U.S. patent application number 13/762601 was filed with the patent office on 2013-07-25 for methods of modulating angiogenesis via trpv4.
This patent application is currently assigned to CHILDREN'S MEDICAL CENTER CORPORATION. The applicant listed for this patent is CHILDREN'S MEDICAL CENTER CORPORATION. Invention is credited to Donald E. Ingber, Charles K. Thodeti.
Application Number | 20130189783 13/762601 |
Document ID | / |
Family ID | 41398519 |
Filed Date | 2013-07-25 |
United States Patent
Application |
20130189783 |
Kind Code |
A1 |
Ingber; Donald E. ; et
al. |
July 25, 2013 |
METHODS OF MODULATING ANGIOGENESIS VIA TRPV4
Abstract
The present invention relates to methods of inhibiting capillary
endothelial (CE) cell migration, the formation of CE networks and
angiogenesis, and uses thereof for the purpose of treating
angiogenesis-related diseases and disorders, particularly when the
diseases or disorders are directly related aberrant angiogenesis.
Inhibition is achieved by inhibiting TRPV4 activity, such as the
levels of TRPV4 expression, calcium influx through TRPV4, and/or
the intracellular signaling from TRPV4 via .beta.1 integrin
activation.
Inventors: |
Ingber; Donald E.; (Boston,
MA) ; Thodeti; Charles K.; (Copley, OH) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
CHILDREN'S MEDICAL CENTER CORPORATION; |
Boston |
MA |
US |
|
|
Assignee: |
CHILDREN'S MEDICAL CENTER
CORPORATION
Boston
MA
|
Family ID: |
41398519 |
Appl. No.: |
13/762601 |
Filed: |
February 8, 2013 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12996069 |
Mar 4, 2011 |
8394779 |
|
|
PCT/US2009/046219 |
Jun 4, 2009 |
|
|
|
13762601 |
|
|
|
|
61058647 |
Jun 4, 2008 |
|
|
|
Current U.S.
Class: |
435/375 |
Current CPC
Class: |
A61K 31/713 20130101;
A61P 3/04 20180101; C07K 16/2842 20130101; A61P 27/02 20180101;
A61K 31/70 20130101; C12N 15/1138 20130101; A61K 45/06 20130101;
A61P 17/06 20180101; C12N 2310/14 20130101; A61P 19/02 20180101;
A61P 25/28 20180101; G01N 33/5064 20130101; A61P 35/00 20180101;
A61P 9/10 20180101 |
Class at
Publication: |
435/375 |
International
Class: |
G01N 33/50 20060101
G01N033/50 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] This invention was made with Government support under Grant
No.: CA45548 awarded by the National Institutes of Health. The
Government has certain rights in the invention.
Claims
1. A method for inhibiting endothelial cell migration, the method
comprising contacting an endothelial cell with a TRPV4 inhibitor.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 12/996,069 filed Mar. 4, 2011, which claims
benefit under 35 U.S.C. .sctn.119(e) of U.S. Provisional
Application No. 61/058,647 filed Jun. 4, 2008, the contents of
which are incorporated herein by reference in their entireties.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Jan. 29, 2013, is named
20130208_SequenceListing-TextFile.sub.--701039.sub.--062453_C and
is 77,333 bytes in size.
BACKGROUND OF INVENTION
[0004] Angiogenesis is the formation, development and growth of new
blood vessels. The normal regulation of angiogenesis is governed by
a fine balance between factors that induce the formation of blood
vessels and those that halt or inhibit the process. When this
balance is upset, it generally results in pathological
angiogenesis. Under normal physiological conditions, angiogenesis
occur in very specific, restricted situations and is highly
regulated through a system of angiogenic stimulators and
inhibitors. For example, angiogenesis is normally observed in wound
healing, fetal and embryonal development, and formation of the
corpus luteum, endometrium and placenta.
[0005] In addition, angiogenesis is regulated by external factors.
Physical forces applied to extracellular matrix (ECM) can influence
the direction of capillary endothelial (CE) cell migration and
oriented sprouting that drive angiogenesis. For example, the
initial step in neovascularization involves reorientation of a
subset of CE cells that spread and migrate perpendicular to the
main axis of the pre-existing vessel towards the angiogenic
stimulus. The growth and development of all living tissues are
influenced by physical forces, and deregulation of this form of
mechanoregulation can lead to various diseases and debilitating
conditions. This is particularly evident in the cardiovascular
system where blood pressure, wall strain and fluid shear stress
elicit biochemical signals in endothelial cells that are required
for normal tissue homeostasis, and when these physical factors are
altered, they produce changes in cell function and vascular wall
remodeling that can contribute to life threatening diseases, such
as hypertension and atherosclerosis. Mechanical forces also play an
important role in the microvasculature. For example,
micromechanical stresses (e.g., cyclical changes in wall strain in
angiogenic atherosclerotic plaques, static stretch in healing
wounds or cancer parenchyma) can be potent inducers of CE
reorientation and capillary ingrowth as chemical factors. Moreover,
physical forces actually dominate and govern the local capillary
response (i.e., whether CE cells will grow, differentiate, die or
move in a specific direction) when stimulated by saturating amounts
of soluble angiogenic factors. Thus, understanding the molecular
mechanism by which CE cells reorient when exposed to mechanical
stress could lead to identification of novel targets for therapy in
angiogenic diseases, such as cancer, arthritis and diabetic
retinopathy.
SUMMARY OF THE INVENTION
[0006] The present invention relates to methods of inhibiting
endothelial cell migration and angiogenesis, and uses thereof for
the purpose of treating angiogenesis-related diseases and
disorders, particularly when the diseases or disorders are directly
related aberrant angiogenesis.
[0007] Embodiments of the present invention are based on the
identification of TRPV4 as the stress-activated (SA) ion channel
responsible for .beta.1 integrin activation in response to
mechanical strain application to microvascular cells. Mechanical
strains imposed upon endothelial cells through their extracellular
matrix adhesions via integrins influence the re-arrangement of the
cells' cytoskeleton which in turn influences the migration
capability of these cells that is needed for re-aligning and/or
reorienting the cells with respect of the orientation of the strain
experienced. The ability of endothelial cells to sense and respond
to mechanical stress is necessary for the formation of new blood
vessels. The inventors have identified TRPV4 as the mechanochemical
`transducer` that detects mechanical strain and transduces the
strain into a chemical signal intracellularly that in turn
chemically activates .beta.1 integrin, which are transmembrane
protein receptors that convey additional migratory and growth
signals to the cell when they link the cytoskeleton to the
extracellular matrix to which they bind. The signal transduction
that leads to integrin activation and subsequent changes in CE cell
behavior is by way of a rapid influx of Ca.sup.2+ via the cell
surface TRPV4 ion channel into the cell. This increase in
intracellular Ca.sup.2+ activates the Ca.sup.2+ dependent PI3/AKT
kinase pathway comprising the phosphorylation of the intracellular
tail domain of the .beta.1 integrin thereby activating the
integrin, and the phosphorylation and translocation of the AKT
protein. The inventors show that siRNAs of TRPV4 can reduce
capillary endothelial (CE) cell migration and formation of CE
vascular networks, and that TRPV4 is required for CE cell migration
and angiogenesis.
[0008] Accordingly, provided herein is a method for inhibiting
endothelial cell migration, the method comprising contacting an
endothelial cell with a TRPV4 inhibitor.
[0009] In one embodiment, provided herein is a method for
inhibiting endothelial cell migration, the method comprising
contacting an endothelial cell with an siRNA directed specifically
against a TRPV4 gene.
[0010] In another embodiment, provided herein is a method for
inhibiting endothelial cell migration, the method comprising
contacting an endothelial cell with an antibody directed
specifically against a .beta.1 integrin, wherein the integrin
function is blocked by the antibody.
[0011] In one embodiment, the antibody is a monoclonal antibody
derived from clone P5D2.
[0012] In one embodiment, provided herein is a method for
inhibiting angiogenesis in a mammal in need thereof, the method
comprising administering a therapeutically effective amount of a
TRPV4 inhibitor and a pharmaceutically acceptable carrier.
[0013] In another embodiment, provided herein is a method of
treating an angiogenesis-related disease in a mammal in need
thereof, the method comprising administering a therapeutically
effective amount of a TRPV4 inhibitor and a pharmaceutically
acceptable carrier.
[0014] In some aspect, a TRPV4 inhibitor is any molecule that
inhibits of the expression or ion transporting function of TRPV4,
or that inhibits the consequences of an activated TRPV4, i.e. the
downstream signal transduction via Ca.sup.2+ influx and the
Ca.sup.2+ dependent PI3/AKT kinase pathway and/or the activity of
the .beta.1 integrin in a CE cell.
[0015] In one embodiment, the TRPV4 inhibitor that inhibits an
expression of TRPV4 in the cell. Such an inhibitor can be an RNA
interference molecule.
[0016] In one embodiment, the TRPV4 inhibitor is an siRNA or dsRNA
that inhibits of expression of TRPV4. The siRNA can be designed in
the form of a small hairpin RNA (shRNA) expressed from a vector.
The vector carrying the shRNA can be transfected into the CE
cells.
[0017] In one embodiment, the TRPV4 inhibitor inhibits an influx of
calcium into the cell via the TRPV4 ion channel. Such compounds
include small molecules ruthenium red and gadolinium chloride.
[0018] In one embodiment, the TRPV4 inhibitor inhibits the
phosphorylation of .beta.1 integrin in the cell.
[0019] In one embodiment, the TRPV4 inhibitor inhibits the
phosphorylation of AKT in the cell.
[0020] In one embodiment, the TRPV4 inhibitor is selected from the
group consisting of an antibody, an RNA interference molecule, a
small molecule, a peptide and an aptamer.
[0021] In one embodiment, the endothelial cell is a mammalian
endothelial cell. In another embodiment, the mammalian endothelial
cell is a human endothelial cell.
[0022] In one embodiment, the angiogenesis-related disease include
but are not limited to cancer, macular degeneration; diabetic
retinopathy; rheumatoid arthritis; Alzheimer's disease; obesity,
psoriasis, atherosclerosis, vascular malformations, angiomata, and
endometriosis.
[0023] In one embodiment, the treatment of angiogenesis-related
diseases related to the eyes, e.g. macular degeneration or diabetic
retinopathy, comprises directly injecting an siRNA, dsRNA, or shRNA
vector directed against a TRPV4 gene into the vitreous cavity of
the affected eye.
[0024] In one embodiment, the treatment of angiogenesis-related
diseases related to the eyes, e.g. macular degeneration or diabetic
retinopathy, comprises directly injecting an antibody specifically
against a .beta.1 integrin function into the vitreous cavity of the
eye, wherein the integrin function is blocked by the antibody.
[0025] In other embodiments, the treatment of angiogenesis-related
diseases having localized aberrant angiogenesis, e.g. solid
non-metastatic tumor, arthritis, and endometriosis, comprises
directly injecting an siRNA, dsRNA, or shRNA vector directed
against a TRPV4 gene to the location or tissue with aberrant
angiogenesis.
[0026] In other embodiments, the treatment of angiogenesis-related
diseases having localized aberrant angiogenesis, e.g. solid
non-metastatic tumor, arthritis, and endometriosis, comprises
directly injecting an antibody specifically against a .beta.1
integrin function into the location or tissue with aberrant
angiogenesis, wherein the integrin function is blocked by the
antibody.
[0027] In one embodiment, the TRPV4 inhibitor is administered in
conjunction with an anti-angiogenic therapy or anti-proliferation
therapy. Such therapies include anti-VEGF therapy, chemotherapy,
radiation, antibody-based therapy and immune system enhancement
therapy, e.g. granulocyte stimulation factor (GSF) therapy.
[0028] In one embodiment, the mammal in need of treatment is a
human.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 shows the percentage of cells oriented
90.+-.30.degree. degrees (aligned) relative to the direction of
applied strain in control and strain exposed cells
(p<0.0006).
[0030] FIG. 2A shows the Western blot analysis of CE cell lysates
showing time-dependent phosphorylation of .beta.1 integrin
cytoplasmic tail at threonine T788/789 in response to mechanical
strain. Histogram shows the corresponding densitometric
quantification of .beta.1 integrin phosphorylation.
[0031] FIG. 2B are the Western blots showing the MAP kinase
(ERK1/2) phosphorylation in CE cells in the absence and presence of
mechanical strain.
[0032] FIG. 2C are the Western blots showing the binding of
GST-FNIII.sub.8-11 in CE cells in the absence and presence of
mechanical strain.
[0033] FIG. 2D are Western blots showing the binding of specific
antibody against activated .beta.1 integrin, 12G10 in CE cells in
the absence and presence of mechanical strain.
[0034] FIG. 2E shows the percentage of cells oriented
90.+-.30.degree. degrees (aligned) relative to the direction of
applied cyclic strain in the absence or presence of the integrin
blocking antibody P5D2 (*p<0.001).
[0035] FIG. 3A shows the quantification of mechanical
strain-induced GFP-AKT-PH domain translocation to the membrane in
the absence or presence of the PI3K inhibitor LY294002, measured as
a fraction of total cell membrane perimeter that is enhanced with
GFP-AKT-PH in randomly selected cells and the ratio of GFP
fluorescence intensity in the membrane vs cytosol (*P<0.05).
[0036] FIG. 3B shows the representative Western blots showing
time-dependent phosphorylation of AKT at ser-473 in response to
mechanical strain in the presence and absence of the PI3K
inhibitor, LY294002.
[0037] FIG. 3C shows the representative Western blots showing
phosphorylation of .beta.1 integrin cytoplasmic tail at T788/789
and AKT at ser-473 in response to mechanical strain in the presence
and absence of the PI3K inhibitor, LY294002.
[0038] FIG. 4A shows the relative change in cytosolic calcium in
Fluo-4 loaded CE cells in response to applied mechanical strain
(15%, 3 sec, arrow) in the absence (.cndot.) and presence
(.smallcircle.) of gadolinium chloride (25 .mu.M; Gd) (F/Fo=ratio
of normalized FLUO-4 fluorescence intensity relative to time 0).
Stretch-activated (SA) calcium channels are upstream of mechanical
strain-induced .beta.1 integrin phosphorylation.
[0039] FIG. 4B are the representative Western blots showing
mechanical strain dependent binding of GST-FNIII.sub.8-11 fragment
in bovine (BCE) and human (HCE) CE cells in the absence and
presence of gadolinium.
[0040] FIG. 4C shows the representative Western blots of
.beta.1-integrin phosphorylation in bovine (BCE) and human (HCE) CE
cells in the absence and presence of gadolinium.
[0041] FIG. 4D shows the percentages of CE cells displaying
GFP-AKT-PH domain translocation to the plasma membrane when
subjected to 0 or 15% mechanical strain in the absence and presence
of gadolinium chloride (*p<0.022).
[0042] FIG. 4E shows the percentage of CE cells oriented
90.+-.30.degree. degrees (aligned) relative to the direction of
applied strain in the absence and presence of gadolinium chloride
(*p<0.0002).
[0043] FIG. 5A shows the Western blotting analysis showing the
expression of TRPV4 in human and bovine CE cells.
[0044] FIG. 5B shows the representative RT-PCR result confirming
knockdown of TRPV2 and TRPV4 mRNA levels in CE cells using specific
siRNAs respectively.
[0045] FIG. 5C shows the suppression of protein expression TRPV4 in
CE cells using specific TRPV4 siRNA.
[0046] FIG. 5D show a histogram of the suppression of TRPV4 protein
levels in bovine CE cells using specific TRPV4 siRNAs
(*P<0.05).
[0047] FIG. 5E shows the relative change in cytosolic calcium in
response to mechanical strain (15%, 4 sec, arrow) in FLUO-4 loaded
CE cells treated with indicated siRNA.
[0048] FIG. 5F shows the average relative increases in cytosolic
calcium induced by mechanical strain in CE cells treated with the
indicated siRNAs (*, p<0.02; n=3 independent experiments).
[0049] FIG. 6A shows the relative changes in cytosolic calcium in
Fluo-4 loaded CE cells in response to mechanical strain (15%, 4
sec, arrow) in the absence (.box-solid.) and presence
(.quadrature.) of the specific TRPV inhibitor ruthenium red
(RR).
[0050] FIG. 6B shows the percentage of cells oriented
90.+-.30.degree. degrees (aligned) relative to the direction of
applied strain in control (white bars) and strain exposed (black
bars) human CE cells treated with the indicated siRNA. Note that
TRPV4 siRNA treated cells failed to reorient fully compared to
TRPV2 or TRPC1 treated cells (*, p<0.0025).
[0051] FIG. 6C shows the phase contrast photomicrographs of CE
cells showing the effects of cyclic strain on cell reorientation in
the absence and presence of ruthenium red. Arrow indicates the
direction of applied strain. Note that ruthenium red inhibits
cyclic strain-induced cell reorientation.
[0052] FIG. 7A shows the representative RT-PCR analysis showing the
efficiency and specificity of siRNA suppression of TRPV4 and TRPC1
mRNA levels in human CE cells.
[0053] FIG. 7B shows the migration of human CE cells treated with
the indicated siRNAs showing that TRPV4 siRNA significantly
inhibited VEGF-induced migration of CE cells compared to control
siRNA treated cells.
[0054] FIG. 7C shows the phase contrast photomicrographs of human
CE cells treated with the indicated siRNA showing that TRPV4 siRNA
completely inhibited capillary tube formation in a MATRIGEL.TM.
angiogenesis assay at 18 hr, whereas treatment with control and
TRPC1 siRNA had no effect.
[0055] FIG. 8 shows the flow cytometric analysis of activated
.beta.1 integrin expression on bovine CE cells detected using the
12G10 antibody in the absence and presence of manganese
(Mn.sup.2+). Note that the expression of activated .beta.1 integrin
is increased following treatment with manganese. The
istoype-matched control IgG is shown as a red peak.
[0056] FIG. 9 shows the representative Western blot showing
mechanical strain (15%, 15 min) dependent tyrosine phosphorylation
of FAK (FAK-pY397 antibody) in bovine CE cells in the absence and
presence of the PI3 kinase inhibitor, LY 294002 (LY, 40 .mu.M).
[0057] FIG. 10A showing a representative RT-PCR results confirming
knockdown of TRPV4, TRPV2 and TRPC1 mRNA levels in human CE cells
using specific siRNAs.
[0058] FIG. 10B showing a representative Western blotting analysis
showing that the same TRPV4 siRNA produced comparable suppression
of protein expression.
[0059] FIG. 11A shows the relative changes in cytosolic calcium
measured in FLUO-4 loaded bovine CE cells in response to the
specific TRPV4 activator 4-.alpha.-PDD (2 .mu.M) or the TRPV4
blocker ruthenium red (RR, 2 .mu.M) in the absence or presence of
extracellular calcium. Arrows denote time drugs were added to
cells. This indicates that TRPV4 channels are functionally
expressed in CE cells.
[0060] FIG. 11B shows the relative changes in cytosolic calcium
measured in FLUO-4 loaded human CE cells in response to the
specific TRPV4 activator 4-.alpha.-PDD (2 .mu.M) or the TRPV4
blocker ruthenium red (RR, 2 .mu.M) in the absence or presence of
extracellular calcium. Arrows denote time drugs were added to
cells. This indicates that TRPV4 channels are functionally
expressed in CE cells.
[0061] FIGS. 12A and B show the representative Western blots and
histogram showing activation of .beta.1 integrins as measured by
binding to 12G10 antibody in response to cyclic strain in the
control and TRPV4 siRNA-transfected CE cells at indicated
times.
[0062] FIGS. 12C and D show the representative Western blots and
histograms showing phosphorylation of AKT at Ser-473 and ERK1/2 in
response to cyclic strain in the control and TRPV4
siRNA-transfected CE cells at indicated times.
Phosphorylation/activation of signaling protein levels were
measured as a percentage of total protein/actin levels and
normalized to basal levels. *P<0.05 for comparison between
control siRNA versus TRPV4 siRNA treated cells.
[0063] FIG. 13 shows phase contrast photomicrographs of human CE
cells transfected with indicated siRNA showing that TRP siRNA
treatment does not affect human CE cell morphology or viability.
Scale bar: 100 .mu.m.
[0064] FIGS. 14A and B show quantification of cell proliferation
(stained using ki 67antibody) and Western blot analysis for PARP
(poly (ADP-ribose) polymerase) cleavage to assess apoptosis in CE
cell, indicating that cyclic strain did not affect CE cell
proliferation or apoptosis.
DETAILED DESCRIPTION OF THE INVENTION
[0065] Unless otherwise explained, all technical and scientific
terms used herein have the same meaning as commonly understood by
one of ordinary skill in the art to which this disclosure belongs.
Definitions of common terms in molecular biology may be found in
Benjamin Lewin, Genes IX, published by Jones & Bartlett
Publishing, 2007 (ISBN-13: 9780763740634); Kendrew et al. (eds.),
The Encyclopedia of Molecular Biology, published by Blackwell
Science Ltd., 1994 (ISBN 0-632-02182-9); and Robert A. Meyers
(ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by VCH Publishers, Inc., 1995 (ISBN
1-56081-569-8). Further, unless otherwise required by context,
singular terms shall include pluralities and plural terms shall
include the singular.
[0066] Unless otherwise stated, the present invention was performed
using standard procedures known to one skilled in the art, for
example, in Maniatis et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,
N.Y., USA (1982); Sambrook et al., Molecular Cloning: A Laboratory
Manual (3rd ed.), Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., USA (2000); Davis et al., Basic Methods in Molecular
Biology, Elsevier Science Publishing, Inc., New York, USA (1986);
Current Protocols in Molecular Biology (CPMB) (Fred M. Ausubel, et
al. ed., John Wiley and Sons, Inc.), Current Protocols in
Immunology (CPI) (John E. Coligan, et. al., ed. John Wiley and
Sons, Inc.), Current Protocols in Cell Biology (CPCB) (Juan S.
Bonifacino et. al. ed., John Wiley and Sons, Inc.), Culture of
Animal Cells: A Manual of Basic Technique by R. Ian Freshney,
Publisher: Wiley-Liss; 5th edition (2005), Animal Cell Culture
Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and
David Barnes editors, Academic Press, 1st edition, 1998), Methods
in Molecular biology, Vol. 180, Transgenesis Techniques by Alan R.
Clark editor, second edition, 2002, Humana Press, and Methods in
Molecular Biology, Vo. 203, 2003, Transgenic Mouse, editored by
Marten H. Hofker and Jan van Deursen, which are all herein
incorporated by reference in their entireties.
[0067] It should be understood that this invention is not limited
to the particular methodology, protocols, and reagents, etc.,
described herein and as such may vary. The terminology used herein
is for the purpose of describing particular embodiments only, and
is not intended to limit the scope of the present invention, which
is defined solely by the claims.
[0068] Other than in the operating examples, or where otherwise
indicated, all numbers expressing quantities of ingredients or
reaction conditions used herein should be understood as modified in
all instances by the term "about." The term "about" when used in
connection with percentages will mean.+-.1%.
[0069] All patents and publications identified are expressly
incorporated herein by reference for the purpose of describing and
disclosing, for example, the methodologies described in such
publications that might be used in connection with the present
invention. These publications are provided solely for their
disclosure prior to the filing date of the present application.
Nothing in this regard should be construed as an admission that the
inventors are not entitled to antedate such disclosure by virtue of
prior invention or for any other reason. All statements as to the
date or representation as to the contents of these documents is
based on the information available to the applicants and does not
constitute any admission as to the correctness of the dates or
contents of these documents.
[0070] The present invention relates to methods of regulating
endothelial cell migration and angiogenesis, and uses thereof for
the purpose of treating angiogenesis-related diseases and
disorders, particularly when the diseases or disorders is directly
related aberrant angiogenesis.
[0071] Embodiments of the present invention are based on the
identification of TRPV4 as the stress-activated (SA) ion channel
responsible for .beta.1 integrin activation in response to
mechanical strain application to microvascular cells. Mechanical
strains imposed upon endothelial cells influence the re-arrangement
of the cells' cytoskeleton which in turn influence the migration
capability of these cells that is needed for re-aligning and/or
reorienting the cells with respect of the orientation of the strain
experienced. The ability of endothelial cells to sense and respond
to mechanical stress is necessary for the formation of new blood
vessels. The inventors have identified TRPV4 as the mechanochemical
"transducer" that detects mechanical strain and transduces the
strain into a chemical signal intracellularly through the
activation of .beta.1 integrin, a transmembrane protein receptor
that links the cytoskeleton to the extracellular matrix. The signal
transduction is by way of a rapid influx of Ca.sup.2+ via the cell
surface TRPV4 ion channel into the cell (FIGS. 4A, 5E and 6A). This
increase in intracellular Ca.sup.2+ activates the Ca.sup.2+
dependent PI3/AKT kinase pathway, which leads to the
phosphorylation of the intracellular tail domain of the .beta.1
integrin, thereby activating this signaling adhesion receptor
(FIGS. 2 and 3), and the phosphorylation and translocation of the
AKT protein (FIGS. 3 and 4).
[0072] The inventors showed that inhibition of the TRPV4 channel
using a TRPV4 inhibitor ruthenium red (FIG. 6A) or a SA ion channel
inhibitor gadolinium chloride (FIG. 4A), or by inhibiting the
expression of TRPV4 by a siRNA specific for TRPV4 (FIG. 5E)
resulted in reduced influx of Ca.sup.2+ into mechanically strained
CE cells. There are also concomitant reduction in phosphorylation
of .beta.1 integrin (FIGS. 2A, 3C and 4C), reduction in the
phosphorylation and translocation of the AKT protein (FIGS. 3 and
4D), consequential reduction in the re-alignment of CE cells (FIGS.
2E, 4E, 6B and C), and inhibition of capillary tube formation (FIG.
7B). Moreover, the inventors showed that the Ca.sup.2+ dependent
PI3/AKT kinase pathway and the activation of the .beta.1 integrin
are necessary for the cytoskeletal rearrangement and alignment of
CE. Inhibition of the Ca.sup.2+ dependent PI3/AKT kinase pathway
using LY 294002 or the function-blocking anti-.beta.1 integrin
antibody (clone P5D2) (Mukhopadhyay, N. K. et al., 2004, Ann.
Thorac. Surg. 78:450; Blaschke, F. et al., 2002, Biochem. Biophys.
Res. Commun. 296:890) greatly reduced CE cell re-alignment when
subjected to mechanical strain (FIGS. 3 and 2E). Therefore,
inhibition of the expression of TRPV4 or inhibiting the
consequences of an activated TRPV4, i.e. the downstream signal
transduction events via blocking of the signal transducing
Ca.sup.2+ dependent PI3/AKT kinase pathways and/or blocking the
activity of an activated .beta.1 integrin, can be used to inhibit
CE cell alignment, CE cell migration, inhibition of capillary tube
formation and overall angiogenesis.
[0073] Accordingly, embodiments of the invention provide methods
for inhibiting endothelial cell migration and angiogenesis, the
method comprising contacting an endothelial cell with a TRPV4
inhibitor.
[0074] In one embodiment, provided herein is a method for
inhibiting endothelial cell migration and angiogenesis, the method
comprising contacting an endothelial cell with an siRNA directed
specifically against a TRPV4 gene.
[0075] In another embodiment, provided herein is a method for
inhibiting endothelial cell migration and angiogenesis, the method
comprising contacting an endothelial cell with an antibody directed
specifically against a .beta.1 integrin, wherein the integrin
function is blocked by the antibody.
[0076] As used herein, when an .beta.1 integrin function is
blocked, there is reduced or no downstream phosphorylation of
various signaling pathways known to be associated with .beta.1
integrin, e.g. the RhoA, Racl, Ras, Raf, MEK, ERK, and JNK
pathways. In one embodiment, the .beta.1 integrin activated,
meaning that the integrin is phosphorylated.
[0077] In one embodiment, the antibody is a monoclonal antibody
derived from clone P5D2. P5D2 has been shown to be a
function-blocking .beta.1 integrin antibody in that it inhibits the
downstream phosphorylation of various signaling pathways known to
be associated with activated .beta.1 integrin.
[0078] Embodiments of the invention also provide methods for
inhibiting angiogenesis in a mammal in need thereof, the method
comprising administering a therapeutically effective amount of a
TRPV4 inhibitor and a pharmaceutically acceptable carrier.
[0079] Embodiments of the invention also provide methods for
treating an angiogenesis-related disease in a mammal in need
thereof, the method comprising administering a therapeutically
effective amount of a TRPV4 inhibitor and a pharmaceutically
acceptable carrier.
[0080] As used herein, inhibiting endothelial cell migration refers
to the reduction in cell migration and/or capillary tube formation
in the presence of a TRPV4 inhibitor. Assays for in vitro cell
migration and capillary tube formation are well known to one
skilled in the art, e.g. in Lingen M W, 2003, Methods Mol Med.
78:337-47 and McGonigle and Shifrin, 2008, Curr. Prot.
Pharmacology, Unit 12.12, and any angiogenesis assays known in the
art or described herein.
[0081] As used herein, the term "inhibiting angiogenesis" means the
reduction or prevention of growth of new blood vessels. Inhibition
include slowing the rate of growth. The growth rate can be reduced
by about 20%, about 30%, about 40%, about 50%, about 60%, about
70%, about 80%, about 90%, about 100%, about 125%, about 150% or
more compared to a control, untreated condition. Inhibition also
means no further growth of new blood vessels from the time of start
of treatment administration. Angiogenesis can be detected by
methods known in the art and/or with any angiogenesis assays known
in the art or described herein.
[0082] As used herein, the term "angiogenic disease or disorder"
refers to diseases or disorders that are the direct result of
aberrant blood vessel proliferation (e.g. diabetic retinopathy,
vascular malformations, angiomata, and hemangiomas) or undesired or
pathological blood vessel proliferation (e.g. in the case cancer
and tumor growth). The term also refers to diseases or disorders
whose pathological progression is dependent on a good blood supply
and thus blood vessel proliferation. Examples include but are not
limited to abnormal vascular proliferation, ascites formation,
psoriasis, age-related macular degeneration, thyroid hyperplasia,
preeclampsia, rheumatoid arthritis and osteo-arthritis, Alzheimer's
disease, obesity, psoriasis, atherosclerosis, vascular
malformations, angiomata, pleural effusion, atherosclerosis,
endometriosis, diabetic/other retinopathies, ocular
neovascularization.
[0083] As used herein, the term "a TRPV4 inhibitor" is any molecule
that inhibits of expression of TRPV4, the ion transport through the
TRPV4channel, or inhibits the consequences of an activated TRPV4,
i.e. the downstream signal transduction via Ca.sup.2+ influx, via
the Ca.sup.2+ dependent PI3/AKT kinase pathway and/or the activity
of the .beta.1 integrin in a CE cell. For example, a TRPV4
inhibitor can be an siRNA or dsRNA that inhibits of expression of
TRPV4, a TRPV4 inhibitor, a TRPV4 antagonist, a SA ion channel
inhibitor or a function-blocking anti .beta.1 integrin antibody.
Examples include but are not limited to small molecules ruthenium
red gadolinium chloride and LY 294002, and the monoclonal antibody
anti-.beta.1 integrin derived from clone P5D2 as described
herein.
[0084] In one embodiment, the TRPV4 inhibitor is selected from the
group consisting of an antibody, an RNA interference molecule, a
small molecule, a peptide and an aptamer.
[0085] As used herein, the term "peptide" refer to a polymer of up
to 20 amino acid residues. The terms apply to amino acid polymers
in which one or more amino acid residue is an artificial chemical
mimetic of a corresponding naturally occurring amino acid, as well
as to naturally occurring amino acid polymers and non-naturally
occurring amino acid polymer. "Peptide" further refer to amino
acids joined to each other by peptide bonds or modified peptide
bonds, i.e., peptide isosteres, and can contain modified amino
acids other than the 20 gene-encoded amino acids.
[0086] As used herein, the term "aptamer" refer to relatively short
RNA or DNA oligonucleotides, which binds to TRPV4 or activated
.beta.1 integrin, and preferably blocks/prevents/inhibits TRPV4
channel Ca.sup.2+ influx or block activated .beta.1 integrin
signally events and cytoskeleton re-organization Methods of
determining integrin activation are known in the art (see U.S.
Patent publication No. 20080274482). "Aptamer" are isolated in
vitro using, for example, the selection procedure known as SELEX
(systematic evolution of ligands by exponential enrichment) (Tuerk
& Gold, 1990; Ellington & Szostak, 1990, U.S. Pat. Nos.
5,475,096 and 5,270,163, which are incorporated herein by reference
in their entirety). Because the selection procedure is driven by
binding of ligands, aptamers bind their ligands with high affinity
and fold into secondary structures which are optimized for ligand
binding (Herman & Patel, 2000, incorporated herein by reference
in its entirety). In this respect aptamers resemble antibodies by
selectively binding corresponding ligand from complex chemical or
biological mixtures. Methods to design and synthesize aptamers and
aptamer binding sequences are known to those of skill in the
art.
[0087] In one embodiment, the TRPV4 inhibitor inhibits an influx of
calcium ions into the cell. In one embodiment, the inhibitor blocks
the channel pore and prevents ion influx. In another embodiment,
the inhibitor prevents the opening of channel pore. The detection,
monitoring and measurement of cellular Ca.sup.2+ influx can be
performed using Ca.sup.2+ sensitive chromophore e.g. FLUO-3, and
FLUO-4 (INVITROGEN.TM. Inc.) as described herein. Such use of
calcium indicators are well known to one skilled in the art.
[0088] In one embodiment, the TRPV4 inhibitor specifically inhibits
the expression of TRPV4 in the cell. In one embodiment, the
inhibitor is an RNA interference molecule specific to a TRPV4 gene
such as an siRNA, shRNA, or dsRNA. The TRPV4 gene is preferably a
mammalian TRPV4 gene.
[0089] As used herein, the term "gene" means the nucleic acid
sequence which is transcribed (DNA) and translated (mRNA) into a
polypeptide in vitro or in vivo when operably linked to appropriate
regulatory sequences. The gene may or may not include regions
preceding and following the coding region, e.g. 5' untranslated (5'
UTR) or "leader" sequences and 3' UTR or "trailer" sequences, as
well as intervening sequences (introns) between individual coding
segments (exons).
[0090] The human TRPV4 gene is located on chromosome 12, location:
12q24.1, 108,705,277-108,755,595 reverse strand (ENSG00000111199)
(Ensembl) assembled in Accession No. NC.sub.--000012.10 (SEQ. ID.
No. 1; GENBANK.TM.) Alternate gene names are OTRPC4; TRP12; VR-OAC;
VRL-2; VRL2; VROAC. This gene encodes a member of the OSM9-like
transient receptor potential channel (OTRPC) subfamily in the
transient receptor potential (TRP) superfamily of ion channels. The
encoded protein is a Ca.sup.2+-permeable, nonselective cation
channel that is thought to be involved in the regulation of
systemic osmotic pressure. Two transcript variants encoding
different isoforms have been found for this gene. Two transcripts
of TRPV4 from this gene are NM.sub.--021625.3 (SEQ. ID. No. 2) and
NM.sub.--147204.1 (SEQ. ID. No. 3) (GENBANK.TM.).
[0091] Public access software programs and methods of predicting
and selecting antisense oligonucleotides and siRNA are known in the
art and are also found on the world wide web sites of
GENSCRIPT.TM., AMBION.RTM., DHARMACON.TM., OLIGOENGINE.TM.
Wadsworth Bioinformatics Center, Whitehead Institute at the
Massachusetts Institute of Technology and are also described in
U.S. Pat. No. 6,060,248. After selecting the antisense
oligonucleotides and siRNA sequences, these molecules can be
produced biologically using an expression vector carrying the
polynucleotides that encode the siRNA or antisense RNA. General
molecular biological methods known in the art can be used to clone
these sequences into the expression vectors. Examples of such are
described herein.
[0092] In other embodiments, the siRNA TRPV4L molecules are
[GCACACCGCCGUACCCUUAUU (sense) (SEQ. ID. No. 4),
5'-PUAAGGGUACGGCGGUGUGCUU (antisense) (SEQ. ID. No. 5],
[GACCAAAUCUGCGCAUGAAUU (sense) (SEQ. ID. No. 6),
5'-PUUCAUGCGCAGAUUUGGUCUU (antisense) (SEQ. ID. No. 7)],
[CAACCGGCCUAUCCUCUUUUU (sense) (SEQ. ID. No. 8),
5'-PAAAGAGGAUAGGCCGGUUGUU (antisense) (SEQ. ID. No. 9)],
[GAACCCGUGUGCCAACAUGUU (sense) (SEQ. ID. No. 10),
5'-PCAUGUUGGCACACGGGUUCUU (antisense) (SEQ. ID. No. 11)]. These
sense and anti-sense strand oligonucleotide can be chemically
synthesized, annealed and formulated for use, e.g. for direct
intravitreal injection into an eye affected with macular
degeneration or diabetic retinopathy. Alternatively, the anti-sense
strand can be designed into short hairpin RNA (shRNA) for plasmid-
or vector-based approaches for supplying siRNAs to cells to produce
stable TRPV4 gene silencing. Examples of vectors for shRNA are
#AM5779: -pSilencer.TM. 4.1-CMV neo; #AM5777: -pSilencer.TM.
4.1-CMV hygro; #AM5775: -pSilencer.TM. 4.1-CMV puro; #AM7209:
-pSilencer.TM. 2.0-U6; #AM7210: -pSilencer.TM. 3.0-H1; #AM5768:
-pSilencer.TM. 3.1-H1 puro; #AM5762: -pSilencer.TM. 2.1-U6 puro;
#AM5770: -pSilencer.TM. 3.1-H1 neo; #AM5764: -pSilencer.TM. 2.1-U6
neo; #AM5766: -pSilencer.TM. 3.1-H1 hygro; #AM5760: -pSilencer.TM.
2.1-U6 hygro; #AM7207: -pSilencer.TM. 1.0-U6 (circular) from
AMBION.RTM..
[0093] Commercial pre-designed RNA interference molecules to TRPV4
are also available, e.g. from INVITROGEN.TM. Inc. (STEALTH.TM.
