U.S. patent application number 10/567958 was filed with the patent office on 2006-10-12 for silencing of tgf-beta receptor type ii expression by sirna.
Invention is credited to Nalin M. Kumar, Asrar B. Malik, Jose S. Pulido, Shahid Siddiqui, Beatrice Yue.
Application Number | 20060229266 10/567958 |
Document ID | / |
Family ID | 34222356 |
Filed Date | 2006-10-12 |
United States Patent
Application |
20060229266 |
Kind Code |
A1 |
Kumar; Nalin M. ; et
al. |
October 12, 2006 |
Silencing of tgf-beta receptor type II expression by sirna
Abstract
The present application is directed to siRNA-based silencing of
the type II receptor of TGF.beta.. siRNAs that target this receptor
abrogate the receptor protein and transcript, TGF.beta.-mediated
processes such as fibronectin assembly and cell migration also are
inhibited and the molecules of the invention are efficacious in
reducing the inflammatory response and matrix deposition. These
findings show that siRNAs can be successfully delivered both in
vitro and in vivo to regulate the TGF.beta. type II receptor level
and modulate wound response. Methods and compositions exploiting
the findings of the present invention have a wide-ranging
application, extending from treatment of disorders of the eye to
other organs and tissues throughout the body.
Inventors: |
Kumar; Nalin M.; (Wilmette,
IL) ; Yue; Beatrice; (Deerfield, IL) ;
Siddiqui; Shahid; (Wilmette, IL) ; Malik; Asrar
B.; (Hinsdale, IL) ; Pulido; Jose S.;
(Brookfield, WI) |
Correspondence
Address: |
LICATA & TYRRELL P.C.
66 E. MAIN STREET
MARLTON
NJ
08053
US
|
Family ID: |
34222356 |
Appl. No.: |
10/567958 |
Filed: |
August 10, 2004 |
PCT Filed: |
August 10, 2004 |
PCT NO: |
PCT/US04/25984 |
371 Date: |
May 10, 2006 |
Current U.S.
Class: |
514/44A ;
536/23.1 |
Current CPC
Class: |
C12N 2310/14 20130101;
A61P 27/02 20180101; A61P 19/04 20180101; A61P 9/00 20180101; C12N
15/1138 20130101; A61P 17/02 20180101; A61P 9/10 20180101; A61P
27/06 20180101 |
Class at
Publication: |
514/044 ;
536/023.1 |
International
Class: |
A61K 48/00 20060101
A61K048/00; C07H 21/02 20060101 C07H021/02 |
Foreign Application Data
Date |
Code |
Application Number |
Aug 13, 2003 |
US |
60495161 |
Nov 6, 2003 |
US |
60517809 |
Apr 9, 2004 |
US |
60561542 |
Claims
1-11. (canceled)
12. An siRNA molecule that reduces expression of the TGF.beta. type
II receptor, wherein the siRNA molecule is 19-25 base pairs in
length and targets at least a portion of the coding sequence of a
nucleic acid molecule comprising the nucleic acid sequence of SEQ
ID NO:159.
13. A composition comprising the siRNA molecule of claim 12 and a
pharmaceutically acceptable carrier.
14. The composition of claim 13, further comprising a wound healing
agent.
15. A method for promoting wound healing in a mammal comprising
administering a therapeutically effective amount of a composition
comprising the siRNA molecule of claim 12 to a mammal in need of
treatment.
16. A method for inhibiting fibrosis in a mammal comprising
administering a therapeutically effective amount of a composition
comprising the siRNA molecule of claim 12 to a mammal in need of
treatment.
17. A method for inhibiting angiogenesis in a mammal comprising
administering a therapeutically effective amount of a composition
comprising the siRNA molecule of claim 12 to a mammal in need of
treatment.
18. A method for preventing glaucoma in a mammal comprising
administering to a mammal in need of treatment a therapeutically
effective amount of a composition comprising the siRNA molecule of
claim 12.
19. A method of preventing restenosis in a mammal comprising
administering to said mammal a therapeutically effective amount of
a composition comprising the siRNA molecule of claim 12.
