0 132
26226 1 660 677 CAAGAGGATGGCAAACAG 0 133 26227 1 669 686
GACCAGCACCAAGAGGAT 57 134 26228 1 671 688 AAGACCAGCACCAAGAGG 35 135
26229 1 673 690 TAAAGACCAGCACCAAGA 13 136 26230 1 676 693
TGATAAAGACCAGCACCA 0 137 26231 1 678 695 TTTGATAAAGACCAGCAC 26 138
26232 2 375 392 ACTCTCTTTGCCCATCCT 0 139 26233 2 377 394
CGACTCTCTTTGCCCATC 31 140 26234 2 380 397 ATGCGACTCTCTTTGCCC 12 141
26235 2 382 399 AAATGCGACTCTCTTTGC 36 142 26236 2 385 402
CTGAAATGCGACTCTCTT 51 143 26237 2 387 404 AACTGAAATGCGACTCTC 0 144
26238 2 406 423 CTTCACTGTCTCTCCCTG 0 145 26239 2 407 424
CCTTCACTGTCTCTCCCT 56 146 26240 2 409 426 AACCTTCACTGTCTCTCC 0 147
26190 3 520 537 GATCACCACAGGCTCTCA 0 148 26191 3 565 582
TGATAAGACAGCACCAAG 9 149 26192 3 584 601 GGTAGTTCTTGCCACTTT 0 150
26193 3 593 610 GGGCCTATGGGTAGTTCT 0 151 26194 3 617 634
ATTATCTCTGGGTCTGCT 9 152 26195 3 646 663 ACTGACACATTTGAGCAG 0 153
26196 3 654 671 GACTCCCTACTGACACAT 0 154 26197 3 689 706
CAAAGAGCGGTTCTCCAC 0 155 26198 3 696 713 AATTCTCCAAAGAGCGGT 0 156
26199 3 728 745 TCTTGACATCCTTTTCAT 0 157 26200 3 736 753
CCCACCTATCTTGACATC 0 158 26201 3 791 808 AGGCCGAGAGTTCAAAAT 0
159
Example 4
Antisense Inhibition of Human CD40
Antisense oligonucleotides targeted to a CD40 nucleic acid were
tested for their effects on CD40 mRNA in vitro. A549 cells at a
density of 5000 cells per well in a 96-well plate were treated with
120 nM of antisense oligonucleotide. After a treatment period of
approximately 24 hours, RNA was isolated from the cells and CD40
mRNA levels were measured by quantitative real-time PCR, as
described herein. CD40 primer probe set LTS37 was used to measure
mRNA levels. CD40 mRNA levels were adjusted according to total RNA
content as measured by RIBOGREEN.RTM.. Results are presented as
percent inhibition of CD40, relative to untreated control cells in
Table 4.
The antisense oligonucleotides were designed as 4-10-4 gapmers,
5-10-5 gapmers, or 2-15-3 gapmers, where the gap segment comprises
2'-deoxynucleotides and each wing segment comprises 2'-MOE
nucleotides. The motif for each compound is indicated by the column
labeled "motif." The antisense oligonucleotides comprise
phosphorothioate backbones (internucleoside linkages) and
5-methylcytosine substitutions throughout. "5' target site"
indicates the 5'-most nucleotide which the antisense
oligonucleotide is targeted to SEQ ID NO: 1 (GENBANK Accession No.
X60592.1) or SEQ ID NO: 4 (nucleotides 9797000 to 9813000 of
GENBANK Accession No. NT_011362.9).
TABLE-US-00004 TABLE 4 Inhibition of human CD40 mRNA levels by
chimeric oligonucleotides having 4-10-4 MOE wings and deoxy gap,
5-10-5 MOE wings and deoxy gap, and 2-15-3 MOE wings and deoxy gap
Target Target Target Oligo SEQ ID Start Stop % SEQ ID ID NO Motif
Site Site Sequence (5' to 3') Inhibition NO 26163 4 4-10-4 2914
2931 AGCAGCCCCAGAGGACGC 74 90 396243 4 5-10-5 2728 2747
CCAGCAATTCACCGCGCAGG 0 160 396320 4 2-15-3 2728 2747
CCAGCAATTCACCGCGCAGG 0 160 396199 4 5-10-5 2892 2911
TGCAGAGGCAGACGAACCAT 75 161 396276 4 2-15-3 2892 2911
TGCAGAGGCAGACGAACCAT 55 161 396200 4 5-10-5 2904 2923
CAGAGGACGCACTGCAGAGG 79 162 396277 4 2-15-3 2904 2923
CAGAGGACGCACTGCAGAGG 69 162 396201 4 5-10-5 2913 2932
AAGCAGCCCCAGAGGACGCA 76 163 396278 4 2-15-3 2913 2932
AAGCAGCCCCAGAGGACGCA 78 163 396202 4 5-10-5 2924 2943
CAGCGGTCAGCAAGCAGCCC 68 164 396279 4 2-15-3 2924 2943
CAGCGGTCAGCAAGCAGCCC 88 164 396244 4 5-10-5 2928 2947
CTCACAGCGGTCAGCAAGCA 86 165 396321 4 2-15-3 2928 2947
CTCACAGCGGTCAGCAAGCA 75 165 396245 4 5-10-5 3349 3368
GCTGGCAAGGAGATGATAAC 51 166 396322 4 2-15-3 3349 3368
GCTGGCAAGGAGATGATAAC 54 166 396246 4 5-10-5 3480 3499
AGGTTGGAACACCCAAGATA 69 167 396323 4 2-15-3 3480 3499
AGGTTGGAACACCCAAGATA 78 167 396247 4 5-10-5 3649 3668
GGAGAAACCCCTGGTTTCTC 45 168 396324 4 2-15-3 3649 3668
GGAGAAACCCCTGGTTTCTC 26 168 396248 4 5-10-5 3860 3879
TCATTCCTGCCCAGGCTTCA 43 169 396325 4 2-15-3 3860 3879
TCATTCCTGCCCAGGCTTCA 39 169 396249 4 5-10-5 3950 3969
TCAGGTGAAAGTGAAAGCTG 68 170 396326 4 2-15-3 3950 3969
TCAGGTGAAAGTGAAAGCTG 69 170 396250 4 5-10-5 4490 4509
TACCATCTTCAAACACATGA 79 171 396327 4 2-15-3 4490 4509
TACCATCTTCAAACACATGA 71 171 396251 4 5-10-5 4604 4623
TTACCCAAAATGGGAAAGGA 86 172 396328 4 2-15-3 4604 4623
TTACCCAAAATGGGAAAGGA 48 172 396252 4 5-10-5 4810 4829
GAAAGAATACATGTATATGG 72 173 396329 4 2-15-3 4810 4829
GAAAGAATACATGTATATGG 10 173 396253 4 5-10-5 4944 4963
AGAGTCAGACAGCTTTAGAC 78 174 396330 4 2-15-3 4944 4963
AGAGTCAGACAGCTTTAGAC 79 174 396254 4 5-10-5 5651 5670
GTACCACCCATGCTATTAAT 79 175 396331 4 2-15-3 5651 5670
GTACCACCCATGCTATTAAT 84 175 396255 4 5-10-5 5740 5759
ACAGTGACAGAGTCCAAATG 85 176 396332 4 2-15-3 5740 5759
ACAGTGACAGAGTCCAAATG 75 176 396256 4 5-10-5 5830 5849
AATGTAAAGCTGGAAGGGTA 52 177 396333 4 2-15-3 5830 5849
AATGTAAAGCTGGAAGGGTA 37 177 396257 4 5-10-5 5964 5983
GGGCTATGTTTAGCACTTGG 79 178 396334 4 2-15-3 5964 5983
GGGCTATGTTTAGCACTTGG 73 178 396258 4 5-10-5 6078 6097
GGGCTTGATGCCTGAGTCAT 73 179 396335 4 2-15-3 6078 6097
GGGCTTGATGCCTGAGTCAT 40 179 396259 4 5-10-5 6251 6270
TGAAGTGCAAGTCAAAACAG 52 180 396336 4 2-15-3 6251 6270
TGAAGTGCAAGTCAAAACAG 44 180 396260 4 5-10-5 6332 6351
GCAATTTGAAGGGATCTTGA 68 181 396337 4 2-15-3 6332 6351
GCAATTTGAAGGGATCTTGA 42 181 396203 4 5-10-5 6374 6393
CATGCAGTGGGTGGTTCTGG 77 182 396280 4 2-15-3 6374 6393
CATGCAGTGGGTGGTTCTGG 83 182 396204 4 5-10-5 6385 6404
GTTTTTCTCTGCATGCAGTG 78 183 396281 4 2-15-3 6385 6404
GTTTTTCTCTGCATGCAGTG 70 183 396205 4 5-10-5 6424 6443
GCTGGCACAAAGAACAGCAC 61 184 396282 4 2-15-3 6424 6443
GCTGGCACAAAGAACAGCAC 65 184 396261 4 5-10-5 6709 6728
CACTAACCACACAATGATCA 85 185 396338 4 2-15-3 6709 6728
CACTAACCACACAATGATCA 62 185 396206 4 5-10-5 6787 6806
TGTGCAGTCACTCACCAGTT 83 186 396283 4 2-15-3 6787 6806
TGTGCAGTCACTCACCAGTT 72 186 396207 4 5-10-5 6838 6857
GTCTAGGAATTCGCTTTCAC 95 187 396284 4 2-15-3 6838 6857
GTCTAGGAATTCGCTTTCAC 85 187 396208 4 5-10-5 6843 6862
CAGGTGTCTAGGAATTCGCT 98 188 396285 4 2-15-3 6843 6862
CAGGTGTCTAGGAATTCGCT 90 188 396209 4 5-10-5 6883 6902
GTCGCAGTATTTGTGCTGGT 84 189 396286 4 2-15-3 6883 6902
GTCGCAGTATTTGTGCTGGT 86 189 396262 4 5-10-5 7154 7173
ACCCGAAGCCCTAGGTCTGA 92 190 396339 4 2-15-3 7154 7173
ACCCGAAGCCCTAGGTCTGA 84 190 396210 4 5-10-5 7158 7177
CTGGACCCGAAGCCCTAGGT 82 191 396287 4 2-15-3 7158 7177
CTGGACCCGAAGCCCTAGGT 90 191 396211 4 5-10-5 7163 7182
TTCTGCTGGACCCGAAGCCC 65 192 396288 4 2-15-3 7163 7182
TTCTGCTGGACCCGAAGCCC 80 192 396212 4 5-10-5 7204 7223
CTTCTTCACAGGTGCAGATG 79 193 396289 4 2-15-3 7204 7223
CTTCTTCACAGGTGCAGATG 72 193 396263 4 5-10-5 7590 7609
AGCCAGTGGCCAGGCAGGAC 70 194 396340 4 2-15-3 7590 7609