Select RNAi, catalog #1299003, set of 3 Oligos; Oligo ID:
HSS126973-5) and from DHARMACON.TM. (SMARTvector Lentiviral
shRNA--Human TRPV4, catalog #SK-004195-00-10, set of 3 constructs).
Human TRPV4 siRNA, shRNA and lentiviral particle gene silencers are
available from Santa Cruz Biotechnology, Inc. catalog #s sc-61726,
sc-61726-SH, and sc-61726-V respectively.
[0094] A reduction in the expression of TRPV4 in a cell can be
determined by any methods known in the art, e.g. measurement of the
messenger RNA by RT-PCR or by Western blots analysis for the
protein as described herein. Commercial antibody to reactive
against to TRPV4 protein are widely available, e.g. from
ABCAM.RTM., catalog #s ab39260 and ab62992.
[0095] In one embodiment, the TRPV4 inhibitor inhibits the
phosphorylation of .beta.1 integrin in the cell. A reduction in the
phosphorylation of .beta.1 integrin can be determined by any
methods known in the art, e.g. by Western blots analysis for a
phosphorylated epitope on .beta.1 integrin, using an antibody
specific for a phosphorylated epitope, such a, the 12G10 antibody
specific for T788/789P as described herein.
[0096] In one embodiment, the TRPV4 inhibitor inhibits a
phosphorylation and membrane translocation of a AKT protein in the
cell. A reduction in the phosphorylation of AKT can be determined
by any methods known in the art, e.g. by Western blots analysis for
a phosphorylated AKT using antibody specific for a phosphorylated
epitope on AKT, such as the antibody specific for Ser-473 in AKT as
described herein.
[0097] For the avoidance of doubt, a reduction will be at least 5%
relative to in the absence of a TRPV4 inhibitor, preferably at
least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more, up to
and including at least 100% or more, in the case of an increase,
for example, at least 2.times., 3.times., 4.times., 5.times., . . .
10.times. or more). All the percentages in between 5-100% as well
as fractions of integer number of folds increase are also
included.
[0098] In one embodiment, the TRPV4 inhibitor comprises an
antibody. In another embodiment, the TRPV4 inhibitor is an
antibody.
[0099] In one embodiment, the antibody is specific for TRPV4. The
binding of an anti-TRPV4 antibody or variant thereof inhibits the
activation of the ion channel, for example be inhibiting channel
opening or ion flux. Commercially antibodies to TRPV4 are available
from MILIPORE.RTM., INVITROGEN.TM., SIGMA ALDRICH.RTM. and R&D
Systems to name a few. Alternatively, antibodies to TRPV4 can be
made by methods well know to one skilled in the art. The antibodies
to TRPV4 can be assayed for the inhibitory function by measuring
the Ca.sup.2+ influx in the presence and absence of mechanical
strain as described herein, or in the presence and absence of a
TRPV4 agonist, 4.alpha.-phorbol 12, 13-didecanoate.
[0100] In one embodiment, the antibody is specific for .beta.1
integrin. In one embodiment, the TRPV4 inhibitor is a
function-blocking antibody of .beta.1 integrin, e.g. monoclonal
antibody derived from clone P5D2. The binding of a
function-blocking anti-.beta.1 integrin antibody or variant thereof
inhibits the downstream events of an activated .beta.1 integrin,
such as focal adhesion kinase (FAK)-mediated signaling by
phosphorylation described in Nakayamada et. al. 2007Arthritis
Rheum. 56:1559-68. Other signaling pathways associated with .beta.1
integrin activation include but are not limited to RhoA, Rac1, Ras,
Raf, MEK, ERK, and JNK. Methods of assessing the activation of
these pathways via integrins are well known in the art, e.g. in S
Miyamoto, et. al. 1995, J. Cell Biol., 131:791-805. Methods of
assaying phosphorylation in signaling pathways are well known to
one skilled in the art, e.g. Western blot analysis for
phosphorylation at specific serine, threonine or tyrosine
residues.
[0101] In one embodiment, provided herein is a method of inhibiting
angiogenesis in a mammal in need thereof, the method comprises
administering a therapeutically effective amount of an siRNA
directed specifically against a TRPV4 gene and a pharmaceutically
acceptable carrier.
[0102] In one embodiment, provided herein is a method of inhibiting
angiogenesis in a mammal in need thereof, the method comprises
administering a therapeutically effective amount of an antibody
directed specifically against a .beta.1 integrin and a
pharmaceutically acceptable carrier, wherein the integrin function
is blocked by the antibody.
[0103] In one embodiment, the mammal is afflicted with an
angiogenesis-related disease or disorder.
[0104] In one embodiment, provided herein is a method for treatment
of angiogenesis-related diseases in a mammal in need thereof, the
method comprises administering a therapeutically effective amount
of an siRNA directed specifically against a TRPV4 gene and a
pharmaceutically acceptable carrier.
[0105] In one embodiment, provided herein is a method for treatment
of angiogenesis-related diseases in a mammal in need thereof, the
method comprises administering a therapeutically effective amount
of an antibody directed specifically against a .beta.1 integrin and
a pharmaceutically acceptable carrier, wherein the integrin
function is blocked by the antibody.
[0106] In one embodiment, the angiogenesis-related disease or
disorder is selected from the group consisting of consisting of:
cancer, macular degeneration; diabetic retinopathy; rheumatoid
arthritis; Alzheimer's disease; obesity, psoriasis,
atherosclerosis, vascular malformations, angiomata, and
endometriosis.
[0107] In one embodiment, the treatment of angiogenesis-related
diseases related to the eyes, e.g. macular degeneration or diabetic
retinopathy, comprises directly injecting an siRNA, dsRNA, or shRNA
vector directed against a TRPV4 gene into the vitreous cavity of
the affected eye. In one embodiment, a mixture of several different
siRNAs is injected directly into the vitreous cavity. In another
embodiment, the siRNA can be combined with other anti-angiogenic
therapy, such as anti-VEGF therapy, and injected directly into the
vitreous cavity. In another embodiment, the siRNA can be combined
with other TRPV4 inhibitors, e.g. an antibody P5D2, and injected
directly into the vitreous cavity.
[0108] In some embodiments, the siRNA, dsRNA, or shRNA vector
directed against TRPV4 gene is administered systemically, such as
intravenously, e.g. via central venous catheter (CVC or central
venous line or central venous access catheter) placed into a large
vein in the neck (internal jugular vein), chest (subclavian vein)
or groin (femoral vein). Methods of systemic delivery of siRNA,
dsRNA, or shRNA vector are well known in the art, e.g. as described
herein and in Mammoto T., et. al, 2007 (J. Biol. Chem.,
282:23910-23918), Gao and Huang, 2008, (Mol. Pharmaceutics, Web
publication December 30) and a review by Rossil, 2006, Gene
Therapy, 13:583-584. The siRNA, dsRNA, or shRNA vector can be
formulated in various ways, e.g. conjugation of a cholesterol
moiety to one of the strands of the siRNA duplex for systemic
delivery to the liver and jejunum (Soutschek J. et. al. 2004,
Nature, 432:173-178), complexing of siRNAs to protamine fused with
an antibody fragment for receptor-mediated targeting of siRNAs
(Song E, et al. 2005, Nat. Biotechnol., 23: 709-717) and the use of
a lipid bilayer system by Morrissey et al. 2005 (Nat. Biotechnol.,
23: 1002-1007). The lipid bilayer system produces biopolymers that
are in the 120 nanometer diameter size range, and are labeled as
SNALPs, for Stable-Nucleic-Acid-Lipid-Particles. The lipid
combination protects the siRNAs from serum nucleases and allows
cellular endosomal uptake and subsequent cytoplasmic release of the
siRNAs (see WO/2006/007712). These references are incorporated by
reference in their entirety.
[0109] In other embodiment, the treatment of angiogenesis-related
diseases related to the eyes, e.g. macular degeneration or diabetic
retinopathy comprises directly injecting an antibody specifically
against a .beta.1 integrin function into the vitreous cavity of the
eye, wherein the integrin function is blocked by the antibody. In
one embodiment, the antibody in a monoclonal antibody derived from
clone P5D2. In one embodiment, the antibody can be combined with
other anti-angiogenic therapy, such as anti-VEGF therapy, and
injected directly into the vitreous cavity. In another embodiment,
the antibody can be combined with other TRPV4 inhibitors, e.g. an
siRNA to TRPV4 as described herein, and injected directly into the
vitreous cavity.
[0110] In some embodiments, the antibody specifically against a
.beta.1 integrin function is administered intravenously, e.g. via
central venous catheter (CVC or central venous line or central
venous access catheter) placed into a large vein in the neck
(internal jugular vein), chest (subclavian vein) or groin (femoral
vein). Systemic delivery of antibodies can be performed according
to any methods known in the art, e.g. as described in Loberg et.
al. 2007, Cancer Research 67:9417 and WO/2000/050008). These
references are incorporated by reference in their entirety. The
antibody or variants or fragments thereof can be formulated for
systemic delivery such as in liposomes.
[0111] In one embodiments, the treatment of angiogenesis-related
diseases having localized aberrant angiogenesis, e.g. solid
non-metastatic tumor, arthritis, and endometriosis, comprises
directly injecting an siRNA, dsRNA, or shRNA vector directed
against a TRPV4 gene to the location or tissue with aberrant
angiogenesis. In one embodiment, a mixture of several different
siRNAs to TRPV4 is used, directly injected into the bodily site
having localized aberrant angiogenesis. In another embodiment, the
siRNA is combined with other anti-angiogenic therapy, such as
anti-VEGF therapy, and injected directly into the bodily site. In
another embodiment, the siRNA can be combined with other TRPV4
inhibitors, e.g. an antibody P5D2, and injected directly into the
bodily site.
[0112] In other embodiments, the treatment of angiogenesis-related
diseases having localized aberrant angiogenesis, e.g. solid
non-metastatic tumor, arthritis, and endometriosis, comprises
directly injecting an antibody specifically against a .beta.1
integrin function into the location or tissue with aberrant
angiogenesis, wherein the integrin function is blocked by the
antibody. In one embodiment, the antibody in a monoclonal antibody
derived from clone P5D2. In one embodiment, the antibody is
combined with other anti-angiogenic therapy, such as anti-VEGF
therapy, and injected directly into the bodily site having
localized aberrant angiogenesis. In another embodiment, the
antibody is combined with other TRPV4 inhibitors, e.g. an siRNA to
TRPV4 as described herein, and injected directly into the bodily
site.
[0113] In other embodiments, the treatment of angiogenesis-related
diseases comprises systemic administration of an siRNA, dsRNA,
shRNA vector directed against a TRPV4 gene and/or an antibody
specifically against a .beta.1 integrin function into the mammal in
need thereof. Such a mammal would have been diagnosed with an
angiogenesis-related disease or disorder by a skilled
physician.
[0114] In one embodiment, the methods described herein can be
administered in conjunction with other anti-angiogenesis
factor/drugs and treatment regime for the afflicted mammals, such
as chemotherapy and radiation therapy.
[0115] As used herein, the term "a therapeutically effective
amount" refers an amount sufficient to achieve the intended
purpose. For example, an effective amount of a TRPV4 inhibitor that
inhibits angiogenesis will cause a reduction or even completely
halt any new blood vessel formation. An effective amount for
treating or ameliorating a disorder, disease, or medical condition
is an amount sufficient to result in a reduction or complete
removal of the symptoms of the disorder, disease, or medical
condition. The effective amount of a given therapeutic agent will
vary with factors such as the nature of the agent, the route of
administration, the size and species of the animal to receive the
therapeutic agent, and the purpose of the administration. The
effective amount in each individual case may be determined
empirically by a skilled artisan according to established methods
in the art.
[0116] In one embodiment, the endothelial cell is a mammalian
endothelial cell. In another embodiment, the endothelial cell is a
human endothelial cell.
[0117] Mammals include but are not limited to human, cat, dog,
horse, monkey, cow, sheep, goats and other ungulates.
[0118] In one embodiment, the mammal is a human.
Integrins as Mechanoreceptors
[0119] Cells within solid tissues sense mechanical forces caused by
physical distortion of the extracellular matrix (ECM) scaffolds
that hold cells together and form the physical supporting framework
of living tissues. Cell surface receptors that support ECM adhesion
might function as mechanoreceptors and mediate cellular
mechanosensation. Integrins, which are the most ubiquitous ECM
receptors, are heterodimeric transmembrane proteins consisting of
.alpha. and .beta. chains. Their extracellular domain binds to
ligands present in the ECM that contain specific peptide sequences,
such as the arg-gly-asp (RGD) sequence from the cell-binding region
of fibronectin. Integrins undergo conformational changes in
response to ligand occupancy that result in receptor `activation`
and initiation of downstream signaling responses in the absence of
external mechanical loads. For example, unbound (inactive)
integrins do not form focal adhesions, whereas integrin ligation as
a result of cell binding to ECM proteins, synthetic RGD peptides,
or activating anti-integrin antibodies induces changes in integrin
conformation that activate the small GTPase Rho and promote `focal
adhesion` formation. Focal adhesions are multi-molecular protein
complexes that mechanically anchor the short cytoplasmic tails of
integrins to the cytoskeleton; they also serve as orienting
scaffolds for various signaling transducing molecules. Because
integrins form a relatively stiff connection between the ECM and
the cytoskeleton, they provide a preferred path for mechanical
force transfer across the cell surface. Importantly, while force
application to bound (already activated) integrins alters the
activity of many signaling molecules inside adherent cells,
including small GTPases (e.g., Rho, Rac), protein kinases (e.g.,
FAK, Fyn, Src), and cAMP, application of similar forces to
unligated integrins (i.e., through bound non-activating
anti-.beta.1 integrin antibodies) fails to produce focal adhesion
formation or induce these signaling responses. Thus, activated
integrins function to focus and concentrate mechanical stresses at
focal adhesions where forces are transferred to molecular partners
of integrins that elicit changes in chemical activities
milliseconds to minutes after force application.
Integrins and Stress-Activated (SA) Ion Channels
[0120] One of the most ubiquitous and rapid mechanosensing
mechanisms used by cells involves force-induced activation of SA
ion channels on the cell surface. Some of these molecules, such as
members of the Transient Receptor Potential (TRP) family of
mechanically-gated ion channels can be triggered to alter calcium
transport as a result of physical distortion of the lipid bilayer
and resulting stress transfer to the channel. For example, the
activation of calcium influx by fluid shear stress in kidney
epithelial cells is mediated by interactions between two members of
the TRP family of mechanoregulated ion channels, polycystin-1 and
polycystin-2, that colocalize with microtubules within the primary
cilium. Other types of SA channels, such as SAKcaC, also have been
identified in mammalian cells that are activated within
milliseconds after mechanical stress is applied to the cell's
surface membrane (e.g., as analyzed in patch-clamp experiments).
Therefore, mechanosignaling through SA channels is often viewed as
a mechanism that is independent of integrin-based mechanosensation.
However, forces that are channeled through transmembrane integrins
to the cytoskeleton also can specifically activate certain SA
channels. For example, many mechanosensitive ion channels lose
their normal regulated activities (and become super-activated) if
the lipid bilayer is separated from the underlying cortical
cytoskeleton, and certain types of TRP channels, such as TRPV2 and
TRPV4, may bind directly to microtubules or actin filaments through
ankyrin repeats within their cytoplasmic domains. Interestingly,
ankyrin domains in integrin-linked kinase (ILK) mediate its
association with integrins via PINCH protein; this raises the
possibility that TRP channels may associate with integrins through
other cytoskeletal-associated adapter proteins present within focal
adhesions. This is supported by the recent finding that enhanced
osmotransduction in cultured nociceptors is mediated by TRPV4, and
requires signaling through integrins and Src in dorsal root
ganglia. In contrast, the finding that amphipathic molecules that
intercalate in the lipid bilayer, such as chlorpromazine and
trinitrophenol, alter the activity of certain SA channels intimates
that they are directly activated by membrane bilayer distortion;
however, these lipidmodifying molecules also can alter cytoskeletal
mechanics in certain cells. Moreover, the SA channel SAKcaC
contains a 59 amino acid long cytoplasmic STREX domain which when
deleted attenuates amphipath-induced SA activity; thus, a
submembranous adapter protein appears to mediate force transmission
from membrane lipids to this channel. Importantly, integrins also
have been shown to co-localize and coimmunoprecipitate with members
of the ENaC channel family and polycystin-1. In the neuromuscular
junction, stimulation of muscle cell contraction triggers calcium
influx within 100 msec at the synapse, and this response can be
prevented by adding inhibitors of integrin binding. Both a rapid
millisecond calcium influx response and a slower wave of calcium
influx on the order of seconds to minutes also can be produced by
applying force directly to cell surface integrins, whereas
application of the same force to other transmembrane receptors is
less effective at eliciting this response.
[0121] Integrins are a large family of proteins with 8 known
.alpha. and 18.beta. subunits that combine to form distinct dimeric
transmembrane receptors. There has been extensive investigation
into the function of the various domains of the .alpha. and .beta.
integrin chains. The extracellular domains of both subunits
contribute to binding of specific adhesive ligands; however, the
.alpha. and .beta. integrin cytoplasmic tails, which are relatively
short, appear to interact independently with intracellular
proteins. The .alpha. subunits are poorly conserved with the
exception of a small domain near the membrane, whereas the .beta.
subunits are much more homologous. Although a integrins can bind
focal adhesion proteins, most known cytoskeletal interactions occur
through the .beta. integrin chains, and the majority of integrin
signaling is mediated by .beta. integrins.
Transient Receptor Potential (TRP) Channels
[0122] TRP channels comprise a large family of cation channels that
provide a pathway for calcium influx into cells. Among the
.about.30 TRP-channel proteins identified in mammals, endothelial
cells express .about.20 members that are classified into six
subfamilies: canonical (TRPC), vanilloid (TRPV), melastatin
(TRPVM), polycystin (TRPP), mucolipin (TRPML) and TRPA.
Structurally, TRP channels consist of six transmembrane
(TM)-spanning helices with a pore region between TM5 and
cytoplasmic N and C termini. Both TRPC and TRPV channels contain
multiple anykyrin domains at their N-terminus that are absent in
TRPM channels. Most of the TRP channels contain PDZ binding motifs
and recognition sites for PKC and PI3K. TRPC subfamily channels
that are ubiquitously expressed in endothelial cells are
responsible for store-operated or receptor-mediated calcium entry;
they also have been implicated in control of endothelial barrier
function and vasorelaxation. Among the vertebrate TRPV and TRPM
channels, TRPV4 and TRPV2 are considered mechanosensitive, and
growing evidence suggests that TRPV4 plays critical role in
mechanical force-induced regulation of endothelial cell function.
For example, in endothelial cells, TRPV4 acts as a calcium entry
channel that is activated by increases in cell volume and
temperature. TRPV4 can also be activated by ligands such as
arachidonic acid and its metabolites, endocannabinoids and a
synthetic phorbol ester, 4-.alpha.-phorbol 12, 13-didecanoate
(PPD), and it can be suppressed by integrin and Src kinase
inhibitors during osmotransduction in dorsal root ganglia. In the
present invention with sensitive calcium imaging (FLUO4-based)
methods, it was found that force application to integrins activates
calcium influx within 5 to 50 msec in CE cells that can be
inhibited by treatment with the SA channel inhibitor gadolinium
chloride, or by suppressing expression of TRPV4, but not TRPV2,
using siRNA. Moreover, this response requires physical distortion
(strain) of integrin-cytoskeleton linkages in the cytoplasm
directly beneath the site of mechanical stress application, and
single chain integrin chimeras that only contain the cytoplasmic
tail of integrin can support force-induced calcium influx. In
addition, the inventors also showed that GFP-TRPV4 is recruited to
focal adhesions that form when cells bind to magnetic microbeads
coated with integrin ligands. Taken together, these data indicate
that force application to transmembrane integrin receptors results
in almost immediate activation of SA channels on the cell surface
as a result of distortion of the supramolecular lattice that links
the cytoplasmic portions of integrins to TRPV4 channels within the
focal adhesion.
Generation of Recombinant TRPV4 or Activated .beta.1 Integrin
Proteins
[0123] In some embodiment, TRPV4 or function-blocking .beta.1
integrin antibodies can be generated by any methods known in the
art, for example, immunizing a mammal with TRPV4 or activated
.beta.1 integrin proteins. Large quantities of such proteins can be
made using standard molecular recombinant protein expression
methods. Protein coding nucleic acid sequences of TRPV4 or .beta.1
integrin protein or fragments thereof can be amplified by
polymerase chain reaction (PCR) and cloned into protein expression
vectors. In humans, there are six isoform of .beta.1 integrin mRNA
(GENBANK.TM. Accession Nos. NM.sub.--002211.3, NM.sub.--033666.2,
NM.sub.--033667.2, NM.sub.--033668.2, NM.sub.--033669.2,
NM.sub.--133376.2). In humans, there are two isoform of TRPV4 mRNA
(GENBANK.TM. Accession Nos. NM.sub.--021625 and NM.sub.--147204).
The resultant expression vectors can be then be transfected into
corresponding host for protein expression.
[0124] Examples of expression vectors and host cells are the pET
vectors (NOVAGEN.RTM.), pGEX vectors (GE Life Sciences), and pMAL
vectors (New England labs. Inc.) for protein expression in E. coli
host cell such as BL21, BL21(DE3) and AD494(DE3)pLysS, Rosetta
(DE3), and Origami(DE3) ((NOVAGEN.RTM.); the strong CMV
promoter-based pcDNA3.1 (INVITROGEN.TM. Inc.) and pClneo vectors
(Promega) for expression in mammalian cell lines such as CHO, COS,
HEK-293, Jurkat, and MCF-7; replication incompetent adenoviral
vector vectors pAdeno X, pAd5F35, pLP-Adeno-X-CMV (CLONTECH.RTM.),
pAd/CMV/V5-DEST, pAd-DEST vector (INVITROGEN.TM. Inc.) for
adenovirus-mediated gene transfer and expression in mammalian
cells; pLNCX2, pLXSN, and pLAPSN retrovirus vectors for use with
the RETRO-X.TM. system from Clontech for retroviral-mediated gene
transfer and expression in mammalian cells; pLenti4/V5-DEST.TM.,
pLenti6/V5-DEST.TM., and pLenti6.2/V5-GW/lacZ (INVITROGEN.TM. Inc.)
for lentivirus-mediated gene transfer and expression in mammalian
cells; adenovirus-associated virus expression vectors such as
pAAV-MCS, pAAV-IRES-hrGFP, and pAAV-RC vector (STRATAGENE.RTM.) for
adeno-associated virus-mediated gene transfer and expression in
mammalian cells; BACpak6 baculovirus (CLONTECH.RTM.) and
pFastBac.TM. HT (INVITROGEN.TM. Inc.) for the expression in
Spodopera frugiperda 9 (Sf9) and Sfl 1 insect cell lines;
pMT/BiP/V5-His (INVITROGEN.TM. Inc.) for the expression in
Drosophila schneider S2 cells; Pichia expression vectors
pPICZ.alpha., pPICZ, pFLD.alpha. and pFLD (INVITROGEN.TM. Inc.) for
expression in Pichia pastoris and vectors pMETcs and pMET for
expression in P. methanolica; pYES2/GS and pYD1 (INVITROGEN.TM.
Inc.) vectors for expression in yeast Saccharomyces cerevisiae.
Recent advances in the large scale expression heterologous proteins
in Chlamydomonas reinhardtii are described by Griesbeck C. et. al.
2006 Mol. Biotechnol. 34:213-33 and Fuhrmann M. 2004, Methods Mol
Med. 94:191-5. Foreign heterologous coding sequences are inserted
into the genome of the nucleus, chloroplast and mitochodria by
homologous recombination. The chloroplast expression vector p64
carrying the most versatile chloroplast selectable marker
aminoglycoside adenyl transferase (aadA), which confer resistance
to spectinomycin or streptomycin, can be used to express foreign
protein in the chloroplast. Biolistic gene gun method is used to
introduced the vector in the algae. Upon its entry into
chloroplasts, the foreign DNA is released from the gene gun
particles and integrates into the chloroplast genome through
homologous recombination.
[0125] In one embodiment, the expression vector is a viral vector,
such as a lentivirus, adenovirus, or adeno-associated virus. A
simplified system for generating recombinant adenoviruses is
presented by He T C. et. al. Proc. Natl. Acad. Sci. USA,
95:2509-2514, 1998. The gene of interest is first cloned into a
shuttle vector, e.g. pAdTrack-CMV. The resultant plasmid is
linearized by digesting with restriction endonuclease Pme I, and
subsequently cotransformed into E. coli BJ5183 cells with an
adenoviral backbone plasmid, e.g. pAdEasy-1 of STRATAGENE.RTM.'s
ADEASY.TM. Adenoviral Vector System. Recombinant adenovirus vectors
are selected for kanamycin resistance, and recombination confirmed
by restriction endonuclease analyses. Finally, the linearized
recombinant plasmid is transfected into adenovirus packaging cell
lines, for example HEK 293 cells (E1-transformed human embryonic
kidney cells) or 911 (E1-transformed human embryonic retinal cells)
(Human Gene Therapy 7:215-222, 1996). Recombinant adenovirus are
generated within the HEK 293 cells.
[0126] In one embodiment, the preferred viral vector is a
lentiviral vector and there are many examples of use of lentiviral
vectors for gene therapy for inherited disorders of haematopoietic
cells and various types of cancer, and these references are hereby
incorporated by reference (Klein, C. and Baum, C. (2004), Hematol.
J., 5:103-111; Zufferey, R et. al. (1997), Nat. Biotechnol.,
15:871-875; Morizono, K. et. al. (2005), Nat. Med., 11:346-352; Di
Domenico, C. et. al. (2005). Hum. Gene Ther., 16:81-90). The HIV-1
based lentivirus can effectively transduce a broader host range
than the Moloney Leukemia Virus (MoMLV)-base retroviral systems.
Preparation of the recombinant lentivirus can be achieved using the
pLenti4/V5-DEST.TM., pLenti6/V5-DEST.TM. or pLenti vectors together
with VIRAPOWER.TM. Lentiviral Expression systems from
INVITROGEN.TM. Inc.
[0127] In one embodiment, the expression viral vector can be a
recombinant adeno-associated virus (rAAV) vector. Using rAAV
vectors, genes can be delivered into a wide range of host cells
including many different human and non-human cell lines or tissues.
Because AAV is non-pathogenic and does not illicit an immune
response, a multitude of pre-clinical studies have reported
excellent safety profiles. rAAVs are capable of transducing a broad
range of cell types and transduction is not dependent on active
host cell division. High titers, >10.sup.8 viral particle/ml,
are easily obtained in the supernatant and 10.sup.11-10.sup.12
viral particle/ml with further concentration. The transgene is
integrated into the host genome so expression is long term and
stable.
[0128] The use of alternative AAV serotypes other than AAV-2
(Davidson et al (2000), PNAS 97(7)3428-32; Passini et al (2003), J.
Virol 77(12):7034-40) has demonstrated different cell tropisms and
increased transduction capabilities. With respect to brain cancers,
the development of novel injection techniques into the brain,
specifically convection enhanced delivery (CED; Bobo et al (1994),
PNAS 91(6):2076-80; Nguyen et al (2001), Neuroreport 12(9):1961-4),
has significantly enhanced the ability to transduce large areas of
the brain with an AAV vector.
[0129] Large scale preparation of AAV vectors is made by a
three-plasmid cotransfection of a packaging cell line: AAV vector
carrying the chimeric DNA coding sequence, AAV RC vector containing
AAV rep and cap genes, and adenovirus helper plasmid pDF6, into
50.times.150 mm plates of subconfluent 293 cells. Cells are
harvested three days after transfection, and viruses are released
by three freeze-thaw cycles or by sonication.
[0130] AAV vectors are then purified by two different methods
depending on the serotype of the vector. AAV2 vector is purified by
the single-step gravity-flow column purification method based on
its affinity for heparin (Auricchio, A., et. al., 2001, Human Gene
therapy 12; 71-6; Summerford, C. and R. Samulski, 1998, J. Virol.
72:1438-45; Summerford, C. and R. Samulski, 1999, Nat. Med. 5:
587-88). AAV2/1 and AAV2/5 vectors are currently purified by three
sequential CsCl gradients.
Generating TRPV4 and Function-Blocking Anti-.beta.1 Integrin
Antibodies
[0131] As used herein, the term "antibody" refers to immunoglobulin
molecules and immunologically active portions of immunoglobulin
molecules, i.e., molecules that contain an antigen binding site
that immunospecifically binds an antigen. The terms also refers to
antibodies comprised of two immunoglobulin heavy chains and two
immunoglobulin light chains as well as a variety of forms besides
antibodies; including, for example, Fv, Fab, and F(ab)'.sub.2 as
well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al.,
Eur. J. Immunol. 17, 105 (1987)) and single chains (e.g., Huston et
al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird
et al., Science 242, 423-426 (1988), which are incorporated herein
by reference). (See, generally, Hood et al., Immunology, Benjamin,
N.Y., 2nd ed. (1984), Harlow and Lane, Antibodies. A Laboratory
Manual, Cold Spring Harbor Laboratory (1988) and Hunkapiller and
Hood, Nature, 323, 15-16 (1986), which are incorporated herein by
reference.).
[0132] In one embodiment, the TRPV4 or function-blocking
anti-.beta.1 integrin antibody is a polyclonal antibody. In one
embodiment, the TRPV4 or function-blocking anti-.beta.1 integrin
antibody is a monoclonal antibody. In preferred embodiment, the
TRPV4 or function-blocking anti-.beta.1 integrin antibody is a
humanized antibody. In preferred another embodiment the TRPV4 or
function-blocking anti-.beta.1 integrin antibody is a chimeric
antibody. In yet another embodiment, the TRPV4 or function-blocking
anti-.beta.1 integrin antibodies include, but are not limited to
multispecific, human, single chain antibodies, Fab fragments,
F(ab)'.sub.2 fragments, fragments produced by a Fab expression
library, domain-deleted antibodies (including, e.g., CH2
domain-deleted antibodies), anti-idiotypic (anti-Id) antibodies
(including, e.g., anti-Id antibodies to antibodies of the
invention), and epitope-binding fragments of any of the above. The
immunoglobulin molecules of the invention can be of any type (e.g.,
IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3,
IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.
[0133] Encompassed in the methods disclosed herein are TRPV4 or
function-blocking anti-.beta.1 integrin antibodies that are, but
are not limited to, engineered forms of antibodies and antibody
fragments such as diabodies, triabodies, tetrabodies, and higher
multimers of scFvs, single-domain antibodies, as well as
minibodies, such as two scFv fragments joined by two constant (C)
domains. See, e.g., Hudson, P. J. and Couriau, C., Nature Med. 9:
129-134 (2003); U.S. Publication No. 20030148409; U.S. Pat. No.
5,837,242.
[0134] In one embodiment, the TRPV4 or function-blocking
anti-.beta.1 integrin antibodies can be obtained from any animal
origin including birds and mammals. Preferably, the antibodies are
human, murine (e.g., mouse and rat), donkey, ship rabbit, goat,
guinea pig, camel, horse, or chicken. As used herein, "human"
antibodies include antibodies having the amino acid sequence of a
human immunoglobulin and include antibodies isolated from human
immunoglobulin libraries or from animals transgenic for one or more
human immunoglobulin and that do not express endogenous
immunoglobulins, as described infra and, for example in, U.S. Pat.
No. 5,939,598 by Kucherlapati et al.
[0135] In a preferred embodiment for use in humans, the TRPV4 or
function-blocking anti-.beta.1 integrin antibodies are human or
humanized antigen-binding antibody fragments of the present
invention and include, but are not limited to, Fab, Fab and
F(ab)'.sub.2, Fd, single-chain Fvs (scFv), single-domain
antibodies, triabodies, tetrabodies, minibodies, domain-deleted
antibodies, single-chain antibodies, disulfide-linked Fvs (sdFv)
and fragments comprising either a variable light chain (VL) or
variable heavy chain VH region. Antigen-binding antibody fragments,
including single-chain antibodies, may comprise the variable
region(s) alone or in combination with the entirety or a portion of
the following: hinge region, CH1, CH2, and CH3 domains. Also
included in the invention are antigen-binding fragments also
comprising any combination of variable region(s) with a hinge
region, CH1, CH2, and CH3 domains.
[0136] Preferred antibodies in the therapeutic methods of the
invention are those containing a deletion of the CH2 domain.
[0137] As used herein, the term "humanized" immunoglobulin or
"humanized" antibody refers to an immunoglobulin comprising a human
framework, at least one complementarity determining regions (CDR)
from a non-human antibody, and in which any constant region present
is substantially identical to a human immunoglobulin constant
region, i.e., at least about 85-90%, preferably at least 95%
identical. Hence, all parts of a humanized immunoglobulin, except
possibly the CDRs, are substantially identical to corresponding
parts of one or more native human immunoglobulin sequences. For
example, a humanized immunoglobulin would not encompass a chimeric
mouse variable region/human constant region antibody.
[0138] As used herein, the term "framework region" refers to those
portions of antibody light and heavy chain variable regions that
are relatively conserved (i.e., other than the CDRs) among
different immunoglobulins in a single species, as defined by Kabat,
et al., op. cit. As used herein, a "human framework region" is a
framework region that is substantially identical (about 85% or
more) to the framework region of a naturally occurring human
antibody.
[0139] As used herein, the term "chimeric" antibody refers to an
antibody whose heavy and light chains have been constructed,
typically by genetic engineering, from immunoglobulin gene segments
belonging to different species. For example, the variable (V)
segments of the genes from a mouse monoclonal antibody may be
joined to human constant (C) segments, such as gamma1 and/or
gamma4. A typical therapeutic or diagnostic chimeric antibody is
thus a hybrid protein comprising at least one V region (e.g., VH or
VL) or the entire antigen-binding domain (i.e., VH and VL) from a
mouse antibody and at least one C (effector) region (e.g., CH(CH1,
CH2, CH3, or CH4) or CL or the entire C domain (i.e., CH and CL)
from a human antibody, although other mammalian species may be
used. In some embodiments, especially for use in the therapeutic
methods of the TRPV4 or function-blocking anti-.beta.1 integrin
antibodies should contain no CH2 domain.
[0140] In one embodiment, a chimeric antibody may contain at least
the TRPV4 or function-blocking anti-.beta.1 integrin antigen
binding Fab or F(ab)'.sub.2 region while the humanized antibody can
contain at least the TRPV4 or function-blocking anti-.beta.1
integrin antigen binding Fv region fused to a human Fc region.
[0141] The terms "antigen" is well understood in the art and refer
to the portion of a macromolecule which is specifically recognized
by a component of the immune system, e.g., an antibody or a T-cell
antigen receptor. The term antigen includes any protein determinant
capable of specific binding to an immunoglobulin. Antigenic
determinants usually consist of chemically active surface groupings
of molecules such as amino acids or sugar side chains and usually
have specific three dimensional structural characteristics, as well
as specific charge characteristics.
[0142] Alternatively, techniques described for the production of
single chain antibodies (U.S. Pat. No. 4,946,778; Bird, Science
242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA
85:5879-5883 (1988); and Ward et al., Nature 334:544-54 (1989)) can
be adapted to produce single chain antibodies. Single chain
antibodies are formed by linking the heavy and light chain
fragments of the Fv region via an amino acid bridge, resulting in a
single chain polypeptide. Techniques for the assembly of functional
Fv fragments in E. coli may also be used (Skerra et al., Science
242:1038-1041 (1988)).
[0143] Recombinant expression of an antibody disclosed herein, or
fragment, derivative or analog thereof, (e.g., a heavy or light
chain of an antibody of the invention or a single chain antibody of
the invention), including a recombinant protein derived from the
antibody antigen-binding region, requires construction of an
expression vector containing a polynucleotide that encodes the
antibody. Once a polynucleotide encoding an antibody molecule or a
heavy or light chain of an antibody or portion thereof (preferably
containing the heavy or light chain variable domain) of the
invention has been obtained, the vector for the production of the
antibody molecule may be produced by recombinant DNA technology
using techniques well known in the art. Thus, methods for preparing
a protein by expressing a polynucleotide containing an
antibody-encoding nucleotide sequence are described herein. Methods
which are well known to those skilled in the art can be used to
construct expression vectors containing antibody coding sequences
and appropriate transcriptional and translational control signals.
These methods include, for example, in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic
recombination. The invention, thus, provides replicable vectors
comprising a nucleotide sequence encoding an antibody molecule of
the invention, or a heavy or light chain thereof, or a heavy or
light chain variable domain, operably linked to a promoter. Such
vectors may include the nucleotide sequence encoding the constant
region of the antibody molecule (see, e.g., PCT publication WO
86/05807; PCT publication WO 89/01036; and U.S. Pat. No. 5,122,464)
and the variable domain of the antibody may be cloned into such a
vector for expression of the entire heavy or light chain. Methods
for generating multivalent and bispecific antibody fragments are
described by Tomlinson I. and Holliger P. (2000) Methods Enzymol,
326, 461-479 and the engineering of antibody fragments and the rise
of single-domain antibodies is described by Holliger P. (2005) Nat.
Biotechnol. September; 23(9):1126-36, and are both hereby
incorporated by reference.
Inhibition of TRPV4 Expression
[0144] In one embodiment, the expression of TRPV4 is inhibited by
an RNA interference molecule.
[0145] RNA interference-inducing molecules include but are not
limited to siRNA, dsRNA, stRNA, shRNA, micro RNAi (mRNAi),
antisense oligonucleotides etc. and modified versions thereof,
where the RNA interference molecule silences the gene expression of
TRPV4. An anti-sense oligonucleic acid, or a nucleic acid analogue,
for example but are not limited to DNA, RNA, peptide-nucleic acid
(PNA), pseudo-complementary PNA (pc-PNA), or locked nucleic acid
(LNA) and the like.
