U.S. patent application number 14/542923 was filed with the patent office on 2015-10-15 for modulation of cd40 expression.
This patent application is currently assigned to ISIS PHARMACEUTICALS, INC.. The applicant listed for this patent is Isis Pharmaceuticals, Inc.. Invention is credited to C. Frank Bennett, Lex M. Cowsert, Susan M. Freier.
Application Number | 20150291960 14/542923 |
Document ID | / |
Family ID | 40419509 |
Filed Date | 2015-10-15 |
United States Patent
Application |
20150291960 |
Kind Code |
A1 |
Bennett; C. Frank ; et
al. |
October 15, 2015 |
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 |
Isis Pharmaceuticals, Inc. |
Carlsbad |
CA |
US |
|
|
Assignee: |
ISIS PHARMACEUTICALS, INC.
Carlsbad
CA
|
Family ID: |
40419509 |
Appl. No.: |
14/542923 |
Filed: |
November 17, 2014 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12743797 |
Feb 15, 2011 |
8916531 |
|
|
PCT/US2008/012998 |
Nov 20, 2008 |
|
|
|
14542923 |
|
|
|
|
60989421 |
Nov 20, 2007 |
|
|
|
Current U.S.
Class: |
514/44A ;
536/24.5 |
Current CPC
Class: |
C12N 2310/315 20130101;
A61P 29/00 20180101; C12N 2310/3231 20130101; A61P 35/00 20180101;
C12N 2310/3341 20130101; C12N 2310/321 20130101; C12N 15/113
20130101; C12N 2310/346 20130101; C12N 2310/11 20130101; C12N
2310/321 20130101; C12N 15/1138 20130101; C12N 2310/3525 20130101;
C12N 2310/341 20130101 |
International
Class: |
C12N 15/113 20060101
C12N015/113 |
Claims
1.-46. (canceled)
47. A compound comprising a modified oligonucleotide consisting of
18 to 24 linked nucleosides having a nucleobase sequence comprising
a portion of at least 18 contiguous nucleobases complementary to an
equal length portion of nucleobases 8492 to 10547, 11244 to 12591
or 13802 to 14016 of SEQ ID NO: 4, wherein the nucleobase sequence
of the modified oligonucleotide is at least 80% complementary to
SEQ ID NO: 4.
48. The compound of claim 47, wherein the nucleobase sequence of
the compound is at least 85%, at least 90%, at least 95%, or 100%
complementary to SEQ ID NO: 4.
49. The compound of claim 47, wherein the modified oligonucleotide
consists of 20 linked nucleosides.
50. The compound of claim 47, wherein at least one internucleoside
linkage is a modified internucleoside linkage.
51. The compound of claim 50, wherein at least one modified
internucleoside linkage is a phosphorothioate internucleoside
linkage.
52. The compound of claim 50, wherein each modified internucleoside
linkage is a phosphorothioate internucleoside linkage.
53. The compound of claim 47, wherein the modified oligonucleotide
comprises at least one modified sugar.
54. The compound of claim 53, wherein at least one modified sugar
is a bicyclic sugar.
55. The compound of claim 54, wherein the bicyclic sugar has a
4'-CH(CH.sub.3)--O-2' bridge or a 4'-(CH.sub.2)n--O-2' bridge,
wherein n is 1 or 2.
56. The compound of claim 53, wherein at least one modified sugar
comprises a 2'-O-methoxyethyl.
57. The compound of claim 47, wherein at least one nucleoside
comprises a modified nucleobase.
58. The compound of claim 57, wherein the modified nucleobase is a
5-methylcytosine.
59. The compound of claim 47, wherein the modified oligonucleotide
is single-stranded.
60. The compound of claim 47, wherein the modified oligonucleotide
comprises: 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.
61. The compound of claim 60, wherein the modified 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, wherein at least one internucleoside
linkage is a phosphorothioate linkage, and wherein each cytosine is
a 5-methylcytosine.
62. The compound of claim 60, wherein the modified oligonucleotide
comprises: a gap segment consisting of ten linked deoxynucleosides;
a 5' wing segment consisting of three 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
modified sugar, wherein at least one internucleoside linkage is a
phosphorothioate linkage, and wherein each cytosine is a
5-methylcytosine.
63. A composition comprising the compound of claim 47, or a salt
thereof, and a pharmaceutically acceptable carrier or diluent.
64. A method comprising administering to an animal the compound of
claim 47 for the treatment of cancer or an inflammatory or immune
associated condition.
65. The method of claim 64, wherein the animal is a human.
66. The method of claim 64, wherein the administering is parenteral
administration.
67. The method of claim 66, wherein the parenteral administration
is subcutaneous or intravenous administration.
68. The method of claim 64, wherein the administering is aerosol,
topical or oral administration.
Description
RELATED APPLICATIONS
[0001] This application is continuation of U.S. patent application
Ser. No. 12/743,797, filed Feb. 15, 2011, which is a U.S. National
Phase filing under 35 U.S.C. .sctn.371 claiming priority to
International Serial No. PCT/US2008/012998 filed Nov. 20, 2008,
which claims priority to U.S. Provisional Application 60/989,421,
filed Nov. 20, 2007, each of which is incorporated herein by
reference in its entirety.
SEQUENCE LISTING
[0002] The present application is being filed along with a Sequence
Listing in electronic format. The Sequence Listing is provided as a
file entitled BIOL0096USC1SEQ_ST25.txt, created Nov. 12, 2014,
which is 72 Kb in size. The information in the electronic format of
the sequence listing is incorporated herein by reference in its
entirety.
FIELD OF THE INVENTION
[0003] 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
[0004] 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.
[0005] 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.
[0006] 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).
[0007] 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).
[0008] 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.
[0009] 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).
[0010] 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
[0011] Provided herein are antisense compounds, compositions, and
methods for the treatment and prevention of inflammatory conditions
and cancer.
[0012] 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.
[0013] 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: [0014] (a) positions 11250-12685, corresponding to intron
6; [0015] (b) positions 2943-6367, corresponding to intron 1,
[0016] (c) positions 6447-6780, corresponding to intron 2, [0017]
(d) positions 6907-7157, corresponding to intron 3, [0018] (e)
positions 7305-7673, corresponding to intron 4, [0019] (f)
positions 7768-11187, corresponding to intron 5, [0020] (g)
positions 12773-12877, corresponding to intron 7, or [0021] (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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] The antisense compound may comprise linked nucleosides.
Preferably, the antisense compound is an antisense
oligonucleotide.
[0027] The antisense compound may be a single-stranded or
double-stranded oligonucleotide. Preferably, the antisense compound
is a single-stranded oligonucleotide.
[0028] 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.
[0029] 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.
[0030] The antisense compound may comprise a modified nucleobase.
The modified nucleobase may be a 5-methylcytosine. Preferably,
every cytosine is a 5-methylcytosine.
[0031] The antisense compound may be a gapmer, for example an
oligonucleotide comprising: [0032] a gap segment consisting of
linked deoxynucleosides; [0033] a 5' wing segment consisting of
linked nucleosides; [0034] a 3' wing segment consisting of linked
nucleosides; [0035] 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.
[0036] 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.
[0037] The antisense oligonucleotide may be a 4-10-4 MOE gapmer.
The antisense oligonucleotide may consist of 18 linked
nucleosides.
[0038] 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.
[0039] The composition may comprise a single-stranded or
double-stranded oligonucleotide.
[0040] 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 [0041] (a) about 15 mol %
POPC, about 45 mol % DOPE, about 20 mol % MoChol, about 20 mol %
Chems [0042] (b) about 60 mol % POPC, about 10 mol % DOTAP, about
30 mol % Chems [0043] (c) about 30 mol % POPC, about 10 mol %
DOTAP, about 20 mol % Chems, about 40 mol % Chol [0044] (d) about
60 mol % POPC, about 20 mol % HistChol, about 20 mol % Chol.
[0045] 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.
[0046] 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.
[0047] 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
[0048] 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.
[0049] 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
[0050] 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.
[0051] Unless otherwise indicated, the following terms have the
following meanings:
[0052] "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.
[0053] "2'-O-methoxyethyl nucleotide" means a nucleotide comprising
a 2'-O-methoxyethyl modified sugar moiety.
[0054] "5-methylcytosine" means a cytosine modified with a methyl
group attached to the 5' position. A 5-methylcytosine is a modified
nucleobase.
[0055] "Acceptable safety profile" means a pattern of side effects
that is within clinically acceptable limits.
[0056] "Active pharmaceutical ingredient" means the substance or
substances in a pharmaceutical composition that provides a desired
effect.
[0057] "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.
[0058] "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.
[0059] "Administering" means providing a pharmaceutical agent to an
individual, and includes, but is not limited to administering by a
medical professional and self-administering.
[0060] "Antisense compound" means an oligomeric compound that is
capable of undergoing hybridization to a target nucleic acid
through hydrogen bonding.
[0061] "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.
[0062] "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.
[0063] "Bicyclic sugar" means a furosyl ring modified by the
bridging of two non-geminal ring atoms. A bicyclic sugar is a
modified sugar.
[0064] "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.
[0065] "Cap structure" or "terminal cap moiety" means chemical
modifications, which have been incorporated at either terminus of
an antisense compound.
[0066] "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.
[0067] "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.
[0068] "Complementarity" means the capacity for pairing between
nucleobases of a first nucleic acid and a second nucleic acid.
[0069] "Comply" means the adherence with a recommended therapy by a
individual.
[0070] "Contiguous nucleobases" means nucleobases immediately
adjacent to each other.
[0071] "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.
[0072] "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.
[0073] "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.
[0074] "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.
[0075] "Efficacy" means the ability to produce a desired
effect.
[0076] "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.
[0077] "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.
[0078] "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.
[0079] "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
[0080] "Individual" means a human or non-human animal selected for
treatment or therapy.
[0081] "Individual compliance" means adherence to a recommended or
prescribed therapy by a individual.
[0082] "Injection site reaction" means inflammation or abnormal
redness of skin at a site of injection in a individual.
[0083] "Internucleoside linkage" refers to the chemical bond
between nucleosides.
[0084] "Linked nucleosides" means adjacent nucleosides which are
bonded together.
[0085] "Modified internucleoside linkage" refers to a substitution
and/or any change from a naturally occurring internucleoside bond
(i.e. a phosphodiester internucleoside bond).