AGCCAGTGGCCAGGCAGGAC 56 194 396214 4 5-10-5 7704 7723
GAAGAAGCCGACTGGGCAGG 76 195 396291 4 2-15-3 7704 7723
GAAGAAGCCGACTGGGCAGG 80 195 396215 4 5-10-5 7709 7728
TTGGAGAAGAAGCCGACTGG 77 196 396292 4 2-15-3 7709 7728
TTGGAGAAGAAGCCGACTGG 80 196 396216 4 5-10-5 7718 7737
GATGACACATTGGAGAAGAA 76 197 396293 4 2-15-3 7718 7737
GATGACACATTGGAGAAGAA 65 197 396264 4 5-10-5 7953 7972
TGTCTATTACCTCAAAGAGA 89 198 396341 4 2-15-3 7953 7972
TGTCTATTACCTCAAAGAGA 72 198 396265 4 5-10-5 8492 8511
ACAGTGTGTTCAGAGGATTG 82 199 396342 4 2-15-3 8492 8511
ACAGTGTGTTCAGAGGATTG 67 199 396266 4 5-10-5 9755 9774
ACAATACACTTTACATGTTT 90 200 396343 4 2-15-3 9755 9774
ACAATACACTTTACATGTTT 63 200 396267 4 5-10-5 10414 10433
ATTGTGTCTTTAGAACCAGA 84 201 396344 4 2-15-3 10414 10433
ATTGTGTCTTTAGAACCAGA 59 201 396268 4 5-10-5 10528 10547
GGGCCCTAAAGGATGTAAAA 34 202 396345 4 2-15-3 10528 10547
GGGCCCTAAAGGATGTAAAA 76 202 396217 4 5-10-5 11218 11237
CAGTCTTGTTTGTGCCTGCC 70 203 396294 4 2-15-3 11218 11237
CAGTCTTGTTTGTGCCTGCC 79 203 396269 4 5-10-5 11244 11263
TGTCCAGGACTCACCACAGA 77 204 396346 4 2-15-3 11244 11263
TGTCCAGGACTCACCACAGA 83 204 396270 4 5-10-5 11801 11820
TATGGCACCTTCTTAAATAT 85 205 396347 4 2-15-3 11801 11820
TATGGCACCTTCTTAAATAT 81 205 396271 4 5-10-5 12248 12267
TGCTTTTGGTATAGAAGAGT 86 206 396348 4 2-15-3 12248 12267
TGCTTTTGGTATAGAAGAGT 76 206 396235 4 5-10-5 12526 12545
AAATGTGGCTGGCAGATGTC 79 207 396312 4 2-15-3 12526 12545
AAATGTGGCTGGCAGATGTC 82 207 396236 4 5-10-5 12572 12591
GTCAGAGCTCATCTACATCA 87 208 396313 4 2-15-3 12572 12591
GTCAGAGCTCATCTACATCA 82 208 396237 4 5-10-5 12754 12773
CTGATAAAGACCAGCACCAA 69 209 396314 4 2-15-3 12754 12773
CTGATAAAGACCAGCACCAA 70 209 396238 4 5-10-5 12762 12781
AGGACTCACTGATAAAGACC 69 210 396315 4 2-15-3 12762 12781
AGGACTCACTGATAAAGACC 43 210 396239 4 5-10-5 12982 13001
CAGACTCTGAATCAGTTTTA 78 211 396316 4 2-15-3 12982 13001
CAGACTCTGAATCAGTTTTA 70 211 396240 4 5-10-5 13021 13040
CAGTCCCCAATTCTGCTGCC 43 212 396317 4 2-15-3 13021 13040
CAGTCCCCAATTCTGCTGCC 70 212 396241 4 5-10-5 13107 13126
CCAGTGTTAGGCTCTGCCAG 76 213 396318 4 2-15-3 13107 13126
CCAGTGTTAGGCTCTGCCAG 85 213 396242 4 5-10-5 13134 13153
GAATGCCAGGAAAGGAGTGA 69 214 396319 4 2-15-3 13134 13153
GAATGCCAGGAAAGGAGTGA 84 214 396272 4 5-10-5 13171 13190
CAGCCCCAAGGCCCAAAGAT 48 215 396349 4 2-15-3 13171 13190
CAGCCCCAAGGCCCAAAGAT 57 215 396220 4 5-10-5 13491 13510
CTGCACTGGAGCAGCAGTGT 81 216 396297 4 2-15-3 13491 13510
CTGCACTGGAGCAGCAGTGT 74 216 396221 4 5-10-5 13517 13536
ACCGGTTGGCATCCATGTAA 61 217 396298 4 2-15-3 13517 13536
ACCGGTTGGCATCCATGTAA 73 217 396222 4 5-10-5 13525 13544
CCTGGGTGACCGGTTGGCAT 71 218 396299 4 2-15-3 13525 13544
CCTGGGTGACCGGTTGGCAT 82 218 396223 4 5-10-5 13802 13821
CAAGTTGGGAGACTGGATGG 65 219 396300 4 2-15-3 13802 13821
CAAGTTGGGAGACTGGATGG 79 219
396224 4 5-10-5 13810 13829 CTTTAATACAAGTTGGGAGA 68 220 396301 4
2-15-3 13810 13829 CTTTAATACAAGTTGGGAGA 64 220 396225 4 5-10-5
13877 13896 TCGGAAGGTCTGGTGGATAT 65 221 396302 4 2-15-3 13877 13896
TCGGAAGGTCTGGTGGATAT 83 221 396226 4 5-10-5 13896 13915
TGGGCACCAAACTGCTGGAT 70 222 396303 4 2-15-3 13896 13915
TGGGCACCAAACTGCTGGAT 76 222 396227 4 5-10-5 13937 13956
TATGGCTTCCTGGGCGCAGG 59 223 396304 4 2-15-3 13937 13956
TATGGCTTCCTGGGCGCAGG 74 223 396228 4 5-10-5 13961 13980
AATGCTGCAATGGGCATCTG 78 224 396305 4 2-15-3 13961 13980
AATGCTGCAATGGGCATCTG 83 224 396229 4 5-10-5 13977 13996
GTTCACTATCACAAACAATG 84 225 396306 4 2-15-3 13977 13996
GTTCACTATCACAAACAATG 67 225 396230 4 5-10-5 13997 14016
CAGTTAAGCAGCTTCCAGTT 84 226 396307 4 2-15-3 13997 14016
CAGTTAAGCAGCTTCCAGTT 85 226 396231 4 5-10-5 14028 14047
AATTTTATTTAGCCAGTCTC 80 227 396308 4 2-15-3 14028 14047
AATTTTATTTAGCCAGTCTC 79 227 396232 4 5-10-5 14046 14065
GTTGTATAAATATATTCTAA 44 228 396309 4 2-15-3 14046 14065
GTTGTATAAATATATTCTAA 25 228 396233 4 5-10-5 14065 14084
ACAGTGTTTTTGAGATTCTG 83 229 396310 4 2-15-3 14065 14084
ACAGTGTTTTTGAGATTCTG 50 229 396273 4 5-10-5 14725 14744
CTCAGGACCCAGAGTGAGGA 37 230 396350 4 2-15-3 14725 14744
CTCAGGACCCAGAGTGAGGA 50 230 396274 4 5-10-5 15073 15092
TGGGTTAAACCTCACCTCGA 59 231 396351 4 2-15-3 15073 15092
TGGGTTAAACCTCACCTCGA 56 231 396275 4 5-10-5 15350 15369
ATTAGGTCCCAAAGTTCCCC 23 232 396352 4 2-15-3 15350 15369
ATTAGGTCCCAAAGTTCCCC 48 232 396234 1 5-10-5 42 61
GGCAGACGAACCATGGCGAG 86 233 396311 1 2-15-3 42 61
GGCAGACGAACCATGGCGAG 82 233 396213 1 5-10-5 435 454
GTAGCAATCTGCTTGACCCC 82 234 396290 1 2-15-3 435 454
GTAGCAATCTGCTTGACCCC 79 234 396218 1 5-10-5 590 609
GACCACAGACAACATCAGTC 89 235 396295 1 2-15-3 590 609
GACCACAGACAACATCAGTC 85 235 396219 1 5-10-5 683 702
CCACCTTTTTGATAAAGACC 65 236 396296 1 2-15-3 683 702
CCACCTTTTTGATAAAGACC 41 236
Example 5
Antisense Inhibition of Human CD40 in HuVEC Cells, Primer Probe Set
LTS37
Several antisense oligonucleotides exhibiting in vitro inhibition
of CD40 (see Example 4) were tested at various doses in HuVEC
cells. Cells were plated at densities of 5000 cells per well and
treated with nM concentrations of antisense oligonucleotide as
indicated in Table 5. After a treatment period of approximately 24
hours, RNA was isolated from the cells and CD40 mRNA levels were
measured by quantitative real-time PCR, as described herein. Human
CD40 primer probe set LTS37 was used to measure mRNA levels. CD40
mRNA levels were adjusted according to total RNA content as
measured by RIBOGREEN.RTM.. Results are presented as percent
inhibition of CD40, relative to untreated control cells. As
illustrated in Table 5, CD40 mRNA levels were reduced in a
dose-dependent manner.
TABLE-US-00005 TABLE 5 Antisense Inhibition of human CD40 in HuVEC
cells, Primer Probe Set LTS37 ISIS 0.2344 0.4688 0.9375 1.875 3.75
7.5 15.0 30.0 No nM nM nM nM nM nM nM nM 26163 17 35 38 51 62 67 82
89 396236 23 49 59 77 86 92 91 89 396266 35 45 58 74 55 72 57 56
396307 21 45 43 56 80 79 82 82 396218 34 47 52 57 78 82 86 86
396279 34 54 59 49 72 82 88 87 396287 31 48 52 50 64 77 85 86
396264 39 34 49 56 71 84 88 86
Example 6
Antisense Inhibition of Human CD40 in AGS Cells
Antisense oligonucleotides exhibiting in vitro inhibition of CD40
(see Example 4) were tested at various doses in AGS cells (human
adenocarcinoma cells). Antisense oligonucleotides of SEQ ID No. 90
and SEQ ID No. 208 were designed as 4-10-4 gapmers or 5-10-5
gapmers, respectively, where the gap segment comprises
2'-deoxynucleotides and each wing segment comprises 2'-MOE or 2'OMe
nucleotides. The antisense oligonucleotides comprise
phosphorothioate backbones (internucleoside linkages) and
5-methylcytosine substitutions throughout.