[0146] RNA interference (RNAi) is an evolutionally conserved
process whereby the expression or introduction of RNA of a sequence
that is identical or highly similar to a target gene results in the
sequence specific degradation or specific post-transcriptional gene
silencing (PTGS) of messenger RNA (mRNA) transcribed from that
targeted gene (see Coburn, G. and Cullen, B. (2002) J. of Virology
76(18):9225), thereby inhibiting expression of the target gene. In
one embodiment, the RNA is double stranded RNA (dsRNA). This
process has been described in plants, invertebrates, and mammalian
cells. In nature, RNAi is initiated by the dsRNA-specific
endonuclease Dicer, which promotes processive cleavage of long
dsRNA into double-stranded fragments termed siRNAs. siRNAs are
incorporated into a protein complex (termed "RNA induced silencing
complex," or "RISC") that recognizes and cleaves target mRNAs. RNAi
can also be initiated by introducing nucleic acid molecules, e.g.,
synthetic siRNAs or RNA interfering agents, to inhibit or silence
the expression of target genes. As used herein, "inhibition of
target gene expression" includes any decrease in expression or
protein activity or level of the target gene or protein encoded by
the target gene as compared to a situation wherein no RNA
interference has been induced. The decrease can be of at least 30%,
40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% or more as compared to the
expression of a target gene or the activity or level of the protein
encoded by a target gene which has not been targeted by an RNA
interfering agent.
[0147] "Short interfering RNA" (siRNA), also referred to herein as
"small interfering RNA" is defined as an agent which functions to
inhibit expression of a target gene, e.g., by RNAi. An siRNA can be
chemically synthesized, can be produced by in vitro transcription,
or can be produced within a host cell. In one embodiment, siRNA is
a double stranded RNA (dsRNA) molecule of about 15 to about 40
nucleotides in length, preferably about 15 to about 28 nucleotides,
more preferably about 19 to about 25 nucleotides in length, and
more preferably about 19, 20, 21, 22, or 23 nucleotides in length,
and can contain a 3' and/or 5' overhang on each strand having a
length of about 0, 1, 2, 3, 4, or 5 nucleotides. The length of the
overhang is independent between the two strands, i.e., the length
of the overhang on one strand is not dependent on the length of the
overhang on the second strand. Preferably the siRNA is capable of
promoting RNA interference through degradation or specific
post-transcriptional gene silencing (PTGS) of the target messenger
RNA (mRNA).
[0148] siRNAs also include small hairpin (also called stem loop)
RNAs (shRNAs). In one embodiment, these shRNAs are composed of a
short (e.g., about 19 to about 25 nucleotide) antisense strand,
followed by a nucleotide loop of about 5 to about 9 nucleotides,
and the analogous sense strand. Alternatively, the sense strand can
precede the nucleotide loop structure and the antisense strand can
follow. These shRNAs can be contained in plasmids, retroviruses,
and lentiviruses and expressed from, for example, the pol III U6
promoter, or another promoter (see, e.g., Stewart, et al. (2003)
RNA April; 9(4):493-501, incorporated by reference herein in its
entirety).
[0149] The target gene or sequence of the RNA interfering agent can
be a cellular gene or genomic sequence, e.g. the TRPV4 (genomic
sequence ENSG00000111199 (Ensembl) assembled in GENBANK.TM.
Accession No. NC.sub.--000012.10 (SEQ. ID. No. 1),
NM.sub.--021625.3 (SEQ. ID. No. 2) and NM.sub.--147204.1 (SEQ. ID.
No. 3)). An siRNA can be substantially homologous to the target
gene or genomic sequence, or a fragment thereof. As used in this
context, the term "homologous" is defined as being substantially
identical, sufficiently complementary, or similar to the target
mRNA, or a fragment thereof, to effect RNA interference of the
target. In addition to native RNA molecules, RNA suitable for
inhibiting or interfering with the expression of a target sequence
include RNA derivatives and analogs. Preferably, the siRNA is
identical to its target.
[0150] The siRNA preferably targets only one sequence. Each of the
RNA interfering agents, such as siRNAs, can be screened for
potential off-target effects by, for example, expression profiling.
Such methods are known to one skilled in the art and are described,
for example, in Jackson et al, Nature Biotechnology 6:635-637,
2003. In addition to expression profiling, one can also screen the
potential target sequences for similar sequences in the sequence
databases to identify potential sequences which can have off-target
effects. For example, according to Jackson et al. (Id.) 15, or
perhaps as few as 11 contiguous nucleotides of sequence identity
are sufficient to direct silencing of non-targeted transcripts.
Therefore, one can initially screen the proposed siRNAs to avoid
potential off-target silencing using the sequence identity analysis
by any known sequence comparison methods, such as BLAST.
[0151] siRNA molecules need not be limited to those molecules
containing only RNA, but, for example, further encompasses
chemically modified nucleotides and non-nucleotides, and also
include molecules wherein a ribose sugar molecule is substituted
for another sugar molecule or a molecule which performs a similar
function. Moreover, a non-natural linkage between nucleotide
residues can be used, such as a phosphorothioate linkage. For
example, siRNA containing D-arabinofuranosyl structures in place of
the naturally-occurring D-ribonucleosides found in RNA can be used
in RNAi molecules according to the present invention (U.S. Pat. No.
5,177,196). Other examples include RNA molecules containing the
o-linkage between the sugar and the heterocyclic base of the
nucleoside, which confers nuclease resistance and tight
complementary strand binding to the oligonucleotidesmolecules
similar to the oligonucleotides containing 2'-O-methyl ribose,
arabinose and particularly D-arabinose (U.S. Pat. No.
5,177,196).
[0152] The RNA strand can be derivatized with a reactive functional
group of a reporter group, such as a fluorophore. Particularly
useful derivatives are modified at a terminus or termini of an RNA
strand, typically the 3' terminus of the sense strand. For example,
the 2'-hydroxyl at the 3' terminus can be readily and selectively
derivatized with a variety of groups.
[0153] siRNA and miRNA molecules having various "tails" covalently
attached to either their 3'- or to their 5'-ends, or to both, are
also known in the art and can be used to stabilize the siRNA and
miRNA molecules delivered using the methods of the present
invention. Generally speaking, intercalating groups, various kinds
of reporter groups and lipophilic groups attached to the 3' or 5'
ends of the RNA molecules are well known to one skilled in the art
and are useful according to the methods of the present invention.
Descriptions of syntheses of 3'-cholesterol or 3'-acridine modified
oligonucleotides applicable to preparation of modified RNA
molecules useful according to the present invention can be found,
for example, in the articles: Gamper, H. B., Reed, M. W., Cox, T.,
Virosco, J. S., Adams, A. D., Gall, A., Scholler, J. K., and Meyer,
R. B. (1993) Facile Preparation and Exonuclease Stability of
3'-Modified Oligodeoxynucleotides. Nucleic Acids Res. 21 145-150;
and Reed, M. W., Adams, A. D., Nelson, J. S., and Meyer, R. B., Jr.
(1991) Acridine and Cholesterol-Derivatized Solid Supports for
Improved Synthesis of 3'-Modified Oligonucleotides. Bioconjugate
Chem. 2 217-225 (1993).
[0154] siRNAs useful for the methods described herein include siRNA
molecules of about 15 to about 40 or about 15 to about 28
nucleotides in length, which are homologous to the TRPV4 gene.
Preferably, the TRPV4 targeting siRNA molecules have a length of
about 19 to about 25 nucleotides. More preferably, the targeting
siRNA molecules have a length of about 19, 20, 21, or 22
nucleotides. The targeting siRNA molecules can also comprise a 3'
hydroxyl group. The targeting siRNA molecules can be
single-stranded or double stranded; such molecules can be blunt
ended or comprise overhanging ends (e.g., 5', 3'). In specific
embodiments, the RNA molecule is double stranded and either blunt
ended or comprises overhanging ends.
[0155] In one embodiment, at least one strand of the TRPV4
targeting RNA molecule has a 3' overhang from about 0 to about 6
nucleotides (e.g., pyrimidine nucleotides, purine nucleotides) in
length. In other embodiments, the 3' overhang is from about 1 to
about 5 nucleotides, from about 1 to about 3 nucleotides and from
about 2 to about 4 nucleotides in length. In one embodiment the
targeting RNA molecule is double stranded--one strand has a 3'
overhang and the other strand can be blunt-ended or have an
overhang. In the embodiment in which the targeting RNA molecule is
double stranded and both strands comprise an overhang, the length
of the overhangs can be the same or different for each strand. In a
particular embodiment, the RNA of the present invention comprises
about 19, 20, 21, or 22 nucleotides which are paired and which have
overhangs of from about 1 to about 3, particularly about 2,
nucleotides on both 3' ends of the RNA. In one embodiment, the 3'
overhangs can be stabilized against degradation. In a preferred
embodiment, the RNA is stabilized by including purine nucleotides,
such as adenosine or guanosine nucleotides. Alternatively,
substitution of pyrimidine nucleotides by modified analogues, e.g.,
substitution of uridine 2 nucleotide 3' overhangs by
2'-deoxythymidine is tolerated and does not affect the efficiency
of RNAi. The absence of a 2' hydroxyl significantly enhances the
nuclease resistance of the overhang in tissue culture medium.
Oligonucleotide Modifications
[0156] Unmodified oligonucleotides may be less than optimal in some
applications, e.g., unmodified oligonucleotides can be prone to
degradation by e.g., cellular nucleases. Nucleases can hydrolyze
nucleic acid phosphodiester bonds. However, chemical modifications
to one or more of the subunits of oligonucleotide can confer
improved properties, and, e.g., can render oligonucleotides more
stable to nucleases.
[0157] Modified nucleic acids and nucleotide surrogates can include
one or more of: [0158] (i) alteration, e.g., replacement, of one or
both of the non-linking phosphate oxygens and/or of one or more of
the linking phosphate oxygens in the phosphodiester backbone
linkage. [0159] (ii) alteration, e.g., replacement, of a
constituent of the ribose sugar, e.g., of the 2' hydroxyl on the
ribose sugar; [0160] (iii) wholesale replacement of the phosphate
moiety with "dephospho" linkers; [0161] (iv) modification or
replacement of a naturally occurring base with a non-natural base;
[0162] (v) replacement or modification of the ribose-phosphate
backbone; [0163] (vi) modification of the 3' end or 5' end of the
oligonucleotide, e.g., removal, modification or replacement of a
terminal phosphate group or conjugation of a moiety, e.g., a
fluorescently labeled moiety, to either the 3' or 5' end of
oligonucleotide; and [0164] (vii) modification of the sugar (e.g.,
six membered rings).
[0165] The terms replacement, modification, alteration, and the
like, as used in this context, do not imply any process limitation,
e.g., modification does not mean that one must start with a
reference or naturally occurring ribonucleic acid and modify it to
produce a modified ribonucleic acid bur rather modified simply
indicates a difference from a naturally occurring molecule.
[0166] As oligonucleotides are polymers of subunits or monomers,
many of the modifications described herein can occur at a position
which is repeated within an oligonucleotide, e.g., a modification
of a nucleobase, a sugar, a phosphate moiety, or the non-bridging
oxygen of a phosphate moiety. It is not necessary for all positions
in a given oligonucleotide to be uniformly modified, and in fact
more than one of the aforementioned modifications may be
incorporated in a single oligonucleotide or even at a single
nucleoside within an oligonucleotide.
[0167] In some cases the modification will occur at all of the
subject positions in the oligonucleotide but in many, and in fact
in most cases it will not. By way of example, a modification may
only occur at a 3' or 5' terminal position, may only occur in the
internal region, may only occur in a terminal regions, e.g. at a
position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10
nucleotides of an oligonucleotide. A modification may occur in a
double strand region, a single strand region, or in both. A
modification may occur only in the double strand region of an
oligonucleotide or may only occur in a single strand region of an
oligonucleotide. E.g., a phosphorothioate modification at a
non-bridging oxygen position may only occur at one or both termini,
may only occur in a terminal regions, e.g., at a position on a
terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of
a strand, or may occur in double strand and single strand regions,
particularly at termini. The 5' end or ends can be
phosphorylated.
[0168] A modification described herein may be the sole
modification, or the sole type of modification included on multiple
nucleotides, or a modification can be combined with one or more
other modifications described herein. The modifications described
herein can also be combined onto an oligonucleotide, e.g. different
nucleotides of an oligonucleotide have different modifications
described herein.
[0169] In some embodiments it is particularly preferred, e.g., to
enhance stability, to include particular nucleobases in overhangs,
or to include modified nucleotides or nucleotide surrogates, in
single strand overhangs, e.g., in a 5' or 3' overhang, or in both.
E.g., it can be desirable to include purine nucleotides in
overhangs. In some embodiments all or some of the bases in a 3' or
5' overhang will be modified, e.g., with a modification described
herein. Modifications can include, e.g., the use of modifications
at the 2' OH group of the ribose sugar, e.g., the use of
deoxyribonucleotides, e.g., deoxythymidine, instead of
ribonucleotides, and modifications in the phosphate group, e.g.,
phosphothioate modifications. Overhangs need not be homologous with
the target sequence.
Specific Modifications to Oligonucleotide
The Phosphate Group
[0170] The phosphate group is a negatively charged species. The
charge is distributed equally over the two non-bridging oxygen
atoms. However, the phosphate group can be modified by replacing
one of the oxygens with a different substituent. One result of this
modification to RNA phosphate backbones can be increased resistance
of the oligoribonucleotide to nucleolytic breakdown. Thus while not
wishing to be bound by theory, it can be desirable in some
embodiments to introduce alterations which result in either an
uncharged linker or a charged linker with unsymmetrical charge
distribution.
[0171] Examples of modified phosphate groups include
phosphorothioate, phosphoroselenates, borano phosphates, borano
phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl
or aryl phosphonates and phosphotriesters. In certain embodiments,
one of the non-bridging phosphate oxygen atoms in the phosphate
backbone moiety can be replaced by any of the following: S, Se,
BR.sub.3 (R is hydrogen, alkyl, aryl), C (i.e. an alkyl group, an
aryl group, etc. . . . ), H, NR.sub.2 (R is hydrogen, alkyl, aryl),
or OR(R is alkyl or aryl). The phosphorous atom in an unmodified
phosphate group is achiral. However, replacement of one of the
non-bridging oxygens with one of the above atoms or groups of atoms
renders the phosphorous atom chiral; in other words a phosphorous
atom in a phosphate group modified in this way is a stereogenic
center. The stereogenic phosphorous atom can possess either the "R"
configuration (herein Rp) or the "S" configuration (herein Sp).
[0172] Phosphorodithioates have both non-bridging oxygens replaced
by sulfur. The phosphorus center in the phosphorodithioates is
achiral which precludes the formation of oligoribonucleotides
diastereomers. Thus, while not wishing to be bound by theory,
modifications to both non-bridging oxygens, which eliminate the
chiral center, e.g. phosphorodithioate formation, may be desirable
in that they cannot produce diastereomer mixtures. Thus, the
non-bridging oxygens can be independently any one of S, Se, B, C,
H, N, or OR (R is alkyl or aryl).
[0173] The phosphate linker can also be modified by replacement of
bridging oxygen, (i.e. oxygen that links the phosphate to the
nucleoside), with nitrogen (bridged phosphoroamidates), sulfur
(bridged phosphorothioates) and carbon (bridged
methylenephosphonates). The replacement can occur at the either
linking oxygen or at both the linking oxygens. When the bridging
oxygen is the 3'-oxygen of a nucleoside, replacement with carbon is
preferred. When the bridging oxygen is the 5'-oxygen of a
nucleoside, replacement with nitrogen is preferred.
Replacement of the Phosphate Group
[0174] The phosphate group can be replaced by non-phosphorus
containing connectors. While not wishing to be bound by theory, it
is believed that since the charged phosphodiester group is the
reaction center in nucleolytic degradation, its replacement with
neutral structural mimics should impart enhanced nuclease
stability. Again, while not wishing to be bound by theory, it can
be desirable, in some embodiment, to introduce alterations in which
the charged phosphate group is replaced by a neutral moiety.
[0175] Examples of moieties which can replace the phosphate group
include methyl phosphonate, hydroxylamino, siloxane, carbonate,
carboxymethyl, carbamate, amide, thioether, ethylene oxide linker,
sulfonate, sulfonamide, thioformacetal, formacetal, oxime,
methyleneimino, methylenemethylimino, methylenehydrazo,
methylenedimethylhydrazo and methyleneoxymethylimino. Preferred
replacements include the methylenecarbonylamino and
methylenemethylimino groups.
[0176] Modified phosphate linkages where at least one of the
oxygens linked to the phosphate has been replaced or the phosphate
group has been replaced by a non-phosphorous group, are also
referred to as "non-phosphodiester backbone linkage."
Replacement of Ribophosphate Backbone
[0177] Oligonucleotide--mimicking scaffolds can also be constructed
wherein the phosphate linker and ribose sugar are replaced by
nuclease resistant nucleoside or nucleotide surrogates. While not
wishing to be bound by theory, it is believed that the absence of a
repetitively charged backbone diminishes binding to proteins that
recognize polyanions (e.g. nucleases). Again, while not wishing to
be bound by theory, it can be desirable in some embodiment, to
introduce alterations in which the bases are tethered by a neutral
surrogate backbone. Examples include the morpholino, cyclobutyl,
pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. A
preferred surrogate is a PNA surrogate.
Sugar Modifications
[0178] An oligonucleotide can include modification of all or some
of the sugar groups of the nucleic acid. E.g., the 2' hydroxyl
group (OH) can be modified or replaced with a number of different
"oxy" or "deoxy" substituents. While not being bound by theory,
enhanced stability is expected since the hydroxyl can no longer be
deprotonated to form a 2'-alkoxide ion. The 2'-alkoxide can
catalyze degradation by intramolecular nucleophilic attack on the
linker phosphorus atom. Again, while not wishing to be bound by
theory, it can be desirable to some embodiments to introduce
alterations in which alkoxide formation at the 2' position is not
possible.
[0179] Examples of "oxy"-2' hydroxyl group modifications include
alkoxy or aryloxy (OR, e.g., R.dbd.H, alkyl, cycloalkyl, aryl,
aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG),
O(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2OR; "locked" nucleic
acids (LNA) in which the 2' hydroxyl is connected, e.g., by a
methylene bridge, to the 4' carbon of the same ribose sugar;
O-AMINE (AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino) and aminoalkoxy,
O(CH.sub.2).sub.nAMINE, (e.g., AMINE=NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, or diheteroaryl amino, ethylene diamine, polyamino). It is
noteworthy that oligonucleotides containing only the methoxyethyl
group (MOE), (OCH.sub.2CH.sub.2OCH.sub.3, a PEG derivative),
exhibit nuclease stabilities comparable to those modified with the
robust phosphorothioate modification.
[0180] "Deoxy" modifications include hydrogen (i.e. deoxyribose
sugars, which are of particular relevance to the overhang portions
of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, diheteroaryl amino, or amino acid);
NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE (AMINE=NH.sub.2;
alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,
heteroaryl amino, or diheteroaryl amino), --NHC(O)R(R=alkyl,
cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto;
alkyl-thio-alkyl; thioalkoxy; thioalkyl; and alkyl, cycloalkyl,
aryl, alkenyl and alkynyl, which may be optionally substituted with
e.g., an amino functionality.
[0181] The sugar group can also contain one or more carbons that
possess the opposite stereochemical configuration than that of the
corresponding carbon in ribose. Thus, an oligonucleotide can
include nucleotides containing e.g., arabinose, as the sugar. The
monomer can have an alpha linkage at the 1' position on the sugar,
e.g., alpha-nucleosides. Oligonucleotides can also include "abasic"
sugars, which lack a nucleobase at C-1'. These abasic sugars can
also be further containing modifications at one or more of the
constituent sugar atoms. Oligonucleotides can also contain one or
more sugars that are in the L form, e.g. L-nucleosides.
[0182] Preferred substitutents are 2'-O-Me (2'-O-methyl), 2'-O-MOE
(2'-.beta.-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl]
(2'-O-NMA), 2'-S-methyl, 2'-O--CH2-(4'-C) (LNA),
2'-O--CH2CH2-(4'-C) (ENA), 2'-O-aminopropyl (2'-O-AP),
2'-.beta.-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl
(2'-O-DMAP) and 2'-.beta.-dimethylaminoethyloxyethyl
(2'-O-DMAEOE).
Terminal Modifications
[0183] The 3' and 5' ends of an oligonucleotide can be modified.
Such modifications can be at the 3' end, 5' end or both ends of the
molecule. They can include modification or replacement of an entire
terminal phosphate or of one or more of the atoms of the phosphate
group. E.g., the 3' and 5' ends of an oligonucleotide can be
conjugated to other functional molecular entities such as labeling
moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3
or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon,
boron or ester). The functional molecular entities can be attached
to the sugar through a phosphate group and/or a linker. The
terminal atom of the linker can connect to or replace the linking
atom of the phosphate group or the C-3' or C-5' 0, N, S or C group
of the sugar. Alternatively, the linker can connect to or replace
the terminal atom of a nucleotide surrogate (e.g., PNAs).
[0184] When a linker/phosphate-functional molecular
entity-linker/phosphate array is interposed between two strands of
a dsRNA, this array can substitute for a hairpin RNA loop in a
hairpin-type RNA agent.
[0185] Terminal modifications useful for modulating activity
include modification of the 5' end with phosphate or phosphate
analogs. E.g., in preferred embodiments antisense strands of
dsRNAs, are 5' phosphorylated or include a phosphoryl analog at the
5' prime terminus. 5'-phosphate modifications include those which
are compatible with RISC mediated gene silencing. Modifications at
the 5'-terminal end can also be useful in stimulating or inhibiting
the immune system of a subject. Suitable modifications include:
5'-monophosphate ((HO)2(O)P--O-5'); 5'-diphosphate
((HO).sub.2(O)P--O--P(HO)(O)--O-5'); 5'-triphosphate
((HO).sub.2(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-guanosine cap
(7-methylated or non-methylated)
(7m-G-O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5'); 5'-adenosine
cap (Appp), and any modified or unmodified nucleotide cap structure
(N--O-5'-(HO)(O)P--O--(HO)(O)P--O--P(HO)(O)--O-5');
5'-monothiophosphate (phosphorothioate; (HO)2(S)P--O-5');
5'-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P--O-5'),
5'-phosphorothiolate ((HO)2(O)P--S-5'); any additional combination
of oxygen/sulfur replaced monophosphate, diphosphate and
triphosphates (e.g. 5'-alpha-thiotriphosphate,
5'-beta-thiotriphosphate, 5'-gamma-thiotriphosphate, etc.),
5'-phosphoramidates ((HO)2(O)P--NH-5', (HO)(NH2)(O)P--O-5'),
5'-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl,
etc., e.g. RP(OH)(O)--O-5'-, (OH).sub.2(O)P-5'-CH.sub.2--),
5'-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-),
ethoxymethyl, etc., e.g. RP(OH)(O)--O-5'-). Other embodiments
include replacement of oxygen/sulfur with BH.sub.3, BH.sub.3--
and/or Se.
[0186] Terminal modifications can also be useful for monitoring
distribution, and in such cases the preferred groups to be added
include fluorophores, e.g., fluorscein or an ALEXA.RTM. dye, e.g.,
ALEXA.RTM. 488. Terminal modifications can also be useful for
enhancing uptake, useful modifications for this include
cholesterol. Terminal modifications can also be useful for
cross-linking an RNA agent to another moiety; modifications useful
for this include mitomycin C.
Nucleobases
[0187] Adenine, guanine, cytosine and uracil are the most common
bases found in RNA. These bases can be modified or replaced to
provide RNA's having improved properties. For example, nuclease
resistant oligoribonucleotides can be prepared with these bases or
with synthetic and natural nucleobases (e.g., inosine, thymine,
xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine)
and any one of the above modifications. Alternatively, substituted
or modified analogs of any of the above bases and "universal bases"
can be employed. Examples include 2-(halo)adenine,
2-(alkyl)adenine, 2-(propyl)adenine, 2 (amino)adenine,
2-(aminoalkyll)adenine, 2 (aminopropyl)adenine, 2 (methylthio)
N.sup.6 (isopentenyl)adenine, 6 (alkyl)adenine, 6 (methyl)adenine,
7 (deaza)adenine, 8 (alkenyl)adenine, 8-(alkyl)adenine, 8
(alkynyl)adenine, 8 (amino)adenine, 8-(halo)adenine,
8-(hydroxyl)adenine, 8 (thioalkyl)adenine, 8-(thiol)adenine,
N.sup.6-(isopentyl)adenine, N.sup.6 (methyl)adenine, N.sup.6,
N.sup.6 (dimethyl)adenine, 2-(alkyl)guanine, 2 (propyl)guanine,
6-(alkyl)guanine, 6 (methyl)guanine, 7 (alkyl)guanine, 7
(methyl)guanine, 7 (deaza)guanine, 8 (alkyl)guanine,
8-(alkenyl)guanine, 8 (alkynyl)guanine, 8-(amino)guanine, 8
(halo)guanine, 8-(hydroxyl)guanine, 8 (thioalkyl)guanine,
8-(thiol)guanine, N (methyl)guanine, 2-(thio)cytosine, 3 (deaza) 5
(aza)cytosine, 3-(alkyl)cytosine, 3 (methyl)cytosine,
5-(alkyl)cytosine, 5-(alkynyl)cytosine, 5 (halo)cytosine, 5
(methyl)cytosine, 5 (propynyl)cytosine, 5 (propynyl)cytosine, 5
(trifluoromethyl)cytosine, 6-(azo)cytosine, N4 (acetyl)cytosine, 3
(3 amino-3 carboxypropyl)uracil, 2-(thio)uracil, 5 (methyl) 2
(thio)uracil, 5 (methylaminomethyl)-2 (thio)uracil, 4-(thio)uracil,
5 (methyl) 4 (thio)uracil, 5 (methylaminomethyl)-4 (thio)uracil, 5
(methyl) 2,4 (dithio)uracil, 5 (methylaminomethyl)-2,4
(dithio)uracil, 5 (2-aminopropyl)uracil, 5-(alkyl)uracil,
5-(alkynyl)uracil, 5-(allylamino)uracil, 5 (aminoallyl)uracil, 5
(aminoalkyl)uracil, 5 (guanidiniumalkyl)uracil, 5
(1,3-diazole-1-alkyl)uracil, 5-(cyanoalkyl)uracil,
5-(dialkylaminoalkyl)uracil, 5 (dimethylaminoalkyl)uracil,
5-(halo)uracil, 5-(methoxy)uracil, uracil-5 oxyacetic acid, 5
(methoxycarbonylmethyl)-2-(thio)uracil, 5
(methoxycarbonyl-methyl)uracil, 5 (propynyl)uracil, 5
(propynyl)uracil, 5 (trifluoromethyl)uracil, 6 (azo)uracil,
dihydrouracil, N3 (methyl)uracil, 5-uracil (i.e., pseudouracil), 2
(thio)pseudouracil, 4 (thio)pseudouraci-1,2,4-(dithio)psuedouracil,
5-(alkyl)pseudouracil, 5-(methyl)pseudouracil,
5-(alkyl)-2-(thio)pseudouracil, 5-(methyl)-2-(thio)pseudouracil,
5-(alkyl)-4 (thio)pseudouracil, 5-(methyl)-4 (thio)pseudouracil,
5-(alkyl)-2,4 (dithio)pseudouracil, 5-(methyl)-2,4
(dithio)pseudouracil, 1 substituted pseudouracil, 1 substituted
2(thio)-pseudouracil, 1 substituted 4 (thio)pseudouracil, 1
substituted 2,4-(dithio)pseudouracil, 1
(aminocarbonylethylenyl)-pseudouracil, 1
(aminocarbonylethylenyl)-2(thio)-pseudouracil, 1
(aminocarbonylethylenyl)-4 (thio)pseudouracil, 1
(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil, 1
(aminoalkylaminocarbonylethylenyl)-pseudouracil, 1
(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil, 1
(aminoalkylaminocarbonylethylenyl)-4 (thio)pseudouracil, 1
(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil,
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl, 7-substituted
1,3-(diaza)-2-(oxo)-phenoxazin-1-yl, 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl, 7-substituted
1,3-(diaza)-2-(oxo)-phenthiazin-1-yl, 7-substituted
1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,
7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,
7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl,
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl,
7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl,
7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl,
1,3,5-(triaza)-2,6-(dioxa)-naphthalene, inosine, xanthine,
hypoxanthine, nubularine, tubercidine, isoguanisine, inosinyl,
2-aza-inosinyl, 7-deaza-inosinyl, nitroimidazolyl, nitropyrazolyl,
nitrobenzimidazolyl, nitroindazolyl, aminoindolyl,
pyrrolopyrimidinyl, 3-(methyl)isocarbostyrilyl,
5-(methyl)isocarbostyrilyl,
3-(methyl)-7-(propynyl)isocarbostyrilyl, 7-(aza)indolyl,
6-(methyl)-7-(aza)indolyl, imidizopyridinyl,
9-(methyl)-imidizopyridinyl, pyrrolopyrizinyl, isocarbostyrilyl,
7-(propynyl)isocarbostyrilyl, propynyl-7-(aza)indolyl,
2,4,5-(trimethyl)phenyl, 4-(methyl)indolyl, 4,6-(dimethyl)indolyl,
phenyl, napthalenyl, anthracenyl, phenanthracenyl, pyrenyl,
stilbenzyl, tetracenyl, pentacenyl, difluorotolyl,
4-(fluoro)-6-(methyl)benzimidazole, 4-(methyl)benzimidazole,
6-(azo)thymine, 2-pyridinone, 5 nitroindole, 3 nitropyrrole,
6-(aza)pyrimidine, 2 (amino)purine, 2,6-(diamino)purine, 5
substituted pyrimidines, N.sup.2-substituted purines,
N.sup.6-substituted purines, O.sup.6-substituted purines,
substituted 1,2,4-triazoles, or any O-alkylated or N-alkylated
derivatives thereof;
[0188] Further purines and pyrimidines include those disclosed in
U.S. Pat. No. 3,687,808, hereby incorporated by reference, those
disclosed in the Concise Encyclopedia Of Polymer Science And
Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley &
Sons, 1990, and those disclosed by Englisch et al., Angewandte
Chemie, International Edition, 1991, 30, 613.
Cationic Groups
[0189] Modifications to oligonucleotides can also include
attachment of one or more cationic groups to the sugar, base,
and/or the phosphorus atom of a phosphate or modified phosphate
backbone moiety. A cationic group can be attached to any atom
capable of substitution on a natural, unusual or universal base. A
preferred position is one that does not interfere with
hybridization, i.e., does not interfere with the hydrogen bonding
interactions needed for base pairing. A cationic group can be
attached e.g., through the C2' position of a sugar or analogous
position in a cyclic or acyclic sugar surrogate. Cationic groups
can include e.g., protonated amino groups, derived from e.g.,
O-AMINE (AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl,
arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino,
ethylene diamine, polyamino); aminoalkoxy, e.g.,
O(CH.sub.2).sub.nAMINE, (e.g., AMINE=NH.sub.2; alkylamino,
dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl
amino, or diheteroaryl amino, ethylene diamine, polyamino); amino
(e.g. NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino,
diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid);
or NH(CH.sub.2CH.sub.2NH).sub.nCH.sub.2CH.sub.2-AMINE
(AMINE=NH.sub.2; alkylamino, dialkylamino, heterocyclyl, arylamino,
diaryl amino, heteroaryl amino, or diheteroaryl amino).
Placement within an Oligonucleotide
[0190] Some modifications may preferably be included on an
oligonucleotide at a particular location, e.g., at an internal
position of a strand, or on the 5' or 3' end of an oligonucleotide.
A preferred location of a modification on an oligonucleotide, may
confer preferred properties on the agent. For example, preferred
locations of particular modifications may confer optimum gene
silencing properties, or increased resistance to endonuclease or
exonuclease activity.
[0191] One or more nucleotides of an oligonucleotide may have a
2'-5' linkage. One or more nucleotides of an oligonucleotide may
have inverted linkages, e.g. 3'-3',5'-5',2'-2' or 2'-3'
linkages.
[0192] An oligonucleotide may comprise at least one
5'-pyrimidine-purine-3' (5'-PyPu-3') dinucleotide wherein the
pyrimidine is modified with a modification chosen independently
from a group consisting of 2'-O-Me (2'-O-methyl), 2'-O-MOE
(2'-.beta.-methoxyethyl), 2'-F, 2'-O-[2-(methylamino)-2-oxoethyl]
(2'-O-NMA), 2'-S-methyl, 2'-O--CH.sub.2-(4'-C) (LNA) and
2'-O--CH.sub.2CH.sub.2-(4'-C) (ENA).
[0193] In one embodiment, the 5'-most pyrimidines in all
occurrences of sequence motif 5'-pyrimidine-purine-3' (5'-PyPu-3')
dinucleotide in the oligonucleotide are modified with a
modification chosen from a group consisting of 2''-O-Me
(2'-O-methyl), 2'-O-MOE (2'-O-methoxyethyl), 2'-F,
2'-O-[2-(methylamino)-2-oxoethyl] (2'-O-NMA), 2'-S-methyl,
2'-O--CH.sub.2-(4'-C) (LNA) and 2'-O--CH.sub.2CH.sub.2-(4'-C)
(ENA).
[0194] A double-stranded oligonucleotide may include at least one
5'-uridine-adenine-3' (5'-UA-3') dinucleotide wherein the uridine
is a 2'-modified nucleotide, or a 5'-uridine-guanine-3' (5'-UG-3')
dinucleotide, wherein the 5'-uridine is a 2'-modified nucleotide,
or a terminal 5'-cytidine-adenine-3' (5'-CA-3') dinucleotide,
wherein the 5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-uridine-uridine-3' (5'-UU-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide, or a terminal
5'-cytidine-cytidine-3' (5'-CC-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-cytidine-uridine-3' (5'-CU-3') dinucleotide, wherein the
5'-cytidine is a 2'-modified nucleotide, or a terminal
5'-uridine-cytidine-3' (5'-UC-3') dinucleotide, wherein the
5'-uridine is a 2'-modified nucleotide. Double-stranded
oligonucleotides including these modifications are particularly
stabilized against endonuclease activity.
General References
[0195] The oligoribonucleotides and oligoribonucleosides used in
accordance with this invention may be synthesized with solid phase
synthesis, see for example "Oligonucleotide synthesis, a practical
approach", Ed. M. J. Gait, IRL Press, 1984; "Oligonucleotides and
Analogues, A Practical Approach", Ed. F. Eckstein, IRL Press, 1991
(especially Chapter 1, Modern machine-aided methods of
oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide
synthesis, Chapter 3,2'-O-Methyloligoribonucleotide-s: synthesis
and applications, Chapter 4, Phosphorothioate oligonucleotides,
Chapter 5, Synthesis of oligonucleotide phosphorodithioates,
Chapter 6, Synthesis of oligo-2'-deoxyribonucleoside
methylphosphonates, and. Chapter 7, Oligodeoxynucleotides
containing modified bases. Other particularly useful synthetic
procedures, reagents, blocking groups and reaction conditions are
described in Martin, P., Helv. Chim. Acta, 1995, 78, 486-504;
Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311
and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,
6123-6194, or references referred to therein. Modification
described in WO 00/44895, WO01/75164, or WO02/44321 can be used
herein. The disclosure of all publications, patents, and published
patent applications listed herein are hereby incorporated by
reference.
Phosphate Group References
[0196] The preparation of phosphinate oligoribonucleotides is
described in U.S. Pat. No. 5,508,270. The preparation of alkyl
phosphonate oligoribonucleotides is described in U.S. Pat. No.
4,469,863. The preparation of phosphoramidite oligoribonucleotides
is described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878.
The preparation of phosphotriester oligoribonucleotides is
described in U.S. Pat. No. 5,023,243. The preparation of borano
phosphate oligoribonucleotide is described in U.S. Pat. Nos.
5,130,302 and 5,177,198. The preparation of 3'-Deoxy-3'-amino
phosphoramidate oligoribonucleotides is described in U.S. Pat. No.
5,476,925. 3'-Deoxy-3'-methylenephosphonate oligoribonucleotides is
described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801.
Preparation of sulfur bridged nucleotides is described in Sproat et
al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.
Tetrahedron Lett. 1989, 30, 4693.
Sugar Group References
[0197] Modifications to the 2' modifications can be found in Verma,
S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references
therein. Specific modifications to the ribose can be found in the
following references: 2'-fluoro (Kawasaki et. al., J. Med. Chem.,
1993, 36, 831-841), 2'-MOE (Martin, P. Helv. Chim. Acta 1996, 79,
1930-1938), "LNA" (Wengel, J. Acc. Chem. Res. 1999, 32,
301-310).
Replacement of the Phosphate Group References
[0198] Methylenemethylimino linked oligoribonucleosides, also
identified herein as MMI linked oligoribonucleosides,
methylenedimethylhydrazo linked oligoribonucleosides, also
identified herein as MDH linked oligoribonucleosides, and
methylenecarbonylamino linked oligonucleosides, also identified
herein as amide-3 linked oligoribonucleosides, and
methyleneaminocarbonyl linked oligonucleosides, also identified
herein as amide-4 linked oligoribonucleosides as well as mixed
backbone compounds having, as for instance, alternating MMI and PO
or PS linkages can be prepared as is described in U.S. Pat. Nos.
5,378,825, 5,386,023, 5,489,677 and in published PCT applications
PCT/US92/04294 and PCT/US92/04305 (published as WO 92/20822 WO and
92/20823, respectively). Formacetal and thioformacetal linked
oligoribonucleosides can be prepared as is described in U.S. Pat.
Nos. 5,264,562 and 5,264,564. Ethylene oxide linked
oligoribonucleosides can be prepared as is described in U.S. Pat.
No. 5,223,618. Siloxane replacements are described in Cormier, J.
F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements
are described in Tittensor, J. R. J. Chem. Soc. C 1971, 1933.
Carboxymethyl replacements are described in Edge, M. D. et al. J.
Chem. Soc. Perkin Trans. 11972, 1991. Carbamate replacements are
described in Stirchak, E. P. Nucleic Acids Res. 1989, 17, 6129.
Replacement of the Phosphate-Ribose Backbone References
[0199] Cyclobutyl sugar surrogate compounds can be prepared as is
described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate
can be prepared as is described in U.S. Pat. No. 5,519,134.
Morpholino sugar surrogates can be prepared as is described in U.S.