20. A method of preventing or treating scarring in a mammal
comprising administering to said mammal a therapeutically effective
amount of a composition comprising the siRNA molecule of claim 12.
Description
FIELD OF THE INVENTION
[0001] The present invention is directed to methods and
compositions for silencing transforming growth factor beta type II
receptor (TGF.beta.RII) expression. More particularly the present
invention describes methods and compositions for reducing such
expression using small interfering RNA (siRNA) molecules.
BACKGROUND OF THE INVENTION
[0002] Transforming growth factor-.beta. (TGF.beta.) comprises a
family of structurally related multifunctional cytokines. They have
a wide variety of biological actions, including cell growth,
differentiation, apoptosis, fibrogenesis and angiogenesis.
(Massague et al., Cancer Surv. 12, 81-103, (1992), Piek et al.,
FASEB J. 13, 2105-2124, (1999), Border & Noble N. Engl. J. Med.
331, 1286-1292 (1994); Govinda and Bhoola, Pharmacol. Ther.
98:257-265 (2003); Cusiefen et al., Cornea 19:526-533; Sakimoto et
al., Gene Therapy 7:1915-1924 (2000)) TGF.beta. is typically
secreted in a biologically latent form. It is activated through a
complex process of proteolytic activation and dissociation of
latency protein subunits. (Massague, Annu. Rev. Biochem. 67,
753-791 (1998)).
[0003] The mechanism of action of TGF.beta. is mediated by its
binding to receptors known as TGF.beta. receptors, types I, II and
III. Receptors I and II are transmembrane glycoproteins of 55 and
70 kDa shown to be important in signal transduction. The TGF.beta.
ligand binding site for these receptors is extracellular. The
mechanism by which the signaling is thought to be achieved is via
activation of phosphorylation of transcription factors known as
Smads. (Massague & Wotton, EMBO J. 19, 1745-1754 (1999)).
[0004] TGF.beta. has emerged as a key component of the fibrogenic
response to wounding and is upregulated during many different types
of wound healing in tissues such as the eye, liver, and skin.
(Border & Noble, N. Engl. J. Med. 331, 1286-1292 (1994), Connor
et al., J. Clin. Invest. 83, 1661-1666 (1989), McCormick et al., J
Immunol. 163, 5693-5699 (1999), Shah et al., J. Cell Sci. 108,
985-1002 (1995)). In the eye, of the three human isoforms
(TGF.beta.1, TGF.beta.2, and TGF.beta.3), TGF.beta.2 is the
predominant one. (Lutty et al., Invest. Ophthalmol. Vis. Sci. 34,
477-487 (1993), Pasquale et al., Invest. Ophthalmol. Vis. Sci. 34,
23-30 (1993)). TGF.beta.s have been implicated in several scarring
processes including proliferative vitreoretinopathy, (Kon et al.,
Invest. Ophthalmol. Vis. Sci. 40, 705-712 (1999)), cataract
formation, (Hales et al., Invest. Ophthalmol. Vis. Sci. 36,
1709-1713 (1989)), corneal opacities, (Chen et al., Invest.
Ophthalmol. Vis. Sci. 41, 4108-4116 (2000)), and conjunctival wound
healing, (Cordeiro, Clin. Sci. 104, 181-187 (2003)) especially that
occurring after filtration surgery for a major blinding disease,
glaucoma. In addition, TGF.beta. in conjunction with connective
tissue growth factor (CTGF) has an important role in angiogenesis
(Abreu et al., Nature Cell Biol. 4:599-604 (2002)). Furthermore,
recent studies have shown that TGF may actually be involved in the
pathogenesis of primary open angle glaucoma (Inatani et al.,
Graefes Arch. Clin. Exp. Ophthalmol. 239(2):109-13, 2001; Ochiai et
al., Jap. J. Ophthalmol. 46(3):249-53, 2002; Gattanka et al.,
Invest. Ophthalmol. Vis. Sci. 45(1):153-8, 2004).
[0005] In glaucoma filtration surgery, excessive postoperative
scarring at the wound site significantly reduces surgical success.