Cells were plated at densities of 5000 cells per well and treated
with nM concentrations of antisense oligonucleotide as indicated in
Table 6. After a treatment period of approximately 24 hours, RNA
was isolated from the cells and relative CD40 mRNA expression
levels were quantified by real time RT-PCR using the QuantiTect.TM.
SYBR.RTM. Green RT-PCR kit (Qiagen). CD40 mRNA levels were adjusted
according to GAPDH content, a housekeeping gene. Results are
presented as percent inhibition of CD40, relative to cells treated
with a scrambled control oligonucleotide ( TCCATTTATTAGTCTAGGAA
(5-10-5 gapmer, where the gap segment comprises 2'-deoxynucleotides
and each wing segment comprises 2'-MOE nucleotides. The
oligonucleotide comprises phosphorothioate backbones
(internucleoside linkages) and 5-methylcytosine substitutions
throughout.
TABLE-US-00006 TABLE 6 Antisense Inhibition of human CD40 in AGS
cells ISIS Wing 12.5 25.0 50.0 No Motif segment nM nM nM Seq ID
26163 4-10-4 2'MOE 80 69 90 90 396236 5-10-5 2'MOE 83 86 94 208 --
4-10-4 2'OMe 51 54 66 90 -- 5-10-5 2'OMe 60 63 69 208
As illustrated in Table 6, CD40 mRNA levels were reduced in a
dose-dependent manner. Antisense oligonucleotides comprising 2'MOE
wing segments are more active than those with 2'OMe wing
segments.
Example 7
Antisense Inhibition of Murine CD40 In Vitro
Chimeric antisense oligonucleotides having 5-10-5 MOE wings and
deoxy gap and 4-10-4 MOE wings and deoxy gap may be designed to
target murine CD40. These antisense oligonucleotides can be
evaluated for their ability to reduce CD40 mRNA in primary mouse
hepatocytes using similar methods as described in the human in
vitro study.
For example, primary mouse hepatocytes may be treated with 0.2344
nM, 0.4688 nM, 0.9375 nM, 1.875 nM, 3.75 nM, 7.5 nM, 15.0 nM, and
30.0 nM of antisense oligonucleotides for a period of approximately
24 hours. RNA can be isolated from the cells and CD40 mRNA levels
can be measured by quantitative real-time PCR, as described herein.
Murine CD40 primer probe sets can be used to measure mRNA levels.
CD40 mRNA levels can then be adjusted according to total RNA
content as measured by RIBOGREEN.RTM..
Example 8
Antisense Inhibition of Murine CD40 In Vivo
Antisense oligonucleotides showing statistically significant
dose-dependent inhibition from an in vitro study can be evaluated
for their ability to reduce CD40 mRNA in vivo.
Treatment
Antisense oligonucleotide can be evaluated in Balb/c mice and
compared to a control group treated with saline. Oligonucleotide or
saline would be administered subcutaneously at a dose of 5 mg/kg,
10 mg/kg, 25 mg/kg, or 50 mg/kg twice a week for three weeks. After
the treatment period, whole liver can be collected for RNA analysis
and protein analysis.
RNA Analysis
Liver RNA can be isolated for real-time PCR analysis of CD40. It is
theorized that an antisense oligonucleotide showing significant
dose-dependent inhibition in vitro may show significant
dose-dependent inhibition in vivo.
Protein Analysis
Liver CD40 protein may be measured by Western blot.
Example 9
Tolerability of Antisense Compounds in Rodents
Male 6 week old Balb/c mice were dosed subcutaneous 2.times. per
week for 4 weeks with 25 or 50 mg/kg of antisense oligonucleotides
Isis 26163 or Isis 396236. Mice were sacrificed 2 days following
last administration. Body weights of the animals were monitored
throughout the study. After sacrification liver, spleen and kidney
weights and liver enzymes ALT and AST from mouse plasma were
determined.
Compared to a saline control treatment body weights of the mice are
not affected by antisense oligonucleotides Isis 26163 or Isis
396236. Liver weight and spleen weight displayed a slight increase
for Isis 26163 but not for Isis 396236. LFT (liver function test)
elevations were small and within the normal range of high dose
mouse studies.
Example 10
Antisense Inhibition of Human CD40 In Vitro on T24
Cells--Comparative Data for ISIS 26163 and ISIS19216
Antisense oligonucleotides ISIS 26163 and ISIS19216 targeted to a
CD40 nucleic acid were tested for their effects on CD40 mRNA in
vitro. The antisense oligonucleotides were designed as 4-10-4
gapmers, where the gap segment comprises 2'-deoxynucleotides and
each wing segment comprises 2'-MOE nucleotides. The antisense
oligonucleotides comprise phosphorothioate backbones
(internucleoside linkages) and 5-methylcytosine substitutions
throughout or in the wings, respectively.
T24 cells at a density of 7000 cells per well in a 96-well plate
were treated with 100 nM or 150 nM, respectively, of antisense
oligonucleotide. After a treatment period of approximately 24
hours, RNA was isolated from the cells and CD40 mRNA levels were
measured by quantitative real-time PCR, as described herein. CD40
mRNA levels were adjusted according to GAPDH content, a
housekeeping gene.
Results are presented in Table 7 as percent inhibition of CD40,
relative to untreated control cells.
TABLE-US-00007 TABLE 7 Oligo ID Target site nM on T24 cells %
Inhibition SEQ ID No. 26163 70-87 100 98 90 19216 73-90 150 66
10
Sequence ISIS 26163 shows a superior activity over ISIS19126, which
overlaps the sequence 15 nucleobases.
SEQUENCE LISTINGS
1
SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 238 <210>
SEQ ID NO 1 <211> LENGTH: 1004 <212> TYPE: DNA
<213> ORGANISM: H. sapiens <400> SEQUENCE: 1 gcctcgctcg
ggcgcccagt ggtcctgccg cctggtctca cctcgccatg gttcgtctgc 60
ctctgcagtg cgtcctctgg ggctgcttgc tgaccgctgt ccatccagaa ccacccactg
120 catgcagaga aaaacagtac ctaataaaca gtcagtgctg ttctttgtgc
cagccaggac 180 agaaactggt gagtgactgc acagagttca ctgaaacgga
atgccttcct tgcggtgaaa 240 gcgaattcct agacacctgg aacagagaga
cacactgcca ccagcacaaa tactgcgacc 300 ccaacctagg gcttcgggtc
cagcagaagg gcacctcaga aacagacacc atctgcacct 360 gtgaagaagg
ctggcactgt acgagtgagg cctgtgagag ctgtgtcctg caccgctcat 420
gctcgcccgg ctttggggtc aagcagattg ctacaggggt ttctgatacc atctgcgagc
480 cctgcccagt cggcttcttc tccaatgtgt catctgcttt cgaaaaatgt
cacccttgga 540 caagctgtga gaccaaagac ctggttgtgc aacaggcagg
cacaaacaag actgatgttg 600 tctgtggtcc ccaggatcgg ctgagagccc
tggtggtgat ccccatcatc ttcgggatcc 660 tgtttgccat cctcttggtg
ctggtcttta tcaaaaaggt ggccaagaag ccaaccaata 720 aggcccccca
ccccaagcag gaaccccagg agatcaattt tcccgacgat cttcctggct 780
ccaacactgc tgctccagtg caggagactt tacatggatg ccaaccggtc acccaggagg
840 atggcaaaga gagtcgcatc tcagtgcagg agagacagtg aggctgcacc
cacccaggag 900 tgtggccacg tgggcaaaca ggcagttggc cagagagcct
ggtgctgctg ctgcaggggt 960 gcaggcagaa gcggggagct atgcccagtc
agtgccagcc cctc 1004 <210> SEQ ID NO 2 <211> LENGTH:
427 <212> TYPE: DNA <213> ORGANISM: H. sapeins
<220> FEATURE: <221> NAME/KEY: misc_feature <222>
LOCATION: 13, 52, 152, 193, 245, 254, 263, 298, 305, 323, 331, 344,
374 <223> OTHER INFORMATION: n = A,T,C or G <400>
SEQUENCE: 2 gataccatct gcnagccctg cccagtcggc ttcttctcca atgtgtcatc
tnctttcgaa 60 aaatgtcacc cttggacaag ctgtgagacc aaagacctgg
ttgtgcaaca ggcaggcaca 120 aacaagactg atgttgtctg tggtccccag
gntcggctga gagccctggt ggtgatcccc 180 atcatcttcg ggntcctgtt
tgccatcctc ttggtgctgg tctttatcaa aaaggtggcc 240 aagangccaa
ccantaaggc ccnccacccc aagcaggaac cccaggagat caattttncc 300
gacgntcttc ctggctccaa cantgctgct ncagtgcagg agantttaca tggatgccaa
360 ccggtcaccc aggnaggatg ggcaaagaga gtcgcatttc agttgcaggg
agagacagtg 420 aaggttg 427 <210> SEQ ID NO 3 <211>
LENGTH: 871 <212> TYPE: DNA <213> ORGANISM: H. sapiens
<400> SEQUENCE: 3 acctcgccat ggttcgtctg cctctgcagt gcgtcctctg
gggctgcttg ctgaccgctg 60 tccatccaga accacccact gcatgcagag
aaaaacagta cctaataaac agtcagtgct 120 gttctttgtg ccagccagga
cagaaactgg tgagtgactg cacagagttc actgaaacgg 180 aatgccttcc
ttgcggtgaa agcgaattcc tagacacctg gaacagagag acacactgcc 240
accagcacaa atactgcgac cccaacctag ggcttcgggt ccagcagaag ggcacctcag
300 aaacagacac catctgcacc tgtgaagaag gctggcactg tacgagtgag
gcctgtgaga 360 gctgtgtcct gcaccgctca tgctcgcccg gctttggtgt
caagcagatt gctacagggg 420 tttctgatac catctgcgag ccctgcccag
tcggcttctt ctccaatgtg tcatctgctt 480 tcgaaaaatg tcacccttgg
acaaggtccc aggatcggct gagagcctgt ggtgatccca 540 tcatcttcgg
atctgtttgc atctcttggt gctgtcttat caaaaagtgg caagaactac 600
ccataggccc ccacccagca gacccagaga taatttctga gatttctgct caaatgtgtc
660 agtagggagt catgagcaca gtcccacggt ggagaaccgc tctttggaga
attgtgccca 720 gattgccatg aaaaggatgt caagataggt gggttttgtg
gggggtaaac cttccccttt 780 tgagctgtga attttgaact ctcggccttt
aagaatgggg ggttaaccaa tttgactcca 840 acagttaaac ttgattatga
ggtttgcctt t 871 <210> SEQ ID NO 4 <211> LENGTH: 16001
<212> TYPE: DNA <213> ORGANISM: H. sapiens <400>
SEQUENCE: 4 cagcttgagg tctgtgttga gattaccagc ttcgcacccc ctgccaccaa
ctctttgtca 60 tgattggagg ctgtacttag gatgagaagt tgctgaaccc
actcattcat ttattcattc 120 attcagcacc catatattgg tgtcctatta
tgtacctagg actgggatag atccagtcct 180 gccctcaggg agctcacagt
caaatgggac caggcagaag gagttatgag ctggcatgct 240 aaatgctgtg
gcaacacagg ggaggaggca gggtgggata gagtacctac ctctgcctca 300
gggagttggg gaaggtgtaa gaggaggcaa catttaaact gtttaagaaa ggagaaggaa