[0086] "Modified oligonucleotide" means an oligonucleotide
comprising a modified internucleoside linkage, a modified sugar,
and/or a modified nucleobase.
[0087] "Modified sugar" refers to a substitution and/or any change
from a natural sugar.
[0088] "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).
[0089] "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.
[0090] "Modified sugar moiety" means a sugar moiety having any
substitution and/or change from a natural sugar moiety.
[0091] "Motif" means the pattern of unmodified and modified
nucleosides in an antisense compound.
[0092] "Naturally occurring internucleoside linkage" means a 3' to
5' phosphodiester linkage.
[0093] "Natural sugar moiety" means a sugar moiety found in DNA
(2'-H) or RNA (2'-OH).
[0094] "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.
[0095] "Nucleoside" means a nucleobase linked to a sugar.
[0096] 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.
[0097] "Nucleobase" means a heterocyclic moiety capable of pairing
with a base of another nucleic acid.
[0098] "Nucleobase sequence" means the order of contiguous
nucleobases independent of any sugar, linkage, and/or nucleobase
modification.
[0099] "Nucleotide" means a nucleoside having a phosphate group
covalently linked to the sugar portion of the nucleoside.
[0100] 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).
[0101] "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.
[0102] "Oligonucleoside" means an oligonucleotide in which the
internucleoside linkages do not contain a phosphorus atom.
[0103] "Oligonucleotide" means a polymer or oligomer of linked
nucleosides each of which can be modified or unmodified,
independent one from another.
[0104] "Parenteral administration," means administration through
injection or infusion. Parenteral administration includes, but is
not limited to, subcutaneous administration, intravenous
administration, or intramuscular administration.
[0105] "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.
[0106] "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.
[0107] "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.
[0108] "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.
[0109] "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.
[0110] "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.
[0111] "Recommended therapy" means a therapeutic regimen
recommended by a medical professional for the treatment,
amelioration, or prevention of a disease.
[0112] "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.
[0113] "Single-stranded modified oligonucleotide" means a modified
oligonucleotide which is not hybridized to a complementary
strand.
[0114] "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.
[0115] 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.
[0116] "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.
[0117] "Subcutaneous administration" means administration just
below the skin. "Intravenous administration" means administration
into a vein.
[0118] "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.
[0119] "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.
[0120] "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.
[0121] "Target region" means a portion of a target nucleic acid to
which one or more antisense compounds is targeted.
[0122] "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.
[0123] "Therapeutically effective amount" means an amount of a
pharmaceutical agent that provides a therapeutic benefit to an
individual.
[0124] "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
[0125] 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.
[0126] 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.
[0127] 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.
[0128] 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.
[0129] 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.
[0130] 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.
[0131] 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.
[0132] 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.
[0133] 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
[0134] 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.
[0135] 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.
[0136] 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.
[0137] 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.
[0138] In one embodiment, antisense compounds targeted to a CD40
nucleic acid possess a 5-10-5 gapmer motif.
[0139] 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.
[0140] 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
[0141] Nucleotide sequences that encode CD40 include, without
limitation, the following:
[0142] 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.sub.--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.
[0143] 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.
[0144] 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.
[0145] 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.
[0146] 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.
[0147] 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.
[0148] 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.
[0149] 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
[0150] 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.
[0151] 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.
[0152] 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
[0153] 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.
[0154] 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.
[0155] 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
[0156] 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).
[0157] 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).
[0158] 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.
[0159] 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).
[0160] 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.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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
[0165] 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.
[0166] 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
[0167] 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.
[0168] 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.
[0169] 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
[0170] 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.
[0171] 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.
[0172] Methods of preparation of phosphorous-containing and
non-phosphorous-containing linkages are well known.
[0173] 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.
[0174] 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.
[0175] 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
[0176] 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).
[0177] 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, 0-allyl, 0-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.
[0178] 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).
[0179] 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.
[0180] 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.
[0181] 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.
[0182] 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.
[0183] 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.
[0184] 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.
[0185] 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.sub.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.
[0186] 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.
[0187] 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
[0188] 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).
[0189] 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.
[0190] 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.
[0191] 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.
[0192] 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
[0193] 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.
[0194] 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
[0195] 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.
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] 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.
[0201] 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.
[0202] 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.
[0203] 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.
[0204] 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.
[0205] 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.
[0206] 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.
[0207] 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.
[0208] 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 Endert et al., WO 07/031333
of Panzner et al., WO 07/107304 of Panzner et al. and WO 08/043575
of Panzner et al. Amphoteric liposomes have an excellent
biodistribution 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.
[0209] By "amphoteric" is meant herein that the liposomes comprise
charged groups of both anionic and cationic character wherein:
[0210] (i) at least one of the charged groups has a pKa between 4
and 7.4, [0211] (ii) the cationic charge prevails at pH 4 and
[0212] (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.
[0213] 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.%.
[0214] 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.)
[0215] 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.
[0216] 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.
[0217] Preferred cationic components include DMTAP, DPTAP, DOTAP,
DC-Chol, MoChol, HisChol, DPIM, CHIM, DORIE, DDAB, DAC-Chol,
TC-Chol, DOTMA, DOGS, (C18).sub.2Gly.sup.+
N,N-dioctadecylamido-glycin, CTAB, CPyC, DODAP and DOEPC.
[0218] 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.
[0219] 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.
[0220] 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.
[0221] 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.
[0222] 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.
[0223] 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.%).
[0224] Preferred phosphatidylethanolamines include DOPE, DMPE and
DPPE.
[0225] In some embodiments said neutral lipid may comprise POPC and
DOPE.
[0226] 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.
[0227] 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: [0228] (a)
about 15 mol.% POPC, about 45 mol.% DOPE, about 20 mol.% MoChol and
about 20 mol.% CHEMS; [0229] (b) about 10 mol.% POPC, about 30
mol.% DOPE, about 30 mol.% MoChol and about 30 mol.% CHEMS; [0230]
(c) about 10 mol.% POPC, about 30 mol.% DOPE, about 20 mol.% MoChol
and about 40 mol.% CHEMS; [0231] (d) about 6 mol.% POPC, about 24
mol.% DOPE, about 47 mol.% MoChol and about 23 mol.% CHEMS.
[0232] 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.%.
[0233] 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.
[0234] 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.
[0235] 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.
[0236] 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.
[0237] 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.
[0238] 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.
[0239] 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.
[0240] 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.
[0241] 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.
[0242] 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.
[0243] 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
[0244] DMPC Dimyristoylphosphatidylcholine [0245] DPPC
Dipalmitoylphosphatidylcholine [0246] DSPC
Distearoylphosphatidylcholine [0247] POPC
Palmitoyl-oleoylphosphatidylcholine [0248] DOPC
Dioleoylphosphatidylcholine [0249] DOPE
Dioleoylphosphatidylethanolamine [0250] DMPE
Dimyristoylphosphatidylethanolamine [0251] DPPE
Dipalmitoylphosphatidylethanolamine [0252] DOPG
Dioleoylphosphatidylglycerol [0253] POPG
Palmitoyl-oleoylphosphatidylglycerol [0254] DMPG
Dimyristoylphosphatidylglycerol [0255] DPPG
Dipalmitoylphosphatidylglycerol [0256] DMPS
Dimyristoylphosphatidylserine [0257] DPPS
Dipalmitoylphosphatidylserine [0258] DOPS
Dioleoylphosphatidylserine [0259] POPS
Palmitoyl-oleoylphosphatidylserine [0260] DMPA
Dimyristoylphosphatidic acid [0261] DPPA Dipalmitoylphosphatidic
acid [0262] DOPA Dioleoylphosphatidic acid [0263] POPA
Palmitoyl-oleoylphosphatidic acid [0264] CHEMS
Cholesterolhemisuccinate [0265] DC-Chol
3-.beta.-[N--(N',N'-dimethylethane) carbamoyl] cholesterol [0266]
CetylP Cetylphosphate [0267] DODAP
(1,2)-dioleoyloxypropyl)-N,N-dimethylammonium chloride [0268] DOEPC
1,2-dioleoyl-sn-glycero-3-ethylphosphocholine [0269] DAC-Chol
3-.beta.-[N--(N,N'-dimethylethane) carbamoyl] cholesterol [0270]
TC-Chol 3-.beta.-[N--(N',N',N'-trimethylaminoethane) carbamoyl]
cholesterol [0271] DOTMA
(1,2-dioleyloxypropyl)-N,N,N-trimethylammoniumchlorid)
(Lipofectin.RTM.) [0272] DOGS ((C18).sub.2GlySper3.sup.+)
N,N-dioctadecylamido-glycyl-spermine (Transfectam.RTM.) [0273] CTAB
Cetyl-trimethylammoniumbromide, [0274] CPyC
Cetyl-pyridiniumchloride [0275] DOTAP
(1,2-dioleoyloxypropyl)-N,N,N-trimethylammonium salt [0276] DMTAP
(1,2-dimyristoyloxypropyl)-N,N,N-trimethylammonium salt [0277]
DPTAP (1,2-dipalmitoyloxypropyl)-N,N,N-trimethylammonium salt
[0278] DOTMA (1,2-dioleyloxypropyl)-N,N,N-trimethylammonium
chloride) [0279] DORIE (1,2-dioleyloxypropyl)-3
dimethylhydroxyethyl ammoniumbromide) [0280] DDAB
Dimethyldioctadecylammonium bromide [0281] DPIM
4-(2,3-bis-palmitoyloxy-propyl)-1-methyl-1H-imidazole [0282] CHIM
Histaminyl-Cholesterolcarbamate [0283] MoChol
4-(2-Aminoethyl)-Morpholino-Cholesterolhemisuccinate [0284] HisChol
Histaminyl-Cholesterolhemisuccinate. [0285] HCChol
N.alpha.-Histidinyl-Cholesterolcarbamate [0286] HistChol
N.alpha.-Histidinyl-Cholesterol-hemisuccinate. [0287] AC
Acylcarnosine, Stearyl- & Palmitoylcarnosine [0288] HistDG
1,2-Dipalmitoylglycerol-hemisuccinate-N.alpha.-Histidinyl-hemisuccinate,
& Distearoyl-, Dimyristoyl-, Dioleoyl- or
palmitoyl-oleoylderivatives [0289] IsoHistSuccDG
1,2-Dipalmitoylglycerol-Oa-Histidinyl-N.alpha.-hemisuccinat, &
Distearoyl-, Dimyristoyl-, Dioleoyl- or palmitoyl-oleoylderivatives
[0290] DGSucc 1,2-Dipalmitoyglycerol-3-hemisuccinate &
Distearoyl-, Dimyristoyl- Dioleoyl- or
palmitoyl-oleoylderivatives
[0291] 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.