Pat. Nos. 5,142,047 and 5,235,033, and other related patent
disclosures. Peptide Nucleic Acids (PNAs) are known per se and can
be prepared in accordance with any of the various procedures
referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties
and Potential Applications, Bioorganic & Medicinal Chemistry,
1996, 4, 5-23. They may also be prepared in accordance with U.S.
Pat. No. 5,539,083.
Terminal Modification References
[0200] Terminal modifications are described in Manoharan, M. et al.
Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and
references therein.
Nuclebases References
[0201] N-2 substituted purine nucleoside amidites can be prepared
as is described in U.S. Pat. No. 5,459,255. 3-Deaza purine
nucleoside amidites can be prepared as is described in U.S. Pat.
No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can
be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl
pyrimidine nucleoside amidites can be prepared as is described in
U.S. Pat. No. 5,484,908. Additional references are disclosed in the
above section on base modifications
Oligonucleotide Production
[0202] The oligonucleotide compounds of the invention can be
prepared using solution-phase or solid-phase organic synthesis.
Organic synthesis offers the advantage that the oligonucleotide
strands comprising non-natural or modified nucleotides can be
easily prepared. Any other means for such synthesis known in the
art may additionally or alternatively be employed. It is also known
to use similar techniques to prepare other oligonucleotides, such
as the phosphorothioates, phosphorodithioates and alkylated
derivatives. The double-stranded oligonucleotide compounds of the
invention may be prepared using a two-step procedure. First, the
individual strands of the double-stranded molecule are prepared
separately. Then, the component strands are annealed.
[0203] Regardless of the method of synthesis, the oligonucleotide
can be prepared in a solution (e.g., an aqueous and/or organic
solution) that is appropriate for formulation. For example, the
oligonucleotide preparation can be precipitated and redissolved in
pure double-distilled water, and lyophilized. The dried
oligonucleotide can then be resuspended in a solution appropriate
for the intended formulation process.
[0204] Teachings regarding the synthesis of particular modified
oligonucleotides may be found in the following U.S. patents or
pending patent applications: U.S. Pat. Nos. 5,138,045 and
5,218,105, drawn to polyamine conjugated oligonucleotides; U.S.
Pat. No. 5,212,295, drawn to monomers for the preparation of
oligonucleotides having chiral phosphorus linkages; U.S. Pat. Nos.
5,378,825 and 5,541,307, drawn to oligonucleotides having modified
backbones; U.S. Pat. No. 5,386,023, drawn to backbone-modified
oligonucleotides and the preparation thereof through reductive
coupling; U.S. Pat. No. 5,457,191, drawn to modified nucleobases
based on the 3-deazapurine ring system and methods of synthesis
thereof; U.S. Pat. No. 5,459,255, drawn to modified nucleobases
based on N-2 substituted purines; U.S. Pat. No. 5,521,302, drawn to
processes for preparing oligonucleotides having chiral phosphorus
linkages; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids;
U.S. Pat. No. 5,554,746, drawn to oligonucleotides having
.beta.-lactam backbones; U.S. Pat. No. 5,571,902, drawn to methods
and materials for the synthesis of oligonucleotides; U.S. Pat. No.
5,578,718, drawn to nucleosides having alkylthio groups, wherein
such groups may be used as linkers to other moieties attached at
any of a variety of positions of the nucleoside; U.S. Pat. Nos.
5,587,361 and 5,599,797, drawn to oligonucleotides having
phosphorothioate linkages of high chiral purity; U.S. Pat. No.
5,506,351, drawn to processes for the preparation of 2'-O-alkyl
guanosine and related compounds, including 2,6-diaminopurine
compounds; U.S. Pat. No. 5,587,469, drawn to oligonucleotides
having N-2 substituted purines; U.S. Pat. No. 5,587,470, drawn to
oligonucleotides having 3-deazapurines; U.S. Pat. No. 5,223,168,
and U.S. Pat. No. 5,608,046, both drawn to conjugated 4'-desmethyl
nucleoside analogs; U.S. Pat. Nos. 5,602,240, and 5,610,289, drawn
to backbone-modified oligonucleotide analogs; and U.S. Pat. Nos.
6,262,241, and 5,459,255, drawn to, inter alia, methods of
synthesizing 2'-fluoro-oligonucleotides.
Angiogenic-Related Disease and Disorder
[0205] The angiogenesis-related disease or disorder can be
selected, for example, from a group consisting of cancer, ascites
formation, psoriasis, age-related macular degeneration, thyroid
hyperplasia, preeclampsia, rheumatoid arthritis and osteoarthritis,
Alzheimer's disease, obesity, psoriasis, atherosclerosis, vascular
malformations, angiomata, pleural effusion, atherosclerosis,
endometriosis, diabetic/other retinopathies, neovascular glaucoma,
age-related macular degeneration, hemangiomas, corneal
neovascularization, sickle cell anemia, sarcoidosis, syphilis,
pseudoxanthoma elasticum, Paget's disease, vein occlusion, artery
occlusion, carotid obstructive disease, chronic uveitis/vitritis,
Mycobacteria infections, Lyme disease, systemic lupus
erythematosis, retinopathy of prematurity, vascular malformations,
angiomata, Eales' disease, Behcet's disease, infections causing
retinitis or choroiditis, presumed ocular histoplasmosis, Best's
disease, myopia, optic pits, Stargardt's disease, pars planitis,
chronic retinal detachment, hyperviscosity syndromes,
toxoplasmosis, histoplasmosis, trauma and post-laser complications.
In one embodiment, the age-related macular degeneration is wet
macular degeneration. Other eye-associated diseases that can
involve inappropriate angiogenesis include, but are not limited to,
diseases associated with rubeosis (neovascularization of the angle)
and diseases caused by the abnormal proliferation of fibrovascular
or fibrous tissue, including all forms of prolific
vitreoretinopathy.
[0206] In one embodiment, the angiogenesis-related disease or
disorder is cancer, where the rapidly dividing neoplastic cancer
cells require an efficient blood supply to sustain the continued
growth of the tumor. As used herein, "cancer" refers to any of
various malignant neoplasms characterized by the proliferation of
anaplastic cells that tend to invade surrounding tissue and
metastasize to new body sites and also refers to the pathological
condition characterized by such malignant neoplastic growths. The
blood vessels provide conduits to metastasize and spread elsewhere
in the body. Upon arrival at the metastatic site, the cancer cells
then work on establishing a new blood supply network.
Administration of a pharmaceutically effective amount of a TRPV4
inhibitor or a pharmaceutical composition comprising a TRPV4
inhibitor and a pharmaceutically acceptable carrier can inhibit
angiogenesis. By inhibiting angiogenesis at the primary tumor site
and secondary tumor site, embodiments of the invention serve to
prevent and limit the progression of the disease.
[0207] It is emphasized that any solid tumor that requires an
efficient blood supply to keep growing is a candidate target. For
example, candidates for the treatment methods described herein
include carcinomas and sarcomas found in the anus, bladder, bile
duct, bone, brain, breast, cervix, colon/rectum, endometrium,
esophagus, eye, gallbladder, head and neck, liver, kidney, larynx,
lung, mediastinum (chest), mouth, ovaries, pancreas, penis,
prostate, skin, small intestine, stomach, spinal marrow, tailbone,
testicles, thyroid and uterus. The types of carcinomas include
papilloma/carcinoma, choriocarcinoma, endodermal sinus tumor,
teratoma, adenoma/adenocarcinoma, melanoma, fibroma, lipoma,
leiomyoma, rhabdomyoma, mesothelioma, angioma, osteoma, chondroma,
glioma, lymphoma/leukemia, squamous cell carcinoma, small cell
carcinoma, large cell undifferentiated carcinomas, basal cell
carcinoma and sinonasal undifferentiated carcinoma. The types of
sarcomas include, for example, soft tissue sarcoma such as alveolar
soft part sarcoma, angiosarcoma, dermatofibrosarcoma, desmoid
tumor, desmoplastic small round cell tumor, extraskeletal
chondrosarcoma, extraskeletal osteosarcoma, fibrosarcoma,
hemangiopericytoma, hemangiosarcoma, Kaposi's sarcoma,
leiomyosarcoma, liposarcoma, lymphangiosarcoma, lymphosarcoma,
malignant fibrous histiocytoma, neurofibrosarcoma,
rhabdomyosarcoma, synovial sarcoma, and Askin's tumor, Ewing's
sarcoma (primitive neuroectodermal tumor), malignant
hemangioendothelioma, malignant schwannoma, osteosarcoma, and
chondrosarcoma. Abnormal build up and growth of blood vessels in
the skin or internal organs in the form of hemangiomas can also be
treated according to the methods described herein.
[0208] In one embodiment, the methods described herein can be used
in preventing blinding blood vessel growth associated with diabetic
eye diseases, namely diabetic retinopathy. The methods described
herein can antagonize the effects of VEGF, a substance naturally
produced in the body that promotes blood vessel formation. Released
by the retina (light-sensitive tissue in back of the eye) when
normal blood vessels are damaged by tiny blood clots due to
diabetes, VEGF turns on its receptor, igniting a chain reaction
that culminates in new blood vessel growth. However, the backup
blood vessels are faulty; they leak, bleed and encourage scar
tissue that detaches the retina, resulting in severe loss of
vision. Such growth is the hallmark of diabetic retinopathy, the
leading cause of blindness among young people in developed
countries.
[0209] In yet another embodiment, the invention can be used in the
treatment of age-related macular degeneration, as it is known that
VEGF also contributes to abnormal blood vessel growth from the
choroid layer of the eye into the retina, similar to what occurs
during the wet or neovascular form of age-related macular
degeneration. Macular degeneration, often called AMD or ARMD
(age-related macular degeneration), is the leading cause of vision
loss and blindness in Americans aged 65 and older. New blood
vessels grow (neovascularization) beneath the retina and leak blood
and fluid. This leakage causes permanent damage to light-sensitive
retinal cells, which die off and create blind spots in central
vision or the macula. Administration of a TRPV4 inhibitor can serve
to inhibit unwanted neovascularization in the choroid layer of the
eye.
[0210] In one embodiment, the angiogenesis-related disease or
disorder is rheumatoid arthritis. Rheumatoid arthritis (RA) is
characterized by synovial tissue swelling, leukocyte ingress and
angiogenesis, or new blood vessel growth. The disease is thought to
occur as an immunological response to an as yet unidentified
antigen. The expansion of the synovial lining of joints in
rheumatoid arthritis (RA) and the subsequent invasion by the pannus
of underlying cartilage and bone necessitate an increase in the
vascular supply to the synovium, to cope with the increased
requirement for oxygen and nutrients. Angiogenesis is now
recognised as a key event in the formation and maintenance of the
pannus in RA (Paleolog, E. M., 2002, Arthritis Res. 4 (Suppl
3):581-S90). Even in early RA, some of the earliest histological
observations are blood vessels. A mononuclear infiltrate
characterizes the synovial tissue along with a luxuriant
vasculature. Angiogenesis is integral to formation of the
inflammatory pannus and without angiogenesis, leukocyte ingress
could not occur (Koch, A. E., 2000, Ann. Rheum. Dis.; 59(Suppl
1):165-i71). Disruption of the formation of new blood vessels would
not only prevent delivery of nutrients to the inflammatory site, it
could also reduce joint swelling due to the additional activity of
VEGF, a potent pro-angiogenic factor in RA, as a vascular
permeability factor. Anti-VEGF hexapeptide RRKRRR (dRK6) (SEQ. ID.
NO. 12) can suppress and mitigate the arthritis severity (Seung-Ah
Yoo, et. al., J. Immunol. 2005, 174:5846-55). Inhibition of
angiogenesis by a pharmaceutically effective amount of TRPV4
inhibitor can also suppress and mitigate the arthritis
severity.
[0211] In one embodiment, the angiogenesis-related disease or
disorder is Alzheimer's disease. Alzheimer's disease (AD) is the
most common cause of dementia worldwide. AD is characterized by an
excessive cerebral amyloid deposition leading to degeneration of
neurons and eventually to dementia. The exact cause of AD is still
unknown. It has been shown by epidemiological studies that
long-term use of non-steroidal anti-inflammatory drugs, statins,
histamine H2-receptor blockers, or calcium-channel blockers, all of
which are cardiovascular drugs with anti-angiogenic effects, seem
to prevent Alzheimer's disease and/or influence the outcome of AD
patients. Therefore, it has been speculated that angiogenesis in
the brain vasculature may play an important role in AD. In
Alzheimer's disease, the brain endothelium secretes the precursor
substrate for the beta-amyloid plaque and a neurotoxic peptide that
selectively kills cortical neurons. Moreover amyloid deposition in
the vasculature leads to endothelial cell apoptosis and endothelial
cell activation which leads to neovascularization. Vessel formation
could be blocked by the VEGF antagonist SU 4312 as well as by
statins, indicating that anti-angiogenesis strategies based on VEGF
inhibition can interfere with endothelial cell activation in AD
(Schultheiss C., el. al., 2006, Angiogenesis. 9(2):59-65; Grammas
P., et. al., 1999, Am. J. Path., 154(2):337-42) and can be used for
preventing and/or treating AD. In the same way, the anti-angiogenic
properties of a pharmaceutically effective amount of TRPV4
inhibitor can be useful preventing and/or treating AD.
[0212] In one embodiment, the pathological angiogenic disease or
disorder is obesity. Adipogenesis in obesity requires close
interplay between differentiating adipocytes, stromal cells, and
blood vessels. There are close spatial and temporal
interrelationships between blood vessel formation and adipogenesis,
and the sprouting of new blood vessels from preexisting vasculature
is coupled to adipocyte differentiation. Adipogenic/angiogenic cell
clusters can morphologically and immunohistochemically be
distinguished from crown-like structures frequently seen in the
late stages of adipose tissue obesity. Administration of anti-VEGF
antibodies inhibited not only angiogenesis but also the formation
of adipogenic/angiogenic cell clusters, indicating that the
coupling of adipogenesis and angiogenesis is essential for
differentiation of adipocytes in obesity. (Satoshi Nishimura et.
al., 2007, Diabetes 56:1517-1526). It has been shown that the
angiogenesis inhibitor, TNP-470 was able to prevent diet-induced
and genetic obesity in mice (Ebba Brakenhielm et. al., Circulation
Research, 2004; 94:1579). TNP-470 reduced vascularity in the
adipose tissue, thereby inhibiting the rate of growth of the
adipose tissue and obesity development.
[0213] In one embodiment, the angiogenesis-related disease or
disorder is endometriosis. Excessive endometrial angiogenesis is
proposed as an important mechanism in the pathogenesis of
endometriosis (Healy, D L., et. al., 1998, Human Reproduction
Update, 4:736-740). The endometrium of patients with endometriosis
shows enhanced endothelial cell proliferation. Moreover there is an
elevated expression of the cell adhesion molecule integrin v.beta.3
in blood vessels in the endometrium of women with endometriosis
when compared with normal women. U.S. Pat. No. 6,121,230 described
the use of anti-VEGF agents in the treatment of endometriosis and
this patent is incorporated hereby reference. Encompassed in the
methods disclosed herein is the treatment of endometriosis with
anti-angiogenic therapy. Encompassed in the methods disclosed
herein is the treatment of obesity with anti-angiogenic therapy,
including the use of a pharmaceutically effective amount of TRPV4
inhibitor.
[0214] Diseases associated with chronic inflammation are
accompanied by angiogenesis and can be treated by the compositions
and methods of the present invention. Diseases with symptoms of
chronic inflammation include obesity, inflammatory bowel diseases
such as Crohn's disease and ulcerative colitis, psoriasis,
sarcoidosis, atherosclerosis including plaque rupture, Sjogrens
disease, acne rosacea, syphilis, chemical burns, bacterial ulcers,
fungal ulcers, Behcet's syndrome, Stevens-Johnson's disease,
Mycobacteria infections, Herpes simplex infections, Herpes zoster
infections, protozoan infections, Mooren's ulcer, leprosy,
Wegener's sarcoidosis, pemphigoid, lupus, systemic lupus
erythematosis, polyarteritis, lyme's disease, Bartonelosis,
tuberculosis, histoplasmosis and toxoplasmosis. Angiogenesis is a
key element that these chronic inflammatory diseases have in
common. The chronic inflammation depends on continuous formation of
capillary sprouts to maintain an influx of inflammatory cells. The
influx and presence of the inflammatory cells sometimes produce
granulomas to help maintain the chronic inflammatory state.
Inhibition of angiogenesis by the compositions and methods of the
present invention would prevent the formation of the granulomas and
alleviate the disease.
[0215] The inflammatory bowel diseases also show extraintestinal
manifestations such as skin lesions. Such lesions are characterized
by inflammation and angiogenesis and can occur at many sites other
than the gastrointestinal tract. The compositions and methods of
the present invention are also capable of treating these lesions by
preventing the angiogenesis, thus, reducing the influx of
inflammatory cells and the lesion formation.
[0216] Sarcoidosis is another chronic inflammatory disease that is
characterized as a multisystem granulomatous disorder. The
granulomas of this disease may form anywhere in the body, and,
thus, the symptoms depend on the site of the granulomas and whether
the disease active. The granulomas are created by the angiogenic
capillary sprouts providing a constant supply of inflammatory
cells.
Anti-Angiogenic Factors/Drugs
[0217] In one embodiment, the pharmaceutically effective amount of
TRPV4 inhibitor can be administer in conjunction with one or more
additional anti-angiogenic factors, drugs or therapeutics. For
example, other potent angiogenesis inhibitors such as angiostatin,
endostatin and cleaved antithrombin III can be incorporated into
the composition.
[0218] There are three main types of anti-angiogenic drugs that are
currently approved by the United States Food and Drug
Administration (FDA) for the treatment of cancer and tumors: (1)
Drugs that stop new blood vessels from sprouting (true angiogenesis
inhibitors); (2) Drugs that attack a tumor's established blood
supply (vascular targeting agents); and (3) Drugs that attack both
the cancer cells as well as the blood vessel cells (the
double-barreled approach).
[0219] In one embodiment, the anti-angiogenic therapy includes but
is not limited to the administration of monoclonal antibody
therapies directed against specific pro-angiogenic growth factors
and/or their receptors. Examples of these are: bevacizumab
(AVASTIN.RTM.), cetuximab (ERBITUX.RTM.), panitumumab
(VECTIBIX.TM.), and trastuzumab (HERCEPTIN.RTM.).
[0220] In another embodiment, the anti-angiogenic therapies include
but are not limited to administration of small molecule tyrosine
kinase inhibitors (TKIs) of multiple pro-angiogenic growth factor
receptors. The three TKIs that are currently approved as
anti-cancer therapies are erlotinib (TARCEVA.RTM.), sorafenib
(NEXAVAR.RTM.), and sunitinib (SUTENT.RTM.).
[0221] In another embodiment, the anti-angiogenic therapies include
but are not limited to administration of inhibitors of mTOR
(mammalian target of rapamycin) such as temsirolimus (TORICEL.TM.),
bortezomib (VELCADE.RTM.), thalidomide (THALOMID.RTM.) and
Doxycyclin.
[0222] Many of the current anti-angiogenesis factors or drugs
attack the VEGF pathway. Bevacizumab (AVASTIN.RTM.) was the first
drug that targeted new blood vessels to be approved for use against
cancer. It is a monoclonal antibody that binds to VEGF, thereby
blocking VEGF from reaching the VEGF receptor (VEGFR). Other drugs,
such as sunitinib (SUTENT.RTM.) and sorafenib (NEXAVAR.RTM.), are
small molecules that attach to the VEGF receptor itself, preventing
it from being turned on. Such drugs are collectively termed VEGF
inhibitors.
[0223] As the VEGF protein interacts with the VEGFRs, inhibition of
either the ligand VEGF, e.g. by reducing the amount that is
available to interact with the receptor; or inhibition of the
receptor's intrinsic tyrosine kinase activity, blocks the function
of this pathway. This pathway controls endothelial cell growth, as
well as permeability, and these functions are mediated through the
VEGFRs.
[0224] "VEGF inhibitors" include any compound or agent that
produces a direct or indirect effect on the signaling pathways that
promote growth, proliferation and survival of a cell by inhibiting
the function of the VEGF protein, including inhibiting the function
of VEGF receptor proteins. These include any organic or inorganic
molecule, including, but not limited to modified and unmodified
nucleic acids such as antisense nucleic acids, RNAi agents such as
siRNA or shRNA, peptides, peptidomimetics, receptors, ligands, and
antibodies that inhibit the VEGF signaling pathway. The siRNAs are
targeted at components of the VEGF pathways and can inhibit the
VEGF pathway. Preferred VEGF inhibitors, include for example,
AVASTIN.RTM. (bevacizumab), an anti-VEGF monoclonal antibody of
Genentech, Inc. of South San Francisco, Calif., VEGF Trap
(Regeneron/Aventis). Additional VEGF inhibitors include CP-547,632
(3-(4-Bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin
1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide
hydrochloride; Pfizer Inc., NY), AG13736, AG28262 (Pfizer Inc.),
SU5416, SU11248, & SU6668 (formerly Sugen Inc., now Pfizer, New
York, N.Y.), ZD-6474 (AstraZeneca), ZD4190 which inhibits VEGF-R2
and -R1 (AstraZeneca), CEP-7055 (Cephalon Inc., Frazer, Pa.), PKC
412 (Novartis), AEE788 (Novartis), AZD-2171), NEXAVAR.RTM. (BAY
43-9006, sorafenib; Bayer Pharmaceuticals and Onyx
Pharmaceuticals), vatalanib (also known as PTK-787, ZK-222584:
Novartis & Schering: AG), MACUGEN.RTM. (pegaptanib octasodium,
NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862 (glufanide
disodium, Cytran Inc. of Kirkland, Wash., USA), VEGFR2-selective
monoclonal antibody DC101 (ImClone Systems, Inc.), angiozyme, a
synthetic ribozyme from Ribozyme (Boulder, Colo.) and Chiron
(Emeryville, Calif.), Sirna-027 (an siRNA-based VEGFR1 inhibitor,
Sirna Therapeutics, San Francisco, Calif.) Caplostatin, soluble
ectodomains of the VEGF receptors, Neovastat (IEterna Zentaris Inc;
Quebec City, Calif.), ZM323881 (CALBIOCHEM.RTM. CA, USA),
pegaptanib (Macugen) (Eyetech Pharmaceuticals), an anti-VEGF
aptamer and combinations thereof.
[0225] VEGF inhibitors are also disclosed in U.S. Pat. Nos.
6,534,524 and 6,235,764. Additional VEGF inhibitors are described,
for example, in WO 99/24440; WO 95/21613; WO 98/50356; WO 99/10349;
WO 97/22596; WO 97/32856; WO 98/54093; WO 98/02438; WO 99/16755; WO
99/61422; WO 99/62890; WO 98/02437; WO 01/02369; WO 01/95353; WO
02/44158; WO 03/106462A1; U.S. Pat. Publ. No. 20060094032; U.S.
Pat. Nos. 6,534,524; 5,834,504; 5,883,113; 5,886,020; 5,792,783;
6,653,308; U.S. Provisional Application No. 60/491,771; and
60/460,695. These references are incorporated herein in their
entirety.
[0226] In one embodiment, the pharmaceutically effective amount of
TRPV4 inhibitor can be administer in conjunction with VEGF
inhibitors.
[0227] In another embodiment, other anti-angiogenic factors include
but are not limited to alpha-2 antiplasmin (fragment), angiostatin
(plasminogen fragment), antiangiogenic antithrombin III,
cartilage-derived inhibitor (CDI), CD59 complement fragment,
endostatin (collagen XVIII fragment), fibronectin fragment,
gro-beta (a C--X--C chemokine), heparinases heparin hexasaccharide
fragment, human chorionic gonadotropin (hCG), interferon
alpha/beta/gamma, interferon inducible protein (IP-10),
interleukin-12, kringle 5 (plasminogen fragment),
beta-thromboglobulin, EGF (fragment), VEGF inhibitor, endostatin,
fibronection (45 kD fragment), high molecular weight kininogen
(domain 5), NK1, NK2, NK3 fragments of HGF, PF-4, serpin proteinase
inhibitor 8, TGF-beta-1, thrombospondin-1, prosaposin, p53,
angioarrestin, metalloproteinase inhibitors (TIMPs),
2-Methoxyestradiol, placental ribonuclease inhibitor, plasminogen
activator inhibitor, prolactin 16 kD fragment, proliferin-related
protein (PRP), retinoids, tetrahydrocortisol-S transforming growth
factor-beta (TGF-b), vasculostatin, and vasostatin (calreticulin
fragment), pamidronate-thalidomide, TNP470, the bisphosphonate
family such as amino-bisphosphonate zoledronic acid.
bombesin/gastrin-releasing peptide (GRP) antagonists such as
RC-3095 and RC-3940-II (Bajol A M, et. al., British Journal of
Cancer (2004) 90, 245-252), and anti-VEGF peptide RRKRRR (dRK6)
(SEQ. ID. NO. 12) (Seung-Ah Yoo, J. Immuno, 2005, 174:
5846-5855).
Anti-Angiogenesis Assay Methods
[0228] The effectiveness of any given TRPV4 inhibitor as described
herein can be evaluated in vitro or in vivo or both, as described,
e.g., in the examples provided herein below. For the avoidance of
doubt, one can also use other assays commonly accepted in the
field. For example, one can use the "CAM" assay. The chick
chorioallantoic membrane (CAM) assay is frequently used to evaluate
the effects of angiogenesis regulating factors because it is
relatively easy and provides relatively rapid results. A TRPV4
inhibitor useful in the methods described herein will decrease the
number of microvessels in the modified CAM assay described by
Iruela-Arispe et al., 1999, Circulation 100: 1423-1431
(incorporated herein by reference in its entirety), relative to
controls with no TRPV4 inhibitor that is added (control). The
method is based on the vertical growth of new capillary vessels
into a collagen gel pellet placed on the CAM. In the assay as
described by Iruela-Arispe et al., the collagen gel is supplemented
with VEGF.sub.165 (250 ng/gel) in the presence or absence of a
TRPV4 inhibitor. The extent of the anti-angiogenic effect is
measured using FITC-dextran (50 .mu.g/mL) (SIGMA ALDRICH.RTM.)
injected into the circulation of the CAM. The degree of
fluorescence intensity parallels variations in capillary density;
the linearity of this correlation can be observed with a range of
capillaries between 5 and 540. Morphometric analyses are performed,
for example, by acquisition of images with a CCD camera. Images are
then analyzed by importing into an analysis package, e.g., NHImage
1.59, and measurements of fluorescence intensity are obtained as
positive pixels. Each data point is compared with its own positive
and negative controls present in the same CAM and interpreted as a
percentage of inhibition, considering the positive control to be
100% (VEGF.sub.165 alone) and the negative control (vehicle alone)
0%. Statistical evaluation of the data is performed to check
whether groups differ significantly from random, e.g., by analysis
of contingency with Yates' correction.
[0229] Additional angiogenesis assays are known in the art and can
be used to evaluate a TRPV4 inhibitor for use in the methods
described herein. These include, for example, the corneal
micropocket assay, hamster cheek pouch assay, the MATRIGEL.TM.
assay and modifications thereof, and co-culture assays. Donovan et
al. describe a comparison of three different in vitro assays
developed to evaluate angiogenesis regulators in a human background
(Donovan et al., 2001, Angiogenesis 4: 113-121, incorporated herein
by reference). Briefly, the assays examined include: 1) a basic
MATRIGEL.TM. assay in which low passage human endothelial cells
(Human umbilical vein endothelial cells, HUVEC) are plated in wells
coated with MATRIGEL.TM. (Becton Dickinson, Cedex, France) with or
without angiogenesis regulator(s); 2) a similar MATRIGEL.TM. assay
using "growth factor reduced" or GFR MATRIGEL.TM.; and 3) a
co-culture assay in which primary human fibroblasts and HUVEC are
co-cultured with or without additional angiogenesis
regulator(s)--the fibroblasts produce extracellular matrix and
other factors that support HUVEC differentiation and tubule
formation. In the Donovan et al. paper the co-culture assay
provided microvessel networks that most closely resembled
microvessel networks in vivo. Other CE cells, such as the bovine CE
cells described herein, can be used instead of HUVEC. In addition,
the basic MATRIGEL.TM. assay and the GFR MATRIGEL.TM. assay can
also be used by one of skill in the art to evaluate whether a given
TRPV4 inhibitor is an angiogenesis inhibitor as necessary for the
methods described herein. Finally, an in vitro angiogenesis assay
kit is marketed by CHEMICON.RTM.. The Fibrin Gel In Vitro
Angiogenesis Assay Kit is CHEMICON.RTM. Catalog No. ECM630. A TRPV4
inhibitor as described herein is considered useful in a method for
the inhibition of angiogenesis and for the treatment of an
angiogenesis-related disease or disorder as described herein if it
reduces angiogenesis in any one of these assays by 10% or more
relative to a control assay performed the absence of any TRPV4
inhibitor. A TRPV4 inhibitor as described herein preferably reduces
angiogenesis in one or more of these assays by at least 10%, at
least 20%, at least 30%, at least 40%, at least 50%, at least 60%,
at least 70%, at least 80%, at least 90% or more, up to and
including 100% inhibition.
[0230] Alternatively, angiogenesis inhibition can be measured
functionally downstream, as a reduction or cessation of tumor
growth or tumor size. For example, if there is zero growth of tumor
mass, or at least 5% reduction in the size of the tumor mass, there
is angiogenesis inhibition by the methods as described herein.
Formulation and Administration
[0231] In one embodiment, the TRPV4 inhibitor is delivered in a
pharmaceutically acceptable carrier.
[0232] In one embodiment, the term "pharmaceutically acceptable"
means approved by a regulatory agency of the Federal or a state
government or listed in the U.S. Pharmacopeia or other generally
recognized pharmacopeia for use in animals, and more particularly
in humans. Specifically, it refers to those compounds, materials,
compositions, and/or dosage forms which are, within the scope of
sound medical judgment, suitable for use in contact with the
tissues of human beings and animals without excessive toxicity,
irritation, allergic response, or other problem or complication,
commensurate with a reasonable benefit/risk ratio.
[0233] The term "carrier" refers to a diluent, adjuvant, excipient,
or vehicle with which the therapeutic is administered. Such
pharmaceutical carriers can be sterile liquids, such as water and
oils, including those of petroleum, animal, vegetable or synthetic
origin, such as peanut oil, soybean oil, mineral oil, sesame oil
and the like. Water is a preferred carrier when the pharmaceutical
composition is administered intravenously. Saline solutions and
aqueous dextrose and glycerol solutions can also be employed as
liquid carriers, particularly for injectable solutions. Suitable
pharmaceutical excipients include starch, glucose, lactose,
sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium
stearate, glycerol monostearate, talc, sodium chloride, dried skim
milk, glycerol, propylene, glycol, water, ethanol and the like. The
composition, if desired, can also contain minor amounts of wetting
or emulsifying agents, or pH buffering agents. These compositions
can take the form of solutions, suspensions, emulsion, tablets,
pills, capsules, powders, sustained-release formulations, and the
like. The composition can be formulated as a suppository, with
traditional binders and carriers such as triglycerides. Oral
formulation can include standard carriers such as pharmaceutical
grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate, etc. Examples of
suitable pharmaceutical carriers are described in Remington's
Pharmaceutical Sciences, 18th Ed., Gennaro, ed. (Mack Publishing
Co., 1990). The formulation should suit the mode of administration.
Additional carrier agents, such as liposomes, can be added to the
pharmaceutically acceptable carrier.
[0234] As used herein, the terms "administering," refers to the
placement of a TRPV4 inhibitor that can inhibit angiogenesis into a
subject by a method or route which results in at least partial
localization of the TRPV4 at a desired site. The TRPV4 inhibitor
can be administered by any appropriate route which results in an
effective treatment in the subject.
[0235] As used herein, the term "comprising" or "comprises" is used
in reference to methods, and respective component(s) thereof, that
are essential to the invention, yet open to the inclusion of
unspecified elements, whether essential or not. The use of
"comprising" indicates inclusion rather than limitation.
[0236] The term "consisting of" refers to methods, and respective
components thereof as described herein, which are exclusive of any
element not recited in that description of the embodiment.
[0237] Therapeutic compositions contain a physiologically tolerable
carrier together with an active agent as described herein,
dissolved or dispersed therein as an active ingredient. In a
preferred embodiment, the therapeutic composition is not
immunogenic when administered to a mammal or human patient for
therapeutic purposes. As used herein, the terms "pharmaceutically
acceptable", "physiologically tolerable" and grammatical variations
thereof, as they refer to compositions, carriers, diluents and
reagents, are used interchangeably and represent that the materials
are capable of administration to or upon a mammal without the
production of undesirable physiological effects such as nausea,
dizziness, gastric upset and the like. A pharmaceutically
acceptable carrier will not promote the raising of an immune
response to an agent with which it is admixed, unless so desired.
The preparation of a pharmacological composition that contains
active ingredients dissolved or dispersed therein is well
understood in the art and need not be limited based on formulation.
Typically such compositions are prepared as injectable either as
liquid solutions or suspensions, however, solid forms suitable for
solution, or suspensions, in liquid prior to use can also be
prepared. The preparation can also be emulsified or presented as a
liposome composition. The active ingredient can be mixed with
excipients which are pharmaceutically acceptable and compatible
with the active ingredient and in amounts suitable for use in the
therapeutic methods described herein. Specifically contemplated
pharmaceutical compositions are active RNAi ingredients in a
preparation for delivery as described herein above, or in
references cited and incorporated herein in that section. Suitable
excipients include, for example, water, saline, dextrose, glycerol,
ethanol or the like and combinations thereof. In addition, if
desired, the composition can contain minor amounts of auxiliary
substances such as wetting or emulsifying agents, pH buffering
agents and the like which enhance the effectiveness of the active
ingredient. The therapeutic composition of the present invention
can include pharmaceutically acceptable salts of the components
therein. Pharmaceutically acceptable salts include the acid
addition salts (formed with the free amino groups of the
polypeptide) that are formed with inorganic acids such as, for
example, hydrochloric or phosphoric acids, or such organic acids as
acetic, tartaric, mandelic and the like. Salts formed with the free
carboxyl groups can also be derived from inorganic bases such as,
for example, sodium, potassium, ammonium, calcium or ferric
hydroxides, and such organic bases as isopropylamine,
trimethylamine, 2-ethylamino ethanol, histidine, procaine and the
like. Physiologically tolerable carriers are well known in the art.
Exemplary liquid carriers are sterile aqueous solutions that
contain no materials in addition to the active ingredients and
water, or contain a buffer such as sodium phosphate at
physiological pH value, physiological saline or both, such as
phosphate-buffered saline. Still further, aqueous carriers can
contain more than one buffer salt, as well as salts such as sodium
and potassium chlorides, dextrose, polyethylene glycol and other
solutes. Liquid compositions can also contain liquid phases in
addition to and to the exclusion of water. Exemplary of such
additional liquid phases are glycerin, vegetable oils such as
cottonseed oil, and water-oil emulsions. The amount of an active
agent used in the methods described herein that will be effective
in the treatment of a particular disorder or condition will depend
on the nature of the disorder or condition, and can be determined
by standard clinical techniques.
[0238] Routes of administration include, but are not limited to,
direct injection, intradermal, intravitreal, intramuscular,
intraperitoneal, intravenous, subcutaneous, intranasal, epidural,
and oral routes. The agent can be administered by any convenient
route, for example by infusion or bolus injection, by absorption
through epithelial or mucocutaneous linings (e.g., oral mucosa,
rectal and intestinal mucosa, etc.) and may be administered
together with other biologically active agents. Administration can
be systemic or local.
[0239] The precise dose and formulation to be employed depends upon
the potency of the inhibitor, and include amounts large enough to
produce the desired effect, e.g., a reduction in invasion of new
blood vessels in the eye or elsewhere. The dosage should not be so
large as to cause unacceptable adverse side effects. Generally, the
dosage will vary with the type of TRPV4 inhibitor (e.g., an
antibody or fragment, small molecule, siRNA, etc.), and with the
age, condition, and sex of the patient are also considered. Dosage
and formulation of the TRPV4 inhibitor will also depend on the
route of administration, and the seriousness of the disease or
disorder, and should be decided according to the judgment of the
practitioner and each patient's circumstances. Effective doses can
be extrapolated from dose-response curves derived from in vitro or
animal model test systems.
[0240] The dosage can be determined by one of skill in the art and
can also be adjusted by the individual physician in the event of
any complication. Typically, the dosage ranges from 0.001 mg/kg
body weight to 5 g/kg body weight. In some embodiments, the dosage
range is from 0.001 mg/kg body weight to 1 g/kg body weight, from
0.001 mg/kg body weight to 0.5 g/kg body weight, from 0.001 mg/kg
body weight to 0.1 g/kg body weight, from 0.001 mg/kg body weight
to 50 mg/kg body weight, from 0.001 mg/kg body weight to 25 mg/kg
body weight, from 0.001 mg/kg body weight to 10 mg/kg body weight,
from 0.001 mg/kg body weight to 5 mg/kg body weight, from 0.001
mg/kg body weight to 1 mg/kg body weight, from 0.001 mg/kg body
weight to 0.1 mg/kg body weight, from 0.001 mg/kg body weight to
0.005 mg/kg body weight. Alternatively, in some embodiments the
dosage range is from 0.1 g/kg body weight to 5 g/kg body weight,
from 0.5 g/kg body weight to 5 g/kg body weight, from 1 g/kg body
weight to 5 g/kg body weight, from 1.5 g/kg body weight to 5 g/kg
body weight, from 2 g/kg body weight to 5 g/kg body weight, from
2.5 g/kg body weight to 5 g/kg body weight, from 3 g/kg body weight
to 5 g/kg body weight, from 3.5 g/kg body weight to 5 g/kg body
weight, from 4 g/kg body weight to 5 g/kg body weight, from 4.5
g/kg body weight to 5 g/kg body weight, from 4.8 g/kg body weight
to 5 g/kg body weight. In one embodiment, the dose range is from 5
.mu.g/kg body weight to 30 .mu.g/kg body weight. Alternatively, the
dose range will be titrated to maintain serum levels between 5
.mu.g/mL and 30 .mu.g/mL.