360 aggaaggaaa catcaagagc aatgagataa accgtaagga aaaaagattg
tgtgcctggg 420 aagtggggag aggctgaagc cagtgtgcat gggaggacgt
ggcaggaagt aagcatagaa 480 agagaggttg gggctgagtg aggaagggtg
tgctttgcca ggataagaaa tttggatcat 540 cctgagggtg cagggaaacc
caaagaggct tttatgcagg aaagtgtccc agtcgatcag 600 atctggagtt
tatcaagaga atcgcagcag tgtaaagagt tgcctggcac atagtaggtg 660
ctcaataaat cctgtggaat gagagaggct gttgggacag tccagatgaa agatggcacc
720 ctctgaatta gggcagtggc agaggtgaca gaaaataagg gatggatcct
agaggcagaa 780 tccacaagac ttgcaaatgt gcccttaaca gcagaagcca
tgagaatggg gttagaagtg 840 tcctcctctt ccatcctccc tgatctgtga
ggtctggcct cttgtcctct tccagaaatt 900 gagagcttgg agggtcatca
cctcaaccgt tgtctagttc aagctcctaa gttttccttg 960 gagaaaaatt
taggcctaag ggaacacagt ccttcagagg cagaggctgc accagaaccc 1020
aggtgttcca tgctgcaaag cagagtgtta acactggttc aagaacatct tgagggccag
1080 gtgcggtggc tcatgcctgt aatctcagca ctttgggagg ccaaggcagg
cggatcacct 1140 gaggtcagga gtttgagacc agcctggcca acgtggagaa
accccgtctc tactaaaaat 1200 acaaaaatta gctgggcgtg gtggcgccag
cctgtaatcc cagctactcg ggaggctgag 1260 gcaggagaat tgcttgaacc
caggaggcag aggttgcagt gagccgagat catgccactg 1320 cactccagcc
tgggcaacag aatgagactc tgtcaaaaaa aacccaaaaa ccaaaaacca 1380
aacaaacacc caaaaaacaa aacgaaacaa aaaacaaaaa aacaaaagca cgtcttcaga
1440 gttcatgaac ccaggaaatg taggcacaag tgtgtgtgtt tctgcaaaat
gagagggtcc 1500 cgagtttcct aacatactta aagtgtttta tgatgccaca
aaaggctggc ccactctttt 1560 tttttttttg agatggagtt ttgcactgtc
gcccaggctg aagtgcagtg atgtaatcat 1620 tgcaatcatg gctcactgca
accttgactt cttgagctca agcgatcctc ctgcctcagc 1680 ctcctgagta
gctgggacta caggtgtttg ccaccatgcc tggctaactt aaaaatttct 1740
ttattttgta gagatggggg tcttgccatg ttgccaggct ggtcttgaac tcctggcctc
1800 aagcaatctc cctttttggc cttccaaagt gttagattac aggcgtaagc
caccgcgcct 1860 ggcccccacc tttattttta tttttattta ttatttattt
tttttttttt tgagactgag 1920 tcttgctctg ccttcgaggc tggagtgcag
tggcacgatc tcggctcatt gcaatctctg 1980 cctctgcggt tcaagcgatt
ctcctgcctc agcctcccga gtagctggga ttacaggcga 2040 acgccactac
atccggttaa tttttgtatt tttagtagag acggagtttc actatgttgg 2100
ccaggttggt ctcgaactcc tgacctcaag tgatctgccc gcctcggcct cctaaagtgc
2160 tggattacag gcgtgagcca ccgcgcccgg ccccactctt aataaatgcc
tgtctccagg 2220 tgctgggtgg gaggtgggat ggaatggaat gaggtgagga
cgcatggatg catggatgaa 2280 tggatgggaa gttgagacga cgcgcccaca
cgagggaatt tcctttgaaa gagagcgaaa 2340 ttctgagttg ggaaactctt
ccttgaaacg cctccccata ccccagctgt ggccttcccg 2400 ttttctgcgt
ggtggtgtgg ggggaacttc ctcaggcctc tccgcagtgg agcctctttc 2460
ggttctgcca ggatacctag aggcagcgga gagcggggca gggaggggaa aaccgtgagg
2520 gtccctgtgg caggccccag cacccatggg atctctctcc ggtcgcagga
agcaggctag 2580 ctcctagccc gcctcggctt ggcctttgtg ggacctgggg
gcaaagaaga agagctgtct 2640 ctgggaccat gcctcctccc gtacacagca
agatgcgtcc ctaaactccc gggggaatta 2700 gacttgtggg aatgttctgg
ggaaactcct gcgcggtgaa ttgctggggg ctccgccccc 2760 ccgataggtg
gaccgcgatt ggtctttgaa gaccccgccc ctttcctggg cggggccaag 2820
gctggggcag gggagtcagc agaggcctcg ctcgggcgcc cagtggtcct gccgcctggt
2880 ctcacctcgc tatggttcgt ctgcctctgc agtgcgtcct ctggggctgc
ttgctgaccg 2940 ctgtgagttg tttttgcccc gaccagacgg gagttgggag
tggggaatga gaaggaaagg 3000 gaaggaagac ttcggggaag aggccttcct
ggctgatttt tgtgggggca ggagggtggg 3060 tgggagctgg gcaaggtgcc
cccgctcctg gctgaatggg gtgggctgcc tctctcttct 3120 cccgggctgg
ggtcccggga gcggcctaca ggggccgctc agggaaggca ctggctgccc 3180
aagcgtgcct agacggcctg gacgggttta gggagcctca gaggctggcc acacagagac
3240 tggtaggggg ttcagagggc gggaagtgag gcggaccaag ggaaggggcg
ggtctggccc 3300 gtttcctgtc cccttcttat tgtggacaga tgccagcctc
tgtaagtagt tatcatctcc 3360 ttgccagctg gggctgcctt cttccagggc
atcttgtggg aacaagagat gggtgcagag 3420 gcccaggtac ttttgtgaga
aggcaaggag cttttaacat cgccttccac cccgaaccgt 3480 atcttgggtg
ttccaaccta ggaggaatcc ccagggcttt gcctttttct cctgaattta 3540
agatgacata ggagacccct ggggagatga acagtttatg ggacacaata aagggttagg
3600 agaccagagt tctggttggc tctgacaggg ctggtgatca gagggctgga
gaaaccaggg 3660 gtttctccag gcaccagagg ggctcagagc caaccaagca
tatctccggg attttcagaa 3720 gcctacactt gactcacttt ttgtttaaat
gtatttttgt agttcctcat tctggaggct 3780 gggaatcccc caagtacctg
gctccttcat cccagcccct ctggcctccc cctactttag 3840
agggctgtag attcctgcct gaagcctggg caggaatgac ccatggtatc aaggaaagca
3900 agggaagcag caagggaaga gagggagtgg ggaggctgct ttggtcccac
agctttcact 3960 ttcacctgaa gcaatggctc ttagggaaca gggaggcagg
gggagggcgg agctggaaag 4020 aggtaaaggg gggcccttgt ggtaggagtg
gagaaagagc cagaggaggt ggggtgaagg 4080 gtgtgatcca ggcttctcaa
gagcagagtt tgccctcata actcccaact ttggctccag 4140 gtagaggctg
ggctgtgaca acaatgtcag aagctatcta ttgagggctt cttgtgtgtc 4200
aggctctgag ccaaacactg cctgttttct ttgtctgatt tctcacaact cccccattat
4260 acagatgggc aaattgaggc tcagaaaggg ggattgtctt gccaaaggtc
tcatagctag 4320 ctaatggaag aacctggttg tgaatctaca tctgcatgat
tcccgagcct gcctctcaga 4380 tagtgagagt ctccaagctc tggtcctgag
ctgttttgtg gcagaaggac cagaactatg 4440 gggagtgaga actggagatt
gacagacttt taggggagcg ttttatttct catgtgtttg 4500 aagatggtat
caaggacttt cctatctttg ggagtgtggg agctccacgt tcacaggatg 4560
gtgtcttgca atgagctggt ggggggcagt agccttttct acttcctttc ccattttggg
4620 taagacacat ttctgtaagt aatttgctga gatacccagg ttgaatgaga
gccaccagtt 4680 aggtaggatt ctggacagcc agccaggtag ccgggctgct
tgccatatat catgcaagca 4740 gaaacaaatg aatgatgatt aaaattgcca
tttaatgagc acctactatg ttcctgacac 4800 tgtgctaggc catatacatg
tattctttct tatcttcgta atccaacctg cagggcaggc 4860 attattactc
ccattttaga gatagagaaa ctgaggctaa gagaagcaaa ataactagta 4920
agtgttacaa agtcaggact ggagtctaaa gctgtctgac tctcaaactt gtgttctttt
4980 cactggctgt tcccaaactg tgggacagtt ttaaggagca catggacata
gaattaaaca 5040 tacacttact ttacagttct tttaaaaatc cttctcattt
tttcaaagag gaagtctctg 5100 gagctagaat agagttaatg cctctcaaag
gcttgctaat ccttctttta aaacaaaaat 5160 caagagcagg cctgggaggg
ccttcaacaa gcaaacaacc agctgggttt taataacctt 5220 gttttgtttc
cccagaattt atttttaggg ttacctttta tttatgagaa gtgatactgg 5280
ttcttgtctc ttggcaatga tgtgaggttt acatttaaag taaatgtacc ggccaggcac
5340 ggtggcttgt gcctgtaatc ccagcacttt gggaggccaa ggcagtcaga
tcacttgagg 5400 tcaggagttt tagatcagcc tggccaacat ggtgaaaccc
tgtctctact aaaaatacat 5460 aaattagccg ggcatagtgg tacacacctg
taatcccagc tactcaggag gctgaggctg 5520 gagaattgct tgaacccagg
agatagaggt tgcagtgggc tgagatgatg ccactgcact 5580 ccagcctggg
cgatggagcg agactctgtc tcaaaaaata aaataaaagt attgaaatta 5640
acaataagta attaatagca tgggtggtac ctggatgtag taaaatggtg aagatgaaac
5700 acaagttgat ggagagagga gcattgagac ctgagttctc atttggactc
tgtcactgtg 5760 agactctggg caagtgaccc tcctctttgg tgctcagtct
caactatctg taaaatgaaa 5820 gtgtgagttt acccttccag ctttacattc
tagcatttta tgagggaagg gctggatgaa 5880 cagatgatga ggagttggag
gaagaaaaca tgatgggctt tggaaaggag caggaaggga 5940 agcagaagaa
taggaggaag aggccaagtg ctaaacatag ccccaaacag cactgggacc 6000
agctgaagtc agccagcttc aggactccag gggagctgct ggagtcccca tatcctatgg
6060 gatctttggg aagaggaatg actcaggcat caagccccaa ggaattctgt
tctgttcaga 6120 gaatattgtg agtttacagt accattgctt tgtaaaaata
ccagaatgat tctctgggtg 6180 cgattataat cagctcagtt gacaatttac
ttgaaaacaa acatgccaaa tatcatgcag 6240 gttccacttt ctgttttgac
ttgcacttca gtttgcagcc tctgtcctgg atgactttta 6300 cctttctgct
gaagaagttg caacggagat ttcaagatcc cttcaaattg cacaattctg 6360
tttttaggtc catccagaac cacccactgc atgcagagaa aaacagtacc taataaacag
6420 tcagtgctgt tctttgtgcc agccaggtga gatgccaacc ctctagcccc
atcatggagt 6480 ccccctttgc tttggtggca gacgcagacc ccatatgtta
actgtaaact caaatctgaa 6540 acgacccatt tcccagccct gcttcactgt
cagaatgttc tggttccctc tctaccaggt 6600 aaaactctgt ctaccctgaa
ctagggatcc cagcttctcc atcttcctcg cctgattatg 6660 aaggatccaa
gactttcatc tttgaatccc ctaccctaaa gcctggcctg atcattgtgt 6720
ggttagtgtc tgactcatgg agttggccag agccctccct catttcctga tgttttccag
6780 gacagaaact ggtgagtgac tgcacagagt tcactgaaac ggaatgcctt
ccttgcggtg 6840 aaagcgaatt cctagacacc tggaacagag agacacactg
ccaccagcac aaatactgcg 6900 accccagtgc gtgcgctgtt gggaaaggga
cgcttgggaa ccgggctgat attcccgaca 6960 atgcagccat tctaatttta
tgtagccagg gtctgctctg attggttgga gtccgggctg 7020 tactgatcat
taaatgattt gattgccatc tctacttgga agagggtctg aggaagaaag 7080
agcaggcaat gtggggagtg aggctcagag catggcccag cagggggttc ccatccttcc
7140 tgcccttctc ttctcagacc tagggcttcg ggtccagcag aagggcacct
cagaaacaga 7200 caccatctgc acctgtgaag aaggctggca ctgtacgagt
gaggcctgtg agagctgtgt 7260 cctgcaccgc tcatgctcgc ccggctttgg
ggtcaagcag attggtaagt ggctcatctg 7320 ggaatcagtt ttggaggggg
acagaggagc ttagggccca aggtgagggg ctgggcagtg 7380 ggcacttagc
cccagaggca gaggaagcag aggctccaac ctatgtcggt atccccactg 7440
gagtgagctg cagacgggac cttgttcatt ctgccttctg ccatggggat ctgcctttga
7500 agggcaatgg gagaagtcct cctggggact gcagctgtcg ggggcagtac
cacatcgggg 7560 gaagagtgct caaggcagga gctcttcccg tcctgcctgg
ccactggctg ccttgtgagc 7620 cggacaggtg gtccactgtg atggttaatg
tccccctccc cacccactcc cagctacagg 7680 ggtttctgat accatctgcg
agccctgccc agtcggcttc ttctccaatg tgtcatctgc 7740 tttcgaaaaa
tgtcaccctt ggacaaggta taagcactca tcccttgtgt ttcctgctct 7800
aagagtggca tggagctgcc tccattctct ccagccacct gtcctgtccc tgctcccaga
7860 ggtccacaca cactcatgta cttgtgaagc atctgcagag tggcctcatg