[0292] One of skill in the art will recognize that formulations are
routinely designed according to their intended use, i.e. route of
administration.
[0293] 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.
[0294] 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. No.
09/108,673 (filed Jul. 1, 1998), Ser. No. 09/315,298 (filed May 20,
1999) and Ser. No. 10/071,822, filed Feb. 8, 2002, each of which is
incorporated herein by reference in their entirety.
[0295] 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.
[0296] 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).
[0297] Anti-inflammatory drugs, including but not limited to
nonsteroidal anti-inflammatory drugs and corticosteroids, and
antiviral drugs, including but not limited to ribivirin,
vidarabine, acyclovir and ganciclovir, may also be combined in
compositions of the invention. 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.
[0298] 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
[0299] 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 commerical 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
[0300] Described herein are methods for treatment of cells with
antisense oligonucleotides, which can be modified appropriately for
treatment with other antisense compounds.
[0301] In general, cells are treated with antisense
oligonucleotides when the cells reach approximately 60-80%
confluency in culture.
[0302] 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.
[0303] 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.
[0304] 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.
[0305] 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
[0306] 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
[0307] 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
[0308] 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.
[0309] 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.
[0310] 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.
[0311] 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
[0312] 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
[0313] 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
[0314] 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.
[0315] 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.
[0316] 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.
[0317] 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.
[0318] 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
[0319] 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.
[0320] 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.
[0321] 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
[0322] 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
[0323] 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.
[0324] 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 Target Target
Oligo SEQ Start Stop Target % SEQ ID ID NO Site Site Region
Sequence (5' to 3') Inhibition ID 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
[0325] 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.
[0326] 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 Target Target Oligo SEQ Start Stop Target % SEQ ID ID NO
Site Site Region Sequence (5' to 3') Inhibition ID 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
[0327] 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.
[0328] 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 Target Target Oligo SEQ Start Stop % SEQ ID ID NO Site Site
Sequence (5' to 3') Inhibition ID 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
[0329] 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.
[0330] 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.sub.--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 Start Stop % SEQ ID ID NO Motif Site
Site Sequence (5' to 3') Inhibition ID 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
[0331] 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
[0332] 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.
[0333] 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
TABLE-US-00006 (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-00007 TABLE 6 Antisense Inhibition of human CD40 in AGS
cells ISIS Wing No Motif segment 12.5 nM 25.0 nM 50.0 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
[0334] 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
[0335] 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.
[0336] 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
[0337] 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
[0338] 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
[0339] 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
[0340] Liver CD40 protein may be measured by Western blot.
Example 9
Tolerability of Antisense Compounds in Rodents
[0341] 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.
[0342] 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 ISIS 19216
[0343] Antisense oligonucleotides ISIS 26163 and ISIS 19216
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.
[0344] 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.
[0345] Results are presented in Table 7 as percent inhibition of
CD40, relative to untreated control cells.
TABLE-US-00008 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
[0346] Sequence ISIS 26163 shows a superior activity over ISIS
19216, which overlaps the sequence by 15 nucleobases.
Sequence CWU 1
1
23611004DNAHomo sapiens 1gcctcgctcg ggcgcccagt ggtcctgccg
cctggtctca cctcgccatg gttcgtctgc 60ctctgcagtg cgtcctctgg ggctgcttgc
tgaccgctgt ccatccagaa ccacccactg 120catgcagaga aaaacagtac
ctaataaaca gtcagtgctg ttctttgtgc cagccaggac 180agaaactggt
gagtgactgc acagagttca ctgaaacgga atgccttcct tgcggtgaaa
240gcgaattcct agacacctgg aacagagaga cacactgcca ccagcacaaa
tactgcgacc 300ccaacctagg gcttcgggtc cagcagaagg gcacctcaga
aacagacacc atctgcacct 360gtgaagaagg ctggcactgt acgagtgagg
cctgtgagag ctgtgtcctg caccgctcat 420gctcgcccgg ctttggggtc
aagcagattg ctacaggggt ttctgatacc atctgcgagc 480cctgcccagt
cggcttcttc tccaatgtgt catctgcttt cgaaaaatgt cacccttgga
540caagctgtga gaccaaagac ctggttgtgc aacaggcagg cacaaacaag
actgatgttg 600tctgtggtcc ccaggatcgg ctgagagccc tggtggtgat
ccccatcatc ttcgggatcc 660tgtttgccat cctcttggtg ctggtcttta
tcaaaaaggt ggccaagaag ccaaccaata 720aggcccccca ccccaagcag
gaaccccagg agatcaattt tcccgacgat cttcctggct 780ccaacactgc
tgctccagtg caggagactt tacatggatg ccaaccggtc acccaggagg
840atggcaaaga gagtcgcatc tcagtgcagg agagacagtg aggctgcacc
cacccaggag 900tgtggccacg tgggcaaaca ggcagttggc cagagagcct
ggtgctgctg ctgcaggggt 960gcaggcagaa gcggggagct atgcccagtc
agtgccagcc cctc 10042427DNAHomo sapiensmisc_feature(13)..(13)n is
a, c, g, or t 2gataccatct gcnagccctg cccagtcggc ttcttctcca
atgtgtcatc tnctttcgaa 60aaatgtcacc cttggacaag ctgtgagacc aaagacctgg
ttgtgcaaca ggcaggcaca 120aacaagactg atgttgtctg tggtccccag
gntcggctga gagccctggt ggtgatcccc 180atcatcttcg ggntcctgtt
tgccatcctc ttggtgctgg tctttatcaa aaaggtggcc 240aagangccaa
ccantaaggc ccnccacccc aagcaggaac cccaggagat caattttncc
300gacgntcttc ctggctccaa cantgctgct ncagtgcagg agantttaca
tggatgccaa 360ccggtcaccc aggnaggatg ggcaaagaga gtcgcatttc
agttgcaggg agagacagtg 420aaggttg 4273871DNAHomo sapiens 3acctcgccat
ggttcgtctg cctctgcagt gcgtcctctg gggctgcttg ctgaccgctg 60tccatccaga
accacccact gcatgcagag aaaaacagta cctaataaac agtcagtgct
120gttctttgtg ccagccagga cagaaactgg tgagtgactg cacagagttc
actgaaacgg 180aatgccttcc ttgcggtgaa agcgaattcc tagacacctg
gaacagagag acacactgcc 240accagcacaa atactgcgac cccaacctag
ggcttcgggt ccagcagaag ggcacctcag 300aaacagacac catctgcacc