[0241] Administration of the doses recited above can be repeated
for a limited period of time. In some embodiments, the doses are
given once a day, or multiple times a day, for example but not
limited to three times a day. In a preferred embodiment, the doses
recited above are administered daily for several weeks or months.
The duration of treatment depends upon the subject's clinical
progress and responsiveness to therapy. Continuous, relatively low
maintenance doses are contemplated after an initial higher
therapeutic dose.
[0242] As exemplary, for the treatment of solid tumors that are
accessible by catheters or needles, the TRPV4 inhibitor and a
pharmaceutically acceptable carrier can be formulated for direct
application by injection into the solid tumor and/or adjacent to
the tumor site, e.g. melanoma and hemangiomas. The inhibitor can
also be formulated for a transdermal delivery, e.g. a skin patch.
For cancers or tumors not so easily accessible, the TRPV4 inhibitor
can be administered to one of the main blood vessel that drains the
cancer site, e.g. into the portal vein for liver cancer. For the
treatment of macular degeneration or retinopathy, the TRPV4
inhibitor can be formulated for direct injection into the vitreous
cavity of the affected eye.
[0243] In one embodiment, the TRPV4 inhibitor is an RNA
interference molecule such as an siRNA. Such siRNA is delivered by
delivering a vector encoding small hairpin RNA (shRNA) in a
pharmaceutically acceptable carrier to the cells in an organ of an
individual. The shRNA is converted by the cells after transcription
into siRNA capable of targeting TRPV4. In one embodiment, the
vector can be a regulatable vector, such as tetracycline inducible
vector. Such vectors with inducible promoters are well known in the
art and are also easily found in the commercial sector, e.g.
pSingle-tTS-shRNA vector from CLONTECH.RTM..
[0244] In one embodiment, the treatment of angiogenesis-related
diseases related to the eyes, e.g. macular degeneration or diabetic
retinopathy, comprises directly injecting an siRNA, dsRNA, or shRNA
vector directed against a TRPV4 gene into the vitreous cavity of
the affected eye.
[0245] In other embodiments, the treatment of angiogenesis-related
diseases having localized aberrant angiogenesis, e.g. solid
non-metastatic tumor, arthritis, and endometriosis, comprises
directly injecting an siRNA, dsRNA, or shRNA vector directed
against a TRPV4 gene to the location of tissue with aberrant
angiogenesis.
[0246] In one embodiment, the RNA interfering molecules used in the
methods described herein are taken up actively by cells in vivo
following intravenous injection, e.g., hydrodynamic injection,
without the use of a vector, illustrating efficient in vivo
delivery of the RNA interfering molecules, e.g., the siRNAs used in
the methods of the invention.
[0247] Other strategies for delivery of the RNA interfering
molecules, e.g., the siRNAs or shRNAs used in the methods of the
invention, can also be employed, such as, for example, delivery by
a vector, e.g., a plasmid or viral vector, e.g., a lentiviral
vector. Such vectors can be used as described, for example, in
Xiao-Feng Qin et al. Proc. Natl. Acad. Sci. U.S.A., 100: 183-188.
Other delivery methods include delivery of the RNA interfering
agents, e.g., the siRNAs or shRNAs of the invention, using a basic
peptide by conjugating or mixing the RNA interfering agent with a
basic peptide, e.g., a fragment of a TAT peptide, mixing with
cationic lipids or formulating into particles.
[0248] As noted, the dsRNA, such as siRNA or shRNA can be delivered
using an inducible vector, such as a tetracycline inducible vector.
Methods described, for example, in Wang et al. Proc. Natl. Acad.
Sci. 100: 5103-5106, using pTet-On vectors (BD Biosciences
Clontech, Palo Alto, Calif.) can be used. In some embodiments, a
vector can be a plasmid vector, a viral vector, or any other
suitable vehicle adapted for the insertion and foreign sequence and
for the introduction into eukaryotic cells. The vector can be an
expression vector capable of directing the transcription of the DNA
sequence of the agonist or antagonist nucleic acid molecules into
RNA. Viral expression vectors can be selected from a group
comprising, for example, reteroviruses, lentiviruses, Epstein Barr
virus-, bovine papilloma virus, adenovirus- and
adeno-associated-based vectors or hybrid virus of any of the above.
In one embodiment, the vector is episomal. The use of a suitable
episomal vector provides a means of maintaining the antagonist
nucleic acid molecule in the subject in high copy number extra
chromosomal DNA thereby eliminating potential effects of
chromosomal integration.
[0249] In some embodiments, the siRNA, dsRNA, or shRNA vector
directed against a TRPV4 gene is administered intravenously, e.g.
via central venous catheter (CVC or central venous line or central
venous access catheter) placed into a large vein in the neck
(internal jugular vein), chest (subclavian vein) or groin (femoral
vein). Methods of systemic delivery of siRNA, dsRNA, or shRNA
vector are well known in the art, e.g. as described herein and in
Gao and Huang, 2008, (Mol. Pharmaceutics, Web publication December
30) and review by Rossil, 2006, Gene Therapy, 13:583-584. The
siRNA, dsRNA, or shRNA vector can be formulated in various ways,
e.g. conjugation of a cholesterol moiety to one of the strands of
the siRNA duplex for systemic delivery to the liver and jejunum
(Soutschek J. et. al. 2004, Nature, 432:173-178), complexing of
siRNAs to protamine fused with an antibody fragment for
receptor-mediated targeting of siRNAs (Song E, et al. 2005, Nat
Biotechnol., 23: 709-717) and the use of a lipid bilayer system by
Morrissey et al. 2005 (Nat. Biotechnol., 23: 1002-1007). The lipid
bilayer system produces biopolymers that are in the 120 nanometer
diameter size range, and are labeled as SNALPs, for
Stable-Nucleic-Acid-Lipid-Particles. The lipid combination protects
the siRNAs from serum nucleases and allows cellular endosomal
uptake and subsequent cytoplasmic release of the siRNAs (see
WO/2006/007712). These references are incorporated by reference in
their entirety.
[0250] In another embodiment, the treatment of angiogenesis-related
diseases related to the eyes, e.g. macular degeneration or diabetic
retinopathy comprises directly injecting an antibody specifically
against a .beta.1 integrin function into the vitreous cavity of the
eye, wherein the integrin function is blocked by the antibody.
[0251] In other embodiments, the treatment of angiogenesis-related
diseases having localized aberrant angiogenesis, e.g. solid
non-metastatic tumor, arthritis, and endometriosis, comprises
directly injecting an antibody specifically against a .beta.1
integrin function into the location or tissue with aberrant
angiogenesis, wherein the integrin function is blocked by the
antibody.
[0252] In one embodiment, the antibody in a monoclonal antibody
derived from clone P5D2.
[0253] In some embodiments, the TRPV4 inhibitor is a an antibody. a
small molecule, a peptide or an aptamer. Such an inhibitor can be
targeted to specific organ or tissue by means of a targeting
moiety, such as e.g., an antibody or targeted liposome technology.
In some embodiments, aTRPV4 inhibitor can be targeted to tissue- or
tumor-specific targets by using bispecific antibodies, for example
produced by chemical linkage of an anti-ligand antibody (Ab) and an
Ab directed toward a specific target. To avoid the limitations of
chemical conjugates, molecular conjugates of antibodies can be used
for production of recombinant bispecific single-chain Abs directing
ligands and/or chimeric inhibitors at cell surface molecules. The
conjugation of an antibody to a TRPV4 inhibitor permits the
inhibitor attached to accumulate additively at the desired target
site. Antibody-based or non-antibody-based targeting moieties can
be employed to deliver a ligand or the inhibitor to a target site.
Preferably, a natural binding agent for an unregulated or disease
associated antigen is used for this purpose.
[0254] For therapeutic applications, the antibodies can be
administered to a mammal, preferably a human, in a pharmaceutically
acceptable dosage form, including those that may be administered to
a human intravenously as a bolus or by continuous infusion over a
period of time, by intramuscular, subcutaneous, intra-articular,
intrasynovial, intrathecal, oral, topical, or inhalation routes.
The antibody is also suitably administered by intratumoral,
peritumoral, intralesional, or perilesional routes, to exert local
as well as systemic therapeutic effects.
[0255] In some embodiments, the antibody is administered
intravenously, e.g. via central venous catheter (CVC or central
venous line or central venous access catheter) placed into a large
vein in the neck (internal jugular vein), chest (subclavian vein)
or groin (femoral vein).
[0256] Such dosage forms encompass pharmaceutically acceptable
carriers that are inherently nontoxic and nontherapeutic. Examples
of such carriers include ion exchangers, alumina, aluminum
stearate, lecithin, serum proteins, such as human serum albumin,
buffer substances such as phosphates, glycine, sorbic acid,
potassium sorbate, partial glyceride mixtures of saturated
vegetable fatty acids, water, salts, or electrolytes such as
protamine sulfate, disodium hydrogen phosphate, potassium hydrogen
phosphate, sodium chloride, zinc salts, colloidal silica, magnesium
trisilicate, polyvinyl pyrrolidone, cellulose-based substances, and
polyethylene glycol. Carriers for topical or gel-based forms of
antibody include polysaccharides such as sodium
carboxymethylcellulose or methylcellulose, polyvinylpyrrolidone,
polyacrylates, polyoxyethylene-polyoxypropylene-block polymers,
polyethylene glycol, and wood wax alcohols. For all
administrations, conventional depot forms are suitably used. Such
forms include, for example, microcapsules, nano-capsules,
liposomes, plasters, inhalation forms, nose sprays, and sublingual
tablets. The antibody will typically be formulated in such vehicles
at a concentration of about 0.1 mg/ml to 100 mg/ml.
[0257] Depending on the type and severity of the disease, about
0.015 to 15 mg/kg of antibody is an initial candidate dosage for
administration to the patient, whether, for example, by one or more
separate administrations, or by continuous infusion. For repeated
administrations over several days or longer, depending on the
condition, the treatment is repeated until a desired suppression of
disease symptoms occurs. However, other dosage regimens may be
useful.
[0258] The effectiveness of the antibody in treating disease can be
improved by administering the antibody serially or in combination
with another agent that is effective for those purposes, such as
another antibody directed against a different epitope or
neutralizing a different protein than the first antibody, or one or
more conventional therapeutic agents such as, for example,
alkylating agents, folic acid antagonists, anti-metabolites of
nucleic acid metabolism, antibiotics, pyrimidine analogs,
5-fluorouracil, purine nucleosides, amines, amino acids, triazol
nucleosides, corticosteroids, calcium, retinoids, lipoxygenase and
cyclooxygenase inhibitors, fumaric acid and its salts, analgesics,
psychopharmaceuticals, local anesthetics, spasmolytics, and
beta-blockers. Such other agents can be present in the composition
being administered or can be administered separately. Also, the
antibody is suitably administered serially or in combination with
radiological treatments, whether involving irradiation or
administration of radioactive substances.
[0259] Efficacy testing can be performed during the course of
treatment using the methods described herein. Measurements of the
degree of severity of a number of symptoms associated with a
particular ailment are noted prior to the start of a treatment and
then at later specific time period after the start of the
treatment. For example, when treating an autoimmune disease such as
rheumatoid arthritis, the severity of joint pain can be scored from
a number of 1-10, with a score of 1 representing mild discomfort
and a score of 10 represent constant unbearable pain with or
without movement; the range of motion of an affected joint can also
are be measured as a degree of angle for which that joint can move.
The joint pain and range of motion are noted before and after a
treatment. The severity of joint pain and range of motion after the
treatment are compared to those before the treatment. A decrease in
the pain score and/or an increase in the degree of angle of joint
movement indicate that the treatment is effective in reducing
inflammation in the affected joint, thereby decreasing pain and
improving joint movement. Other methods of efficacy testing
includes evaluating for visual problems, new blood vessel invasion,
rate of vessel growth, angiogenesis, etc.: (1) inhibiting the
disease, e.g., arresting, or slowing the pathogenic growth of new
blood vessels; or (2) relieving the disease, e.g., causing
regression of symptoms, reducing the number of new blood vessels in
a tissue exhibiting pathology involving angiogenesis (e.g., the
eye); and (3) preventing or reducing the likelihood of the
development of a neovascular disease, e.g., an ocular neovascular
disease).
[0260] The present invention can be defined in any of the following
alphabetized paragraphs: [0261] [A] The use of a TRPV4 inhibitor
for inhibiting endothelial cell migration. [0262] [B] The use of
paragraph [A], wherein the TRPV4 inhibitor is selected from the
group consisting of an antibody, an RNA interference molecule, a
small molecule, a peptide and an aptamer. [0263] [C] The use of an
siRNA directed specifically against a TRPV4 gene for inhibiting
endothelial cell migration. [0264] [D] The use of an antibody
directed specifically against a .beta.1 integrin for inhibiting
endothelial cell migration, wherein the integrin function is
blocked by the antibody. [0265] [E] The use of paragraph [D],
wherein the antibody is a monoclonal antibody derived from clone
P5D2. [0266] [F] The use of any of paragraphs [A]-[E], wherein the
endothelial cell is a mammalian endothelial cell. [0267] [G] The
use of paragraph [F], wherein the mammalian endothelial cell is a
human endothelial cell. [0268] [H] The use of a therapeutically
effective amount of a TRPV4 inhibitor and a pharmaceutically
acceptable carrier for inhibiting angiogenesis in a mammal in need
thereof. [0269] [I] The use of a therapeutically effective amount
of a TRPV4 inhibitor and a pharmaceutically acceptable carrier for
the manufacture of a medicament for inhibiting angiogenesis in a
mammal in need thereof. [0270] [J] The use of paragraph [H] or [I]
wherein the TRPV4 inhibitor is selected from the group consisting
of an antibody, an RNA interference molecule, a small molecule, a
peptide and an aptamer. [0271] [K] The use of an siRNA directed
specifically against a TRPV4 gene for inhibiting angiogenesis in a
mammal in need thereof. [0272] [L] The use of an siRNA directed
specifically against a TRPV4 gene for the manufacture of a
medicament for inhibiting angiogenesis in a mammal in need thereof.
[0273] [M] The use of an antibody directed specifically against a
.beta.1 integrin for inhibiting angiogenesis in a mammal in need
thereof, wherein the integrin function is blocked by the antibody.
[0274] [N] The use of an antibody directed specifically against a
.beta.1 integrin for the manufacture of a medicament for inhibiting
angiogenesis in a mammal in need thereof, wherein the integrin
function is blocked by the antibody. [0275] [O] The use of
paragraph [M] or [N], wherein the antibody is a monoclonal antibody
derived from clone P5D2. [0276] [P] The use of any of paragraph
[K]-[O], wherein the mammal is afflicted with an
angiogenesis-related disease or disorder. [0277] [Q] The use of
paragraph [P], the angiogenesis-related disease is selected from
the group consisting of cancer, macular degeneration; diabetic
retinopathy; rheumatoid arthritis; Alzheimer's disease; obesity,
psoriasis, atherosclerosis, vascular malformations, angiomata, and
endometriosis. [0278] [R] The use of any of paragraph [K]-[Q],
wherein the mammal is a human. [0279] [S] The use of a
therapeutically effective amount of a TRPV4 inhibitor and a
pharmaceutically acceptable carrier for the treatment of an
angiogenesis-related disease in a mammal in need thereof. [0280]
[T] The use of a therapeutically effective amount of a TRPV4
inhibitor and a pharmaceutically acceptable carrier for the
manufacture of a medicament for the treatment of an
angiogenesis-related disease in a mammal in need thereof. [0281]
[U] The use of paragraph [S] or [T], wherein the TRPV4 inhibitor is
selected from the group consisting of an antibody, an RNA
interference molecule, a small molecule, a peptide and an aptamer.
[0282] [V] The use of an siRNA directed specifically against a
TRPV4 gene for the treatment of an angiogenesis-related disease in
a mammal in need thereof. [0283] [W] The use of an siRNA directed
specifically against a TRPV4 gene for the manufacture of a
medicament for the treatment of an angiogenesis-related disease in
a mammal in need thereof. [0284] [X] The use of an antibody
directed specifically against a .beta.1 integrin for the treatment
of an angiogenesis-related disease in a mammal in need thereof,
wherein the integrin function is blocked by the antibody. [0285]
[Y] The use of an antibody directed specifically against a .beta.1
integrin for the manufacture of a medicament for the treatment of
an angiogenesis-related disease in a mammal in need thereof,
wherein the integrin function is blocked by the antibody. [0286]
[Z] The use of any of paragraph [S]-[Y], wherein the
angiogenesis-related disease is selected from the group consisting
of cancer, macular degeneration; diabetic retinopathy; rheumatoid
arthritis; Alzheimer's disease; obesity, psoriasis,
atherosclerosis, vascular malformations, angiomata, and
endometriosis. [0287] [AA] The use of any of paragraph [S]-[Y],
wherein the mammal is a human. [0288] [BB] The use of any of
paragraph [S]-[Y], wherein an anti-angiogenic therapy is included.
[0289] [CC] The use of any of paragraph [S]-[Y], wherein
chemotherapy or radiation therapy is included. [0290] [DD] A method
for inhibiting endothelial cell migration, the method comprising
contacting an endothelial cell with a TRPV4 inhibitor. [0291] [EE]
The method of paragraph [DD], wherein the TRPV4 inhibitor is
selected from the group consisting of an antibody, an RNA
interference molecule, a small molecule, a peptide and an aptamer.
[0292] [FF] The method of paragraph [EE], wherein the TRPV4
inhibitor inhibits an influx of calcium into the cell. [0293] [GG]
The method of paragraph [EE], wherein the TRPV4 inhibitor is an RNA
interference molecule that inhibits TRPV4 expression in the cell.
[0294] [HH] The method of paragraph [GG], wherein the TRPV4
inhibitor is an siRNA directed specifically against a TRPV4 gene.
[0295] [II] The method of paragraph [EE], wherein the TRPV4
inhibitor inhibits a phosphorylation of .beta.1 integrin in the
cell. [0296] [JJ] The method of paragraph [EE], wherein the TRPV4
inhibitor inhibits a phosphorylation of AKT in the cell. [0297]
[KK] The method of paragraph [EE], wherein the TRPV4 inhibitor is
an antibody directed specifically against a .beta.1 integrin.
[0298] [LL] The method of paragraph [DD], wherein the endothelial
cell is a mammalian endothelial cell. [0299] [MM] The method of
paragraph [DD], wherein the mammalian endothelial cell is a human
endothelial cell. [0300] [NN] A method for inhibiting endothelial
cell migration, the method comprising contacting an endothelial
cell with an siRNA directed specifically against a TRPV4 gene.
[0301] [OO] A method for inhibiting endothelial cell migration, the
method comprising contacting an endothelial cell with an antibody
directed specifically against a .beta.1 integrin, wherein the
integrin function is blocked by the antibody. [0302] [PP] The
method of paragraph [OO], wherein the antibody is a monoclonal
antibody derived from clone P5D2. [0303] [QQ] A method for
inhibiting angiogenesis in a mammal in need thereof, the method
comprising administering a therapeutically effective amount of a
TRPV4 inhibitor and a pharmaceutically acceptable carrier. [0304]
[RR] A method of treating an angiogenesis-related disease in a
mammal in need thereof, the method comprising administering a
therapeutically effective amount of a TRPV4 inhibitor and a
pharmaceutically acceptable carrier. [0305] [SS] The method of
paragraph [QQ] or [RR], wherein the TRPV4 inhibitor is selected
from the group consisting of an antibody, an RNA interference
molecule, a small molecule, a peptide and an aptamer. [0306] [TT] A
method for inhibiting angiogenesis in a mammal in need thereof, the
method comprising administering a therapeutically effective amount
of an siRNA directed specifically against a TRPV4 gene and a
pharmaceutically acceptable carrier. [0307] [UU] A method for
inhibiting angiogenesis in a mammal in need thereof, the method
comprising administering a therapeutically effective amount of an
antibody directed specifically against a .beta.1 integrin and a
pharmaceutically acceptable carrier, wherein the integrin function
is blocked by the antibody. [0308] [VV] The method of any of
paragraph [QQ], [TT] and [UU], wherein the mammal is afflicted with
an angiogenesis-related disease or disorder. [0309] [WW] A method
treating an angiogenesis-related disease in a mammal in need
thereof, the method comprising administering a therapeutically
effective amount of an siRNA directed specifically against a TRPV4
gene and a pharmaceutically acceptable carrier. [0310] [XX] A
method of treating an angiogenesis-related disease in a mammal in
need thereof, the method comprising administering a therapeutically
effective amount of an antibody directed specifically against a
.beta.1 integrin and a pharmaceutically acceptable carrier, wherein
the integrin function is blocked by the antibody. [0311] [YY] The
method of paragraph [UU] or [XX], wherein the antibody is a
monoclonal antibody derived from clone P5D2. [0312] [ZZ] The method
of any of paragraph [RR], [VV]-[XX], wherein the
angiogenesis-related disease is selected from the group consisting
of cancer, macular degeneration; diabetic retinopathy; rheumatoid
arthritis; Alzheimer's disease; obesity, psoriasis,
atherosclerosis, vascular malformations, angiomata, and
endometriosis. [0313] [AAA] The method of any of paragraph
[RR]-[XX], wherein the mammal is a human. [0314] [BBB] The method
of any of paragraph [RR]-[XX], wherein an anti-angiogenic therapy
is administered in conjunction with the method. [0315] [CCC] The
method of any of paragraph [RR]-[XX], wherein a chemotherapy and/or
radiation therapy is administered in conjunction with the
method.
[0316] This invention is further illustrated by the following
example which should not be construed as limiting. The contents of
all references cited throughout this application, as well as the
figures and table are incorporated herein by reference.
EXAMPLE
Materials and Methods
Cell Cultures
[0317] Bovine CE cells (passage 10 to 15) were maintained at
37.degree. C. in 10% CO.sub.2 on gelatin-coated tissue culture
dishes in low glucose Dulbecco's Modified Eagle's Medium (DMEM;
INVITROGEN.TM. Inc.) supplemented with 10% fetal calf serum (FCS)
(HyClone), 10 mM HEPES (JRH-Biosciences) and L-glutamine (0.292
mg/ml), penicillin (100 U/ml), streptomycin (100 .mu.g/ml) (GPS),
as described in Matthews, et. al. (J Cell Sci., 119:508-518
(2006)). Human microvascular endothelial cells from dermis (HMVECs)
(Cambrex, Walkersville, Md.) were cultured in EBM-2 (Cambrex),
supplemented with 5% FBS and growth factors (bFGF, IGF, VEGF)
according to the manufacturer's instructions.
Materials
[0318] Antibodies against activated .beta.1 integrin (clone 12G10)
were from ABD Serotec and those directed against T788/789 of
.beta.1 integrin cytoplasmic tail, and phosho FAK pY-397 were from
Biosource.TM. International/MILLIPORE.RTM.. ALEXA.RTM.-conjugated
phalloidin and secondary antibodies were from MOLECULAR
PROBES.RTM./INVITROGEN.TM. Inc. Antibodies against phospho AKT
Ser-473, AKT, phospho-ERK1/2, ERK1/2 and FAK were from CELL
SIGNALING TECHNOLOGY.RTM., and those against vinculin and actin
were from SIGMA ALDRICH.RTM.. Human fibronectin was obtained from
BD.TM. Biosciences. Gadolinium Chloride, 4-.alpha.-PDD and
ruthenium red were purchased from SIGMA ALDRICH.RTM.; LY294002 was
from CALBIOCHEM.RTM..
Mechanical Strain Application
[0319] CE cells cultured on fibronectin-coated 6 well UNIFLEX.TM.
(FLEXCELL.RTM. International) plates for 24 hours were subjected to
uniaxial cyclic stretch (10% elongation; 1 Hz frequency) for 1-2 h
using a FLEXERCELL.RTM. TENSION PLUS.TM. System (FLEXCELL.RTM.
International). In some experiments, CE cells were plated on
fibronectin-coated 6 well BIOFLEX.RTM. (FLEXCELL.RTM.
International) for 1 h and subjected to static stretch (15%
elongation) for 1-15 min. Control cells were maintained under
identical conditions in the absence of strain application. Calcium
Imaging: CE cells adherent to the flexible substrates were loaded
with Fluo-4/AM (1 .mu.M) for 30 min, washed 3 times in calcium
medium (136 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO.sub.4, 1.1 mM
CaCl.sub.2, 1.2 mM KH.sub.2PO.sub.4, 5 mM NaHCO.sub.3, 5.5 mM
glucose, and 20 mM Hepes. pH 7.4) and then exposed to mechanical
strain (15% elongation) for 3-4 sec using a Stage Flexer
(FLEXCELL.RTM. International) apparatus that is fixed on a Nikon
upright microscope equipped with CCD camera (Spot-RT slider,
Diagnostics Corp, USA). Images were acquired for every 4 seconds
and analyzed using IP lab software and Microsoft Excel as described
in Matthews, et. al. (2006) (J. Cell Sci., 119:508-518). Calcium
imaging with TRPV channel activators was performed on cells
cultured on MatTek glass bottomed dishes on LEICA.RTM. Confocal
Microscope and analyzed using LEICA.RTM. software and
MICROSOFT.RTM. EXCEL.RTM.. siRNA knock down of TRPV channels: smart
pool siRNAs (10 nM) of TRPV2, TRPV4 (both from Dharmacon), TRPC1
(AMBION.RTM.) or control (QIAGEN.RTM.) siRNAs was transfected into
CE cells using SILENTFECT.TM. reagent (BIORAD.RTM.) as described in
Mammoto et. al. (2007) (J. Cell Sci., 120:456-467). Three days
later cells were used for calcium imaging or reorientation
experiments. The knock down of TRPV channel expression was assessed
using RT-PCR with species-specific primers and Western blotting.
The primers used for RT-PCR were: TRPV2 (human:
Forward-CAAACCGATTTGACCGAGAT (SEQ. ID. NO. 13);
Reverse-GTTCAGCACAGCCTTCATCA (SEQ. ID. NO. 14) and bovine:
Forward-CAGCTGGGAGGAAAACTCAG (SEQ. ID. NO. 15);
Reverse-GGGAGGAAGTCCTTTTCCAG (SEQ. ID. NO. 16)), TRPV4 (human:
Forward-GACGGGGACCTATAGCATCA (SEQ. ID. NO. 17);
Reverse-AACAGGTCCAGGAGGAAGGT (SEQ. ID. NO. 18) and bovine:
Forward-GACTACCTGCGGCTGGC (SEQ. ID. NO. 19);
Reverse-TTCATCCAGCCCAGGAC (SEQ. ID. NO. 20)), TRPC1 (human:
Forward-CACTCGTTCATTGGCACCTGCTTT (SEQ. ID. NO. 21);
Reverse-GCAGCTTCGTCAGCACAATCACA (SEQ. ID. NO. 22); bovine:
Forward-CCATTCGTTCATCGGCACTTGCTT (SEQ. ID. NO. 23);
Reverse-TTATGAAGCATTGCCACCAGCAGC (SEQ. ID. NO. 24)) and GAPDH
(Forward-ACCACAGTCCATGCCATCAC (SEQ. ID. NO. 25);
Reverse-TCCACCACCCTGTTGCTGTA (SEQ. ID. NO. 26)).
Morphological and Immuno Fluorescence Studies
[0320] Cells were transfected with GFP-AKT-PH domain (kind gift of
Dr. Martin Schwartz) using EFFECTENE.RTM. (QIAGEN.RTM., Chatsworth,
Calif.). Cells adherent to flexible ECM substrates and subjected to
mechanical stretch were washed in PBS, fixed in 4% paraformaldehyde
for 30 min either mounted on glass slides (for visualizing
GFP-AKT-PH translocation) or permeabilized with 0.25% TRITON.RTM.-X
100/PBS for 5 min for immuno staining. After blocking with DMEM
containing 10% FBS, cells were incubated for 1 h with
ALEXA.RTM.-phalloidin to visualize stress fibers, washed and
mounted on glass slides using FLUOROMOUNT-G.TM. (Southern Biotech).
For staining focal adhesions, cells were incubated with antibodies
against vinculin for 1 h followed by rinsing and incubation with
ALEXA.RTM.-conjugated secondary antibodies; activated .beta.1
integrins were detected using 12G10 antibody. Images were acquired
on a LEICA.RTM. Confocal SP2 microscope and processed using
LEICA.RTM. software and Adobe Photoshop. GFP-AKT-PH domain
translocation to the plasma membrane was quantified measuring
either ratio of the perimeter of whole cell membrane and the
membrane that contains the GFP-AKT-PH domain or the ratio of GFP
fluorescence intensity of translocated GFP-AKT-PH domain and
cytosol adjacent to the membrane. CE cell reorientation in response
to cyclic strain was measured by quantitating the angle of
orientation of cells relative to the direction of applied strain
using ImageJ software and MICROSOFT.RTM. EXCEL.RTM.. Cells on
substrates exposed to uniaxial cyclic strain with their longest
axis oriented between 60 and 120 degrees relative to the direction
of the applied strain field were considered to be aligned.
Statistical differences between experimental groups were determined
using the student t-test.
Biochemical Analysis
[0321] Western blotting were performed according to methods
published in Mammoto et. al. (2007) (J. Cell Sci., 120:456-467).
Membranes containing transferred protein were blocked in 3%
BSA/TBST for 1 h and incubated overnight with primary antibodies
against AKT and phospho Ser-473 AKT (1:1000), phospho-Thr 788/789
of .beta.1 integrin cytoplasmic tail (1:1000), ERK1/2 and phospho
ERK1/2 (1:1000), FAK and phospho FAK-pY397 (1:1000), actin (1:1000)
and TRPV4 (1:1000) at 4.degree. C. The membranes were subsequently
washed incubated with HRP-conjugated secondary antibodies (1:5000)
for 1 h and washed and incubated with SUPERSIGNAL.RTM. West Pico
ECL reagent from Pierce Biotechnology Inc. (USA) and exposed to
Kodak X-ray film (SIGMA ALDRICH.RTM.).
Integrin Activation Assay
[0322] .beta.1 integrin activation was measured using a glutathione
S-transferase (GST) fusion protein consisting of FN III repeat 8-11
domains or 12G10 antibodies (Orr, A. W. et. al., 2006, Mol. Biol.
Cell 17:4686-4697). Briefly, cells subjected to mechanical stretch
were incubated with either 5 .mu.g/ml of GST-FN III.sub.8-11
protein or 12G10 antibodies in PBS containing Ca.sup.2+ and
Mg.sup.2+for 30 min at 37.degree. C., washed and lysed in
SDS-sample buffer. The samples were separated on SDS-PAGE and the
bound reagents were assessed on Western blots using anti
GST-antibodies and HRP-conjugated secondary antibodies.
FACS Analysis
[0323] Activated .beta.1 integrin expression was measured with
specific antibodies (12G10) using flow cytometry (Kawaguchi et al.,
2003, J. Cell Sci., 116:3893-3904). Briefly, cells were incubated
with 12G10 antibody in FACS buffer (PBS containing 1% bovine serum
albumin) for 20 min on ice and fixed in 4% paraformaldehyde for 15
min. After fixation, the cells were washed twice with FACS buffer
and incubated with PE-conjugated secondary antibodies (Vector
Laboratories, USA) for 20 min on ice. The cells were then washed
twice and analyzed on GUAVA.RTM. Personal Cytometer (GUAVA.RTM.
Technologies). Isotype-matched IgG and secondary antibodies alone
were used as controls.
Whole-Cell Patch Clamp Experiments
[0324] One day after bovine CE cells were transfected with
TRPV4-EGFP, they were plated on gelatin-coated glass coverslips and
allowed to grow for .about.24 h prior to recording. Cells were
recorded in the whole-cell mode using borosilicate glass pipettes
(1-3 M.OMEGA.) containing: (in mM) 120 CsMeSO.sub.3, 10 EGTA, 2
MgCl.sub.2, 10 HEPES; pH and osmolarity were adjusted with CsOH or
HMeSO.sub.3, as needed, to 7.2 (23.degree. C.) and .about.300 mOsm,
respectively. Cells were bathed in a solution containing (in mM):
138 NaCl, 5 KCl, 1.8 CaCl.sub.2, 1 MgCl.sub.2, 10 HEPES; pH and
osmolarity were adjusted with NaOH or HCl, as needed, to 7.4
(23.degree. C.) and .about.310 mOsm, respectively. Cells were held
at -40 mV for 3-5 min to allow for intracellular dialysis. Bath
superfusion was stopped prior to initiating the recording of
currents resulting from the indicated voltage protocol applied
every 5 s. Immediately before use, a 4-.alpha.-PDD stock solution
(2 mM in EtOH, on ice) was diluted 1:50 in bath solution under
vortex and 50 .mu.l was subsequently added to a still bath (0.5 ml)
by pipette (final concentrations of 4 .mu.M 4-.alpha.-PDD and 0.2%
EtOH) and mixed by 3 gentle up/down pipette strokes. Once a clearly
discernable 4-.alpha.-PDD-induced current was observed (typically
after a 10-30 sec delay), the bath solution was changed to a
Na.sup.+- and Ca.sup.2+-free solution (in mM: 145
N-methyl-D-glucamine.Cl, 10 HEPES, adjusted to pH 7.4 and
.about.310 mOsm with HCl) by re-starting the superfusion. In some
cases, the original bath solution was later re-introduced as a
wash.
Cell Migration and In Vitro Angiogenesis Assay
[0325] Cell migration assay was performed using Transwell assay.
Briefly, cells were plated on to gelatin coated (0.5%) transwell
membranes (Coster) in EBM2 supplemented with 0.3% FBS and their
migration in response to VEGF (10 ng/ml) was monitored. The
migrated cells were stained with Giemsa solution for 16 h and ten
random fields were counted. To measure in vitro angiogenesis, CE
cells were plated on MATRIGEL.TM. (BD Biosciences) and incubated in
the presence of VEGF (10 ng/ml) at 37.degree. C. After 18 h, tube
formation was assessed in ten random fields (Mamotto, 2009, Nature
457:1103-1109).
Results
Capillary Cell Reorientation Induced by Cyclic Strain
[0326] To begin to analyze the molecular mechanism by which
mechanical strain can influence CE cell motility and angiogenesis
as observed in ECM gels and living tissues (Korff and Augustin,
1999, J. Cell Sci. 112: 3249-3258; Jounget. al., 2005, Micro vasc
Res.; Matsumoto et al. 2007, Tissue Eng 13:207-217; Pietramaggiori,
et al. 2007, Ann. Surg. 246:896-902), bovine CE cells were cultured
on flexible fibronectin-coated substrates and subjected to 10%
uniaxial cyclical strain (1 Hz) using a FLEXERCELL.RTM. TENSION
PLUS.TM. system. Fluorescence microscopic analysis of cells labeled
with ALEXA.RTM. 488-phalloidin combined with computerized
morphometry revealed that stress fibers thickened in these cells,
and almost 90% of them realigned perpendicular to the main axis of
the applied strain within 2 hr after force application (FIG. 1).
Immunofluorescence micrographs of CE cells subjected to 0 or 10%
uniaxial cyclic strain and stained for vinculin and actin stress
fibers showing that application of strain causes enhanced
recruitment of vinculin to large focal adhesions that colocalize
with the ends of reinforced stress fibers, indicating that stress
fiber realignment was accompanied by redistribution and
reorientation of focal adhesions containing vinculin (data not
shown), focal adhesion kinase (FAK) and talin (not shown), which
appeared in close association with the ends of newly aligned stress
fibers (data not shown).
Strain-Induced Capillary Cell Reorientation Requires .beta.1
Integrin Activation
[0327] The effects of fluid shear on large vessel endothelium
(Tzima et. al. 2001, Embo J. 20:4639-4647) and mechanical strain on
fibroblasts (Katsumi, A. et. al. 2005, Biol. Chem. 280:16546-1
6549) are mediated by stress-dependent activation of integrin
receptors within minutes after force application. When CE cells
cultured on flexible fibronectin-coated substrates were exposed to
static biaxial strain (15%), .beta.1 integrin activation increased
within 1 min after strain application, as indicated by increased
phosphorylation of the T788/789 site of the .beta.1 integrin
cytoplasmic tail in Western blots (FIG. 2A), which has been shown
to correlate with integrin activation (Nilsson, S. et. al. 2006,
Exp. Cell. Res. 312:844-853; Stawowy, P. et al., 2005, Cardiovasc.
Res. 67:50-59; Wennerberg, K., 1998, J Cell Sci. 111:1117-1126).
Immunofluorescence staining using 12G10 antibodies that only
recognize the activated conformation of .beta.1 integrins
(Humphries, M. J. 2004 Biochem. Soc. Trans. 32:407-411; Thodeti, C.
K. et al. 2003, J. Biol. Chem. 278:9576-9584) also showed increased
clustering of activated integrins within large streak-like focal
adhesions at the cell periphery within 15 min after force
application (data not shown). The ability of the 12G10 antibody to
detect activated integrins in the CE cells was confirmed using flow
cytometry, which demonstrated a significantly increased 12G10
signal after globally activating integrins by treatment with
manganese (FIG. 8). Mechanical strain-induced activation of
integrin signaling was confirmed independently by demonstrating
increased phosphorylation of MAP kinase (ERK1/2) (FIG. 2B) and FAK
within 5-15 min after exposure to mechanical strain (FIG. 9).
Application of uniaxial cyclic strain (10%; 1 Hz) also induced
.beta.1 integrin activation within minutes, as measured by
increased T788/789 phosphorylation (FIG. 4C) and enhanced binding
of both a fibronectin fragment (GST-FNIII.sub.8-11) (FIGS. 2C and
4B) and the 12G10 antibody that only ligate the activated form of
the .beta.1 integrin receptor (FIG. 2D) (Orr, et. al. 2006, Mol.
Biol. Cell. 17:4686-4697). Cyclic strain also increased .beta.1
integrin activation in human CE cells as measured by enhanced
binding of GST-FNIII.sub.8-11 (FIG. 4B), and thus, this appears to
be a generalized response in CE cells.