gccaaccaga 7920 caggcacatt tccacatttt ttttgcctgc tgtctctttg
aggtaataga cactgttgat 7980 ctctcgcttc atgagagcct cctatcttgg
gggtattggg acacttattt tagctttcct 8040 tctgcccctc ctgcttctcc
tcagttttcc tcgtcttgct ttcaccttac ctggctttct 8100 agggctttct
gggctctggg tgctcaccct gagggcctcc ctctcttacc tccaactcca 8160
aacccacacc aggtcctgcc actggctgtc tacgtgtttt gggaacttac tgtctccact
8220 gttgtcactt tagtttgggc ctcatcactg tggtctgggt gatgcctttt
ctgcctcctg 8280 gcctccctgc ctctgtctct cccctcctgc tggttctgtc
tccatcctct tgccaacatg 8340 agcgttcgac agtttctttc aaatcatgac
actctcctat ttgagatgct tcctgtctct 8400 ctgttggaac taagactcct
tagcatggca cccaaccttc ctgttgcatt tcctgctctc 8460 tttcctgcat
cgcatagctt catgctactt gcaatcctct gaacacactg ttcattctct 8520
tccatcaaac tcatctgcct ggaatacctt aaacatgggc cccaggccag gcgcggtggc
8580 tcttgcctgt aatctcagca ctttggatgc caaggcgggt ggatcacttg
aggtcaggag 8640 ttcaagacca gccagcacaa catggtaaaa acccatctct
actaaaaata ccaaaaaatt 8700 agctgggtgt ggtggtgggc gcctgtaatc
ccagctcctc gggaggctga ggcaggagaa 8760 tcacttgaac ccggaaggtg
gagtttgcag tgagccaaga tagcgccact gcactccagc 8820 ctgggcaaca
gagcgacatt ctgtctcaaa aaacaaacac ctgccccatt aactttttgc 8880
atttgatttt taaaaatggg caagataggc acatgggaca gaaggcacaa aagagccaaa
8940 gtgatgtctt tctcccatcc ctgcccctta ggctcccagt tctttctgga
gggagccatt 9000 gttccttgca tatccttcca gagattctac atataaacaa
accaacacac acacacacac 9060 acacaaacac acacaaaatt tccctccttt
tacttttgca caaataggag tatacatttt 9120 atttgttaac tgtctgcctt
tccctaatag attgaaaatt ccttaaatgt agaaacttgg 9180 cctttttttt
ttcttccatt gatacatccc ctatacctgg aacagtacct gacgcatggt 9240
aggtgcttaa atttttactg ataaatgttg actgataact ggaggcacca ctggtatagt
9300 tttttttttt tttttttttt tttttttttt ttgagacaga gtctcactct
gtcgcccagg 9360 ctggagtgca gtggcgcaat ctcggctcac tgcaagctct
gcctcccagg ttcacgccat 9420 tctcctgcct cagcctcctg agtagctggg
actataggcg cccgccacca cacccggcta 9480 atttttttgt atttttagta
gagacggcgt ttcaccgtgt tagccaggat ggtcttgatc 9540 tcctgacctc
gtgatccgtc tgccttggcc tcccaaagtg ctgggattac aggcgtgagc 9600
caccgtgccc ggccaccagt ggtatagtat taatggaatc agtgcattgg cttacgtatc
9660 tgattacagc tcagtaagtg tgtgaccctc actgagcctc agtctcctca
tctgaaaaat 9720 gggaatgacc ttcatttcac aaggcttgag ctaaaaacat
gtaaagtgta ttgtaaattc 9780 ctgaatgctc tactcatgta agactaaagt
aggccgggcg tggtggctca cacctgtaat 9840 tgcagcactt tgggaggccg
aggagggcag atcatgaggt caagagatcg agaccatcct 9900 ggctaatatg
gtaaaaccct gtctctacta aaaatacaaa aattagctgg gcgtggtggc 9960
gcacatctgt agtcccagct actcaggagg cggaggcagg agaattgctt gaacctggga
10020 ggtggaggtt gcagtgagct gagatcgcgc cactgcattc cagccagtct
ggcgaaagag 10080 caagactctg tctcaaaaaa aaaaaaaaaa aaaaaaaaag
actaaagtac atggtttctt 10140 caaagcttct ctctctttct cccaccttag
atgatttttc ctttgcaatg tcctgtgtcc 10200 attccgcccc actcctcctg
gggccacctg gaccaggtct tcatcatctc atatctatat 10260 gtttgctgtg
tctcctggct ggccactctt ctgtaatttc tcctcctctg agctctctgg 10320
gcagctgaat cttctcacta gtgaagtcgc ctggttggat gctgatgaga ctgaccagct
10380 gaatccagtt gaaaacttca cacttggcag tgatctggtt ctaaagacac
aattttccat 10440 agtttcctaa caccatcctg catgccacct gccttatttc
cccacatcac atcgtcccac 10500 ttagcgggac tgcactgctg atccaaattt
tacatccttt agggcccact caggtcatat 10560 gtcctcaggg aagtctttct
ggaagaacct taaaccagag gttctcaaca gggggcagtt 10620 ttgctccctg
tggaacgttt gccaatgtct ggacacattt cattcgtcac aaacggagag 10680
ggggatgcta cagggatctg gcggatagag gccagggatg ctgctgaaca tctgcaatgc
10740 ataggacagc ccacccccac ccccacaccc ccagtaaata atgatccagc
ccaagtgtca 10800 ctggtgctga cgttgagtaa ccctatctta agctgaactc
atcatctctc cattccagcc 10860 ttggtggatt ctgtctcctc tgaaccattc
ccatctcact ttagcctacc tagatcacaa 10920 agcttggcac tcattataga
ctcccctatt tattactcct tcaagatgtg caagaatctt 10980 ttctctgcac
ttttaagttc tgtaagaaga gtctgtgtcg ttcctataat aaccagcata 11040
ggacgttgca cgtgttgtgt gctcagtgaa cctggatttg ttgattgttg actgactcac
11100 tctagagttg gaaatcttat gcttggggaa acttaatatc tctttctttc
tctgtgtgtg 11160 tgcatttgtg cacgtgtctg tgcatagctg tgagaccaaa
gacctggttg tgcaacaggc 11220 aggcacaaac aagactgatg ttgtctgtgg
tgagtcctgg acaatgggcc ctggagaaag 11280 cctaggaagg tgggaactga
agggggagat gaggcacaca ggaacactgg atgggaaaaa 11340
ggggagggga ggcagtttgg gggtgtggta tcacagctct gccacttatc ttgggagtct
11400 gggcaaatca cttcccctct cttagcctca gtttcttcat ctgtaaaatg
ggatgataac 11460 agcacttcct tagtaggttt tgattttaga gtgagaaggt
tggcctacag taaagatcag 11520 ataatgtaaa tcagtgaaaa aggtcagggg
taagaaaatt acattctctt tacctaacgc 11580 taaatgacca gttaatgggt
gcagcacacc aacatggtac atgtatacat atgtaacaaa 11640 cctgcacatt
atgcacatgt accctaaagc ttaaagtata ataataataa aatttaaaaa 11700
aacgaaaaat acattctctt tgctttttct caaaatgtac tttcctcttt gtagggctgg
11760 gactagaatg aggtgagcaa ggcacttgcc ctcgggcgca atatttaaga
aggtgccata 11820 aaagtgtagt aatcaaggta aattcatttt gatgcaatat
ttttaaaaat aaaaattaat 11880 gcaaagaaat ccatgatgag caagatagca
acattttaaa taaagaacag gatccgaccc 11940 tgtgtttgca tgaccctgcc
tcactcacct caccctaatc ctggccctgg ttccagtaaa 12000 aggaataggc
agccagcctg caggccgtag tttgctgact tggtgtccgc ctgatgattt 12060
tcaaaatatg gcattaaaag aatgtttacc ttgatgactg agtgttttgg acatcctttt
12120 caattttgtc ctgaaacaat ttcatccctt gcctcacgct agtctccgcc
ctgccttttg 12180 gtctttcttt tattttccca ctttgaaaaa aaaattcggc
atgagaaata ctttaccttt 12240 cccctccact cttctatacc aaaagcaaca
tgcagacatg aatcatgcta gacctcggca 12300 ttgggcagag agcagggagt
ggcggggagc atggtgagca ggtggtgaca gccactgcca 12360 ccactcgctt
ctagatggtt cccaggtggg gaggctgcca actggaaccc agtcttccca 12420
gtttgtaaga gaaatcagat gtctaggttt gaatatgtga tctcccagtt taaaaatgtc
12480 ggcaaatatt tccaaacgtt aagaaaatgt tctggctcct ttaaagacat
ctgccagcca 12540 catttcccca aggaccgcgg tttgaacctt ctgatgtaga
tgagctctga cattggaaga 12600 ttctggagtc tgacaagtca cagcaggttg
agggtaggga gaaactgcag gtgaggggtg 12660 catgctgaag tcctgatttc
tccaggtccc caggatcggc tgagagccct ggtggtgatc 12720 cccatcatct
tcgggatcct gtttgccatc ctcttggtgc tggtctttat cagtgagtcc 12780
tcaggtgggg aggtgttggg ggagggaggg gagaccacct gtttcttatc tggcctctcc
12840 aactccccat cctttttttt tttttttttt tttttagaaa aggtggccaa
gaagccaacc 12900 aataaggtag gtcacccctg agaacccggg acagagtttt
gacaaactgg gaagatggcc 12960 tcacggttgc ctatggggca gtaaaactga
ttcagagtct gtctctgcag ccagtggggt 13020 ggcagcagaa ttggggactg
tcatccccac ccaccatgct ccttccatcc agagctcaat 13080 cccccacaga
actgcccctg gcaccactgg cagagcctaa cactggctgt tcttcactcc 13140
tttcctggca ttcaacgcgt ggggagctgc atctttgggc cttggggctg ggtcaaatgg
13200 gtgggagcaa atgtggcagc cccttaagcc cactggctcc cactctggaa
gctcttcgtc 13260 gcccttggtg tggccagcag ggggcaggag gcacccgagg
aatcagcact gacccgccgt 13320 ctgggaaagg ggggagggct tggggaaggg
atccgcttcc cagggagggg ctcctcagag 13380 gcacagctgc ccctgctgct
gggggtgacc tcacaccttg cctctccagg ccccccaccc 13440 caagcaggaa
ccccaggaga tcaattttcc cgacgatctt cctggctcca acactgctgc 13500
tccagtgcag gagactttac atggatgcca accggtcacc caggaggatg gcaaagagag
13560 tcgcatctca gtgcaggaga gacagtgagg ctgcacccac ccaggagtgt
ggccacgtgg 13620 gcaaacaggc agttggccag agagcctggt gctgctgctg
ctgtggcgtg agggtgaggg 13680 gctggcactg actgggcata gctccccgct
tctgcctgca cccctgcagt ttgagacagg 13740 agacctggca ctggatgcag
aaacagttca ccttgaagaa cctctcactt caccctggag 13800 cccatccagt
ctcccaactt gtattaaaga cagaggcaga agtttggtgg tggtggtgtt 13860
ggggtatggt ttagtaatat ccaccagacc ttccgatcca gcagtttggt gcccagagag
13920 gcatcatggt ggcttccctg cgcccaggaa gccatataca cagatgccca
ttgcagcatt 13980 gtttgtgata gtgaacaact ggaagctgct taactgtcca
tcagcaggag actggctaaa 14040 taaaattaga atatatttat acaacagaat
ctcaaaaaca ctgttgagta aggaaaaaaa 14100 ggcatgctgc tgaatgatgg
gtatggaact ttttaaaaaa gtacatgctt ttatgtatgt 14160 atattgccta
tggatatatg tataaataca atatgcatca tatattgata taacaagggt 14220
tctggaaggg tacacagaaa acccacagct cgaagagtgg tgacgtctgg ggtggggaag
14280 aagggtctgg gggagggttg gttaaaggga gatttggctt tcccataatg
cttcatcatt 14340 tttcccaaaa ggagagtgaa ttcacataat gcttatgtaa
ttaaaaaatc atcaaacatg 14400 taaaaagaaa aacgggggtg aacatgctgg
gtgacatgag ctatttaacc tgctgtcagg 14460 ctcacggaat gagggcattt
tctgtagata aataagaatg tccccaggct gctgcccctc 14520 cagggtggtt
tccatgtgtg ctcacatgtg gtattgagat tgcaaagtgc tcttcccatt 14580
tgattcatgt tcacaaaaat agccttcccc agcagggtgg gtctgcatcc ctccctcttt
14640 tacagaggtg gaaatgaggt ccagagaggc gaagtgactt gcctgtggtc
acacagcggt 14700 gtctggtggt gccatgctca gagctcctca ctctgggtcc
tgagctcctc ccagatctgc 14760 ctgctgtctg gaactgacat ctgtagctcc
ttcccaggga gatcctgggt ctctcaccgc 14820 cttctgacct ccctgtggct
gcaggagcta ctgcttctcg gtcagatggt atccccttcc 14880 cccaagcagt
atctcaggat aaaaataaac catcctgttc tcttttcctg ctgagccaac 14940
tgagggggag cctagccagg aggagcggca gatggagtgg gagcagaggg gaggggaggg
15000 gaggaaggtt ggaggacaag gaggagaagg aagagcaatg caaagtgcca
tgcagaaccc 15060 cggcacaaag gttcgaggtg aggtttaacc caggggtctc
agcctttccc aggaaagaac 15120 ctgctttgga tttccccaac acatcctgct
ggctggaggg tgtggggcga tggggggcaa 15180 ggggagatgt ggagagggct
tatcccagtg gaggctgtga gggcagctgc gagccaagag 15240 aggggcacct
ggcttggcag gcttcccaga agagcttcaa gaaccatttg gatacaccag 15300
gtggaggcct ttggtttaga aagtggagca aagtggtggt ggcggggaag gggaactttg
15360 ggacctaatt cttgtctctt atctagagcc taagagtgag tgttgttcac