tgtgaagaag gctggcactg tacgagtgag gcctgtgaga 360gctgtgtcct
gcaccgctca tgctcgcccg gctttggtgt caagcagatt gctacagggg
420tttctgatac catctgcgag ccctgcccag tcggcttctt ctccaatgtg
tcatctgctt 480tcgaaaaatg tcacccttgg acaaggtccc aggatcggct
gagagcctgt ggtgatccca 540tcatcttcgg atctgtttgc atctcttggt
gctgtcttat caaaaagtgg caagaactac 600ccataggccc ccacccagca
gacccagaga taatttctga gatttctgct caaatgtgtc 660agtagggagt
catgagcaca gtcccacggt ggagaaccgc tctttggaga attgtgccca
720gattgccatg aaaaggatgt caagataggt gggttttgtg gggggtaaac
cttccccttt 780tgagctgtga attttgaact ctcggccttt aagaatgggg
ggttaaccaa tttgactcca 840acagttaaac ttgattatga ggtttgcctt t
871416001DNAHomo sapiens 4cagcttgagg tctgtgttga gattaccagc
ttcgcacccc ctgccaccaa ctctttgtca 60tgattggagg ctgtacttag gatgagaagt
tgctgaaccc actcattcat ttattcattc 120attcagcacc catatattgg
tgtcctatta tgtacctagg actgggatag atccagtcct 180gccctcaggg
agctcacagt caaatgggac caggcagaag gagttatgag ctggcatgct
240aaatgctgtg gcaacacagg ggaggaggca gggtgggata gagtacctac
ctctgcctca 300gggagttggg gaaggtgtaa gaggaggcaa catttaaact
gtttaagaaa ggagaaggaa 360aggaaggaaa catcaagagc aatgagataa
accgtaagga aaaaagattg tgtgcctggg 420aagtggggag aggctgaagc
cagtgtgcat gggaggacgt ggcaggaagt aagcatagaa 480agagaggttg
gggctgagtg aggaagggtg tgctttgcca ggataagaaa tttggatcat
540cctgagggtg cagggaaacc caaagaggct tttatgcagg aaagtgtccc
agtcgatcag 600atctggagtt tatcaagaga atcgcagcag tgtaaagagt
tgcctggcac atagtaggtg 660ctcaataaat cctgtggaat gagagaggct
gttgggacag tccagatgaa agatggcacc 720ctctgaatta gggcagtggc
agaggtgaca gaaaataagg gatggatcct agaggcagaa 780tccacaagac
ttgcaaatgt gcccttaaca gcagaagcca tgagaatggg gttagaagtg
840tcctcctctt ccatcctccc tgatctgtga ggtctggcct cttgtcctct
tccagaaatt 900gagagcttgg agggtcatca cctcaaccgt tgtctagttc
aagctcctaa gttttccttg 960gagaaaaatt taggcctaag ggaacacagt
ccttcagagg cagaggctgc accagaaccc 1020aggtgttcca tgctgcaaag
cagagtgtta acactggttc aagaacatct tgagggccag 1080gtgcggtggc
tcatgcctgt aatctcagca ctttgggagg ccaaggcagg cggatcacct
1140gaggtcagga gtttgagacc agcctggcca acgtggagaa accccgtctc
tactaaaaat 1200acaaaaatta gctgggcgtg gtggcgccag cctgtaatcc
cagctactcg ggaggctgag 1260gcaggagaat tgcttgaacc caggaggcag
aggttgcagt gagccgagat catgccactg 1320cactccagcc tgggcaacag
aatgagactc tgtcaaaaaa aacccaaaaa ccaaaaacca 1380aacaaacacc
caaaaaacaa aacgaaacaa aaaacaaaaa aacaaaagca cgtcttcaga
1440gttcatgaac ccaggaaatg taggcacaag tgtgtgtgtt tctgcaaaat
gagagggtcc 1500cgagtttcct aacatactta aagtgtttta tgatgccaca
aaaggctggc ccactctttt 1560tttttttttg agatggagtt ttgcactgtc
gcccaggctg aagtgcagtg atgtaatcat 1620tgcaatcatg gctcactgca
accttgactt cttgagctca agcgatcctc ctgcctcagc 1680ctcctgagta
gctgggacta caggtgtttg ccaccatgcc tggctaactt aaaaatttct
1740ttattttgta gagatggggg tcttgccatg ttgccaggct ggtcttgaac
tcctggcctc 1800aagcaatctc cctttttggc cttccaaagt gttagattac
aggcgtaagc caccgcgcct 1860ggcccccacc tttattttta tttttattta
ttatttattt tttttttttt tgagactgag 1920tcttgctctg ccttcgaggc
tggagtgcag tggcacgatc tcggctcatt gcaatctctg 1980cctctgcggt
tcaagcgatt ctcctgcctc agcctcccga gtagctggga ttacaggcga
2040acgccactac atccggttaa tttttgtatt tttagtagag acggagtttc
actatgttgg 2100ccaggttggt ctcgaactcc tgacctcaag tgatctgccc
gcctcggcct cctaaagtgc 2160tggattacag gcgtgagcca ccgcgcccgg
ccccactctt aataaatgcc tgtctccagg 2220tgctgggtgg gaggtgggat
ggaatggaat gaggtgagga cgcatggatg catggatgaa 2280tggatgggaa
gttgagacga cgcgcccaca cgagggaatt tcctttgaaa gagagcgaaa
2340ttctgagttg ggaaactctt ccttgaaacg cctccccata ccccagctgt
ggccttcccg 2400ttttctgcgt ggtggtgtgg ggggaacttc ctcaggcctc
tccgcagtgg agcctctttc 2460ggttctgcca ggatacctag aggcagcgga
gagcggggca gggaggggaa aaccgtgagg 2520gtccctgtgg caggccccag
cacccatggg atctctctcc ggtcgcagga agcaggctag 2580ctcctagccc
gcctcggctt ggcctttgtg ggacctgggg gcaaagaaga agagctgtct
2640ctgggaccat gcctcctccc gtacacagca agatgcgtcc ctaaactccc
gggggaatta 2700gacttgtggg aatgttctgg ggaaactcct gcgcggtgaa
ttgctggggg ctccgccccc 2760ccgataggtg gaccgcgatt ggtctttgaa
gaccccgccc ctttcctggg cggggccaag 2820gctggggcag gggagtcagc
agaggcctcg ctcgggcgcc cagtggtcct gccgcctggt 2880ctcacctcgc
tatggttcgt ctgcctctgc agtgcgtcct ctggggctgc ttgctgaccg
2940ctgtgagttg tttttgcccc gaccagacgg gagttgggag tggggaatga
gaaggaaagg 3000gaaggaagac ttcggggaag aggccttcct ggctgatttt
tgtgggggca ggagggtggg 3060tgggagctgg gcaaggtgcc cccgctcctg
gctgaatggg gtgggctgcc tctctcttct 3120cccgggctgg ggtcccggga
gcggcctaca ggggccgctc agggaaggca ctggctgccc 3180aagcgtgcct
agacggcctg gacgggttta gggagcctca gaggctggcc acacagagac
3240tggtaggggg ttcagagggc gggaagtgag gcggaccaag ggaaggggcg
ggtctggccc 3300gtttcctgtc cccttcttat tgtggacaga tgccagcctc
tgtaagtagt tatcatctcc 3360ttgccagctg gggctgcctt cttccagggc
atcttgtggg aacaagagat gggtgcagag 3420gcccaggtac ttttgtgaga
aggcaaggag cttttaacat cgccttccac cccgaaccgt 3480atcttgggtg
ttccaaccta ggaggaatcc ccagggcttt gcctttttct cctgaattta
3540agatgacata ggagacccct ggggagatga acagtttatg ggacacaata
aagggttagg 3600agaccagagt tctggttggc tctgacaggg ctggtgatca
gagggctgga gaaaccaggg 3660gtttctccag gcaccagagg ggctcagagc
caaccaagca tatctccggg attttcagaa 3720gcctacactt gactcacttt
ttgtttaaat gtatttttgt agttcctcat tctggaggct 3780gggaatcccc
caagtacctg gctccttcat cccagcccct ctggcctccc cctactttag
3840agggctgtag attcctgcct gaagcctggg caggaatgac ccatggtatc
aaggaaagca 3900agggaagcag caagggaaga gagggagtgg ggaggctgct
ttggtcccac agctttcact 3960ttcacctgaa gcaatggctc ttagggaaca
gggaggcagg gggagggcgg agctggaaag 4020aggtaaaggg gggcccttgt
ggtaggagtg gagaaagagc cagaggaggt ggggtgaagg 4080gtgtgatcca
ggcttctcaa gagcagagtt tgccctcata actcccaact ttggctccag
4140gtagaggctg ggctgtgaca acaatgtcag aagctatcta ttgagggctt
cttgtgtgtc 4200aggctctgag ccaaacactg cctgttttct ttgtctgatt
tctcacaact cccccattat 4260acagatgggc aaattgaggc tcagaaaggg
ggattgtctt gccaaaggtc tcatagctag 4320ctaatggaag aacctggttg
tgaatctaca tctgcatgat tcccgagcct gcctctcaga 4380tagtgagagt
ctccaagctc tggtcctgag ctgttttgtg gcagaaggac cagaactatg
4440gggagtgaga actggagatt gacagacttt taggggagcg ttttatttct
catgtgtttg 4500aagatggtat caaggacttt cctatctttg ggagtgtggg
agctccacgt tcacaggatg 4560gtgtcttgca atgagctggt ggggggcagt
agccttttct acttcctttc ccattttggg 4620taagacacat ttctgtaagt
aatttgctga gatacccagg ttgaatgaga gccaccagtt 4680aggtaggatt
ctggacagcc agccaggtag ccgggctgct tgccatatat catgcaagca
4740gaaacaaatg aatgatgatt aaaattgcca tttaatgagc acctactatg
ttcctgacac 4800tgtgctaggc catatacatg tattctttct tatcttcgta
atccaacctg cagggcaggc 4860attattactc ccattttaga gatagagaaa
ctgaggctaa gagaagcaaa ataactagta 4920agtgttacaa agtcaggact
ggagtctaaa gctgtctgac tctcaaactt gtgttctttt 4980cactggctgt
tcccaaactg tgggacagtt ttaaggagca catggacata gaattaaaca
5040tacacttact ttacagttct tttaaaaatc cttctcattt tttcaaagag
gaagtctctg 5100gagctagaat agagttaatg cctctcaaag gcttgctaat
ccttctttta aaacaaaaat 5160caagagcagg cctgggaggg ccttcaacaa
gcaaacaacc agctgggttt taataacctt 5220gttttgtttc cccagaattt
atttttaggg ttacctttta tttatgagaa gtgatactgg 5280ttcttgtctc
ttggcaatga tgtgaggttt acatttaaag taaatgtacc ggccaggcac
5340ggtggcttgt gcctgtaatc ccagcacttt gggaggccaa ggcagtcaga
tcacttgagg 5400tcaggagttt tagatcagcc tggccaacat ggtgaaaccc
tgtctctact aaaaatacat 5460aaattagccg ggcatagtgg tacacacctg
taatcccagc tactcaggag gctgaggctg 5520gagaattgct tgaacccagg
agatagaggt tgcagtgggc tgagatgatg ccactgcact 5580ccagcctggg
cgatggagcg agactctgtc tcaaaaaata aaataaaagt attgaaatta
5640acaataagta attaatagca tgggtggtac ctggatgtag taaaatggtg
aagatgaaac 5700acaagttgat ggagagagga gcattgagac ctgagttctc
atttggactc tgtcactgtg 5760agactctggg caagtgaccc tcctctttgg
tgctcagtct caactatctg taaaatgaaa 5820gtgtgagttt acccttccag
ctttacattc tagcatttta tgagggaagg gctggatgaa 5880cagatgatga