[0328] To explore if this mechanical strain-induced wave of .beta.1
integrin activation is required for CE cell reorientation, cells
were pre-incubated with function-blocking anti-.beta.1 integrin
(P5D2) antibody for 30 min, and then the cells were subjected to
uniaxial cyclical strain (10%) for 2 hr. Treatment with this
inhibitory antibody inhibited strain-induced cell realignment by
almost 70% (p<0.001) (FIG. 2E), and it also prevented associated
reorientation of stress fibers and focal adhesions (data not
shown). These results indicate that application of mechanical
strain to CE cells through existing integrins that are bound to
substrate-immobilized ECM molecules (and hence activated) induces
focal adhesion remodeling, stress fiber realignment, and cell
reorientation through a mechanism that requires activation of
additional integrin receptors.
PI3K is Upstream of .beta.1 Integrin Activation in this Mechanical
Signaling Cascade
[0329] PI3K has been implicated in the activation of .beta.1
integrins by fluid shear stress in large vessel endothelium (Tzima,
E. et al. 2005 Nature 437:426-431), however, it also can act
downstream of integrin activation (Berrier, A. L., et. al., 2000 J.
Cell Biol. 151:1549-1 560). To explore whether PI3K is involved in
early mechanical signaling in microvascular endothelium, CE cells
were transfected with GFP-fused to an AKT-PH domain that
translocates to the plasma membrane when it binds to the PI3K
product, phosphatidyl inositol-3-phosphate (Watton and Downward,.
1999 Curr. Biol. 9:433-436). Bright linear AKT-PH-GFP staining was
detected at the peripheral membrane within 1 min after application
of mechanical strain (15%), whereas it remained diffusely
distributed throughout the cytoplasm in control (unstrained) CE
cells (data not shown). Quantification of AKT-PH-GFP translocation
by two independent parameters (fraction of AKT-PH-GFP in total
perimeter of the membrane, or GFP-fluorescence intensity ratio
between membrane and cytosol) revealed a significant increase in
response to mechanical strain that was inhibited by treatment with
the PI3K inhibitor, LY294002 (FIG. 3A). Mechanical strain also
activated PI3K as determined by enhanced phosphorylation of its
downstream target AKT at Ser-473 within minutes after force
application, as detected in Western blots (FIG. 3B). Moreover,
strain-induced translocation of AKT-PH-GFP to the membrane and AKT
phosphorylation were both abolished by inhibiting PI3K with
LY294002 (FIGS. 3A and C). LY294002 treatment also prevented
.beta.1 integrin activation (FIG. 3C) and suppressed FAK activation
(FIG. 9). Thus, force application through ECM-integrin adhesions
activates additional cell surface 01 integrin receptors by
stimulating PI3K activity.
Strain-Induced Cytoskeletal Reorientation is Mediated by
Stress-Activated Ion Channels
[0330] SA ion channels have been implicated in force-dependent
alignment of large vessel endothelial cells (Naruse, K., et. al.
1998 Am. J. Physiol. 274:H1 532-1538). Direct force application to
cell surface .beta.1 integrins using magnetic tweezers also results
in rapid (within 2-5 sec) calcium influx in the bovine CE cells,
and this response can be blocked using the general SA channel
inhibitor, gadolinium chloride (Yang and Sachs, 1989 Science
243:1068-1071; Matthews, B. D., et. al., 2006, J. Cell Sci.
119:508-518). To confirm that mechanical strain also activates SA
channels in these CE cells, cells adherent to flexible ECM
substrates were loaded with the calcium reporter dye FLUO-4,
subjected to static mechanical strain (15%) and calcium influx was
measured using microfluorimetry (Matthews, B. D., et. al.,
2006).
[0331] Stretching CE cells for as little as 3 sec induced rapid
calcium influx, and this response could be almost completely
abolished by treatment with gadolinium chloride (FIG. 4A).
Pretreatment of bovine and human CE cells for 30 min with
gadolinium chloride also significantly inhibited .beta.1 integrin
activation in response to mechanical strain, as measured by
decreased binding of GST-FNIII.sub.8-11 (FIG. 4B) and 12G10
antibody (not shown) that specifically bind to activated .beta.1
integrins and reduced .beta.1 integrin phosphorylation (FIG. 4C).
In addition, gadolinium chloride inhibited PI3K activity, as
measured by membrane translocation of GFP-AKT-PH (FIG. 4D).
Finally, the cell and cytoskeletal reorientation that are normally
induced by mechanical strain were also greatly suppressed in the
presence of this SA ion channel blocker (FIG. 4E). Thus,
strain-dependent activation of mechanosensitive calcium channels
appears to be required for activation of both PI3K and .beta.1
integrins, as well as subsequent cytoskeletal reorientation in CE
cells.
TRPV4 Channels Mediate Cyclic Strain-Induced Capillary Cell
Reorientation
[0332] The specific type of mechanosensitive ion channel that
mediates the effects of mechanical stretching on CE cell
orientation was identified. TRPV4 is an interesting potential
candidate because it mediates cell sensitivity to osmotic stresses
(Liedtke, W. 2005 J. Physiol. 567:53-58) and shear stress-induced
vasodilation (Kohler, R. et al. 2006 Arterioscler. Thromb. Vasc.
Biol. 26:1495-1502), and it was found that TRPV4 is activated
within milliseconds after mechanical stresses are applied directly
to apical cell surface .beta.1 integrin receptors using magnetic
cytometry. vasodilation. To determine whether TRPV4 is the
candidate mechanosensitive channel, its expression was measured in
CE cells. Western blot analysis showed a strong band around 85 kDa
(and a fainter band at .about.100 kDa) in both bovine and human CE
cells (FIG. 5A). RT-PCR analysis also confirmed the presence of
TRPV4 mRNA in both bovine and human CE cells (FIGS. 5B and 10).
Moreover, a specific activator of TRPV4 channels, 4-.alpha.-PDD,
induced a robust calcium signal in bovine as well as human CE
cells, thus strongly indicating that both cell types express
functional TRPV4 channels (FIG. 11).
[0333] Next, the TRPV4 channel activation was measured directly by
whole-cell clamp using bovine CE cells transiently transfected with
TRPV4-EGFP that gave robust TRPV4 currents in response to
4-.alpha.-PDD, and it was found that substitution of
N-methyl-D-glucamine for cations in the bathing solution, inhibited
activation of inward, but not outward, currents by 4-.alpha.-PDD in
these cells (data not shown). This approach was used because
TRPV4-like currents in primary endothelial cells are small,
transient, and difficult to characterize, as previously described
in Vriens J et. al., 2005 (Circ Res., 97:908-915) and as was
observed herein as well. Thus, taken together, these findings
strongly suggest that CE cells express functional TRPV4 channels,
although at a low level.
[0334] To confirm that calcium influx through TRPV4 channels
mediates the effects of mechanical strain on CE cell orientation,
the expression of TRPV4 in bovine and human CE cells were knocked
down using specific siRNA; sham siRNA and siRNA directed against
the closely related channel, TRPV2, were used as controls. Sequence
analysis of smart pool siRNAs confirmed that both siRNA sequences
exhibit 80-100% homology with human and bovine TRPV4. RT-PCR
analysis revealed that TRPV2 and TRPV4 mRNA levels were knocked
down by 90% and 70%, respectively, in bovine CE cells using this
approach, whereas use of a sham control siRNA had no effect (FIGS.
5B and 10). It was found that TRPV4 protein expression was also
knocked down by .about.60% and 80% in bovine and human CE cells,
respectively (FIGS. 5C, 5D and 10).
[0335] Importantly, microfluorimetric analysis revealed that
application of mechanical strain (15%) for 4 sec induced a large
wave of calcium influx in bovine CE cells transfected with control
siRNAs, whereas this response was significantly inhibited
(p<0.02) in cells treated with TRPV4 siRNA (FIG. 5E, F). In
contrast, use of siRNA directed against the closely related SA
channel TRPV2 had no effect (FIG. 5 E, F). siRNA knock down of
TRPV4 also inhibited cyclic strain-induced activation of .beta.1
integrins, AKT, and ERK1/2, further confirming that TRPV4
activation is upstream of integrin activation (FIG. 12).
[0336] Pretreatment of CE cells with the general TRPV inhibitor,
ruthenium red, or with TRPV4 siRNA also significantly suppressed
calcium signaling and cell reorientation induced by application of
cyclic strain in CE cells, whereas addition of siRNA against two
different related SA channels, TRPV2 or TRPC1 (FIG. 10), were
ineffective (FIG. 6). This inhibition was specific for
reorientation as transfection of cells with TRPV4 siRNA did not
alter the number of viable adherent CE cells when they were
cultured on standard tissue culture substrates (FIG. 13). Moreover,
it was found that application of similar cyclic strain, in the
presence or absence of ruthenium red, did not effect CE cell
proliferation or apoptosis, as measured by Ki 67 staining and
poly(ADPribosyl) polymerase cleavage (FIG. 14). Taken together,
these results indicate that TRPV4 channels are mechanosensitive
calcium channels in CE cells that are activated by mechanical
strain applied through the integrin-mediated cell-ECM adhesions,
and that calcium influx through these channels is required for
downstream signaling events that drive the cell and cytoskeletal
reorientation response triggered by cell stretching.
TRPV4 Channels are Required for Angiogenesis In Vitro
[0337] To determine if activation of TRPV4 channels by physical
interactions between cells and ECM are physiologically relevant,
these specific siRNAs were used to knock down the expression of
TRPV4 and TRPC1 in human CE cells and tested their ability to form
three-dimensional tubular capillary networks on MATRIGEL.TM.
substrates. RT-PCR analysis confirmed that transfection of TRPV4
and TRPC1 siRNAs resulted in knock down of TRPV4 and TRPC1 mRNA
levels by more than 70% and 90%, respectively (FIG. 7A), and, as
described above, this resulted in suppression of TRPV4 protein
levels by 90% in these human cells (FIG. 5C). TRPV4 siRNA also had
no significant effect on expression of TRPC1, and vice versa (FIG.
7A). Importantly, knockdown of TRPV4 attenuated CE cell migration
(FIG. 7B) and completely abolished tube formation in the
MATRIGEL.TM. angiogenesis assay at 18 hr, whereas tubular
differentiation proceeded normally when cells were transfected with
sham control or TRPC1 siRNA at equal concentrations (FIG. 7C). This
inhibition was specific for tube formation as transfection of cells
with TRPV4 siRNA did not alter the number of viable adherent CE
cells when they were cultured on standard, rigid tissue culture
substrates (FIGS. 13 and 14). Thus, these findings indicate that
TRPV4 channels mediate mechanical signaling through integrin-ECM
adhesions in CE cells, and that this mechanotransduction response
is required for angiogenesis in vitro.
[0338] The inventors have shown that application of mechanical
strain to bound integrins on the CE cell surface stimulates calcium
influx through mechanosensitive TRPV4 ion channels, which activates
additional .beta.1 integrins and subsequent downstream cytoskeletal
reorientation responses that are required for formation of tubular
capillary networks during angiogenesis.
[0339] The references cited herein and throughout the specification
are incorporated herein by reference.
Sequence CWU 1
1
26150319DNAHomo sapiens 1ccggccggga ttcaggaagc gcggatctcc
cggccgccgg cgcccagccg tcccggaggt 60aagtggggcc cgggcccggg aggggcggct
cagccgaggt cccctcgcgc ccctggggac 120atctccgtgg cccgtccggc
tccggggggt cccgaggctc caaaatgcgg gccaggggcg 180cgggagacgg
aaggcaccag gttccgcagg cgccagcctc tcagctaagt gcccttgggc
240cggacaccgg ctctttgggt ctcggtttta tcacctgtga aatgggcacc
aacgtggggc 300ctggggtagc aggtctgggg ggagggcggt tcggttccct
gagaccccaa gccagggaac 360caaaggcccg gagccttgca gcccacctta
ggagacttgg aagagggatt tcgggggacc 420taaggtttgc ccttggcccc
tgagcatgtc ggagggaatt tggagtctgg agcttccaaa 480ggcttcttct
tggttactga gtcccggaga gacggctgtt tcctccaaga ggcatgaaaa
540tctttaacct ctagttctgc cctggactct caggacgtcc cgggggcggg
tggctcctgg 600gggtgggtag cgggggtggg ggtgggggag agagagactc
ccacactccc cgcttgcctg 660gaaacaccaa ccacagatgc attcatcgag
ccacccactg ctccagcctg ccccagctgt 720tccctctgtc tgtcctctct
gttttgcaga tggggaaact gaggcttagg tcggggatct 780agacaattgg
gatttaaacc cagggactat ccagccccaa agcccttccc accacaccag
840gtggcctgtc ctggggccag ctctgcacac agggcctggt gcccccgggg
tgcttgggaa 900gtggcagggc agaggtgggc cctgtggctg ttctggctca
gcttctaaaa caagagcctc 960tgctgggggc agaggggccg tgaacccctg
aaatgttagg cagataccct gtgggagctt 1020tgttctggga tgctaagaac
cgcttgagga tttaagcttt gccactttgg ctccggagca 1080agggcagagg
gtaagtcggg actccccggg ctcctgagga gggtgacgag gtgggctttt
1140gggggaacaa gggtagaaag gtcaggcctg ggttatctgc ggggctaaga
gcgggcctgt 1200gtggggcccg gggtgtgctc tgcagtcccc tgctgtgtga
ccttggcctg gtcccttagc 1260tgtctgagcc tttgtgttct ctcctctgta
atactggggt gcctgagggc aagcccccag 1320ggctgtcgtg aggaccgatg
accttccagg aacctggcac agccactgtt ggctgccatc 1380aatgtttaac
cagttgtcgt tgccccaaac attttcttaa caaagagggt gaaaaaagtc
1440aattggccat ttccacattt ctctagttca ttctgtttga agaatgatgg
gaaccagaaa 1500gcctggacac ccaccttgat tggtgaattg cacgcggaag
gggtcccaga cacaaatggc 1560aaatggcagt catagttcag ggcggttcac
tttgtaagat caaggtgggg ctgttttgaa 1620atggaggtaa ccagggaatg
gttgcctgaa caaaaagggt gtgatgcccc caggagggat 1680gtttcacatg
agctccggtg aggatggctg ggattcccca ggtgaaaccc agatacctta
1740tatctgggga aggggctgtg gaggttcctc ctgccttatc tggtgccaac
tgggcctctg 1800ccacttactc cctcctccag caaatgctct ctgcaagctc
acctgtgcca gctctgggct 1860gggcactgga gcagtgagat gcttccaatc
ccagcttcgc tgtagcctga ggcgtgtgaa 1920ggcaagaagg attttccctt
gtggcccccc ggggaggagc tcccacacct gacttgtgag 1980catctagcaa
ggtgtgcatg gatggccgac gcttagtaaa tgtatgctgt ggaatggatg
2040gggtcagggg agaatgccgt agggtaggag gactggccca ggaaggaccc
acgagggctt 2100ggtcaaggta gtgtttaagc cgcaccttaa agaataaatg
cagaagtaga ggaaagagta 2160ttcaggcaaa gagaatggca ctagcagaga
tatgactttt acctgttacc aggtggctac 2220acctggggtt tatagagcag
agaaatagtt ctgtttgtct ggagtttgtg acgtgactgg 2280aaagggtgag
ggatcagaca ggcaggagct aaatcagacc cagaggccca gaagtcctgc
2340ctgggagtga cacggttgtt tgtctgttct agaaaggtcc ctgtggctgc
agagtgggaa 2400agattggagg agctcagaca gagaggctgg ccccaaggct
ggggaagtca tccaagctct 2460gagtggagag cggcagctgg aaggagatgt
aggagggaag gaaacacaca agactgagcc 2520tcatgtgcct ggcacatagt
aggtgctcag tcagtatcta tggcatggtt gtctgaacaa 2580atacttttga
gatggtcaaa gtcctttgag ttccttcctc tgttcctagg gggatggggt
2640ctacatgaag tgggtgtctc tactccatgc aggcccttga ccctgctgtc
tgcctcagtt 2700tcccccattt gactttggga aagacactcc agtccgttct
ccaaattata gggacccagg 2760taacagctcc tcttgttgtc acagataggg
tctggggagc caagataaca tttatttcat 2820gccagtgggc cctctggaat
gctccacatc gcccatttgg agcagaattc acctgttaaa 2880gattaattcc
gatctcttga gataagtggc tgctgttatg aactctgggt acaaggttct
2940gtctggcttt agccaaccct gggttgcatt aacaactatt atgatggtgg
caagccttta 3000ttgagcactt actgtgtgcc aagcactgag tcaagcaccc
tcagcacatt tggatacaaa 3060actatttttg tctctatttt tcagagggca
gaatgaggtc tagagggtga aatctcttgc 3120ctgggttctc tcagctgaga
aggggcagag ctgggacctg aatcccattt gtgtgattgc 3180aaatccatcc
cttcacctgt aaggtggccc gtccccattt gtctggaccc tcattggggt
3240gcctggggtc atgggggagc tactctgcag tccagagata ggaacctgag
ttggccgggt 3300gcagtggccc acacctgtaa tcccaacact ctgggaggcc
aaggcaggtg gatcggtcga 3360gcccgtgagt ttgagaccag cctgggcaac
atggtgaaac tctgtctcta gaaaaaatac 3420aaaaattagc cgaacgtggt
agcacgcacc tgtagtgcca gctacttgag aggctgaggt 3480gggaggatcg
cttaagccca ggagtgagag gttgcagtga actgcgattg tgccactgca
3540gtccagagca agagatcctg tctcaaaaca aacaaagagc ctgaactgca
gacaggcctg 3600ggcttaaact gcatccgcat gtttaagaca ggcctgctat
tccccgctgg aggagctcag 3660ggaagccacc tggcctctct gggcttccat
ttcctcctgt gtcaaaggga ctctcatccc 3720actttgcagg gatgacccaa
ggcacgtgga tggggctctg aagcacagct cttctgcctc 3780agcccgtgtc
agctcccctt gtccccaggc ccattccagg gcatgttttc ttccagctta
3840tcccccagac atcctcagag agcccctcca tgccaggtgg gtgggtggaa
ggtattgggg 3900gcaaagctag acttgcattt gggcaaataa tgatatgttt
tcagcaatga aatatttatg 3960agcattgact gagtgccagg agctgtcaac
tttttttact ttcctgattt aatgcttaaa 4020aactagggct ggagcccagc
tgcccaggtt caaatcccag cttccaataa gttccattat 4080tcctttggcc
tcagtttcct catctggaca atggggataa ggtaatgcct gccttgctgg
4140gaggttgtgg acagtcttca gcacaacctt gagtgtttag tttaaaacca
gcatctattg 4200cctgttgttc actttttttt ttatttatta tttttagaga
cagggtctca gtctgttgct 4260caggctggaa tgcagtggtg tgatcatagc
tcacagcagc ctcgaactcc tgggctcaag 4320agatcctcct acttcagcct
cccaagtagt tgggacacac ccagcttatt aaaaaaaaag 4380gtttttttca
gacaggggtc tcactatgtt gcccaggttg gtctggaatt ccagagctca
4440agtaaccctc ctgccttggc ctcccaaagt gctcagatta caggtgtgag
ggctgatgtt 4500cattttgatg gtgtcacagc ttccccactg ccgggggtgg
gcaggggcag gaatgctgag 4560ccccatttta cagctgagga aatcgagact
cagagaggaa gcaatttacc cagggatgcc 4620cagctggctc atggcagagc
tggagctgga agccagatct gagtgacact ggtgtccttg 4680ggtccacgaa
aagcccagtg gccagcttag cttcttgcgt ggaaagctac gctaggctca
4740cccaccttgg cgtctgagcc accttagctt acctcccagc tccgccagtt
gctgctctgt 4800gacctcagaa agtggcttaa gctctctgtt cctgggcttt
gctgaggtca gagctctgcc 4860aacttgagga gggatttggg ttattctggc
tgctgctctt gttctggcct gggctccttc 4920aacctcgggt gcccttgcag
cccatgagct ggcaccccag tggccggaca ggaatgagga 4980agggacaggg
cctcactttc aatgtgactc tgacttcctt cccaccccta gccccttcct
5040gagcattggt ttcccctttc tgccccgagc cctggccact gccggctgtg
tgaccccaag 5100gaggttacat cacttctctg agcatgtttc ctcatcggtg
agggaggatc acacgagacc 5160acaccaggcg cacaggaagc ccttgccggt
ctccagatga ggtcagggca ttggaaaggc 5220cacagaagga ggcgggggct
caggggacag acaggcgggc cattgaccaa acgggaatat 5280ccttctattt
ctttggttag tgagtaacag cagcccctct tgtccaccca ccctgcccct
5340tgcacaaaga gctcctcagt ctcaccaggc gcccgttttc ccctgagggt
ggagtggcct 5400gggttccacc cggttgaccg agtgggctgg ccaccttctg
ggaccattcc ctgtgtgtgt 5460gtttgtgtac gtgtgtatgt gtgtgtatat
tgatgtgcgt gtgtatgtgc gtatgtgtgt 5520ctgtgtgtgt atgtgtatgt
gtgtatatgt atgtctatat gtgtatgtgt atatgtgtat 5580atatgtgtat
gtgtgtatat gtatgactat atgtttatgt gtatgtttgt gtatatatgt
5640gtgtatgtgt gtatatgtat gtctgtgtgt atgtgtgtgc atatgtgtat
gtatatacat 5700gtgtatgtgt gtatgtgaat gtctatgtgt gcatgtgtgt
atgtgtgtgt atgtttgcgt 5760gtatatgtat gtatgtgtgt atattgtgtg
tatgtatgtg tatatgtgtg taatgtgtat 5820atgtatttgt gtgtagtgtg
tgtgtgtgtg tgtgtttatg gagggtgggt tccatgggct 5880ttgggctgag
accccagcag gtgagggtgg gacagggggc atggggttga ctgacccatg
5940ggtctggctc actcctgtcc ctgcttggca cacaggaggg gctcaggaat
acctgtagat 6000agagtgaagg gatgaatgaa ggaaggaatg agtgactgga
caagccagcc aggcagggca 6060tcagaaggaa gcacctgctc agatacaggc
tctggctctg agaccaagtg tgcttgagtg 6120agtgtcctgg tcttgttgag
cctcaatttc ttcatctata aaatgggctc acgatagttg 6180tcctcatgag
aggtgttggt cttataggcc agaaaacccc acacatagca ggtgctccgt
6240aagctgtctg ttaaaaaatg gatgggagga agctaattag ttagctgggc
agaggttcgc 6300tttggagacc tgtctgccct ggagagaatg actaagcccg
tctttcactc ctttcctctg 6360ttcattcatt cattcattca ttcatccagc
aaatacatct tgagcagctg ctatgtgcca 6420ggacctgttc caggcatctg
ggcctcagca gttaccagaa gggagtccct ccgccctaga 6480gcttttgtcc
gagtgacatc attccacacg cagtgctttg aggccacttt gtgagactct
6540gatacccaca atgacaagta atccagcagt gggaactgtg atgagctccc
ttttacaggt 6600gaggaaactg aagctcggag aggtcaagtg atccgtctgg
ggccacgcag ttactaaatg 6660gcaaagtcaa gattcagact ccaagcctgt
caccactgtg ccatcttgtc ctgctgagtt 6720ccaagggata tggcgttgag
aaggggggtc ctagggagag ggcatggtag cccccaaagg 6780gatgaatgaa
ctcaaatgga tttaaaagca gatggttgga agtctagcca cattcacttc
6840ctgtagcgtt gtggactcag ttaaatctca ttctttctga tcttcagttt
gttcatctgt 6900gaaatgataa caataacacc gacctcaaat gagagagaaa
tgtgaaagtg cttagcatat 6960gataggtatt ggataaatgt cagcccttac
ctcctttccc ccgggactga ctttatacat 7020ctgggaatgg agacaataat
atttaatgtc tatagttatt atttggatta aaatagatag 7080aaggggccag
ccatggtggt tcacgcctgt aatcacaaca ctttgggagg ccaaggtgtg
7140tggatcacgt gagcccgggc attggagact aacctgggca gtagagtgag
actccatctc 7200tacaaaaaat aaattttaaa agtaggaatg gtggtacatg
cctgtggtcc cagcttctgg 7260ggaggctgag gtgggaggat tgcttgagcc
tgggaggtcg aggctgcagt gagctatgat 7320tgtgccactg cactccagcc
tgggtgacaa agccataccc tgtctcaaaa caaaaaacaa 7380ccaaatggga
ggtgctgtgt gaagtgtgcc atggagggta ggcattactg atggtacatt
7440gcaggcatta gcctcagtca taattccaag cagttaaccc ctgaggcagc
agcttactgt 7500tggcacagac tggtgttgag gtgggctggg acagaggaag
gatctcaaac tggttttctg 7560ctgccttggg ctcccttccc cgactcctgc
cccatccact cataacgggc acctgccctc 7620ttccccctcc tctgatttgg
caaactcggg acttttgctc ctagaagtgg ggtagaggaa 7680cgtatgcatg
gacgagtgtc ctgtcccttc tggtccttag tctcccagct gggaggcagc
7740ccttgtcctg acagccttct aaaggtgaca gaagatggaa atgaggtggc
gaggaaaccc 7800tttccaaagg tgaggtctct gcagtcacga gccctggttt
tggagccggg aagtctgggt 7860tccagtgcct gcccagttct gacctccctg
gacctcaatg tcatcatccc ccacctccca 7920ctgcaaactg agggaatgtt
agcaagaatg gcaaagcagc cattgaacat ccttatctgt 7980aaaatggggg
taataattgc tacgacctgg gtggggttaa gctgtatttg ctccatagtt
8040atttggtata cagtacattc atgctttaaa ctaaaaaata agttcaagtt
cattgaacac 8100ttggtgtttg ttgggcactg cactgaaggc ttgaattatc
acatttaatc ctcacaatag 8160ctctttgggg taggagctgt tattttattt
tttaatattt taaaaaataa ataaatttat 8220ttattttttg tagagatggg
atctcaccat tttgcccagg ctggtctcga actcctgggc 8280tcaagcgatc
ctcccacctt ggcctcccaa agtgttggga ttacaggtgt gagccactgc
8340acccagcctt attttttagt ttaaagcatg aatgtgctat acaccaaata
actatggagc 8400aaatacggct taaccccacc caggtcgtag taattattac
tcccatttta cagataagga 8460aaccaaggcc cagagatgag atgtgacttg
tgcaagacca tacagccagt gagtggcaga 8520tgagatgtaa agcattgatc
aggcaacgtt ctcagactat agaatcctct ctggagttct 8580ccgtggctgt
gaccatcctc taggagaggg gctaggccag agagaaggcg ggcctaaggt
8640tttgttttgt tgagatggag tctcgctctt gtcgcccccg ctggagtgca
gtggtgctat 8700ctctgctcac tgcaatctct gcctcctggg ttcaagagat
tctcatgcct cagcctcccg 8760agtagctggg attacaggtg catgctacca
cacccggcta atttttgtat ttttggtaga 8820gatggggttt caccatgttg
gccagtctgg tcttgaactt ctgacctcaa gtgatccgcc 8880cacctcggtc
tcccaaagtg ctgggattac aggcgtgagc cactgcgccc ggccggacta
8940agaatttgat gcctccctcc tcctgggctg gagctgagta gcatgttctc
aagcagggct 9000ggctgagagg gtggaatcct ggggctatgc tctgatggga
aggaagcgag ataagggaag 9060aaggacctgg agcctcctgg ggtagtgact
ttgtatcctt gagctcctac tacgtgccag 9120accttgtttg aaacccttta
tgtggctggg cacggtggcc gacgcctgta atcccagcat 9180tttgagaggc
cgaggcggtc ggatcacctg aggtcaggag ttcgagacca gcctggccaa
9240catggtgaaa ccccgtctct actaaaaata caaaaattag ctggcgcagc
ggtgggcacc 9300tgtaatccca gctacttggg aggctgaggc aggaggattg
cttgaacctg ggagacggag 9360gttgcagtga gccaagattg tgccacttat
actctagcct gggtgacaga gcaagatcct 9420gtctctctct gtcacacaca
catgcgcaca cacacacaca cacacaagaa agaaaaaaaa 9480gaaacccttt
atgtgtgctt aatctccatg acattcccat gttgcaacaa ggaaactgag
9540gttctgggag ggaagtgacc tgcctgaggt cactaagcct ggaagggtca
tttagatgta 9600attcacatac tcataactca gtcttttaaa acgtacaatt
cagttggttt cttagtgtat 9660tcagagttgt acagacatca gcactatctc
attccagaac attttcatca ccccaaaaaa 9720ggactctgta cccattagca
gtcaccactc cccactccca gcccctggca acaactaatc 9780tgctttctgt
ctctgtggat ttgcctgttc tgggcatttc atataaatgg aatcctgcaa
9840tatgtggcct tttgtgtcta caaggtgcat gctcttaacc attaagccac
accgcttctt 9900ggtggctcct acccaggggc cgggaccctt ggaaatgact
tggtccctgt gaaagctcag 9960aggggcatct gtgtgtgctg tcagccggcc
tccctccagc caagggcttg cagaatgacc 10020aaatgactgc cgtggccacg
ctggccttgc cctccgtggg gcgcggtgag tcaccccaga 10080cctgccctgc
ctgggtggtg tgagtgcatg gggtgggggc ccagcaggac actggggctc
10140tgtctgtcct ggacaggcgg ccactgtgca gcaaacagga agcgggctgg
gccgagacct 10200gctaggtaaa tacaggctgg ccagggaagg tgctgggcat
ggcatgatgc ccccctactg 10260ctgccgcctg ccaagtgcct gggagccgtg
gggtagcctg gcaggcagta ggtttcgcct 10320ggccaaggag ttgagctccc
agccggctgt tcccagcagc cgccacttcg aggtggctgc 10380cagagtctac
ctccaaggct ctggatctgg ccgctctgcc ttctctagga gtcattgcct
10440tccttggtcc tcagtccttt tgttcatctg ttcccctaat gatactaata
aatagtacac 10500tgatactgat gattccaaca gcagccatgc ttgactgaat
gcatgctctg taccaggcac 10560tgggctcagt gttttgcatg ctttatctca
tgtaaccctc acgacagttc ccttggggta 10620ggtgctagga tcattgccat
ttttcaggtg aggaaaccga ggcatgggca ggccaggtga 10680cttgcccaga
gctggtgaac tggtggttcg tcagggactg cgacccatcc cccactccat
10740tctttctcca gcttctccct cccagagttc ccttgtcttt gtaaagtgtc
catcaaaggc 10800gcaggtcccg gggatgcatg ggttctaatc ctgcctcccc
ctttctggag caaggcaatg 10860ccagacaaat gacttcgcct ctctgagcct
cagttttctt acctggaagg tggggagggg 10920gtggacaggt ctgggatttt
ctctctagta acagcaaagc tcagagtcct tgtcttccta 10980gtaccttcct
atccacatgt ccccggcaag actgatttgg aatggcaggc actcacagtt
11040gtgtttattt gagggtacca caggaagtgg gggcccccaa attggctaag
gagccccagg 11100gtggagggag gtagaagcac actgggtctg tctgggagcc
tgagcacctt ctctgtgggc 11160tccttcctac ccatggattc caacccccac
ccacctcccc actgcccacc cactcaccca 11220tctcttctgc tttccttctc
cctgtgcccc aggccacacc attcctctgt gcaccctgca 11280ctttcccctc
tacacctttg tccttgacac ctcctctgcc ctttatcttc ctcctcctcc
11340tcctcacagc ctttccccct ccccatcagg attctggctg cccttgctct
gcaaccctgc 11400ctggaacttc aggtccctga tcttacagcc taaaaaaacc
aaatattcat tgacacctca 11460ttttgcttca gcaccatgct gaatgctggg
gacacagggg acacgaaata gtccctacca 11520catggaacta aaactctgga
ggtgaagaga gaagatgaat caaagaataa tatataggcc 11580gggtgcggtg
gctcacacct ataatcccaa cattttggga ggctgaggca ggtggatcat
11640ctgaggtcag gggttcaaga ccagactgac caacatggca aaaccctgcc
tctactaaaa 11700atacgaaaaa ttagccgggt gttgtggtgc atgcctgtaa
tcccagctac tcgaggaggc 11760tgaggcacga gaattgcttg aacccaggag
gcggaggttg cagtgagctg agatcatgcc 11820actgcactcc aacctggggg
acagagcgag actctcttga aaaaaaaaat acatatatag 11880aaaagtttaa
tcgtgggaag tgctgtaaag gaaggcacat gttactctaa gagtaaagag
11940caggggaaac aggagggctt ttcagaggaa gtgcccttta acatgaggcc
tgaggagtaa 12000gtgggagcca gccagcgaag ggaagggcat tccaggcgga
gggaacagcc taggcaaagg 12060cctggaggca gagagagcca tcagccttcc
ctcctggact gcgagttctc tgaggttcca 12120cacttttatg tctcccctgt
gccctgcaaa caggtgccac tgcattgatg cctggtggaa 12180tgaattgatt
ccagaaggga gggaaccggg agaaaccagc tccctggatt cctccccgct
12240ggggcggagc aggtttgcat gcccaaactt gcccccagat ttaagcagct
ttccttggtc 12300acgtaatcag gaggatggga tttgacaaat gtttgccagg
gtttccatga gatcaggcaa 12360agccgcgggt aaccccgagg atgggcccct
ttttccaccc tccgcaagtt ggggcatggg 12420gacggagcca gagacctcca
ccgcctttga atccgggatt ctcagagaga agccatctgt 12480tgtgcaatct
gctgtttatt gaggctttct ttatgccagg cacttgactc aaggtatata
12540cctctcacaa cgaccctatt acgatccctt aatagggcag ctgagggccg
ggcgtagtgg 12600ctcacgccta taatcccagc actttgggag gctgaggcag
gcggatcacg aggtcaggag 12660atcgagacca tcctggctaa caccatgaaa
ccccgtctgt actaaaaaaa tacaaaaaat 12720tagccgggcg tggtggcggg
tgcctgtagt cccagctact caggaggctg agcagaagaa 12780tggcgtgaac
ccgggaggcg gagcttgcag tgagtggaga tgcgccactg cactccagcc
12840taggcgacag agggagactc cgtctcaaaa aaaaaaaaaa aaaaaaaaaa
atagggcagc 12900tgaagaaagt gaagatcaga gaggttgggt aacttgctca
tactcaccca gcaagtgagc 12960tgtgggtctg ggatctgacc ctctggtctg
cctgacgtca aaatgtgccc caccggctca 13020gggggccatg agagcagaaa