ttctttggtg 15420 gtatggtgga aagagaccaa agacccacgc ctgccacaga
tcatctgtgt ggccttggat 15480 gagtcacttt cactgcttca cttctcagtt
tccctatctg acaaatggtg acaatatctg 15540 cagcagacgc tattgatgcc
tcctgtgttc tcttctaggt cagagcttct caaatttgag 15600 tgtgggtcag
gcgcagtggc tcacgtctgt aatcccagca ctctgggagg cttaggtggg 15660
cagataacct aaggtcagga gttcgagacc agcctgacca acagggcaaa accccgtctc
15720 tactaaaaat acaaaaatta gccgggcatg atggcgggtg cctgtaatcc
cagctacttg 15780 ggaggctgag gcagaagaat cacttgaacc cgggaggtgg
aggttgcagt gagccgagat 15840 catgccactg tactccagcc tgggcaacag
agtgaaactc catctaaaat aacaacaaca 15900 acaatagcaa caacaacaac
aacaatagca acaacaacaa aacttgagcg tgtatctgga 15960 ccacctgcaa
ggtggtcctc tccactattt tcagaactgg a 16001 <210> SEQ ID NO 5
<211> LENGTH: 18 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 5
ccaggcggca ggaccact 18 <210> SEQ ID NO 6 <211> LENGTH:
18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 6 gaccaggcgg caggacca 18
<210> SEQ ID NO 7 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 7 aggtgagacc aggcggca 18 <210> SEQ ID
NO 8 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
8 cagaggcaga cgaaccat 18 <210> SEQ ID NO 9 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 9 gcagaggcag
acgaacca 18 <210> SEQ ID NO 10 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 10 gcaagcagcc ccagagga 18
<210> SEQ ID NO 11 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 11 ggtcagcaag cagcccca 18 <210> SEQ ID
NO 12 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
12 gacagcggtc agcaagca 18
<210> SEQ ID NO 13 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 13 gatggacagc ggtcagca 18 <210> SEQ ID
NO 14 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
14 tctggatgga cagcggtc 18 <210> SEQ ID NO 15 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 15 ggtggttctg
gatggaca 18 <210> SEQ ID NO 16 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 16 gtgggtggtt ctggatgg 18
<210> SEQ ID NO 17 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 17 gcagtgggtg gttctgga 18 <210> SEQ ID
NO 18 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
18 cacaaagaac agcactga 18 <210> SEQ ID NO 19 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 19 ctggcacaaa
gaacagca 18 <210> SEQ ID NO 20 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 20 tcctggctgg cacaaaga 18
<210> SEQ ID NO 21 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 21 ctgtcctggc tggcacaa 18 <210> SEQ ID
NO 22 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
22 ctcaccagtt tctgtcct 18 <210> SEQ ID NO 23 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 23 tcactcacca
gtttctgt 18 <210> SEQ ID NO 24 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 24 gtgcagtcac tcaccagt 18
<210> SEQ ID NO 25 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 25 actctgtgca gtcactca 18 <210> SEQ ID
NO 26 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
26 cagtgaactc tgtgcagt 18 <210> SEQ ID NO 27 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 27 attccgtttc
agtgaact 18 <210> SEQ ID NO 28 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 28 gaaggcattc cgtttcag 18
<210> SEQ ID NO 29 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 29 ttcaccgcaa ggaaggca 18 <210> SEQ ID
NO 30 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
30 ctctgttcca ggtgtcta 18 <210> SEQ ID NO 31 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 31 ctggtggcag
tgtgtctc 18 <210> SEQ ID NO 32 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 32 tggggtcgca gtatttgt 18
<210> SEQ ID NO 33 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 33
ggttggggtc gcagtatt 18 <210> SEQ ID NO 34 <211> LENGTH:
18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 34 ctaggttggg gtcgcagt 18
<210> SEQ ID NO 35 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 35 ggtgcccttc tgctggac 18 <210> SEQ ID
NO 36 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
36 ctgaggtgcc cttctgct 18 <210> SEQ ID NO 37 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 37 gtgtctgttt
ctgaggtg 18 <210> SEQ ID NO 38 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 38 tggtgtctgt ttctgagg 18
<210> SEQ ID NO 39 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 39 acaggtgcag atggtgtc 18 <210> SEQ ID
NO 40 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
40 ttcacaggtg cagatggt 18 <210> SEQ ID NO 41 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 41 gtgccagcct
tcttcaca 18 <210> SEQ ID NO 42 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 42 tacagtgcca gccttctt 18
<210> SEQ ID NO 43 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 43 ggacacagct ctcacagg 18 <210> SEQ ID
NO 44 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
44 tgcaggacac agctctca 18 <210> SEQ ID NO 45 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 45 gagcggtgca
ggacacag 18 <210> SEQ ID NO 46 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 46 aagccgggcg agcatgag 18
<210> SEQ ID NO 47 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 47 aatctgcttg accccaaa 18 <210> SEQ ID
NO 48 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
48 gaaacccctg tagcaatc 18 <210> SEQ ID NO 49 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 49 gtatcagaaa
cccctgta 18 <210> SEQ ID NO 50 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 50 gctcgcagat ggtatcag 18
<210> SEQ ID NO 51 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 51 gcagggctcg cagatggt 18 <210> SEQ ID
NO 52 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
52 tgggcagggc tcgcagat 18 <210> SEQ ID NO 53 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 53 gactgggcag
ggctcgca 18 <210> SEQ ID NO 54 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 54
cattggagaa gaagccga 18 <210> SEQ ID NO 55 <211> LENGTH:
18 <212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 55 gatgacacat tggagaag 18
<210> SEQ ID NO 56 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 56 gcagatgaca cattggag 18 <210> SEQ ID
NO 57 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
57 tcgaaagcag atgacaca 18 <210> SEQ ID NO 58 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 58 gtccaagggt
gacatttt 18 <210> SEQ ID NO 59 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 59 cacagcttgt ccaagggt 18
<210> SEQ ID NO 60 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 60 ttggtctcac agcttgtc 18 <210> SEQ ID
NO 61 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
61 caggtctttg gtctcaca 18 <210> SEQ ID NO 62 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 62 ctgttgcaca
accaggtc 18 <210> SEQ ID NO 63 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 63 gtttgtgcct gcctgttg 18
<210> SEQ ID NO 64 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 64 gtcttgtttg tgcctgcc 18 <210> SEQ ID
NO 65 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
65 ccacagacaa catcagtc 18 <210> SEQ ID NO 66 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 66 ctggggacca
cagacaac 18 <210> SEQ ID NO 67 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 67 tcagccgatc ctggggac 18
<210> SEQ ID NO 68 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 68 caccaccagg gctctcag 18 <210> SEQ ID
NO 69 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
69 gggatcacca ccagggct 18 <210> SEQ ID NO 70 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 70 gaggatggca
aacaggat 18 <210> SEQ ID NO 71 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 71 accagcacca agaggatg 18
<210> SEQ ID NO 72 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 72 ttttgataaa gaccagca 18 <210> SEQ ID
NO 73 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
73 tattggttgg cttcttgg 18 <210> SEQ ID NO 74 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 74 gggttcctgc
ttggggtg 18 <210> SEQ ID NO 75 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide
<400> SEQUENCE: 75 gtcgggaaaa ttgatctc 18 <210> SEQ ID
NO 76 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
76 gatcgtcggg aaaattga 18 <210> SEQ ID NO 77 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 77 ggagccagga
agatcgtc 18 <210> SEQ ID NO 78 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 78 tggagccagg aagatcgt 18
<210> SEQ ID NO 79 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 79 tggagcagca gtgttgga 18 <210> SEQ ID
NO 80 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
80 gtaaagtctc ctgcactg 18 <210> SEQ ID NO 81 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 81 tggcatccat
gtaaagtc 18 <210> SEQ ID NO 82 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 82 cggttggcat ccatgtaa 18
<210> SEQ ID NO 83 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 83 ctctttgcca tcctcctg 18 <210> SEQ ID
NO 84 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
84 ctgtctctcc tgcactga 18 <210> SEQ ID NO 85 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 85 ggtgcagcct
cactgtct 18 <210> SEQ ID NO 86 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 86 aactgcctgt ttgcccac 18