ggagttggag gaagaaaaca tgatgggctt tggaaaggag caggaaggga
5940agcagaagaa taggaggaag aggccaagtg ctaaacatag ccccaaacag
cactgggacc 6000agctgaagtc agccagcttc aggactccag gggagctgct
ggagtcccca tatcctatgg 6060gatctttggg aagaggaatg actcaggcat
caagccccaa ggaattctgt tctgttcaga 6120gaatattgtg agtttacagt
accattgctt tgtaaaaata ccagaatgat tctctgggtg 6180cgattataat
cagctcagtt gacaatttac ttgaaaacaa acatgccaaa tatcatgcag
6240gttccacttt ctgttttgac ttgcacttca gtttgcagcc tctgtcctgg
atgactttta 6300cctttctgct gaagaagttg caacggagat ttcaagatcc
cttcaaattg cacaattctg 6360tttttaggtc catccagaac cacccactgc
atgcagagaa aaacagtacc taataaacag 6420tcagtgctgt tctttgtgcc
agccaggtga gatgccaacc ctctagcccc atcatggagt 6480ccccctttgc
tttggtggca gacgcagacc ccatatgtta actgtaaact caaatctgaa
6540acgacccatt tcccagccct gcttcactgt cagaatgttc tggttccctc
tctaccaggt 6600aaaactctgt ctaccctgaa ctagggatcc cagcttctcc
atcttcctcg cctgattatg 6660aaggatccaa gactttcatc tttgaatccc
ctaccctaaa gcctggcctg atcattgtgt 6720ggttagtgtc tgactcatgg
agttggccag agccctccct catttcctga tgttttccag 6780gacagaaact
ggtgagtgac tgcacagagt tcactgaaac ggaatgcctt ccttgcggtg
6840aaagcgaatt cctagacacc tggaacagag agacacactg ccaccagcac
aaatactgcg 6900accccagtgc gtgcgctgtt gggaaaggga cgcttgggaa
ccgggctgat attcccgaca 6960atgcagccat tctaatttta tgtagccagg
gtctgctctg attggttgga gtccgggctg 7020tactgatcat taaatgattt
gattgccatc tctacttgga agagggtctg aggaagaaag 7080agcaggcaat
gtggggagtg aggctcagag catggcccag cagggggttc ccatccttcc
7140tgcccttctc ttctcagacc tagggcttcg ggtccagcag aagggcacct
cagaaacaga 7200caccatctgc acctgtgaag aaggctggca ctgtacgagt
gaggcctgtg agagctgtgt 7260cctgcaccgc tcatgctcgc ccggctttgg
ggtcaagcag attggtaagt ggctcatctg 7320ggaatcagtt ttggaggggg
acagaggagc ttagggccca aggtgagggg ctgggcagtg 7380ggcacttagc
cccagaggca gaggaagcag aggctccaac ctatgtcggt atccccactg
7440gagtgagctg cagacgggac cttgttcatt ctgccttctg ccatggggat
ctgcctttga 7500agggcaatgg gagaagtcct cctggggact gcagctgtcg
ggggcagtac cacatcgggg 7560gaagagtgct caaggcagga gctcttcccg
tcctgcctgg ccactggctg ccttgtgagc 7620cggacaggtg gtccactgtg
atggttaatg tccccctccc cacccactcc cagctacagg 7680ggtttctgat
accatctgcg agccctgccc agtcggcttc ttctccaatg tgtcatctgc
7740tttcgaaaaa tgtcaccctt ggacaaggta taagcactca tcccttgtgt
ttcctgctct 7800aagagtggca tggagctgcc tccattctct ccagccacct
gtcctgtccc tgctcccaga 7860ggtccacaca cactcatgta cttgtgaagc
atctgcagag tggcctcatg gccaaccaga 7920caggcacatt tccacatttt
ttttgcctgc tgtctctttg aggtaataga cactgttgat 7980ctctcgcttc
atgagagcct cctatcttgg gggtattggg acacttattt tagctttcct
8040tctgcccctc ctgcttctcc tcagttttcc tcgtcttgct ttcaccttac
ctggctttct 8100agggctttct gggctctggg tgctcaccct gagggcctcc
ctctcttacc tccaactcca 8160aacccacacc aggtcctgcc actggctgtc
tacgtgtttt gggaacttac tgtctccact 8220gttgtcactt tagtttgggc
ctcatcactg tggtctgggt gatgcctttt ctgcctcctg 8280gcctccctgc
ctctgtctct cccctcctgc tggttctgtc tccatcctct tgccaacatg
8340agcgttcgac agtttctttc aaatcatgac actctcctat ttgagatgct
tcctgtctct 8400ctgttggaac taagactcct tagcatggca cccaaccttc
ctgttgcatt tcctgctctc 8460tttcctgcat cgcatagctt catgctactt
gcaatcctct gaacacactg ttcattctct 8520tccatcaaac tcatctgcct
ggaatacctt aaacatgggc cccaggccag gcgcggtggc 8580tcttgcctgt
aatctcagca ctttggatgc caaggcgggt ggatcacttg aggtcaggag
8640ttcaagacca gccagcacaa catggtaaaa acccatctct actaaaaata
ccaaaaaatt 8700agctgggtgt ggtggtgggc gcctgtaatc ccagctcctc
gggaggctga ggcaggagaa 8760tcacttgaac ccggaaggtg gagtttgcag
tgagccaaga tagcgccact gcactccagc 8820ctgggcaaca gagcgacatt
ctgtctcaaa aaacaaacac ctgccccatt aactttttgc 8880atttgatttt
taaaaatggg caagataggc acatgggaca gaaggcacaa aagagccaaa
8940gtgatgtctt tctcccatcc ctgcccctta ggctcccagt tctttctgga
gggagccatt 9000gttccttgca tatccttcca gagattctac atataaacaa
accaacacac acacacacac 9060acacaaacac acacaaaatt tccctccttt
tacttttgca caaataggag tatacatttt 9120atttgttaac tgtctgcctt
tccctaatag attgaaaatt ccttaaatgt agaaacttgg 9180cctttttttt
ttcttccatt gatacatccc ctatacctgg aacagtacct gacgcatggt
9240aggtgcttaa atttttactg ataaatgttg actgataact ggaggcacca
ctggtatagt 9300tttttttttt tttttttttt tttttttttt ttgagacaga
gtctcactct gtcgcccagg 9360ctggagtgca gtggcgcaat ctcggctcac
tgcaagctct gcctcccagg ttcacgccat 9420tctcctgcct cagcctcctg
agtagctggg actataggcg cccgccacca cacccggcta 9480atttttttgt
atttttagta gagacggcgt ttcaccgtgt tagccaggat ggtcttgatc
9540tcctgacctc gtgatccgtc tgccttggcc tcccaaagtg ctgggattac
aggcgtgagc 9600caccgtgccc ggccaccagt ggtatagtat taatggaatc
agtgcattgg cttacgtatc 9660tgattacagc tcagtaagtg tgtgaccctc
actgagcctc agtctcctca tctgaaaaat 9720gggaatgacc ttcatttcac
aaggcttgag ctaaaaacat gtaaagtgta ttgtaaattc 9780ctgaatgctc
tactcatgta agactaaagt aggccgggcg tggtggctca cacctgtaat
9840tgcagcactt tgggaggccg aggagggcag atcatgaggt caagagatcg
agaccatcct 9900ggctaatatg gtaaaaccct gtctctacta aaaatacaaa
aattagctgg gcgtggtggc 9960gcacatctgt agtcccagct actcaggagg
cggaggcagg agaattgctt gaacctggga 10020ggtggaggtt gcagtgagct
gagatcgcgc cactgcattc cagccagtct ggcgaaagag 10080caagactctg
tctcaaaaaa aaaaaaaaaa aaaaaaaaag actaaagtac atggtttctt
10140caaagcttct ctctctttct cccaccttag atgatttttc ctttgcaatg
tcctgtgtcc 10200attccgcccc actcctcctg gggccacctg gaccaggtct
tcatcatctc atatctatat 10260gtttgctgtg tctcctggct ggccactctt
ctgtaatttc tcctcctctg agctctctgg 10320gcagctgaat cttctcacta
gtgaagtcgc ctggttggat gctgatgaga ctgaccagct 10380gaatccagtt
gaaaacttca cacttggcag tgatctggtt ctaaagacac aattttccat
10440agtttcctaa caccatcctg catgccacct gccttatttc cccacatcac
atcgtcccac 10500ttagcgggac tgcactgctg atccaaattt tacatccttt
agggcccact caggtcatat 10560gtcctcaggg aagtctttct ggaagaacct
taaaccagag gttctcaaca gggggcagtt 10620ttgctccctg tggaacgttt
gccaatgtct ggacacattt cattcgtcac aaacggagag 10680ggggatgcta
cagggatctg gcggatagag gccagggatg ctgctgaaca tctgcaatgc
10740ataggacagc ccacccccac ccccacaccc ccagtaaata atgatccagc
ccaagtgtca 10800ctggtgctga cgttgagtaa ccctatctta agctgaactc
atcatctctc cattccagcc 10860ttggtggatt ctgtctcctc tgaaccattc
ccatctcact ttagcctacc tagatcacaa 10920agcttggcac tcattataga
ctcccctatt tattactcct tcaagatgtg caagaatctt 10980ttctctgcac
ttttaagttc tgtaagaaga gtctgtgtcg ttcctataat aaccagcata
11040ggacgttgca cgtgttgtgt gctcagtgaa cctggatttg ttgattgttg
actgactcac 11100tctagagttg gaaatcttat gcttggggaa acttaatatc
tctttctttc tctgtgtgtg 11160tgcatttgtg cacgtgtctg tgcatagctg
tgagaccaaa gacctggttg tgcaacaggc 11220aggcacaaac aagactgatg
ttgtctgtgg tgagtcctgg acaatgggcc ctggagaaag 11280cctaggaagg
tgggaactga agggggagat gaggcacaca ggaacactgg atgggaaaaa
11340ggggagggga ggcagtttgg gggtgtggta tcacagctct gccacttatc
ttgggagtct 11400gggcaaatca cttcccctct cttagcctca gtttcttcat
ctgtaaaatg ggatgataac 11460agcacttcct tagtaggttt tgattttaga
gtgagaaggt tggcctacag taaagatcag 11520ataatgtaaa tcagtgaaaa
aggtcagggg taagaaaatt acattctctt tacctaacgc 11580taaatgacca
gttaatgggt gcagcacacc aacatggtac atgtatacat atgtaacaaa
11640cctgcacatt atgcacatgt accctaaagc ttaaagtata ataataataa
aatttaaaaa 11700aacgaaaaat acattctctt tgctttttct caaaatgtac
tttcctcttt gtagggctgg 11760gactagaatg aggtgagcaa ggcacttgcc
ctcgggcgca atatttaaga aggtgccata 11820aaagtgtagt aatcaaggta
aattcatttt gatgcaatat ttttaaaaat aaaaattaat 11880gcaaagaaat
ccatgatgag caagatagca acattttaaa taaagaacag gatccgaccc
11940tgtgtttgca tgaccctgcc tcactcacct caccctaatc ctggccctgg
ttccagtaaa 12000aggaataggc agccagcctg caggccgtag tttgctgact
tggtgtccgc ctgatgattt 12060tcaaaatatg gcattaaaag aatgtttacc
ttgatgactg agtgttttgg acatcctttt 12120caattttgtc ctgaaacaat
ttcatccctt gcctcacgct agtctccgcc ctgccttttg 12180gtctttcttt
tattttccca ctttgaaaaa aaaattcggc atgagaaata ctttaccttt