gccaggtttc tggaaagcct ggggtctctg tctagggcag 13080aaaggaaggt
ctggggaccc tggctcagcc ctgtatctca tcaccctact caggactcct
13140gcacccctta acttgcccac tcctgagccc ttctggtcta tgcagttctt
aacaccatcc 13200gacatgactc tgctgcttga tttgtctagt ttattgtttc
cccaccagaa ggtctgcccc 13260caaagggatt gatatctgtt ctgttcacat
ctgtgtccct agtctagcac agtgcctggc 13320acatagtagg tgctcaataa
atgtgtggaa taaataggta gctggcttct actgctgccc 13380cttgggccct
ttccagagga agatgcagat tctttgtcca tcctacacaa ctgccatctc
13440tgtgtggacc accattcagt atatatattc taaagaagag tttgaatata
aacctgcttt 13500tgaatatata ggatttaaaa ccaacatatt caaaaaccgc
tactttggct gggcgcagtg 13560gctcatgcct gtaattccag cactttggga
ggccgaggcg ggtagatcat ttgaggtcag 13620gagttcaaga ccagcctggc
caacatgtga aaccccgtct gtactaaaaa tacaaaaatt 13680agctgagtgg
tagtggcatg tttctgtaat cccagctact tgggaggctg aggcaggaga
13740attgcttgag cctgggagga ggaggttgca gtgagctgag attgtgccac
tgcactgcag 13800tctgggtgag agagtgagac cctgtctcaa caaacaaaca
cccgctgctc tgaatgaacg 13860ctgttgtgaa gatgtcagtg tgtgaggatg
ttcacagtcg tgcaggaacg ttcttgggtg 13920cctccctgat gcacacctgg
tgcagctggg agcttcccca ctgtggaggg gctcctggta 13980ggaggcattg
tgagcccgtg cgggaacgaa gggcccacca tgcctgctca cagtgggcac
14040acacacatgc acacgcccat ggaggaaggt gggaagaaat gtacatgaca
gtcacacgca 14100gaggagacac atgtggactc acacccaggc tctcagcctc
tctccctgcc tgcatccccg 14160acttccctcc ctcctaaaac tggcatgagt
ttggaatgtg taataacagc ctttagggca 14220aattagaaca gggagtaaac
tctttctctg accacagaga acaggacagt gtttggggag 14280ggaattgcca
cagagcagca gagccgggtg tgtgtgtgtg tgtgtacaca tgcctatgtg
14340tatgtgtaca cgtgagctag tgtgtacaca ctcctgagtg ctgtgatgtg
gaaggagccc 14400agggctagga gtctagaggt tttcttcaaa gccatttgct
ccctagctgg cctctcagag 14460cctccgtttc cttagctgta aagtgggagc
aatagcctag ctttgttcag gttcttcggg 14520ggattgaatg agagaggggc
ttggtggatt ttagcatggg ctataaaatg tcaggctgta 14580gtgctggcac
gtgtgtgcat gtgtgttttg gggaagggga gtaaggagga caggtaaatc
14640aggaccatat catagtggtg atgatgataa ttaaatgtga caagatccaa
catttatcaa 14700atatttctta ggtgcccaag aaacacttga cctgccttat
ctcccagaag ccctacaagg 14760gaggtgctat tattgcaatc cccattttac
aagtagagaa accgaggctc agagagatga 14820gaatgggcgg cagtaatcat
cccttcctcc tgggggtggt tgtgaagctt gaaggagaca 14880ctgtaagcgc
tcgggtggta ccctggtact ccatcactaa cctgctgtgt gacctcaggt
14940aagtcgctta gcctctctga gcctcagttt cctcatctgt aaaatgggaa
tgattgcaat 15000acctacctcc agggcagatt ttctcaacct cagcgctgtt
gatgttgttg actgggttat 15060tctgtgtggt gcggggctgt cttttgtttt
ggaggatgtt tactaccatc cggcatggcg 15120gatcccagca aaggcccctg
tgcaggcctg gtgaggtggc cgagcttcct ggtggggagg 15180ccttccccct
ctcctccctg gtcaatctat ttgagggtga ggatggctcc ccttcaccct
15240cacctggcac ccaggacatc cccagtccat ggagggattc aatctgggct
ctgcagccag 15300ctgcccgcat ttcagtcctg gctctgtcac taacctgctg
tgtgacctca ggcaagtcac 15360ttagcctctc tgagcctcag tttcctcatc
tgtagaatgg gaacgatcgc agtacctact 15420tccagggcag atgttctcag
tctcgacgct attgacattg tgggctgggt catttttttg 15480tggtgtgggg
ctgtcttttg ctttggagga tgtttagtgc catccctggc cttgatctac
15540cagatgccag tagtgctccc tctcctgtac agttgtgaca acaaaacatg
tcatcagata 15600ttgccaaatg tccttagagg aggagcagaa ctgtccttgg
ttgagaacca ctggtctgga 15660gtcatgatga gggctggatg aggccctgtg
ctgggcacag cctgagctac gtcatcaggc 15720gaggatggtg atttagttca
taattattat ttcatcctaa ttagaatgct aatcatgatc 15780gcggcgtgac
ttgccgaagg ccacacagca gccatacagg ttgacagttg ctgggagagg
15840atttgaacct gggattggct ggcaccagag cccccatttt ggagtcccca
ggaacctggg 15900gtgctgtcca ggcttagggg agggctatcc tcctgagggg
aggagggtgc aggcatggtg 15960ggggatggaa ggaccttctt ggctgccttc
tctggccttg ggagctccct gggatggggt 16020tccattagcc cctgaggtgc
catggtgagg ggtgcagtgg atagaagggt actggaaaca 16080cagagggtgc
gcctgatgcc tcgtctcccc ctgacctcag ggcccccagt gaggtggaca
16140gggccatgcc caggagagtc taatccacct cccgccaatg cctgtgcccc
tgcagatgcc 16200cctcctgcct cagcaacccc ctggccccca ttcctgcaga
gcaaagccca aatgcatgtg 16260gcagccatga gggactaggg atccagatcc
ccctctcctg tctggccagt cgaaaggact 16320tcgtgtctcc agaggtccta
ctaggtgctg gaactgagta ggtgcctcac atgctggagg 16380accctaggag
gaggcgtggt cactccagtc ttacaggtgg gaaaaccacg gggcagacag
16440aggaagtgat ttgcccatgg tcaaacggct aatgaccagg gccaagagag
gaactgggtc 16500tcatttcaga acctggcctc ctaacttctc tttcttcgtt
tggttataat aactgaaagt 16560cccactctat ttacagatga ggaaactgag
gctcagagag gctaagcaac ttacctgagg 16620tatttatttt tagatagagt
cttgctctgt tgccctggct ggagtgcagt ggcgccatct 16680tggctcactg
caacctctgc ctcctgggtt caagtgattc tcctgcctca gcctcctaag
16740tagctgggac tacagtcacg tgccaccacg cctggctaat ttttgtattt
tttatagaga 16800cggggttttg ccatgttagc caggctggtc tcgaactcct
gacttcaagt gatccacccg 16860cctcggcctc ccaaagtgct ggggttacag
gcgtgagctg ctgcactgag ccaaaacccc 16920actttaatcc cagaagtggc
actggatata tctgacctca cttcccactg cctgcaaccc 16980tatgaagaag
tgactacaat tgtacccatt ttgcagagga ggaaactgag gcttggggag
17040ctcaagtaac ttacctaaga tcacactaca tgtaaatagc agagctggga
ttagaaccca 17100ggcctgaatg actccaaagg ccaggctgtc tctccctttt
ggtgtccaaa gggaagccca 17160cccccagtgg gagctctgac cctctgtgtc
ctgctgcgcc cactaaggga ggcctcttgc 17220tgtgtcccca cctctctggt
ccaagtcttc cctcctggaa gccacagaac aaacaaggtg 17280ggaactagtt
tatttgtttt tctacgtgcg tattgggtgg gaagggtgag atgtacaaga
17340gagggctttt cagacatgcc cctgcctccc gggtggggtg gtaagagttc
caggaaactc 17400acccttggtg cccagccctg cctggctggc accatgctac
agagagcagg gcactgacag 17460ccaaccagtg gggccttgcc cctcccttgc
cctggctcct ggctaagcac tggacccggg 17520agccagagag acatggttca
agtccagctc ttcttcctgc aagctgtttg ctgcctttga 17580aagctgcttc
ctcatctgag aaatgggaac aaatgacatc tttgtcataa agtttttcat
17640ttgtgtgagg actcagggat ggacaagaca gatacatttc ctgcctcctg
gcacccacag 17700cctgggaacg aaccatcccg tgaacagctg ggataaagct
tctgaggaga ggagcatgga 17760tcctgggagc gagtgtgtgc aggccaggga
gggctttcca gaggagccca gttgagctgg 17820aacaccagtg gggaggagtt
gaccagcaaa ggtgcaggga gggatcagca ctttgcactg 17880gggagcagag
tttgtgcact ggggaagtca actcaagtat tggagcctca gtttcctgtt
17940ctgtaaaatg ggttcatcat gacagtgttt gatgaggaaa aggactgccg
gcctacacag 18000caagtccaca tggattttct gagcccctcc tgtgcctgaa
gcccacggtt aatggttctg 18060ccttagcagg tgcttaccac gtgccaggca
ctgcactgca ctggccactg gactgcatgt 18120tctgtccatg aggcttggat
atccccatct tacagatcag gaagctgagg ctatgaaatg 18180tcgacttgct
caatgtcatg gaatgactaa gtgtggagcc tggatttgaa cttggctctc
18240tggggctcca aagctggctt tcttggtcag cagtagggtc tgggatccaa
gtatggggtc 18300ccagcttgac cctgaagtcc accctctttc agctaatgcc
cagggtagtt ggacctgggg 18360ccaatttgtg tttccaggtt cgtgaaagag
gctcctgttg cagttcccgc ctgaggctgg 18420cggccaacca catctgggag
tggcctccct gtgcccctgt cattacaacg gtggctttga 18480agcagctggc
agcactgctg cttgtccacg tgggaggggg cttcctggag cccccgcccc
18540tggccgggtt ctgcctgact cccctttcat tcccttgcag gctgagcagt
gcagacgggc 18600ctggggcagg catggcggat tccagcgaag gcccccgcgc
ggggcccggg gaggtggctg 18660agctccccgg ggatgagagt ggcaccccag
gtggggaggc ttttcctctc tcctccctgg 18720ccaatctgtt tgagggggag
gatggctccc tttcgccctc accggctgat gccagtcgcc 18780ctgctggccc
aggcgatggg cgaccaaatc tgcgcatgaa gttccagggc gccttccgca
18840agggggtgcc caaccccatc gatctgctgg agtccaccct atatgagtcc
tcggtggtgc 18900ctgggcccaa gaaagcaccc atggactcac tgtttgacta
cggcacctat cgtcaccact 18960ccagtgacaa caagaggtgg aggaagaaga
tcatagagtg agtattgtta gcttcctggc 19020ctgtggtctc ctcctctgta
tccattcacc cattcatcat ccacccctct atctattatc 19080cacccatccg
tcgattcatc catccatcca tctgtccatc caaccatcca tacatctatc
19140catccatcta ttcatctccc tatccatata tcatccgtcc atccatccat
ccaaccaccc 19200atctattcgt ccatccatcc aaccatccac acatctatcc
atcaatccat tcatctctcc 19260atctctatat cctccatcca tccatccaac
catccataca tctatccatc gatccattca 19320tctctccatc catatatcat
ccatccatat atcatccatc catacatcca tccatcatcc 19380agccatctat
catctatcca tctatccaac catccatcat ccatcatcca tccattcatc
19440tattcattta tacatcatcc ttccatctgt ccatctattc atctatccat
cattattcca 19500tctgtccatc tatctactat ccatccatct atccatctac
ccatcatcca tccatccatt 19560catccatcat ccttccatct gtccatctac
tcgtctattc atcattccat atgtccatct 19620atccattaat ctatccatcc
atccatatat catctatcta tccacccatc atccatccat 19680ccatctaccc
atctatccat catccatcca tctattcatc atccatctat ctattaatcc
19740atcatccatt actctaccca tttacccctc tgtccaccct tccataggac
atttgtctgt 19800tcatccattc atccatccac ccactcattt atacaatgca
tccatccacc cactcattta 19860tacaaatgca aatgcattta tacaaatgca
aactcagcca tttacccatt tacccattca 19920cccttccacc cactcattta
tacaactatt tactcatcta actctccatt cattcatctg 19980cttttttttt
ctttttgaga tggagtcttg ctctgttgcc caggctggag tgcaatggca
20040tgatctcggc tcactgaaac ctccacctcc tgggttcaag caattctcca
gtctcagcct 20100cccgagtagc gggattacag gcacccgcca ccacacccag
ctaatttttt ttgtattttt 20160agtagagaca ggttttcacc atcttggcca
ggctggtctt gaactcctga cctcgtgatc 20220cacctgcctt ggcctcccaa
agtgctggga ttataggtgt gagccaccgc atcccgcctc 20280atcttttttt
attcacctac atatccacgt actcatctgt cccccattaa tctacttgtc
20340tgtccatctc ttcacctaca catccagcct tccatttgtt tatcttctta
tttatacatc 20400tgttcatcca tccatccatc catccattta tccatctatc
atctactcat tcatttaccc 20460atctttacac ttttttgtcc acctatccaa
tctatagatc cattgtccac tcattaaaat 20520atctatctac tcatctaccc
atctgtcagc catctgtcca gccatacacc catataacca 20580accttccatt
catctaccat tttctcatct gaatgtcatt tcatcttctc acctacccac
20640tgtttctagt catctagcca ttcagctatc aaactattca tcattcattc
atttattcat 20700tcattcattc catttctcat gcttgattgc tcacctgcct
gctgtccttc tttccttcct 20760ctccttctct cttctaacca ccaattcatt
cacccagatc tctgtccatc catttgtcca 20820ttcactcttt tgtttgctta
gtcactgact catttattca tttgctcatc ttttcttcca 20880tttttgcatc
tattcatcca tccatctccc taccttctct tcctggcaca catccagctg
20940cccctcctgg gtcttcttca atcatcacct ccattcctgg ccatccagtg
gctttcctga 21000ggcttagaaa gagctggaaa ccccaaggca ttcaaccttc
tttcccataa tgacagcctt 21060gctgaaagaa aagacagact ggagagaagc
cctggatggg gccacagcat gtggccccta 21120tgacgcattc catgccaggc
ctagctgggg acagggagcc tccagccccc ttgaccacct 21180tgtctggtcc
ccatcctccc ccagtgtgtt atgctggcat ctgcatgtgg tttgtgtggc
21240actctgcaaa ctcagacatc ccgggttctg tttccagcat gactctattg
tctgtcacct 21300gcagactccc tgctttgtga ggttgagccc tccctcctgg
tcctttagga agcccaggga 21360cttccacaag cttttggcag tttggaggag
ggagcagggc ttgcagtctc ctagccagct 21420cctcctgagc ctcagcaggc
cctgctgaac tgtatccttt ctatgtcccg gccctgctgt 21480ccctaatgtg
ccctgaattg accctttcct caactgctcc atgaaatctc ctctcggtgg
21540cctgtgtgtg tgtcctggag gatggtgctg aggtctggag atgacctgga
cttgcgtgtc 21600ctcctctgtc cccctgctgg ccacagattc atttatctct
tggatggagc cgattcacct 21660tcacagccct tgcttggtca agagccgccc
cctttattta ttcacagtca ttgattcatt 21720cagatgtctc tttttctttt
tttttttttt gagacaggtt ctttctctgc cactcaggct 21780ggagtgcagt
ggcgctatct tggcttactg caacctccac ctcccaggtt caagcaattc
21840tccagcctca gcctcctggg tagctggagc tacaggtgtg cacgaccaca
accggctaat 21900ttttgtattt ttagtagaga cagggtttta ccatgttggc
caggctagtc tcgaactact 21960gtcctcaagt gacctgccca cctcggcctc
ccaaaatgct gggattacag gcgtgagcca 22020ccgcacccag ccagtggatg
tttcttgggt gcccactgtc tgccagtcag tggggttggc 22080cctgaggctt
caggctgagc cagaccagat ccacttctac tcaggcggca ctttccgttt
22140agagggtgag acagacaaga agcaagtagc tgagcacatg gatgtgccat
gtgagttggg 22200ggaggtgctg aagttagaga gtgggtggat acaagaaagg
taatccagga gggcttcttg 22260gaggagttga catttaagcc caaagctaaa
ggtcatgaag gggcagtcac agagagggag 22320ggaagggtgt tgtaggtgga
gagaagtgtg agtgcaaggg cctaaggctg gagaggctct 22380gcagatgtcg
ccaaggaata gggcctcaca gttgtctgtg ctgaaaaatc tgtacaacct
22440ctgctcagag gtggaaacta agggccagag cagcagagtg cccagtgttc
ccatccttct 22500gtccccaagc cttcccaggt cctgttgaaa gctggtgggc
tctgtatctc ccattcccct 22560cccgcaagcc ctcctcccca cccctacccc
aggaagtcca ggctggtgga gtaagtgggg 22620cacggctgag ccctgaaggc
tgcctcagtg agatctggtg ggggcagtat tgctttttat 22680tttttggctc
aaaggtcaaa gttcaggaag aggtaaagtg ggggctgcct aagtgccctc
22740attagaggct gtatctgtgc ctgtgcccct gtcaacagga gagcacgtct
taccctactg 22800tggatgctaa tgatcccctt tgccagtacc tgccctgaga
tggaaaatct caacccctgg 22860cgaagaggaa gggaggagag tggctgtccc
cttctcatgg caagacaggc tgggtacagc 22920cagatgccag ctttgtccac
ccttgcaacc ccgtcccacc tgcttccaca acctgccctg 22980gcctcagttt
ccccaactgc attccccttc ccctctggcc ctgctgcctc ttgttagggt
23040tttctctgtt cccctactct cccctcttct ccctcccaga ccctgtgcct
ccatcttcct 23100ccttctcttt agtctcttat tcattgaaca gagtcttact
gcatcccaag atgtgccagg 23160cactgggcca ggtaagaatg tgacagagag
agcattcctg cctcatggtg gccctgggct 23220agctctttct ctctcagaac
ctagcacata ataggtgccg aaataatgtt tgttgaatga 23280ctgaatgagt
gtctccaagc cctccagctc ttagtgtctg tttccttcct ttctctctct
23340atctcttctc ttcccagccc tctctgtgcc acacccttcc gccatctgcc
tctgtagccc 23400cacatctctc ccatgcacat acaagctcca gacacacagt
aggcactcat taataatggt 23460aatatcaact aacatttatt gaatgtttac
cgtgtggtgg gccatgggca ggtacatatg 23520agcatggcct catttgtctc
aaatgtttgt tgaatgaata aattaatggg ttagttatgg 23580aatgaatgga
tgaaatgcct catcttttag tctgctgttt ctttatttta tctttgtttt
23640tgtctctttc agtttctctg ccttagtaag tactcaataa atgactaata
tatgagtggg 23700tgggtgagtg gatgggcagt tggatggatg gatggatgga
tggatggatg gatgggtggg 23760tgggtggata ggcagatgga tggatagacg
agtgatggat ggatggatgg atggatgggt 23820gagtggatgg atgggcagat
gggtggatgg atgagtgggt ggatggatgg atgagtagtg 23880gatggatggg
tggacagatg gaggagtgag tggatgggta gatgaatgga tgagtgatgg
23940atggatggat ggatggatga gtgggtggat ggatggatgg atcagtgagg
gatggatgga 24000tggatgagtg atggatggat gaatgggtgg atggatgatg
ggtgggtagg tggatgggtg 24060gatgctgggt gggtggatgt atggatgagg
gatggatgga tggatgggcg ggtaggtgga 24120tggatggatg ggcgggtagt
tagatgggtg gatgaatgga tggatgggtg ggtggatggg 24180tagatggatg
gatgagtgat ggacggatgg ataagtggat ggatgggtgg atggatggat
24240gagtgatgga tggatgagtg atgaatggat ggatgggtgg gtaggtggat
gggtgaatgg 24300tgggtgggtg gatggatgga tggatgagtg ggcagatgga
tggatgagtg atggatggat 24360ggatggatgg gtgggtggat gggtagatgg
atggatggat gagtgatgga tggatggatg 24420ggtgagtgga tgggtagatg
gatggatgga tggatgaatg atggatggat gggtggatag 24480atggaggagt
gggtggatgg gtagatggat ggatgagtga tggttggatg gatagatggg
24540tggatgggta gatggatgga tggatggatg agtgatggat ggatgggtgg
atagatggat 24600gagtgggtgg ataggtagat ggatagatga gtgatggatg
gatggatgga tagatggatg 24660ggtcggtgag tggatgtgtg gatggatggg
tggatagatg tatgggcagt ctgtgcattt 24720ctttctgttt gtctccaccc
aacacacagt aggtactagc tatagtttgt tggataaaac 24780attacttagt
ttttacatct gccccaacta cctcaccctg ttccttgtaa ggcttcagct
24840gcctgcccgc caccacagtc tctgggtccc taaggccagg gacagtgggg
caggcagggg 24900atgagccctc ccatcaactt gcctccctac ctcctccagg
aagcagccgc agagccccaa 24960agcccctgcc cctcagccgc cccccatcct
caaagtcttc aaccggccta tcctctttga 25020catcgtgtcc cggggctcca
ctgctgacct ggacgggctg ctcccattct tgctgaccca 25080caagaaacgc
ctaactgatg aggagtttcg aggtgagcca cccagatggg catagccagt
25140gggacagcca ggggtgtggg ggaagcctgg cattgggggc cccctttccc
ctcagcttct 25200ttctttgggt cggtggactg cattggcctg gaaagtgcac
tggacaggga gtctggtcct 25260gtgtgtcctt caccatgtta cttaacctct
ctgtgcctca gctacctcca tttattcatt 25320ctttcattca ttcagccctt
atgtatgaaa aggttagtgt agtgggtaag cagagtccac 25380ctacctgggt
tcagattcta cctttaccag ttaagcgatg tgtgacctct ctgagcctcc
25440gtttcctcat ctgtaaactg gggaataatc atagcatact cctggctctc
atcccacaga 25500gagcccagcg caggcagctg gagtcctgga gctcctgctc
ccctgaggga aggtctggag 25560ggatgggcag gtgtctgggc tggtagtcct
gattctactt cttggggtct gctccacccc 25620agcctagctt tagggctcca
cttcctaggc tgaagcccca gcccagagag ctaacccttc 25680agccttgtcc
agattcaaaa cacccacctc aggacaccgg caccctccac agccccaggc
25740cttacctgtg aacacctgca cccaaatcag ccacctgcaa tgtgctgggt
tctgggtaag 25800ccattattaa actggccgtg atctcacaag tcaagatacc
atgtcaagaa gtgtgacacc 25860aaggctgggc atggtggctc acacctgtaa
tcccagcact ttgggaggcc aaggcaggag 25920ggttgcatga gcctgggagg
ctcaggctgc agtgagctgt gatcatgcca ctgcactcca 25980gcctgggtga
cagacaggaa aaaaaaaaaa aaaagaattg tgatacctgc tatgaagaag
26040ggcccatctt ggaaggcgga ccatggttgg tctacagcct aagtctgagg
aggcttcagg 26100gatgtcagaa gaggcttttt tttttttttt tgagacagag
tttcattctt gttgcccagg 26160ctggagtgca atggcatgat ctctgctcac
tgcaacctcc gcctcctggg ttcaagtgat 26220tctcctgcct cagcctccca
agtagctggg attacaggca tgcgccacca cgcccggcta 26280attttgtatt
tttagtagag acggggtcgc tccatgttgg tcaggttggt cttgaactct
26340cgacctcagg tgatctgccc gccttggcct cccgaagtgc tgggattata
ggcatgagcc 26400actgtgccca gccagaagag gacatttttt aagacttcag
ttactttaag taaattaagt 26460tcccaaacag ggtgaacaag tctgtgctac
atcatccaca tataccttta tcaacctgtt 26520tttttttttt ttttcttttt
tttttttgag atagggtctc attctgtcac ccaggctgga 26580atgctgtggt
gtgatcacag ctccctgcag ccttgaactt ctggcctcaa gcaatcctcc
26640tgcttcggcc tcgagagtag ttgggactac aggtgcaagc taccatgcct
ggctaaattt 26700ttttttttct ttttttttaa gagacaggtc tcactatgtt
gcccaggctg gtatcaaact 26760cctggcttca agcgatcctc ctgcctcaac
ctcccaaagt gctgggatta caggcatgag 26820ccactgaggc tggcctcaac
ctcattctta ccctgaaaca atacgatgca gtatcattgt 26880gtccatcagg
agacaattac tggggccggg tgcggtggct catgcctata atcccagcac
26940tttgggaggc tgaggcgggt ggatcaccta aggtcaggag tttgagacca
gccttgccaa 27000catggtaaaa acctgtctct actaaaaata caaaaaaaaa
aaaaaaaaag ccaggcttag 27060tggcacacac ctgtaatttc agctacttag
gaagctgagc caggagaatc acttgaacct 27120gggaggtggg ggttgcagtg
agccgagatt gcaccactgc actccagcct gggcgaaaga 27180gcgagactct
gtctcaaaaa aaaaaagaaa atacaattgc tggatttatg aaaaatattc
27240attcatggtt cctggccacg cgacgtggcc tcgtttggag gcacaagttt
agagctgtgg 27300gaggacgggg cttctctctg ctcctggagt agctcagtga
tggcatgagt aatctcattc 27360ggagataacc catgtttaag ccctggccaa
atggcctctc ctggtccacc aagtacgtga 27420ggcaaaagtg cggaatcttg
gggtagagcg aatcctggga gatggatgct ggcacctgtg 27480ccttcagcac
caggctagct tgtcaaggcc tctggcttct tctgaattca ggactgagtt
27540gggggcttct agcatagtcc aggaacccag atgcatgtgt gtctgtgcat
gagtgtgggt 27600gggggactct aagataggct ttggaggtgg gtctctgaaa
ttgcagaggc tagcgtatgt 27660gcatagaggg agacttactg tgaggtccat
gatactaatt aagtgccagg gcccctggct 27720aaggcccttt ctgaggtcct
cgacctagtc tacgtcccac aaaacctgga tccgtccctg 27780catggctaaa
aggtcatgag ctcatctgat tatcagggaa gtctgggacg ccctcctgtg
27840cccccacctc ccctccgcag agtttcacaa cctccaattt ggcaattgtc
aatttagagc 27900ccctggctac agcaccccac cctgggcaga gccacttcgc
cacctggtgg ctggttcctg 27960gaactgcatg ttccacctca tctctgggaa
gatgctgctc ctgacatctt ctcccaggac 28020ccaggcgtcc cctccctggg
tctataacct gtgtctgaaa gcccgaatcc agggtctcta 28080gttccacttt
gggtcaccta tggtttggaa ttacctgggg tgcccagctc tcgccttcat
28140tcaatgtgtg ttaactcagc aaatggttgt tgggcaccta ccacatacca
ggcactgtgc 28200ttggcagagg ccgaatgata gtgagcaaag cagacatcac
cctgccctca aggtgcccac 28260aggctcctgg acaggaccat cattgaccaa
gcaaccacat aaataagcac acagttatga 28320actgtaacag gtgtcaacaa
aaggctgtac tggaggattt gacctagtca gggaaaatcc 28380aggagagctt
cctggaggag gtaacacttg agcagagtct gagtgagttg cagttaacca
28440ggtgaaaaga ggagggaaga atgtttcaga ttggggtggg ggttcagaga
cagcaggtgc 28500aaagaccctg tagttcaccg tggtctattg cagtctgaaa
tgaccattcc agctgctatg 28560tggaaagtgg ataaaggaag gccagagagg
ctgggggaaa tgagggaaat aaaggaggtg 28620tcttaggcat gtgagaggga
tggtggctgg gccacattat tccatgagag gtttggtgtc 28680gcctctgagc
tgggcgccag gtggacagcg ggaaagcaac tgggaacaag acttgtccca
28740aacaaccctg ctcctggaag ggctgagact agaattcagg tctcttgagt
ctgccccact 28800gattcccggc atttagggag acggttccta aatcccgtct
catggattga taacagcaac 28860agtaatattt atcaaagact gctatctgcc
tggtactggg ctaatcaaca aacactgatt 28920agctctttga tttcccttca
tagctttaat ttagccagaa aggaccctaa gcagagaaat 28980ggtcatacag
ctgtggggag caaagccagg cctcaaacct gaggtgcctg gcttcagaac
29040gggcacctga acccacacct gcgtctccca ccctctggcc attcctgagc
ccctggctga 29100ttttgccctg tccttgattc acaggggagt tttcaactta
ctcttttgag atataaatca 29160actcaggtaa ccataaaaat agctacaaca
gaaagaaact tgcaaagaga catcagcaag 29220cgttagtgag aggtgttaag
gacccgctag taccaaacgg agagttgact tcatgagaaa 29280gattcgatag
tggatgaaac cttcagggtt attattgttg caccccctag aacccccaag
29340attctctcct ggatccccta aggtcagttc acatccactg tggcacacag
tcccacactt 29400tggggaacgc tgcacccctc cactcccttc taggggcacc
agtgtaaatg cttcatggga 29460aaaggattcg atgctctgac aggtgggtga
atgctgccta gtgtgtcttg cttttacagg 29520ttcacagtcc ctgttagtgt
atgaaaggct ctgagaagtc ctgtagtaag gaattatgat 29580tggctttgtt
taacccagca ttctaaccat ttatttgttt atgaagcact ttacataaat
29640cttttcgagg gccaggtgtg gtggctcaca tctgtaatcc cagcactttg
ggaggctgag 29700gtgggaggat cacttgaggc caggtgtttg agaccagcct
gggtaacaga gtgagatccc 29760ttctctacga aaataaaaat taaaaaatta
gttgagcatg gaggtgcaca cctgtagtct 29820tagctgctac ttgggaggct
gaggtgggag gatcacttga gcccaggaat cggaggctgc 29880aatgagctag
gattgcacaa ctatacccca ggctggatga cagagcgaga ccctctctct
29940caaaaaaaag gatattaata aataaaaata aatacaaaat cttcccaaat
aagattaatt 30000aaataaatac caatctgtag gctgaagcct ctcacatttc
aaagcaccta accccatgcg 30060tttattgtac cctctcagca ccactggagg
cagaaagata
gagtggccct gggtccccac 30120tggacagatg aggaaacagg cttggagagg
agatgttgac agccaggaca tctgaccccc 30180tacccagtgt tctgtacccc
gatgcctgga agttcaaggt catgggctgg agcactgcgt 30240catcttgtgt
gtctctcttg ctagagccat ctacggggaa gacctgcctg cccaaggcct
30300tgctgaacct gagcaatggc cgcaacgaca ccatccctgt gctgctggac
atcgcggagc 30360gcaccggcaa catgagggag ttcattaact cgcccttccg
tgacatctac tatcgaggtg 30420gggccccggg ctgggcaggg gtgccacggg
ggctgatgga gacgctgtcc tttgcttgtc 30480tgactcctga gacttttgat
ctgggcctaa gtgccagcat gtacccagga cctgacaaat 30540ggatggatgg
atggatggat ggatggatgc atggatgcat ggatagatgg atggatgaag
30600gaacggtaac taccccttcc aactttgttt cgagtttcag aatagaagat
tccactgggt 30660gcggtggctc ttacctgtaa tcccagcact ttgggaggcc
aaggggggca gatcgcttga 30720gcccaggagt tcaagaccag catgggcaac
atggtgaaat cctgtcttga caaaagatac 30780aaaaaaaaaa aaataaaata
aaagaaggcc aggtgcggtg gctcacacct gtaatcccag 30840cactttggga
ggctgaggtg ggcagatcac ttgaggtcag gtgttcaaga ccagcctggc
30900caacatggtg aaaccctgtc tctgctgaaa atgcaaaaat tagccaggcg
tggtggtgcg 30960cacctataat ctcagctact tgggaggctg aggcaggaga
atcgcttgaa cctgggaggt 31020ggaggttgca gtgagccaat atcgcgccat
tgcactccag cttgggcaac aagagcaaga 31080ctcatctcaa aaaaaaaaaa
aagatgcaaa aaaagccagg cttgttggtg cacacctgta 31140gtcccagcta
cttgggaggc tggggtggga agatcacttg agctcagagg gtcaaggctt
31200cagtgagcta tgattgtgcc actgcactcc agcctgggtg acagagtgag
accctgtctc 31260aaaaaaaaaa aaaaaaagat tccaagattc tgagatgcag
cgagtctagt gatcatgaac 31320tcgggatctg gccctaagat gctgtggtgg
cggatactct aaggctctat gataataagt 31380cctgagttca caagttctaa
gaggcaaagg cagcaggggg ccaagaactg ggccaatccc 31440cagttgagat
tctgtaaatc agagttgacc ttcagatgcc tggatattaa gattcaaaat
31500ccatgaatat tatatcttga gtccaaaatt ctgggaggcc aaggtactgt
tgtttcctct 31560aggagttttc ttttttttga gacaaggtct tgctctgtta
cccaagctgg agtgcagtgg 31620tgtgatcatg gctcactgca gccttgactt
cctgggctca agggagtctc ccacctcacc 31680ctcccaagta gctgggacca
ccggcatgga ccaccatgcc aggctaattt ttaaaatttc 31740tgtagagaca
gggtctcact atgttgttgt attagcccgt tctcatgctg ctataaagaa
31800ctgcccaaga ctggataatt tataaaggaa agaggcttaa ttgactcaca
gttccgcagg 31860gctggggagg catcaggaaa cttacaatca ttgtggaagg
ggaagcaaac atgtccttct 31920tcacatggtg gtaggaagaa gaagtgccaa
gcaaaagggg gaaaagcccc ttatagaatc 31980atcagatctc ttgaggactc
actcactatc atgaaaacag catgagggta actgccccca 32040tgattaaatt
acctttcaca gggtccctcc catgatacat ggggattatg ggaactacaa
32100ttcaaggtga gatttaggtg tggacacaga gccaaaccat atcagttgcc
caggctggta 32160ctgaactgct aggcttcagc aatcctcctg cctcagcctc
tggagtagct gggaccacag 32220gtgtaagtca ccaggcccag ctaattttta
gcatttctat agagatggga tctcactgtg 32280ttgcccaggc ttgcctcaaa
cgcctgggct caggtgatct ccctccttgg tctcccaaat 32340tgctgggatt
acaggcgtga gcctgcgcct ggcctcctct aggattttta aagcatctta
32400tgaatctaaa aacttctcac attcaggatt ccacaaacta ggtatccttc
aggcttgaga 32460ttctgcttct gcgatggatc ccagggaata tccaaggacc
tatttgctgc ccttgggtgt 32520cgctgggcag gactctgcct gcatccccca
ccccccaatt tctacgtcct gcaccctacc 32580cccaccccca gcaagcctgg
ctaggtctct gctccgccag gaccctggat gaccgtcccc 32640tgcccccagg
tcagacagcc ctgcacatcg ccattgagcg tcgctgcaaa cactacgtgg
32700aacttctcgt ggcccaggga gctgatgtcc acgcccaggc ccgtgggcgc
ttcttccagc 32760ccaaggatga ggggggctac ttctactttg gtaaggaggg
gcctggtggg ggctgacagc 32820atgctggaga agcatggcgg gagatagcat
gatactggtt ggtgtctgca gccctgacca 32880tcacccagac acccagggcc
actctggcca tgagcgcagg cagcactctg gaccacaggc 32940tgcacgttgg
tcttggtcac agggccgttg cctctgaggt gtaagtgcca tggggagtac
33000catggacctg gattcagatc ctcactccag ccaggcacgg tgtctcacac
ctgtcatccc 33060agcactttgg gaggtcaagg caggaggatc gcttgagggc
aggagtttga gaccagccta 33120ggcaacatag caataccctg tctcttttaa
acatttaaaa aatggctggg cacggtggct 33180catgcctgta atcccagcac
tttgggagga tgaggcaggt ggatcacctg aggtcaagag 33240ttcaagacca
gcctggccaa caagatggtg aaacccatct ctacaaaaat aacaaaaatt
33300agccgggcat ggtggtgggt gcctgtaatc ccagctactt gggaggctga
ggcaggagaa 33360ttgcttgaac ctgggggaca gaggttgcaa tgagccgaga
tttccccatt gcactccagc 33420ttgggtgaca gagtgagact ctgtctcaaa
aaataaataa ataaataaat aaataaataa 33480gttagatggc atggtggcat
gtgcttgaag tcccagctgc ttgggaggcc gaggctggag 33540gatcacttga
acctaggagt tcgaggctgc agtaagccat gattgcacca ctgcactcca
33600gcctggaaaa tagagcaaga ccccgtcttt aaaaagcaaa aagaaaagaa
caaaaaaaaa 33660gccaaatcct cactctgcac tttccaggca tgtgacctca
cttccctgag cctcaccttc 33720cccagctgtg cagtggggat cacgagaggc
ccttgggctg tgatgtcagc gcccagctct 33780gtattgtctg tgtgctttaa
tctggtttat gctgggacca acagccccat caccaggccc 33840aaagcccacc
actgccgttg tcatcactgg actcatgaaa tattttgaat tttgcccctg
33900cttaaaggca ttcattatgt gcagctcagg gacaaagtca cacaaagaat
tccatcatca 33960gctgggcgcg gtggctcacg cctgtaatcc cagcactttg
ggaggccgag gtcggcggat 34020cacttgaggt caggagtttg agaccagcct
ggccaacatg gcgaaatcct gcctctacta 34080aaaatacaaa aattagccag
gtgtggtggc gtgcgcctgt aatcccagct actcggctga 34140ggcacaagaa
ctgcttgcac ccgggaggtg gaggttgcaa tgagccgaga ttgtgccact
34200gcactccagc ctaggcgaca gagtgagact ttgtctcaaa aaaaaaaaaa
aaaagaattc 34260catcattgga tgtgtccagt ccctcacagc ctccaaatcg
cgtggctgtg cccttaacta 34320gccacacccc atctccctgg catcacccag
agaaacgtgc agttcatatc cactgctggt 34380gctgtctccg tcattatcct
cagagcgcca cggtgtccgt cccccgagtg tctgcaggca 34440gagtcccacc
ctgggccccc ttcgctgacc tcccaccctt caccctggcc cgcaggggag
34500ctgcccctgt cgctggctgc ctgcaccaac cagccccaca ttgtcaacta
cctgacggag 34560aacccccaca agaaggcgga catgcggcgc caggactcgc
gaggcaacac agtgctgcat 34620gcgctggtgg ccattgctga caacacccgt
gagaacacca agtttgttac caagatgtac 34680gacctgctgc tgctcaagtg
tgcccgcctc ttccccgaca gcaacctgga ggccgtgctc 34740aacaacgacg
gcctctcgcc cctcatgatg gctgccaaga cgggcaagat tggggtgagt
34800gtgcggctgg gggcacagct gatccaccta ctcgtacccc tctgcacaca
cacacgaggg 34860tctgctgctg gtattcatta ttaatacgtg catgcacctc
ccaacatgcc aaccccagtg 34920tgcagatgtc ccagctcaag aacctgctat
ggctccctag tgtccagcca agctccttag 34980cttcatcccc gagcctctcg
cctcatgtca ccctactgga tgttcaccac tccaccccca 35040ccagccagga
tgagccactg ataagaaact gagtccccgg gccctagccc agcggtgctc
35100ccagcctcaa acctgttgaa tggccctgcc ccagttcctt cccttttctg
ggccgcgcag 35160catggggcct ggagaagggt ttctcaactt cagcatgatt
attttgggcg gcaggctctt 35220tgtggtgggg accgtcttgt gctattgcca
gacattgatc tgcatcccgg gcctctaccc 35280accagatgcc aggaacacct
ccccttacac ttgtgaccac caaaaatatc tccagaacat 35340tgctgaattg
tcctctgggg ggcaccatcg ctggattggg aaccaagtct ggagtatgct
35400agaggcagga gcacctgtcc aagccctggc gctaccactt cccagtgtgt
gacctcagac 35460atctcatttc ccttttctgt gcctcagttt ccttgtctgt
aaaatgggga tataagagtg 35520tcctctggtg tggtgttgtg acaaatgcgg
tgatccacat aaggctctta ggaaggagcc 35580tgggagaagc agtaagtgtc
acttacatgt ctgtaatgcc agctagttgg gaggctgagg 35640tgagaggatc