<210> SEQ ID NO 87 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 87 cttctgcctg cacccctg 18 <210> SEQ ID
NO 88 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
88 actgactggg catagctc 18 <210> SEQ ID NO 89 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 89 gccccagagg
acgcactg 18 <210> SEQ ID NO 90 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 90 agcagcccca gaggacgc 18
<210> SEQ ID NO 91 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 91 agcaagcagc cccagagg 18 <210> SEQ ID
NO 92 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
92 gcggtcagca agcagccc 18 <210> SEQ ID NO 93 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 93 ggttctggat
ggacagcg 18 <210> SEQ ID NO 94 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 94 agtgggtggt tctggatg 18
<210> SEQ ID NO 95 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 95 gcactgactg tttattag 18 <210> SEQ ID
NO 96 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 96 ggcacaaaga acagcact 18 <210> SEQ ID
NO 97 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
97 tgtcctggct ggcacaaa 18 <210> SEQ ID NO 98 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 98 cagtttctgt
cctggctg 18 <210> SEQ ID NO 99 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 99 gtcactcacc agtttctg 18
<210> SEQ ID NO 100 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 100 aaggcattcc gtttcagt 18 <210> SEQ ID
NO 101 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
101 ctttcaccgc aaggaagg 18 <210> SEQ ID NO 102 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 102 tgtgtctctc
tgttccag 18 <210> SEQ ID NO 103 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 103 gtggcagtgt gtctctct 18
<210> SEQ ID NO 104 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 104 cccttctgct ggacccga 18 <210> SEQ ID
NO 105 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
105 tgaggtgccc ttctgctg 18 <210> SEQ ID NO 106 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 106 tctgtttctg
aggtgccc 18 <210> SEQ ID NO 107 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 107 gatggtgtct gtttctga 18
<210> SEQ ID NO 108 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 108 aaacccctgt agcaatct 18 <210> SEQ ID
NO 109 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
109 cgcagatggt atcagaaa 18 <210> SEQ ID NO 110 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 110 ggcagggctc
gcagatgg 18 <210> SEQ ID NO 111 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 111 gagaagaagc cgactggg 18
<210> SEQ ID NO 112 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 112 tggagaagaa gccgactg 18 <210> SEQ ID
NO 113 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
113 attggagaag aagccgac 18 <210> SEQ ID NO 114 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 114 acattggaga
agaagccg 18 <210> SEQ ID NO 115 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 115 acacattgga gaagaagc 18
<210> SEQ ID NO 116 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 116 tgacacattg gagaagaa 18 <210> SEQ ID
NO 117 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 117 atgacacatt ggagaaga 18 <210> SEQ ID
NO 118 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
118 aaagcagatg acacattg 18 <210> SEQ ID NO 119 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 119 aggtctttgg
tctcacag 18 <210> SEQ ID NO 120 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 120 ttgcacaacc aggtcttt 18
<210> SEQ ID NO 121 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 121 ttgtttgtgc ctgcctgt 18 <210> SEQ ID
NO 122 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
122 tcttgtttgt gcctgcct 18 <210> SEQ ID NO 123 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 123 agtcttgttt
gtgcctgc 18 <210> SEQ ID NO 124 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 124 cagtcttgtt tgtgcctg 18
<210> SEQ ID NO 125 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 125 tcagtcttgt ttgtgcct 18 <210> SEQ ID
NO 126 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
126 catcagtctt gtttgtgc 18 <210> SEQ ID NO 127 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 127 gaccacagac
aacatcag 18 <210> SEQ ID NO 128 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 128 gggaccacag acaacatc 18
<210> SEQ ID NO 129 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 129 tcaccaccag ggctctca 18 <210> SEQ ID
NO 130 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
130 gatcaccacc agggctct 18 <210> SEQ ID NO 131 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 131 agaggatggc
aaacagga 18 <210> SEQ ID NO 132 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 132 aagaggatgg caaacagg 18
<210> SEQ ID NO 133 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 133 caagaggatg gcaaacag 18 <210> SEQ ID
NO 134 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
134 gaccagcacc aagaggat 18 <210> SEQ ID NO 135 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 135 aagaccagca
ccaagagg 18 <210> SEQ ID NO 136 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 136 taaagaccag caccaaga 18
<210> SEQ ID NO 137 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 137 tgataaagac cagcacca 18 <210> SEQ ID
NO 138 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 138 tttgataaag accagcac 18
<210> SEQ ID NO 139 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 139 actctctttg cccatcct 18 <210> SEQ ID
NO 140 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
140 cgactctctt tgcccatc 18 <210> SEQ ID NO 141 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 141 atgcgactct
ctttgccc 18 <210> SEQ ID NO 142 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 142 aaatgcgact ctctttgc 18
<210> SEQ ID NO 143 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 143 ctgaaatgcg actctctt 18 <210> SEQ ID
NO 144 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
144 aactgaaatg cgactctc 18 <210> SEQ ID NO 145 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 145 cttcactgtc
tctccctg 18 <210> SEQ ID NO 146 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 146 ccttcactgt ctctccct 18
<210> SEQ ID NO 147 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 147 aaccttcact gtctctcc 18 <210> SEQ ID
NO 148 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
148 gatcaccaca ggctctca 18 <210> SEQ ID NO 149 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 149 tgataagaca
gcaccaag 18 <210> SEQ ID NO 150 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 150 ggtagttctt gccacttt 18
<210> SEQ ID NO 151 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 151 gggcctatgg gtagttct 18 <210> SEQ ID
NO 152 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
152 attatctctg ggtctgct 18 <210> SEQ ID NO 153 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 153 actgacacat
ttgagcag 18 <210> SEQ ID NO 154 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 154 gactccctac tgacacat 18
<210> SEQ ID NO 155 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 155 caaagagcgg ttctccac 18 <210> SEQ ID
NO 156 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
156 aattctccaa agagcggt 18 <210> SEQ ID NO 157 <211>
LENGTH: 18 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 157 tcttgacatc
cttttcat 18 <210> SEQ ID NO 158 <211> LENGTH: 18
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 158 cccacctatc ttgacatc 18
<210> SEQ ID NO 159 <211> LENGTH: 18 <212> TYPE:
DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 159 aggccgagag ttcaaaat 18 <210> SEQ ID
NO 160 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
160 ccagcaattc accgcgcagg 20 <210> SEQ ID NO 161 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 161 tgcagaggca
gacgaaccat 20 <210> SEQ ID NO 162 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 162 cagaggacgc actgcagagg 20
<210> SEQ ID NO 163 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 163 aagcagcccc agaggacgca 20 <210> SEQ
ID NO 164 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
164 cagcggtcag caagcagccc 20 <210> SEQ ID NO 165 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 165 ctcacagcgg
tcagcaagca 20 <210> SEQ ID NO 166 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 166 gctggcaagg agatgataac 20
<210> SEQ ID NO 167 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 167 aggttggaac acccaagata 20 <210> SEQ
ID NO 168 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
168 ggagaaaccc ctggtttctc 20 <210> SEQ ID NO 169 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 169 tcattcctgc
ccaggcttca 20 <210> SEQ ID NO 170 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 170 tcaggtgaaa gtgaaagctg 20
<210> SEQ ID NO 171 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 171 taccatcttc aaacacatga 20 <210> SEQ
ID NO 172 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
172 ttacccaaaa tgggaaagga 20 <210> SEQ ID NO 173 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 173 gaaagaatac
atgtatatgg 20 <210> SEQ ID NO 174 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 174 agagtcagac agctttagac 20