12240cccctccact cttctatacc aaaagcaaca tgcagacatg aatcatgcta
gacctcggca 12300ttgggcagag agcagggagt ggcggggagc atggtgagca
ggtggtgaca gccactgcca 12360ccactcgctt ctagatggtt cccaggtggg
gaggctgcca actggaaccc agtcttccca 12420gtttgtaaga gaaatcagat
gtctaggttt gaatatgtga tctcccagtt taaaaatgtc 12480ggcaaatatt
tccaaacgtt aagaaaatgt tctggctcct ttaaagacat ctgccagcca
12540catttcccca
aggaccgcgg tttgaacctt ctgatgtaga tgagctctga cattggaaga
12600ttctggagtc tgacaagtca cagcaggttg agggtaggga gaaactgcag
gtgaggggtg 12660catgctgaag tcctgatttc tccaggtccc caggatcggc
tgagagccct ggtggtgatc 12720cccatcatct tcgggatcct gtttgccatc
ctcttggtgc tggtctttat cagtgagtcc 12780tcaggtgggg aggtgttggg
ggagggaggg gagaccacct gtttcttatc tggcctctcc 12840aactccccat
cctttttttt tttttttttt tttttagaaa aggtggccaa gaagccaacc
12900aataaggtag gtcacccctg agaacccggg acagagtttt gacaaactgg
gaagatggcc 12960tcacggttgc ctatggggca gtaaaactga ttcagagtct
gtctctgcag ccagtggggt 13020ggcagcagaa ttggggactg tcatccccac
ccaccatgct ccttccatcc agagctcaat 13080cccccacaga actgcccctg
gcaccactgg cagagcctaa cactggctgt tcttcactcc 13140tttcctggca
ttcaacgcgt ggggagctgc atctttgggc cttggggctg ggtcaaatgg
13200gtgggagcaa atgtggcagc cccttaagcc cactggctcc cactctggaa
gctcttcgtc 13260gcccttggtg tggccagcag ggggcaggag gcacccgagg
aatcagcact gacccgccgt 13320ctgggaaagg ggggagggct tggggaaggg
atccgcttcc cagggagggg ctcctcagag 13380gcacagctgc ccctgctgct
gggggtgacc tcacaccttg cctctccagg ccccccaccc 13440caagcaggaa
ccccaggaga tcaattttcc cgacgatctt cctggctcca acactgctgc
13500tccagtgcag gagactttac atggatgcca accggtcacc caggaggatg
gcaaagagag 13560tcgcatctca gtgcaggaga gacagtgagg ctgcacccac
ccaggagtgt ggccacgtgg 13620gcaaacaggc agttggccag agagcctggt
gctgctgctg ctgtggcgtg agggtgaggg 13680gctggcactg actgggcata
gctccccgct tctgcctgca cccctgcagt ttgagacagg 13740agacctggca
ctggatgcag aaacagttca ccttgaagaa cctctcactt caccctggag
13800cccatccagt ctcccaactt gtattaaaga cagaggcaga agtttggtgg
tggtggtgtt 13860ggggtatggt ttagtaatat ccaccagacc ttccgatcca
gcagtttggt gcccagagag 13920gcatcatggt ggcttccctg cgcccaggaa
gccatataca cagatgccca ttgcagcatt 13980gtttgtgata gtgaacaact
ggaagctgct taactgtcca tcagcaggag actggctaaa 14040taaaattaga
atatatttat acaacagaat ctcaaaaaca ctgttgagta aggaaaaaaa
14100ggcatgctgc tgaatgatgg gtatggaact ttttaaaaaa gtacatgctt
ttatgtatgt 14160atattgccta tggatatatg tataaataca atatgcatca
tatattgata taacaagggt 14220tctggaaggg tacacagaaa acccacagct
cgaagagtgg tgacgtctgg ggtggggaag 14280aagggtctgg gggagggttg
gttaaaggga gatttggctt tcccataatg cttcatcatt 14340tttcccaaaa
ggagagtgaa ttcacataat gcttatgtaa ttaaaaaatc atcaaacatg
14400taaaaagaaa aacgggggtg aacatgctgg gtgacatgag ctatttaacc
tgctgtcagg 14460ctcacggaat gagggcattt tctgtagata aataagaatg
tccccaggct gctgcccctc 14520cagggtggtt tccatgtgtg ctcacatgtg
gtattgagat tgcaaagtgc tcttcccatt 14580tgattcatgt tcacaaaaat
agccttcccc agcagggtgg gtctgcatcc ctccctcttt 14640tacagaggtg
gaaatgaggt ccagagaggc gaagtgactt gcctgtggtc acacagcggt
14700gtctggtggt gccatgctca gagctcctca ctctgggtcc tgagctcctc
ccagatctgc 14760ctgctgtctg gaactgacat ctgtagctcc ttcccaggga
gatcctgggt ctctcaccgc 14820cttctgacct ccctgtggct gcaggagcta
ctgcttctcg gtcagatggt atccccttcc 14880cccaagcagt atctcaggat
aaaaataaac catcctgttc tcttttcctg ctgagccaac 14940tgagggggag
cctagccagg aggagcggca gatggagtgg gagcagaggg gaggggaggg
15000gaggaaggtt ggaggacaag gaggagaagg aagagcaatg caaagtgcca
tgcagaaccc 15060cggcacaaag gttcgaggtg aggtttaacc caggggtctc
agcctttccc aggaaagaac 15120ctgctttgga tttccccaac acatcctgct
ggctggaggg tgtggggcga tggggggcaa 15180ggggagatgt ggagagggct
tatcccagtg gaggctgtga gggcagctgc gagccaagag 15240aggggcacct
ggcttggcag gcttcccaga agagcttcaa gaaccatttg gatacaccag
15300gtggaggcct ttggtttaga aagtggagca aagtggtggt ggcggggaag
gggaactttg 15360ggacctaatt cttgtctctt atctagagcc taagagtgag
tgttgttcac ttctttggtg 15420gtatggtgga aagagaccaa agacccacgc
ctgccacaga tcatctgtgt ggccttggat 15480gagtcacttt cactgcttca
cttctcagtt tccctatctg acaaatggtg acaatatctg 15540cagcagacgc
tattgatgcc tcctgtgttc tcttctaggt cagagcttct caaatttgag
15600tgtgggtcag gcgcagtggc tcacgtctgt aatcccagca ctctgggagg
cttaggtggg 15660cagataacct aaggtcagga gttcgagacc agcctgacca
acagggcaaa accccgtctc 15720tactaaaaat acaaaaatta gccgggcatg
atggcgggtg cctgtaatcc cagctacttg 15780ggaggctgag gcagaagaat
cacttgaacc cgggaggtgg aggttgcagt gagccgagat 15840catgccactg
tactccagcc tgggcaacag agtgaaactc catctaaaat aacaacaaca
15900acaatagcaa caacaacaac aacaatagca acaacaacaa aacttgagcg
tgtatctgga 15960ccacctgcaa ggtggtcctc tccactattt tcagaactgg a
16001518DNAArtificial sequenceSynthetic oligonucleotide 5ccaggcggca
ggaccact 18618DNAArtificial sequenceSynthetic oligonucleotide
6gaccaggcgg caggacca 18718DNAArtificial sequenceSynthetic
oligonucleotide 7aggtgagacc aggcggca 18818DNAArtificial
sequenceSynthetic oligonucleotide 8cagaggcaga cgaaccat
18918DNAArtificial sequenceSynthetic oligonucleotide 9gcagaggcag
acgaacca 181018DNAArtificial sequenceSynthetic oligonucleotide
10gcaagcagcc ccagagga 181118DNAArtificial sequenceSynthetic
oligonucleotide 11ggtcagcaag cagcccca 181218DNAArtificial
sequenceSynthetic oligonucleotide 12gacagcggtc agcaagca
181318DNAArtificial sequenceSynthetic oligonucleotide 13gatggacagc
ggtcagca 181418DNAArtificial sequenceSynthetic oligonucleotide
14tctggatgga cagcggtc 181518DNAArtificial sequenceSynthetic
oligonucleotide 15ggtggttctg gatggaca 181618DNAArtificial
sequenceSynthetic oligonucleotide 16gtgggtggtt ctggatgg
181718DNAArtificial sequenceSynthetic oligonucleotide 17gcagtgggtg
gttctgga 181818DNAArtificial sequenceSynthetic oligonucleotide
18cacaaagaac agcactga 181918DNAArtificial sequenceSynthetic
oligonucleotide 19ctggcacaaa gaacagca 182018DNAArtificial
sequenceSynthetic oligonucleotide 20tcctggctgg cacaaaga
182118DNAArtificial sequenceSynthetic oligonucleotide 21ctgtcctggc
tggcacaa 182218DNAArtificial sequenceSynthetic oligonucleotide
22ctcaccagtt tctgtcct 182318DNAArtificial sequenceSynthetic
oligonucleotide 23tcactcacca gtttctgt 182418DNAArtificial
sequenceSynthetic oligonucleotide 24gtgcagtcac tcaccagt
182518DNAArtificial sequenceSynthetic oligonucleotide 25actctgtgca
gtcactca 182618DNAArtificial sequenceSynthetic oligonucleotide
26cagtgaactc tgtgcagt 182718DNAArtificial sequenceSynthetic
oligonucleotide 27attccgtttc agtgaact 182818DNAArtificial
sequenceSynthetic oligonucleotide 28gaaggcattc cgtttcag
182918DNAArtificial sequenceSynthetic oligonucleotide 29ttcaccgcaa
ggaaggca 183018DNAArtificial sequenceSynthetic oligonucleotide
30ctctgttcca ggtgtcta 183118DNAArtificial sequenceSynthetic
oligonucleotide 31ctggtggcag tgtgtctc 183218DNAArtificial
sequenceSynthetic oligonucleotide 32tggggtcgca gtatttgt
183318DNAArtificial sequenceSynthetic oligonucleotide 33ggttggggtc
gcagtatt 183418DNAArtificial sequenceSynthetic oligonucleotide
34ctaggttggg gtcgcagt 183518DNAArtificial sequenceSynthetic
oligonucleotide 35ggtgcccttc tgctggac 183618DNAArtificial
sequenceSynthetic oligonucleotide 36ctgaggtgcc cttctgct
183718DNAArtificial sequenceSynthetic oligonucleotide 37gtgtctgttt
ctgaggtg 183818DNAArtificial sequenceSynthetic oligonucleotide
38tggtgtctgt ttctgagg 183918DNAArtificial sequenceSynthetic
oligonucleotide 39acaggtgcag atggtgtc 184018DNAArtificial
sequenceSynthetic oligonucleotide 40ttcacaggtg cagatggt
184118DNAArtificial sequenceSynthetic oligonucleotide 41gtgccagcct
tcttcaca 184218DNAArtificial sequenceSynthetic oligonucleotide
42tacagtgcca gccttctt 184318DNAArtificial sequenceSynthetic
oligonucleotide 43ggacacagct ctcacagg 184418DNAArtificial
sequenceSynthetic oligonucleotide 44tgcaggacac agctctca
184518DNAArtificial sequenceSynthetic oligonucleotide 45gagcggtgca