gtttaagtcc agcctggatt atttgagatc ccaaataaat aaaaaccaaa
35700ataaaaaaaa atgtagcaaa aattactaaa attgcaatta taccatggcc
ctgaggcagt 35760agaaaggctc cagtttaaat cctggctttg gctaggctca
gtggctcgtg tctgtaatcc 35820tagcactttg ggaggctgag gtgggaggag
cacttgagcc caggaggttg aggctgcaat 35880gagctgtgat agtgccattg
cactccagcc tgggcaacag agccagaccc tgtctcaaaa 35940aaccccaaaa
tcctggctgt gcacagtggc tcatgcctgt aatcccagca ctttgggagg
36000ccgaggtggg cggatcacct gaggtcagga gttcaagacc agcctggcca
acatggtgaa 36060accccgtctc tactaaaaat acaaaaatat tagctgggtg
tgatagtggg cacctgtaat 36120cccagctact caggaggctg aggcaggaga
attgcttgaa cctaggagga gagggttgca 36180gtgtgctgag atcgcgccac
tgcactccag cctgggtgac agagagagac tctgtctcaa 36240aacaaaaacc
ccaaaatcct aacattgcct gaccttgggc aattcccacc cctctctgtg
36300cctccgcttc cctgtgtgta agccagggaa ggaggagtgg taacaggaag
gaccgcatgt 36360gatcgtcagt attataattc acatgtcatt aagatggatt
cattgctatt ttaacggcat 36420gaagaagata ttcattacaa ttaagatgtt
gaaggctcat ccaggttctg tctggctcca 36480gctgactggt taagtggctt
ctggaacacc cttcgttttt cccagcctcc attctttatc 36540tgtaaaccaa
gataataaga gcaagaatta aacaagatga ggaaagggtc ctccagcagc
36600cggtgcttag ctgttttctg gggaggcgtc atgtgctgag ctgggcccag
ccctgggggt 36660ctttccagtg tgagccccct gaccctcctg gccccctgtg
cagatctttc agcacatcat 36720ccggcgggag gtgacggatg aggacacacg
gcacctgtcc cgcaagttca aggactgggc 36780ctatgggcca gtgtattcct
cgctttatga cctctcctcc ctggacacgt gtggggaaga 36840ggcctccgtg
ctggagatcc tggtgtacaa cagcaagatt gaggtgggct ccaggagggg
36900gcatggggtg tgaggaagga ggggcagggc tgggagtagg gacagggccc
tggggctggg 36960ctgggggaga cagccccagc ctctaggacc caacgtggtg
ggtctgttgg ctgaatgcaa 37020gagaaggtgc caactaagtg cccagaacag
tgtctgccat gtgaggaaat gagcaagact 37080tgtgggaaga tggacttttg
gggcaggtag gactgggttg ggatcctggc tctgtaactt 37140acatgctgtg
tgtctcagga caagtggctc cacctctctg ggtctcagct tcttcatttg
37200taacctgggg ataaggatca tggatatccc atggggttct tgggaggacc
aaagaggtta 37260attttagaat ggcttggcac agagtaaaca ttatgtcagc
ttttactact catggttgtt 37320atatgaactt ttgaaagctc ttttttttcg
gtcaggcatg gtggcttatg cctgtaattc 37380tagcattttg gggagccaag
gtgggcggat tgcttgaagc caggagtttg agactagcct 37440gggcaatatg
gcgaaacccc gtctctacca aaaaaaaata ataaaaatga aaattagcta
37500ggcatggtgg tgcgagcctg tagtcccagc tacttgggag gcgaggtggg
aggatcacct 37560gagcctgggg aggtcgaggc ttcagtgagc cgagatcatg
ccactgcact ccagcctggg 37620tgacagagtg agatcctgtc tcaaaataaa
caaacaaaca aacaaacaaa aaaccaagct 37680tttttttccc tacttagcag
tattttgtga acgtcgttct gtacttttaa atattctttg 37740gtaatattgt
cgctgcatgt aaacatacca tattcaaatg aactgttctc ttattgctgg
37800tcgcttgggt ttgtttctgc ctttatcagg aataacacta taagagcatc
tctgtggctg 37860ctttattgca caaatcatga ttatagagaa actcaagtta
ataaagggct aggaagctat 37920aaaaatggag cagaaaatgc tggggactgg
gtgcaggtgg tcttccttct agccctgccc 37980tgtggcttac tggtctgtga
gcttggcttc cttgcaagac ctcatttccc tcattttttg 38040aattagtgac
catcttgatt taaattagtg gtggcaggag gtttagcatc tcaggtgcaa
38100actgattgat tagtagtgtc tacccaggga actgtatgaa tacaattgtg
cagggctcaa 38160ttattgatgt ctgccctggg ttcagaattg gagaattgtg
gtgagtcaat catttatcat 38220ctttgggcaa aatttctcaa tctttctctc
tgaatcagaa tttctgagag cggaggccag 38280gatttatact tgcagcaagt
tacataaatt attttgcggc cagcagcttg gtactatcca 38340cagattggag
tttgggaacc atgagaataa atgtcttttt ttcttttttg agatggagtc
38400tcactcttgt tgcccaggct ggagtgcagt agtgcgatct aggctcactg
caatctccgc 38460ctcccaggtt caagtgattc tcctgcctca gccttaggag
tagctgggat tacaggtgtg 38520caccaccacg cccggctaat tgttgtgttt
ttagtggaga cagggtttca ccacattggc 38580caggctgatc ttgaactgct
gaccttaggt gatccgcctg tcttggcctc ccaaagtgct 38640aggattacag
gcatgagcca ctgcggccgg cctgaaaata aatgtctttt aagggacttt
38700ccagttccga gttctgtgat gctagaatag ggtggaaagg tacattgagg
cagaattggg 38760cagctgaatc cattcatgaa tccgtgaatg cagctgagga
atggatggaa agagaaagcg 38820ctgtccgggt ggagggtggg ggaaggcaca
cccgaggcag cctgcctgga ccccccaccc 38880atctcaggaa ggcagccccc
gaccaccctg cctctcttag aaccgccacg agatgctggc 38940tgtggagccc
atcaatgaac tgctgcggga caagtggcgc aagttcgggg ccgtctcctt
39000ctacatcaac gtggtctcct acctgtgtgc catggtcatc ttcactctca
ccgcctacta 39060ccagccgctg gagggcacag tgagtgcccg gggaccgggc
aggggctggg gcaggcactg 39120ggctgagcca tgcaggactg gggcacaacc
tcatccttct gggtcccctg tagggggacc 39180cggagaaggt ttaggaacag
gttggggagg cgccctccag catccacggg tggccctgag 39240ctgggaggag
gaagactcag gaggaagaga gtgaaggagg aggctccatg ggatgccgat
39300gtttcgggcc tgggggaaca tctggattgg gggccagatg ttggaggggc
tgggtgacga 39360cctgtgtgcc cttgctgctc cccagccgcc gtacccttac
cgcaccacgg tggactacct 39420gcggctggct ggcgaggtca ttacgctctt
cactggggtc ctgttcttct tcaccaacgt 39480aagtgcctgg cccccgtgcc
ccccaccctg cctgccctcc tcttctcttc ctgcacctct 39540tttctctccc
tctttttctc ttctctctcc tccaaatggt ccttctcctt tccttcctcc
39600ttttattttc ccatttctct tctacctcct ccaatcccac gttctccatc
tctgcttttt 39660tctccttttc taacaggaaa gagtcctttg tcttttctct
gtaccagcct ctgcctccct 39720ctcttctatc cctttctctg tctcctctct
cctcctctcc tctcctgctg gccccacccc 39780tttcacgtgc cccctcctgt
cttccagatc aaagacttgt tcatgaagaa atgccctgga 39840gtgaattctc
tcttcattga tggctccttc cagctgctct agtgagtaga ggtccctggg
39900ccggcagctt ctgggtgagg aaggtgggtt tgggctgcta ggtcgtccag
attcaggaga 39960gaggtgattc tgttagaaat gaatgcaccc aacctgggca
gctctaaccc aaagtcagga 40020caaagccaca aacaataggg tcttctggtt
caagaaaaat tctgattgct agggagttat 40080catatacaac atggatagaa
ataattttaa cctcttatgc tggtgaatga ttgctaatgt 40140ctgcctgaag
tgccatgtta agaattttgc acctgtgtct gtgtttagtg ggaaagagtt
40200gggattgatt aatgatgtct gcctgggaca gagaatgaga gagttactga
gtgtgtgtta 40260cctctttcac atttccctta gagttggaga atgatgaaat
tcacttagcc acaaaatctc 40320cactgaacac cactaaatct ttctttagca
attccaaggg gcatcacttc aggatcctgt 40380gggacctcag gattaattgc
cagctaattg aatgttcttg ttaacagaat gctggggaac 40440cctgctaatc
cactttgttt cttttctggg ggtctagaag gcattgggaa gtcctgatac
40500cctggagggc ttggaggcgt gtcttcccct ccagagcctc attgtcccct
ctcccctctg 40560gcctctcttg tggtctctgc tgcactgcag cttcatctac
tctgtcctgg tgatcgtctc 40620agcagccctc tacctggcag ggatcgaggc
ctacctggcc gtgatggtct ttgccctggt 40680cctgggctgg atgaatgccc
tttacttcac ccgtgggctg aagctgacgg ggacctatag 40740catcatgatc
cagaaggtac gggctgggag gaccctctgg actcgtgtgt tcctggaggg
40800agtcacacac acaccacatg cacacttatg cacctgcaga cctgaggatg
ctgggtaccg 40860tgggggcagg aggcatggtc tggacactgg ggtgggacca
gggtggagat ggaggaatgg 40920ggaaagtgag aaaccatgtg tctcctcttt
gcctccataa tcccgctggg gtctttagat 40980tctcttcaag gaccttttcc
gattcctgct cgtctacttg ctcttcatga tcggctacgc 41040ttcaggtgag
ctctgggtgc tcaggtggtc ctggcagggg tggtgcaagg acgggaacag
41100tagccatgat gtatagggga caatagcagc cttgctgagt tctttttttt
tttttttttt 41160ttttgaggtg gagttttgct cctgttgccc gggctggagt
gcaatggcac aatctcagct 41220cactgtaacc tccacctccc aggttcaagt
aattctgcct cagcctccca cgtagttggg 41280attacgggct cctgccacca
cgccccacta ttttttaatt tttttttttt tgtattttta 41340gtagagatgg
ggtttcacca tgttggccat gctggtcttg aactcctgac ctcaggtgat
41400ccaccctcct tggcctccca aaatggtggg attacaggtg tgagccaacg
cgcctggcca 41460ctgagttctt atttgtgtgc caggcaccag actaagtacg
tttccatcct tgcctcccaa 41520caagcctgta agctaagtgc ctttattatc
ccctattata gaaaaggaaa ctgaggctta 41580ggagggttaa ataactagca
caagttcaaa agccagtgaa tggtggccgg gcgcagtggc 41640tcacatctgt
aatcccagca cttcaggagg ctgaggcggg tagatcactt gaggtcagga
41700gtttgagacc agcctggcca acatggcaaa accccgtctc taccaaaaaa
tacaaaaatt 41760agacaggcat ggtggcgcgc aactatagac ccagctactc
gggaggctga ggcacaagaa 41820tcgcttgaac ccagggggtg gaggttgcag
taagccaaga tggcaccact gcactccagc 41880ctgggcaaca cagcgagact
ctgtctcaaa aaaaaaaaaa aaaaaaaagc tggtgaatgg 41940caaaactgga
gtattagcta ttgctacaaa ggaagccacc taaagtgggg cttaaaatag
42000cacccactta ttattattat tttttttgtg gcaaggtctc actctgtcac
caaggctgga 42060gggcagtggt gctcttggct cactgcaatc tctgcctcct
gggttcaagc gattctcttg 42120cctcagcctc tcgaatagct gggattacag
gcgtgtgcca ccacgcatgg ctaagtttta 42180tatttttggt agagatgggg
tttcactatg ttggccaggc tggtctcgaa ctcctgaccc 42240caagtgatcc
acccaccttg gcctcccaaa gtgctgggat tacaggcgtg agccactgta
42300ccaggccaac ttactattgc ttatgagtca atgggtcagc tgggtggctg
tgctgccctg 42360ggccaggttt ggctgatccc tacaggctct tgtgcctgtg
gtcagctggc aggctggctg 42420ctggctgact ggtctagcta gtatggactt
ggttagaggg ggtttgctat ctatcttcca 42480catggtcact catttctgga
cagctagctt gggcttgtcc acatggtggc cttagggttc 42540caagaacatg
agcagaagtg ttcaaggaca cctcctggca tggcatcact gctgccttac
42600tctgttggcc aaagcaagtc acaaggccag cctcgattca agggatgggg
aaatagacgc 42660cacttctttt tttccatttt ttttttttgg tggaggcagg
gtcttgctat gttgcccagg 42720ctggtcttga actcctgggc tcaagcagtc
ctcctgcctc agcctcccaa agtgcaggat 42780tataggcgcg agccactgca
cctggcctag actccacttc ttagtgggag aagctgtaaa 42840gccacattga
aggaggatgg atacatggaa ggataaaatt taataattaa actagcagcc
42900caggtatttt cttaaagccc aacatcagct cccttgatat accttcttcg
tgatgtcatg 42960atgccactaa gaagcattct gggacctcag tggcatgggt
catgggagaa ggaccaggga 43020ccagggctgg agaaactgac caagtccttg
ggctgatccc cgaggtgtgg gtaatctgac 43080atcaatgccc agcatccaaa
ggggaatggc caactcaggt tagacctgga cacatacaca 43140tctgagcagg
ttcatctggg gacgagcctg gcacaggaat ctgacaaggg tgatatttta
43200acccacgata gtggccaagt ctctaggtag attattggtg ggaagtggaa
ttgtagtttt 43260cactggggag atagggacag ggtacagctt tcccttccca
gggatcacac tggacaaact 43320agacagtgtc aagggttcaa caacagtttg
ggattcaggt cagggatcta gccctactgc 43380agtcctagtt attgaaacta
aggagaaaca agggttagag tagcagctta gcagtcagct 43440tggttgcagg
tcttctcctt ataggatgca ctagatctca attctgaggc tgacatgaca
43500taaacaggac acacatgctt ctgttcccac actttggtgt ccatagcaga
catcactaat 43560ggatcacaga acagtttttc cggtgagtcc agacaaggtc
tcagaatcct tctcaacaca 43620gtgtttcacc aggtactata caactgaaga
acattagcat atgagttgaa atctgcatac 43680tctaatccct ggttgcaggc
agtatgacat tctgtgcccc actgactgat gggactgagt 43740cttcttcatg
ctcccctccc tggctggttt agggacaggt tgttgctgcc aaactgtgtt
43800gagaaggaat ctgaagctcc acctgggctt agtaggaaag gagaaccatg
atgagtttga 43860gatgtctaga gcaggagagg ccccttagag aacacggctt
tggggacagt caggactgat 43920gcctggtttc agcccccact tttcctctct
gggttggggt ggagtgggaa actgtcagag 43980agagagtcta cccctgcgaa
taggggcatt ccagagcccc caagacctct aagaatggta 44040ggccgcatta
ggatttccca acactattga catttgagac cagatcattc ttcgtggcgg
44100ggaccatcct gtgcattgta gggtgtcagg cagcacccct ggcctccacc
cacctgatgc 44160cagtagcacc cccaccccca gttgtaacaa ccaaatatgt
ctctagacat tgtcaaatgt 44220cccctggtgg gaaaaactga gaccctccct
ggactagatc ctgaaaacct tttccctccc 44280ggtagcaggg aaccaccagg
ttgtgtaagg aggggtgaac tgtcccaggt tggaaacaga 44340gcaggtcaaa
actcctacgc ttatcagtag tgggattgag cctgtgagta gccactgcac
44400tccagcctgg gcaacatagc aagaccccat ctcagaaaga aagagagaga
gagagagata 44460acctcttcct tatcagcaag agtttatgga aaggcttcaa
aggttttggg atcaggagga 44520cctgggtctg gtgtggcttg gctgatgtgt
agggtgacat gggtcagcgt gtgggccagc 44580gtgctccgct cactaggcct
cagctttctc ctccagcaaa tgagaatgac accctccacc 44640tctctgtggg
tgtgctcaca ctcagtgagt gccaccccta tcctcccaca gccctggtct
44700ccctcctgaa cccgtgtgcc aacatgaagg tgtgcaatga ggaccagacc
aactgcacag 44760tgcccactta cccctcgtgc cgtgacagcg agaccttcag
caccttcctc ctggacctgt 44820ttaagctgac catcggcatg ggcgacctgg
agatgctgag cagcaccaag taccccgtgg 44880tcttcatcat cctgctggtg
acctacatca tcctcacctt tgtgctgctc ctcaacatgc 44940tcattgccct
catgggcgag acagtgggcc aggtctccaa ggagagcaag cacatctgga
45000agctgcaggt gaggccccag ggcccccagc cccactctac cagccaccct
cgtccttccg 45060aggagaccca cccgactggc ttcctcggcc ttccactctg
agcaagcagt gtcttctcat 45120tctccctcca cttctctgtc ggtaaaatgg
gcatcagagg
ctgctgctga cttcccagag 45180ctgcctagtg aattttgagg tggcttcttg
cctggctgcc agcagtgggc tcagattctc 45240cttgtggtcc agctcacagg
acacagtcgg ctccacctag catgactgag tgacttctgc 45300cggctcagaa
agtgctccgt gggcatcagc gtttcagcag gggtcccacg ctgagcccag
45360gggcagcttt tgtgctctag cactccagca cagggatgtc agcattctca
tgggggtctt 45420tttctagcaa ctgtgagcat gcttcctaga ggagaaacca
atggggacag gggccgcctt 45480ggcccgggga gctgcagaaa gtttttactt
accaaaagct ttctatgcaa tcctggaaag 45540aacaggccct gggatcacag
ccctcccagg ccaggtgcag cctacaaacc tggtgtccta 45600ataaacagtg
ctgccccacg gcccaggaat acagcacccg tgtttacctt gactgtgcat
45660ttcaggaaaa tggtgcagtg agaaaaaagg gcccaaggtt gtcctcccag
gactgggtct 45720gagggaaaca gctctggcct gctcttaacc tttatctata
ttgcttactt cattgggcct 45780cagtttcccc actgggcatt actcacagga
tgcaaactgg gcatcctgtg ggctggagtg 45840caatggtgtg atcttggctc
actgcaacct ctgcctccca ggctcaagtg attctctgtc 45900ctcagcctcc
cgagtagctg gggctaaggt gtgcaccacc tcacccggct aatttttgta
45960ttttttgtaa agatggggtt tcaccatgtt gcccaggctg gtcttgaact
tctgagttca 46020agctatccac ctgccttggc ctcctaaagt gctgagattg
caggtgtgag ccactgcacc 46080cagcatcaga ccattatatt aaaaacaaca
acaacaacaa aaaactgcaa aatttaaaaa 46140ttaagagatt ccctatgaaa
acccagattt ctggcttttc ttgaaaagtt ggaagctcca 46200ggcagtgcag
ggtccggatt cctgcctggc agccctcagg tggggtgagt ggcagctgcc
46260cccttacacg tgggcatttg ctccctgttc tccccagtct ctacccctcc
ctcactgaga 46320ggcaactgct gtgtaagtgt gatagggctt gggcggcagc
ctttctgcac ctgatcctgt 46380ttcacccatt tgttttcctg cctgccacct
gtgggtattt gagtttataa accctgtgtc 46440tagtggggag taggagtcta
aatgcctagt tctgggccca ccctggcccg ttgtctcatt 46500tctgccacca
gagcggcagg cgcaggctgt gaggctcacc gatgtccctc ctgaccctcc
46560ctccccgcag tgggccacca ccatcctgga cattgagcgc tccttccccg
tattcctgag 46620gaaggccttc cgctctgggg agatggtcac cgtgggcaag
agctcggacg gcactcctga 46680ccgcaggtgg tgcttcaggt gaggctgggg
cagtggggcc aggatggcag ggcggaactg 46740tccccactgg ctcggggccc
ctgctgctcc agcgtcgtct acccattgca atttctggag 46800ccactgaggc
cccaagaggc ccgagcagag ttcatttcca ccaggcgatc tcaggcaagt
46860cagcccacct ctctcagcct cagtttcttc atctgtggaa tgagacaatg
atctcacggg 46920ctcctaggct ggcggtgcgg attagacagg tcagcacgtg
agaagtgctc agccagtgcc 46980cgacgcgcat cgggacgcca caggctcccc
tgttcttgct cagggggaag cagagacttg 47040gatggtgggt tcattttaga
gcatcaccct gtatctacca aacagtgggg tgagctctag 47100tgccctcggt
gaaatgggaa gctgaggaat gtgcagtttc cagggctgga gacctcaccc
47160agcccatctg cagaatgact ccgtgttcca gaggcacagg gagtgccagc
ttcttagggg 47220aaccccttca tgaatctttt ctttccagtt gattcattca
ttgtaaagtg gttgtcagca 47280tggagcataa gcagaatgtt tggagtcaga
caaagtcagt ttcttcatct gtgaaatggg 47340aacaataata gaacccacct
cctaggccat ccgatcagtt caattccatt ttttgtttgt 47400ttttagaggc
agggttgctc aggctggagt gcagtggtgt gatcatagct gactgcagcc
47460tccaactcct ggtctcaagc gatcctccca cctcagcctc ctcatgccac
catgtccgac 47520caatttttaa tttttttgta gagatggggt tcttgctatg
ttgcccaggc tggtctcaaa 47580ctcctggtct caagcgatct tcccacctca
gcctcccaaa atgctgggat tacaggcgtg 47640agccaccaca cccagcctct
gttcagtttg aatccaaagc tcaaatcttg gctgggtgtg 47700gtggctcgtg
cttgtaatcc cagcactttg ggaggccaag gtgggtggat cacctgaggt
47760caggagtttg agaccaccct gggcaacatg gtgaaaccct gtctccacta
aaaatacaaa 47820aattagtcgg gagtggtggc atgcacctgt aatcccagct
actcgggagg ctgaggcagg 47880agaatcgctt gaacctggta ggtggaggtt
gcagtgagcc gagattgcac tactgcactc 47940cagtctggcg gacagagcaa
gactctgcct taaaacaaac aaacaaaaat gaatagatag 48000gataaaaaat
taaagataat atgtaaaaaa aaaagataat atataaaaaa atgagtggat
48060aaacaaaatg tgggctatgt gtacagtgaa atattatctg ggccaggcgt
ggtgactgta 48120ataccaacac tttgggaagc gaggcaagag gattgcttga
gcccaggagt tcaagaccaa 48180catagtgaaa ccccatctct acaaaacgtt
tttaaaaaat tagccaggtg tggtggcata 48240tccctgtaat cccagctaca
tggggggctg aagtgggcgg atcacttgag cctgggaggt 48300caaggttgca
gtgagctgtg atcgtgccac tgcaccctag cctgggcaat ggagtgagac
48360cctgtctcaa aagaaggtta aaaaaaataa tttctgcttg ggggaatgga
agaattttgg 48420aaatagatag ctgtgatggt cacacaatgt ggatgtgctt
aatgccactg aattgtatac 48480tcaaaaatga ttaagatggc aaattttttg
tgtgtgtata cacacacaca cacacacaca 48540cacacacgtc tttttttttt
tttttttgag atggagtctt gctttgtccc ccaggttgga 48600gtgcagtggc
gtgatctcgg ctcactgcaa ctgcctccca ggttcaagtg attttcctgc
48660ctcagcctct cgggtcactg ggactatagg cacccgccac catgccccgc
taattttttt 48720tttaattttt tttaattttt agtagaggtg gggtttcacc
atgttggcca ggctggtctc 48780gaactcctga cctcaaatga cccacccacc
tcggcctccc aaagtgctgg gattgcaggc 48840atgagccacc acgcccagcc
ttgttatgta tatgttacca taataaaaaa ataggagggt 48900gtgctgggga
tggtggcatg gagagcgtct ctgaggggtt ccccatgacc ctctgcttgg
48960gccctgccag ggtggatgag gtgaactggt ctcactggaa ccagaacttg
ggcatcatca 49020acgaggaccc gggcaagaat gagacctacc agtattatgg
cttctcgcat accgtgggcc 49080gcctccgcag gggtgagtgg aggggcgggt
gcggagggga gccccagtcc attctcatca 49140cgaatttgct ttgagcagtc
ctgcttcttt ccgggactca gtgtccgtgt ctacagaatg 49200agagagtgtg
cccctgagct tcctctgctg cttgtgaatg cctggggcac ctcacctacg
49260tggcttcatc ctggccctcc ctactgttga cataagctgg cctggggagg
gaggacgagc 49320tcctcagcca tccctgtgtg gagaaaacct tcctgtttgt
ccagtaagcc atgagcacgc 49380gagacgcagc tggagaggac acaggcactc
agggcattcg gctccaggct tgaatcctgg 49440ttcctccatt ctctcctggg
gaggcacctt gacctctctt gagccccagt ttccacctct 49500gtaaaatgag
catagtcaca tccccccttc agaacactgt ggagtgccca gtacacagta
49560ggcactcaat atacgcacgc tctctctcca ccaaccccca cccctccctc
tgatgtgctc 49620tcggtgcaga tcgctggtcc tcggtggtac cccgcgtggt
ggaactgaac aagaactcga 49680acccggacga ggtggtggtg cctctggaca
gcatggggaa cccccgctgc gatggccacc 49740agcagggtta cccccgcaag
tggaggactg atgacgcccc gctctaggga ctgcagccca 49800gccccagctt
ctctgcccac tcatttctag tccagccgca tttcagcagt gccttctggg
49860gtgtcccccc acaccctgct ttggccccag aggcgaggga ccagtggagg
tgccagggag 49920gccccaggac cctgtggtcc cctggctctg cctccccacc
ctggggtggg ggctcccggc 49980cacctgtctt gctcctatgg agtcacataa
gccaacgcca gagcccctcc acctcaggcc 50040ccagcccctg cctctccatt
atttatttgc tctgctctca ggaagcgacg tgacccctgc 50100cccagctgga
acctggcaga ggccttagga ccccgttcca agtgcactgc ccggccaagc
50160cccagcctca gcctgcgcct gagctgcatg cgccaccatt tttggcagcg
tggcagcttt 50220gcaaggggct ggggccctcg gcgtggggcc atgccttctg
tgtgttctgt agtgtctggg 50280atttgccggt gctcaataaa tgtttattca
ttgacggtg 5031923254DNAHomo sapiens 2ccggccggga ttcaggaagc
gcggatctcc cggccgccgg cgcccagccg tcccggaggc 60tgagcagtgc agacgggcct
ggggcaggca tggcggattc cagcgaaggc ccccgcgcgg 120ggcccgggga
ggtggctgag ctccccgggg atgagagtgg caccccaggt ggggaggctt
180ttcctctctc ctccctggcc aatctgtttg agggggagga tggctccctt
tcgccctcac 240cggctgatgc cagtcgccct gctggcccag gcgatgggcg
accaaatctg cgcatgaagt 300tccagggcgc cttccgcaag ggggtgccca
accccatcga tctgctggag tccaccctat 360atgagtcctc ggtggtgcct
gggcccaaga aagcacccat ggactcactg tttgactacg 420gcacctatcg
tcaccactcc agtgacaaca agaggtggag gaagaagatc atagagaagc
480agccgcagag ccccaaagcc cctgcccctc agccgccccc catcctcaaa
gtcttcaacc 540ggcctatcct ctttgacatc gtgtcccggg gctccactgc
tgacctggac gggctgctcc 600cattcttgct gacccacaag aaacgcctaa
ctgatgagga gtttcgagag ccatctacgg 660ggaagacctg cctgcccaag
gccttgctga acctgagcaa tggccgcaac gacaccatcc 720ctgtgctgct
ggacatcgcg gagcgcaccg gcaacatgcg ggagttcatt aactcgccct
780tccgtgacat ctactatcga ggtcagacag ccctgcacat cgccattgag
cgtcgctgca 840aacactacgt ggaacttctc gtggcccagg gagctgatgt
ccacgcccag gcccgtgggc 900gcttcttcca gcccaaggat gaggggggct
acttctactt tggggagctg cccctgtcgc 960tggctgcctg caccaaccag
ccccacattg tcaactacct gacggagaac ccccacaaga 1020aggcggacat
gcggcgccag gactcgcgag gcaacacagt gctgcatgcg ctggtggcca
1080ttgctgacaa cacccgtgag aacaccaagt ttgttaccaa gatgtacgac
ctgctgctgc 1140tcaagtgtgc ccgcctcttc cccgacagca acctggaggc
cgtgctcaac aacgacggcc 1200tctcgcccct catgatggct gccaagacgg
gcaagattgg gatctttcag cacatcatcc 1260ggcgggaggt gacggatgag
gacacacggc acctgtcccg caagttcaag gactgggcct 1320atgggccagt
gtattcctcg ctttatgacc tctcctccct ggacacgtgt ggggaagagg
1380cctccgtgct ggagatcctg gtgtacaaca gcaagattga gaaccgccac
gagatgctgg 1440ctgtggagcc catcaatgaa ctgctgcggg acaagtggcg
caagttcggg gccgtctcct 1500tctacatcaa cgtggtctcc tacctgtgtg
ccatggtcat cttcactctc accgcctact 1560accagccgct ggagggcaca
ccgccgtacc cttaccgcac cacggtggac tacctgcggc 1620tggctggcga
ggtcattacg ctcttcactg gggtcctgtt cttcttcacc aacatcaaag
1680acttgttcat gaagaaatgc cctggagtga attctctctt cattgatggc
tccttccagc 1740tgctctactt catctactct gtcctggtga tcgtctcagc
agccctctac ctggcaggga 1800tcgaggccta cctggccgtg atggtctttg
ccctggtcct gggctggatg aatgcccttt 1860acttcacccg tgggctgaag
ctgacgggga cctatagcat catgatccag aagattctct 1920tcaaggacct
tttccgattc ctgctcgtct acttgctctt catgatcggc tacgcttcag
1980ccctggtctc cctcctgaac ccgtgtgcca acatgaaggt gtgcaatgag
gaccagacca 2040actgcacagt gcccacttac ccctcgtgcc gtgacagcga
gaccttcagc accttcctcc 2100tggacctgtt taagctgacc atcggcatgg
gcgacctgga gatgctgagc agcaccaagt 2160accccgtggt cttcatcatc
ctgctggtga cctacatcat cctcaccttt gtgctgctcc 2220tcaacatgct
cattgccctc atgggcgaga cagtgggcca ggtctccaag gagagcaagc
2280acatctggaa gctgcagtgg gccaccacca tcctggacat tgagcgctcc
ttccccgtat 2340tcctgaggaa ggccttccgc tctggggaga tggtcaccgt
gggcaagagc tcggacggca 2400ctcctgaccg caggtggtgc ttcagggtgg
atgaggtgaa ctggtctcac tggaaccaga 2460acttgggcat catcaacgag
gacccgggca agaatgagac ctaccagtat tatggcttct 2520cgcataccgt
gggccgcctc cgcagggatc gctggtcctc ggtggtaccc cgcgtggtgg
2580aactgaacaa gaactcgaac ccggacgagg tggtggtgcc tctggacagc
atggggaacc 2640cccgctgcga tggccaccag cagggttacc cccgcaagtg
gaggactgat gacgccccgc 2700tctagggact gcagcccagc cccagcttct
ctgcccactc atttctagtc cagccgcatt 2760tcagcagtgc cttctggggt
gtccccccac accctgcttt ggccccagag gcgagggacc 2820agtggaggtg
ccagggaggc cccaggaccc tgtggtcccc tggctctgcc tccccaccct
2880ggggtggggg ctcccggcca cctgtcttgc tcctatggag tcacataagc
caacgccaga 2940gcccctccac ctcaggcccc agcccctgcc tctccattat
ttatttgctc tgctctcagg 3000aagcgacgtg acccctgccc cagctggaac
ctggcagagg ccttaggacc ccgttccaag 3060tgcactgccc ggccaagccc
cagcctcagc ctgcgcctga gctgcatgcg ccaccatttt 3120tggcagcgtg
gcagctttgc aaggggctgg ggccctcggc gtggggccat gccttctgtg
3180tgttctgtag tgtctgggat ttgccggtgc tcaataaatg tttattcatt
gacggtgaaa 3240aaaaaaaaaa aaaa 325433057DNAHomo sapiens 3ccggccggga
ttcaggaagc gcggatctcc cggccgccgg cgcccagccg tcccggaggc 60tgagcagtgc
agacgggcct ggggcaggca tggcggattc cagcgaaggc ccccgcgcgg
120ggcccgggga ggtggctgag ctccccgggg atgagagtgg caccccaggt
ggggaggctt 180ttcctctctc ctccctggcc aatctgtttg agggggagga
tggctccctt tcgccctcac 240cggctgatgc cagtcgccct gctggcccag
gcgatgggcg accaaatctg cgcatgaagt 300tccagggcgc cttccgcaag
ggggtgccca accccatcga tctgctggag tccaccctat 360atgagtcctc
ggtggtgcct gggcccaaga aagcacccat ggactcactg tttgactacg
420gcacctatcg tcaccactcc agtgacaaca agaggtggag gaagaagatc
atagagaagc 480agccgcagag ccccaaagcc cctgcccctc agccgccccc
catcctcaaa gtcttcaacc 540ggcctatcct ctttgacatc gtgtcccggg
gctccactgc tgacctggac gggctgctcc 600cattcttgct gacccacaag
aaacgcctaa ctgatgagga gtttcgagag ccatctacgg 660ggaagacctg
cctgcccaag gccttgctga acctgagcaa tggccgcaac gacaccatcc
720ctgtgctgct ggacatcgcg gagcgcaccg gcaacatgcg ggagttcatt
aactcgccct 780tccgtgacat ctactatcga ggtcagacag ccctgcacat
cgccattgag cgtcgctgca 840aacactacgt ggaacttctc gtggcccagg
gagctgatgt ccacgcccag gcccgtgggc 900gcttcttcca gcccaaggat
gaggggggct acttctactt tggggagctg cccctgtcgc 960tggctgcctg
caccaaccag ccccacattg tcaactacct gacggagaac ccccacaaga
1020aggcggacat gcggcgccag gactcgcgag gcaacacagt gctgcatgcg
ctggtggcca 1080ttgctgacaa cacccgtgag aacaccaagt ttgttaccaa
gatgtacgac ctgctgctgc 1140tcaagtgtgc ccgcctcttc cccgacagca
acctggaggc cgtgctcaac aacgacggcc 1200tctcgcccct catgatggct
gccaagacgg gcaagattgg gaaccgccac gagatgctgg 1260ctgtggagcc
catcaatgaa ctgctgcggg acaagtggcg caagttcggg gccgtctcct
1320tctacatcaa cgtggtctcc tacctgtgtg ccatggtcat cttcactctc
accgcctact 1380accagccgct ggagggcaca ccgccgtacc cttaccgcac
cacggtggac tacctgcggc 1440tggctggcga ggtcattacg ctcttcactg
gggtcctgtt cttcttcacc aacatcaaag 1500acttgttcat gaagaaatgc
cctggagtga attctctctt cattgatggc tccttccagc 1560tgctctactt
catctactct gtcctggtga tcgtctcagc agccctctac ctggcaggga
1620tcgaggccta cctggccgtg atggtctttg ccctggtcct gggctggatg
aatgcccttt 1680acttcacccg tgggctgaag ctgacgggga cctatagcat
catgatccag aagattctct 1740tcaaggacct tttccgattc ctgctcgtct
acttgctctt catgatcggc tacgcttcag 1800ccctggtctc cctcctgaac
ccgtgtgcca acatgaaggt gtgcaatgag gaccagacca 1860actgcacagt
gcccacttac ccctcgtgcc gtgacagcga gaccttcagc accttcctcc
1920tggacctgtt taagctgacc atcggcatgg gcgacctgga gatgctgagc
agcaccaagt 1980accccgtggt cttcatcatc ctgctggtga cctacatcat
cctcaccttt gtgctgctcc 2040tcaacatgct cattgccctc atgggcgaga
cagtgggcca ggtctccaag gagagcaagc 2100acatctggaa gctgcagtgg
gccaccacca tcctggacat tgagcgctcc ttccccgtat 2160tcctgaggaa
ggccttccgc tctggggaga tggtcaccgt gggcaagagc tcggacggca
2220ctcctgaccg caggtggtgc ttcagggtgg atgaggtgaa ctggtctcac
tggaaccaga 2280acttgggcat catcaacgag gacccgggca agaatgagac
ctaccagtat tatggcttct 2340cgcataccgt gggccgcctc cgcagggatc
gctggtcctc ggtggtaccc cgcgtggtgg 2400aactgaacaa gaactcgaac
ccggacgagg tggtggtgcc tctggacagc atggggaacc 2460cccgctgcga
tggccaccag cagggttacc cccgcaagtg gaggactgat gacgccccgc
2520tctagggact gcagcccagc cccagcttct ctgcccactc atttctagtc
cagccgcatt 2580tcagcagtgc cttctggggt gtccccccac accctgcttt
ggccccagag gcgagggacc 2640agtggaggtg ccagggaggc cccaggaccc
tgtggtcccc tggctctgcc tccccaccct 2700ggggtggggg ctcccggcca
cctgtcttgc tcctatggag tcacataagc caacgccaga 2760gcccctccac
ctcaggcccc agcccctgcc tctccattat ttatttgctc tgctctcagg
2820aagcgacgtg acccctgccc cagctggaac ctggcagagg ccttaggacc
ccgttccaag 2880tgcactgccc ggccaagccc cagcctcagc ctgcgcctga
gctgcatgcg ccaccatttt 2940tggcagcgtg gcagctttgc aaggggctgg
ggccctcggc gtggggccat gccttctgtg 3000tgttctgtag tgtctgggat
ttgccggtgc tcaataaatg tttattcatt gacggtg 3057421RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 4gcacaccgcc guacccuuau u 21521RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 5uaaggguacg gcggugugcu u 21621RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 6gaccaaaucu gcgcaugaau u 21721RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 7uucaugcgca gauuuggucu u 21821RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 8caaccggccu auccucuuuu u 21921RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 9aaagaggaua ggccgguugu u 211021RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 10gaacccgugu gccaacaugu u 211121RNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 11cauguuggca cacggguucu u 21126PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 12Arg
Arg Lys Arg Arg Arg 1 5 1320DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 13caaaccgatt tgaccgagat
201420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 14gttcagcaca gccttcatca 201520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
15cagctgggag gaaaactcag 201620DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 16gggaggaagt ccttttccag
201720DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 17gacggggacc tatagcatca 201820DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
18aacaggtcca ggaggaaggt 201917DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 19gactacctgc ggctggc
172017DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 20ttcatccagc ccaggac 172124DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
21cactcgttca ttggcacctg cttt 242223DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
22gcagcttcgt cagcacaatc aca 232324DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 23ccattcgttc atcggcactt
gctt 242424DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 24ttatgaagca ttgccaccag cagc 242520DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
25accacagtcc atgccatcac 202620DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 26tccaccaccc tgttgctgta 20
* * * * *