<210> SEQ ID NO 175 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 175 gtaccaccca tgctattaat 20 <210> SEQ
ID NO 176 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
176 acagtgacag agtccaaatg 20 <210> SEQ ID NO 177 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 177 aatgtaaagc
tggaagggta 20 <210> SEQ ID NO 178 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 178 gggctatgtt tagcacttgg 20
<210> SEQ ID NO 179 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 179 gggcttgatg cctgagtcat 20 <210> SEQ
ID NO 180 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 180 tgaagtgcaa gtcaaaacag 20
<210> SEQ ID NO 181 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 181 gcaatttgaa gggatcttga 20 <210> SEQ
ID NO 182 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
182 catgcagtgg gtggttctgg 20 <210> SEQ ID NO 183 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 183 gtttttctct
gcatgcagtg 20 <210> SEQ ID NO 184 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 184 gctggcacaa agaacagcac 20
<210> SEQ ID NO 185 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 185 cactaaccac acaatgatca 20 <210> SEQ
ID NO 186 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
186 tgtgcagtca ctcaccagtt 20 <210> SEQ ID NO 187 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 187 gtctaggaat
tcgctttcac 20 <210> SEQ ID NO 188 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 188 caggtgtcta ggaattcgct 20
<210> SEQ ID NO 189 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 189 gtcgcagtat ttgtgctggt 20 <210> SEQ
ID NO 190 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
190 acccgaagcc ctaggtctga 20 <210> SEQ ID NO 191 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 191 ctggacccga
agccctaggt 20 <210> SEQ ID NO 192 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 192 ttctgctgga cccgaagccc 20
<210> SEQ ID NO 193 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 193 cttcttcaca ggtgcagatg 20 <210> SEQ
ID NO 194 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
194 agccagtggc caggcaggac 20 <210> SEQ ID NO 195 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 195 gaagaagccg
actgggcagg 20 <210> SEQ ID NO 196 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 196 ttggagaaga agccgactgg 20
<210> SEQ ID NO 197 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 197 gatgacacat tggagaagaa 20 <210> SEQ
ID NO 198 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
198 tgtctattac ctcaaagaga 20 <210> SEQ ID NO 199 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 199 acagtgtgtt
cagaggattg 20 <210> SEQ ID NO 200 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 200 acaatacact ttacatgttt 20
<210> SEQ ID NO 201
<211> LENGTH: 20 <212> TYPE: DNA <213> ORGANISM:
Artificial Sequence <220> FEATURE: <223> OTHER
INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE: 201
attgtgtctt tagaaccaga 20 <210> SEQ ID NO 202 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 202 gggccctaaa
ggatgtaaaa 20 <210> SEQ ID NO 203 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 203 cagtcttgtt tgtgcctgcc 20
<210> SEQ ID NO 204 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 204 tgtccaggac tcaccacaga 20 <210> SEQ
ID NO 205 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
205 tatggcacct tcttaaatat 20 <210> SEQ ID NO 206 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 206 tgcttttggt
atagaagagt 20 <210> SEQ ID NO 207 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 207 aaatgtggct ggcagatgtc 20
<210> SEQ ID NO 208 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 208 gtcagagctc atctacatca 20 <210> SEQ
ID NO 209 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
209 ctgataaaga ccagcaccaa 20 <210> SEQ ID NO 210 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 210 aggactcact
gataaagacc 20 <210> SEQ ID NO 211 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 211 cagactctga atcagtttta 20
<210> SEQ ID NO 212 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 212 cagtccccaa ttctgctgcc 20 <210> SEQ
ID NO 213 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
213 ccagtgttag gctctgccag 20 <210> SEQ ID NO 214 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 214 gaatgccagg
aaaggagtga 20 <210> SEQ ID NO 215 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 215 cagccccaag gcccaaagat 20
<210> SEQ ID NO 216 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 216 ctgcactgga gcagcagtgt 20 <210> SEQ
ID NO 217 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
217 accggttggc atccatgtaa 20 <210> SEQ ID NO 218 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 218 cctgggtgac
cggttggcat 20 <210> SEQ ID NO 219 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 219 caagttggga gactggatgg 20
<210> SEQ ID NO 220 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 220 ctttaataca agttgggaga 20 <210> SEQ
ID NO 221 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
221 tcggaaggtc tggtggatat 20
<210> SEQ ID NO 222 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 222 tgggcaccaa actgctggat 20 <210> SEQ
ID NO 223 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
223 tatggcttcc tgggcgcagg 20 <210> SEQ ID NO 224 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 224 aatgctgcaa
tgggcatctg 20 <210> SEQ ID NO 225 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 225 gttcactatc acaaacaatg 20
<210> SEQ ID NO 226 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 226 cagttaagca gcttccagtt 20 <210> SEQ
ID NO 227 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
227 aattttattt agccagtctc 20 <210> SEQ ID NO 228 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 228 gttgtataaa
tatattctaa 20 <210> SEQ ID NO 229 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 229 acagtgtttt tgagattctg 20
<210> SEQ ID NO 230 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 230 ctcaggaccc agagtgagga 20 <210> SEQ
ID NO 231 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
231 tgggttaaac ctcacctcga 20 <210> SEQ ID NO 232 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 232 attaggtccc
aaagttcccc 20 <210> SEQ ID NO 233 <211> LENGTH: 20
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: Synthetic
Oligonucleotide <400> SEQUENCE: 233 ggcagacgaa ccatggcgag 20
<210> SEQ ID NO 234 <211> LENGTH: 20 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 234 gtagcaatct gcttgacccc 20 <210> SEQ
ID NO 235 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Synthetic Oligonucleotide <400> SEQUENCE:
235 gaccacagac aacatcagtc 20 <210> SEQ ID NO 236 <211>
LENGTH: 20 <212> TYPE: DNA <213> ORGANISM: Artificial
Sequence <220> FEATURE: <223> OTHER INFORMATION:
Synthetic Oligonucleotide <400> SEQUENCE: 236 ccaccttttt
gataaagacc 20 <210> SEQ ID NO 237 <211> LENGTH: 1570
<212> TYPE: DNA <213> ORGANISM: H. sapiens <400>
SEQUENCE: 237 ggcaggggag tcagcagagg cctcgctcgg gcgcccagtg
gtcctgccgc ctggtctcac 60 ctcgccatgg ttcgtctgcc tctgcagtgc
gtcctctggg gctgcttgct gaccgctgtc 120 catccagaac cacccactgc
atgcagagaa aaacagtacc taataaacag tcagtgctgt 180 tctttgtgcc
agccaggaca gaaactggtg agtgactgca cagagttcac tgaaacggaa 240
tgccttcctt gcggtgaaag cgaattccta gacacctgga acagagagac acacttccac
300 cagcacaaat actgcgaccc caacctaggg cttcgggtcc agcagaaggg
cacctcagaa 360 acagacacca tctgcacctg tgaagaaggc tggcactgta
cgagtgaggc ctgtgagagc 420 tgtgtcctgc accgctcatg ctcgcccggc
tttggggtca agcagattga catctgccag 480 ccacatttcc ccaaggaccg
cggtttgaac cttctgatgt agatgagctc tgacattgga 540 agattctgga
gtctgacaag tcacagcagg ttgagggtag ggagaaactg caggtgaggg 600
gtgcatgctg aagtcctgat ttctccaggt ccccaggatc ggctgagagc cctggtggtg
660 atccccatca tcttcgggat cctgtttgcc atcctcttgg tgctggtctt
tatcaaaaag 720 gtggccaaga agccaaccaa taaggccccc caccccaagc
aggaacccca ggagatcaat 780 tttcccgacg atcttcctgg ctccaacact
gctgctccag tgcaggagac tttacatgga 840 tgccaaccgg tcacccagga
ggatggcaaa gagagtcgca tctcagtgca ggagagacag 900 tgaggctgca
cccacccagg agtgtggcca cgtgggcaaa caggcagttg gccagagagc 960
ctggtgctgc tgctgctgtg gcgtgagggt gaggggctgg cactgactgg gcatagctcc
1020 ccgcttctgc ctgcacccct gcagtttaga caggagacct ggcactggat
gcagaaacag 1080 ttcaccttga agaacctctc acttcaccct ggagcccatc
cagtctccca acttgtatta 1140 aagacagagg cagaagtttg gtggtggtgg
tgttggggta tggtttagta atatccacca 1200 gaccttccga tccagcagtt
tggtgcccag agaggcatca tggtggcttc cctgcgccca 1260 ggaagccata
tacacagatg cccattgcag cattgtttgt gatagtgaac aactggaagc 1320
tgcttaactg tccatcagca ggagactggc taaataaaat tagaatatat ttatacaaca
1380 gaatctcaaa aacactgttg agtaaggaaa aaaaggcatg ctgctgaatg
atgggtatgg 1440 aactttttaa aaaagtacat gcttttatgt atgtatattg
cctatggata tatgtataaa 1500 tacaatatgc atcatatatt gataaaaaaa
aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1560 aaaaaaaaaa 1570 <210>
SEQ ID NO 238 <211> LENGTH: 20 <212> TYPE: DNA
<213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Synthetic Oligonucleotide
<400> SEQUENCE: 238
tccatttatt agtctaggaa 20
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