ggacacag 184618DNAArtificial sequenceSynthetic oligonucleotide
46aagccgggcg agcatgag 184718DNAArtificial sequenceSynthetic
oligonucleotide 47aatctgcttg accccaaa 184818DNAArtificial
sequenceSynthetic oligonucleotide 48gaaacccctg tagcaatc
184918DNAArtificial sequenceSynthetic oligonucleotide 49gtatcagaaa
cccctgta 185018DNAArtificial sequenceSynthetic oligonucleotide
50gctcgcagat ggtatcag 185118DNAArtificial sequenceSynthetic
oligonucleotide 51gcagggctcg cagatggt 185218DNAArtificial
sequenceSynthetic oligonucleotide 52tgggcagggc tcgcagat
185318DNAArtificial sequenceSynthetic oligonucleotide 53gactgggcag
ggctcgca 185418DNAArtificial sequenceSynthetic oligonucleotide
54cattggagaa gaagccga 185518DNAArtificial sequenceSynthetic
oligonucleotide 55gatgacacat tggagaag 185618DNAArtificial
sequenceSynthetic oligonucleotide 56gcagatgaca cattggag
185718DNAArtificial sequenceSynthetic oligonucleotide 57tcgaaagcag
atgacaca 185818DNAArtificial sequenceSynthetic oligonucleotide
58gtccaagggt gacatttt 185918DNAArtificial sequenceSynthetic
oligonucleotide 59cacagcttgt ccaagggt 186018DNAArtificial
sequenceSynthetic oligonucleotide 60ttggtctcac agcttgtc
186118DNAArtificial sequenceSynthetic oligonucleotide 61caggtctttg
gtctcaca 186218DNAArtificial sequenceSynthetic oligonucleotide
62ctgttgcaca accaggtc 186318DNAArtificial sequenceSynthetic
oligonucleotide 63gtttgtgcct gcctgttg 186418DNAArtificial
sequenceSynthetic oligonucleotide 64gtcttgtttg tgcctgcc
186518DNAArtificial sequenceSynthetic oligonucleotide 65ccacagacaa
catcagtc 186618DNAArtificial sequenceSynthetic oligonucleotide
66ctggggacca cagacaac 186718DNAArtificial sequenceSynthetic
oligonucleotide 67tcagccgatc ctggggac 186818DNAArtificial
sequenceSynthetic oligonucleotide 68caccaccagg gctctcag
186918DNAArtificial sequenceSynthetic oligonucleotide 69gggatcacca
ccagggct 187018DNAArtificial sequenceSynthetic oligonucleotide
70gaggatggca aacaggat 187118DNAArtificial sequenceSynthetic
oligonucleotide 71accagcacca agaggatg 187218DNAArtificial
sequenceSynthetic oligonucleotide 72ttttgataaa gaccagca
187318DNAArtificial sequenceSynthetic oligonucleotide 73tattggttgg
cttcttgg 187418DNAArtificial sequenceSynthetic oligonucleotide
74gggttcctgc ttggggtg 187518DNAArtificial sequenceSynthetic
oligonucleotide 75gtcgggaaaa ttgatctc 187618DNAArtificial
sequenceSynthetic oligonucleotide 76gatcgtcggg aaaattga
187718DNAArtificial sequenceSynthetic oligonucleotide 77ggagccagga
agatcgtc 187818DNAArtificial sequenceSynthetic oligonucleotide
78tggagccagg aagatcgt 187918DNAArtificial sequenceSynthetic
oligonucleotide 79tggagcagca gtgttgga 188018DNAArtificial
sequenceSynthetic oligonucleotide 80gtaaagtctc ctgcactg
188118DNAArtificial sequenceSynthetic oligonucleotide 81tggcatccat
gtaaagtc 188218DNAArtificial sequenceSynthetic oligonucleotide
82cggttggcat ccatgtaa 188318DNAArtificial sequenceSynthetic
oligonucleotide 83ctctttgcca tcctcctg 188418DNAArtificial
sequenceSynthetic oligonucleotide 84ctgtctctcc tgcactga
188518DNAArtificial sequenceSynthetic oligonucleotide 85ggtgcagcct
cactgtct 188618DNAArtificial sequenceSynthetic oligonucleotide
86aactgcctgt ttgcccac 188718DNAArtificial sequenceSynthetic
oligonucleotide 87cttctgcctg cacccctg 188818DNAArtificial
sequenceSynthetic oligonucleotide 88actgactggg catagctc
188918DNAArtificial sequenceSynthetic oligonucleotide 89gccccagagg
acgcactg 189018DNAArtificial sequenceSynthetic oligonucleotide
90agcagcccca gaggacgc 189118DNAArtificial sequenceSynthetic
oligonucleotide 91agcaagcagc cccagagg 189218DNAArtificial
sequenceSynthetic oligonucleotide 92gcggtcagca agcagccc
189318DNAArtificial sequenceSynthetic oligonucleotide 93ggttctggat
ggacagcg 189418DNAArtificial sequenceSynthetic oligonucleotide
94agtgggtggt tctggatg 189518DNAArtificial sequenceSynthetic
oligonucleotide 95gcactgactg tttattag 189618DNAArtificial
sequenceSynthetic oligonucleotide 96ggcacaaaga acagcact
189718DNAArtificial sequenceSynthetic oligonucleotide 97tgtcctggct
ggcacaaa 189818DNAArtificial sequenceSynthetic oligonucleotide
98cagtttctgt cctggctg 189918DNAArtificial sequenceSynthetic
oligonucleotide 99gtcactcacc agtttctg 1810018DNAArtificial
sequenceSynthetic oligonucleotide 100aaggcattcc gtttcagt
1810118DNAArtificial sequenceSynthetic oligonucleotide
101ctttcaccgc aaggaagg
1810218DNAArtificial sequenceSynthetic oligonucleotide
102tgtgtctctc tgttccag 1810318DNAArtificial sequenceSynthetic
oligonucleotide 103gtggcagtgt gtctctct 1810418DNAArtificial
sequenceSynthetic oligonucleotide 104cccttctgct ggacccga
1810518DNAArtificial sequenceSynthetic oligonucleotide
105tgaggtgccc ttctgctg 1810618DNAArtificial sequenceSynthetic
oligonucleotide 106tctgtttctg aggtgccc 1810718DNAArtificial
sequenceSynthetic oligonucleotide 107gatggtgtct gtttctga
1810818DNAArtificial sequenceSynthetic oligonucleotide
108aaacccctgt agcaatct 1810918DNAArtificial sequenceSynthetic
oligonucleotide 109cgcagatggt atcagaaa 1811018DNAArtificial
sequenceSynthetic oligonucleotide 110ggcagggctc gcagatgg
1811118DNAArtificial sequenceSynthetic oligonucleotide
111gagaagaagc cgactggg 1811218DNAArtificial sequenceSynthetic
oligonucleotide 112tggagaagaa gccgactg 1811318DNAArtificial
sequenceSynthetic oligonucleotide 113attggagaag aagccgac
1811418DNAArtificial sequenceSynthetic oligonucleotide
114acattggaga agaagccg 1811518DNAArtificial sequenceSynthetic
oligonucleotide 115acacattgga gaagaagc 1811618DNAArtificial
sequenceSynthetic oligonucleotide 116tgacacattg gagaagaa
1811718DNAArtificial sequenceSynthetic oligonucleotide
117atgacacatt ggagaaga 1811818DNAArtificial sequenceSynthetic
oligonucleotide 118aaagcagatg acacattg 1811918DNAArtificial
sequenceSynthetic oligonucleotide 119aggtctttgg tctcacag
1812018DNAArtificial sequenceSynthetic oligonucleotide
120ttgcacaacc aggtcttt 1812118DNAArtificial sequenceSynthetic
oligonucleotide 121ttgtttgtgc ctgcctgt 1812218DNAArtificial
sequenceSynthetic oligonucleotide 122tcttgtttgt gcctgcct
1812318DNAArtificial sequenceSynthetic oligonucleotide
123agtcttgttt gtgcctgc 1812418DNAArtificial sequenceSynthetic
oligonucleotide 124cagtcttgtt tgtgcctg 1812518DNAArtificial
sequenceSynthetic oligonucleotide 125tcagtcttgt ttgtgcct
1812618DNAArtificial sequenceSynthetic oligonucleotide
126catcagtctt gtttgtgc 1812718DNAArtificial sequenceSynthetic
oligonucleotide 127gaccacagac aacatcag 1812818DNAArtificial
sequenceSynthetic oligonucleotide 128gggaccacag acaacatc
1812918DNAArtificial sequenceSynthetic oligonucleotide
129tcaccaccag ggctctca 1813018DNAArtificial sequenceSynthetic
oligonucleotide 130gatcaccacc agggctct 1813118DNAArtificial
sequenceSynthetic oligonucleotide 131agaggatggc aaacagga
1813218DNAArtificial sequenceSynthetic oligonucleotide
132aagaggatgg caaacagg 1813318DNAArtificial sequenceSynthetic
oligonucleotide 133caagaggatg gcaaacag 1813418DNAArtificial
sequenceSynthetic oligonucleotide 134gaccagcacc aagaggat
1813518DNAArtificial sequenceSynthetic oligonucleotide
135aagaccagca ccaagagg 1813618DNAArtificial sequenceSynthetic
oligonucleotide 136taaagaccag caccaaga 1813718DNAArtificial
sequenceSynthetic oligonucleotide 137tgataaagac cagcacca
1813818DNAArtificial sequenceSynthetic oligonucleotide
138tttgataaag accagcac 1813918DNAArtificial sequenceSynthetic
oligonucleotide 139actctctttg cccatcct 1814018DNAArtificial
sequenceSynthetic oligonucleotide 140cgactctctt tgcccatc
1814118DNAArtificial sequenceSynthetic oligonucleotide
141atgcgactct ctttgccc 1814218DNAArtificial sequenceSynthetic
oligonucleotide 142aaatgcgact ctctttgc 1814318DNAArtificial
sequenceSynthetic oligonucleotide 143ctgaaatgcg actctctt
1814418DNAArtificial sequenceSynthetic oligonucleotide
144aactgaaatg cgactctc 1814518DNAArtificial sequenceSynthetic
oligonucleotide 145cttcactgtc tctccctg 1814618DNAArtificial
sequenceSynthetic oligonucleotide 146ccttcactgt ctctccct
1814718DNAArtificial sequenceSynthetic oligonucleotide
147aaccttcact gtctctcc 1814818DNAArtificial sequenceSynthetic
oligonucleotide 148gatcaccaca ggctctca 1814918DNAArtificial
sequenceSynthetic oligonucleotide 149tgataagaca gcaccaag
1815018DNAArtificial sequenceSynthetic oligonucleotide
150ggtagttctt gccacttt 1815118DNAArtificial sequenceSynthetic
oligonucleotide 151gggcctatgg gtagttct 1815218DNAArtificial
sequenceSynthetic oligonucleotide 152attatctctg ggtctgct
1815318DNAArtificial sequenceSynthetic oligonucleotide
153actgacacat ttgagcag 1815418DNAArtificial sequenceSynthetic
oligonucleotide 154gactccctac tgacacat 1815518DNAArtificial
sequenceSynthetic oligonucleotide 155caaagagcgg ttctccac
1815618DNAArtificial sequenceSynthetic oligonucleotide
156aattctccaa agagcggt 1815718DNAArtificial sequenceSynthetic
oligonucleotide 157tcttgacatc cttttcat 1815818DNAArtificial
sequenceSynthetic oligonucleotide 158cccacctatc ttgacatc
1815918DNAArtificial sequenceSynthetic oligonucleotide
159aggccgagag ttcaaaat 1816020DNAArtificial sequenceSynthetic
oligonucleotide 160ccagcaattc accgcgcagg 2016120DNAArtificial
sequenceSynthetic oligonucleotide 161tgcagaggca gacgaaccat
2016220DNAArtificial sequenceSynthetic oligonucleotide
162cagaggacgc actgcagagg 2016320DNAArtificial sequenceSynthetic
oligonucleotide 163aagcagcccc agaggacgca 2016420DNAArtificial
sequenceSynthetic oligonucleotide 164cagcggtcag caagcagccc
2016520DNAArtificial sequenceSynthetic oligonucleotide
165ctcacagcgg tcagcaagca 2016620DNAArtificial sequenceSynthetic
oligonucleotide 166gctggcaagg agatgataac 2016720DNAArtificial
sequenceSynthetic oligonucleotide 167aggttggaac acccaagata
2016820DNAArtificial sequenceSynthetic oligonucleotide
168ggagaaaccc ctggtttctc 2016920DNAArtificial sequenceSynthetic
oligonucleotide 169tcattcctgc ccaggcttca 2017020DNAArtificial
sequenceSynthetic oligonucleotide 170tcaggtgaaa gtgaaagctg
2017120DNAArtificial sequenceSynthetic oligonucleotide
171taccatcttc aaacacatga 2017220DNAArtificial sequenceSynthetic
oligonucleotide 172ttacccaaaa tgggaaagga 2017320DNAArtificial
sequenceSynthetic oligonucleotide 173gaaagaatac atgtatatgg
2017420DNAArtificial sequenceSynthetic oligonucleotide
174agagtcagac agctttagac 2017520DNAArtificial sequenceSynthetic
oligonucleotide 175gtaccaccca tgctattaat 2017620DNAArtificial
sequenceSynthetic oligonucleotide 176acagtgacag agtccaaatg
2017720DNAArtificial sequenceSynthetic oligonucleotide
177aatgtaaagc tggaagggta 2017820DNAArtificial sequenceSynthetic
oligonucleotide 178gggctatgtt tagcacttgg 2017920DNAArtificial
sequenceSynthetic oligonucleotide 179gggcttgatg cctgagtcat
2018020DNAArtificial sequenceSynthetic oligonucleotide
180tgaagtgcaa gtcaaaacag 2018120DNAArtificial sequenceSynthetic
oligonucleotide 181gcaatttgaa gggatcttga 2018220DNAArtificial
sequenceSynthetic oligonucleotide 182catgcagtgg gtggttctgg
2018320DNAArtificial sequenceSynthetic oligonucleotide
183gtttttctct gcatgcagtg 2018420DNAArtificial sequenceSynthetic
oligonucleotide 184gctggcacaa agaacagcac 2018520DNAArtificial
sequenceSynthetic oligonucleotide 185cactaaccac acaatgatca
2018620DNAArtificial sequenceSynthetic oligonucleotide
186tgtgcagtca ctcaccagtt 2018720DNAArtificial sequenceSynthetic
oligonucleotide 187gtctaggaat tcgctttcac 2018820DNAArtificial
sequenceSynthetic oligonucleotide 188caggtgtcta ggaattcgct
2018920DNAArtificial sequenceSynthetic oligonucleotide
189gtcgcagtat ttgtgctggt 2019020DNAArtificial sequenceSynthetic
oligonucleotide 190acccgaagcc ctaggtctga 2019120DNAArtificial
sequenceSynthetic oligonucleotide 191ctggacccga agccctaggt
2019220DNAArtificial sequenceSynthetic oligonucleotide
192ttctgctgga cccgaagccc 2019320DNAArtificial sequenceSynthetic
oligonucleotide 193cttcttcaca ggtgcagatg 2019420DNAArtificial
sequenceSynthetic oligonucleotide 194agccagtggc caggcaggac
2019520DNAArtificial sequenceSynthetic oligonucleotide
195gaagaagccg actgggcagg 2019620DNAArtificial sequenceSynthetic
oligonucleotide 196ttggagaaga agccgactgg 2019720DNAArtificial
sequenceSynthetic oligonucleotide 197gatgacacat tggagaagaa
2019820DNAArtificial sequenceSynthetic oligonucleotide
198tgtctattac ctcaaagaga 2019920DNAArtificial sequenceSynthetic
oligonucleotide 199acagtgtgtt cagaggattg 2020020DNAArtificial
sequenceSynthetic oligonucleotide 200acaatacact ttacatgttt
2020120DNAArtificial sequenceSynthetic oligonucleotide
201attgtgtctt tagaaccaga 2020220DNAArtificial sequenceSynthetic
oligonucleotide 202gggccctaaa ggatgtaaaa 2020320DNAArtificial
sequenceSynthetic oligonucleotide 203cagtcttgtt tgtgcctgcc
2020420DNAArtificial sequenceSynthetic oligonucleotide
204tgtccaggac tcaccacaga 2020520DNAArtificial sequenceSynthetic
oligonucleotide 205tatggcacct tcttaaatat 2020620DNAArtificial
sequenceSynthetic oligonucleotide 206tgcttttggt atagaagagt
2020720DNAArtificial sequenceSynthetic oligonucleotide
207aaatgtggct ggcagatgtc 2020820DNAArtificial sequenceSynthetic
oligonucleotide 208gtcagagctc atctacatca 2020920DNAArtificial
sequenceSynthetic oligonucleotide 209ctgataaaga ccagcaccaa
2021020DNAArtificial sequenceSynthetic oligonucleotide
210aggactcact gataaagacc 2021120DNAArtificial sequenceSynthetic
oligonucleotide 211cagactctga atcagtttta 2021220DNAArtificial
sequenceSynthetic oligonucleotide 212cagtccccaa ttctgctgcc
2021320DNAArtificial sequenceSynthetic oligonucleotide
213ccagtgttag gctctgccag 2021420DNAArtificial sequenceSynthetic
oligonucleotide 214gaatgccagg aaaggagtga 2021520DNAArtificial
sequenceSynthetic oligonucleotide 215cagccccaag gcccaaagat
2021620DNAArtificial sequenceSynthetic oligonucleotide
216ctgcactgga gcagcagtgt 2021720DNAArtificial sequenceSynthetic
oligonucleotide 217accggttggc atccatgtaa 2021820DNAArtificial
sequenceSynthetic oligonucleotide 218cctgggtgac cggttggcat
2021920DNAArtificial sequenceSynthetic oligonucleotide
219caagttggga gactggatgg 2022020DNAArtificial sequenceSynthetic
oligonucleotide 220ctttaataca agttgggaga 2022120DNAArtificial
sequenceSynthetic oligonucleotide 221tcggaaggtc tggtggatat
2022220DNAArtificial sequenceSynthetic oligonucleotide
222tgggcaccaa actgctggat 2022320DNAArtificial sequenceSynthetic
oligonucleotide 223tatggcttcc tgggcgcagg 2022420DNAArtificial
sequenceSynthetic oligonucleotide 224aatgctgcaa tgggcatctg
2022520DNAArtificial sequenceSynthetic oligonucleotide
225gttcactatc acaaacaatg 2022620DNAArtificial sequenceSynthetic
oligonucleotide 226cagttaagca gcttccagtt 2022720DNAArtificial
sequenceSynthetic oligonucleotide 227aattttattt
agccagtctc 2022820DNAArtificial sequenceSynthetic oligonucleotide
228gttgtataaa tatattctaa 2022920DNAArtificial sequenceSynthetic
oligonucleotide 229acagtgtttt tgagattctg 2023020DNAArtificial
sequenceSynthetic oligonucleotide 230ctcaggaccc agagtgagga
2023120DNAArtificial sequenceSynthetic oligonucleotide
231tgggttaaac ctcacctcga 2023220DNAArtificial sequenceSynthetic
oligonucleotide 232attaggtccc aaagttcccc 2023320DNAArtificial
sequenceSynthetic oligonucleotide 233ggcagacgaa ccatggcgag
2023420DNAArtificial sequenceSynthetic oligonucleotide
234gtagcaatct gcttgacccc 2023520DNAArtificial sequenceSynthetic
oligonucleotide 235gaccacagac aacatcagtc 2023620DNAArtificial
sequenceSynthetic oligonucleotide 236ccaccttttt gataaagacc 20
* * * * *