(Migdal et al, Ophthalmology 101, 1651-1656 (1994), Addicks et al.,
Ophthalmol. 101, 795-798 (1983)). Although anti-scarring agents
such as mitomycin-C and 5-fluorouracil could help prevent
postsurgical scarring and improve glaucoma surgical outcome, (Khaw
et al., Arch. Ophthalmol. 111, 263-267 (1993), Cordeiro et al.,
Invest. Ophthalmol. Vis. Sci. 40, 1975-1982 (1999)) they do so by
causing widespread fibroblast cell death and are associated with
severe and potentially blinding complications. (Crowston et al.
449-454 (1998), Stamper et al., Am. J. Ophthalmol. 114, 544-553
(1992)). In light of the role of TGF.beta. in the wound repair
process, alternative strategies (Codeiro, Prog. Retin. Eye Res. 21,
75-89 (2002)) such as antibodies (Cordeior et al., Invest.
Ophthalmol. Vis. Sci. 40, 2225-2234 (1999), Mead et al., Invest.
Ophthalmol. Vis. Sci. 44, 3394-3401 (2003)) to TGF.beta. and
antisense oligonucleotides (Cordeior, et al., Gene Therapy 10,
59-70 (2003)) have been used to block TGF.beta. action. However
these techniques remain inadequate for the treatment of the
debilitating scarification that occurs in many glaucoma. For
example, use of antisense therapy is poorly effective in treating
various disorders because antisense molecules are known to induce
an interferon response in the patient. Use of antibody-based
therapies are marred by the need to generate specific antibodies
against particular epitopes of a given antigen. Thus, there remains
a need to identify new methods of intervening in disorders that
result from an over-expression or even mere presence of TGF.beta.
type II receptor.
SUMMARY OF THE INVENTION
[0006] The present invention is directed to the use of siRNA both
in vitro and in vivo to regulate the TGF.beta. type II receptor
(TGF.beta. RII) level and modulate wound responses and angiogenesis
in a mammal. The RNA interference-based methods of the present
invention have a wide-ranging application, extending from the eye
to other organs and tissues throughout the body.
[0007] In certain embodiments, the invention is directed to methods
and compositions for promoting wound healing, reducing fibrosis
and/or reducing angiogenesis in a mammal by administering to the
mammal a composition comprising siRNA molecules that target the
type II receptor of TGF.beta..
[0008] The siRNA molecules of the present invention may be
delivered, in a therapeutically effective amount, locally at the
site of the wound or alternatively may be administered
systemically. In certain embodiments, therapeutically effective
siRNA compositions may be administered alone or alternatively, the
siRNA molecule-based therapeutic compositions may be administered
as part of a therapeutic regimen that comprises other wound-healing
compositions.
[0009] In particularly preferred embodiments, the disorder to be
treated by the siRNA based therapeutic compositions of the present
invention is glaucoma. However, it should be understood that the
siRNA compositions of the present invention may be used in the
treatment of any disorder in which signaling through the TGF.beta.
type II receptor is implicated. In addition to glaucoma filtration
surgery, the compositions of the present invention may be used to
promote healing, with a reduction in scarring, of any other
ophthalmic surgery, which may include but is not limited to,
cataract extraction, with or without lens replacement; corneal
transplants, to treat viral infection or penetrating keratoplasty
(PKP); and radial keratotomy and other types of surgery to correct
refraction. The compositions and methods of the invention also may
be used to treat ocular disorders such as, e.g., retinal wounds
such as retinal detachments and tears, retinal vacuolar disorder,
retinal neovascularization, diabetic retinopathy, corneal wounds
such as corneal epithelial wounds, corneal neovascularization,
corneal ulcers, macular holes, macular degeneration, secondary
cataracts, corneal disease, dry eye/Sjogren's syndrome and uveitis.
These disorders include wound healing disorders, proliferative
disorders, anti-degenerative disorders and anti-angiogenesis
disorders that effect the eye.
[0010] In each of the above methods, the method involves
administering to the mammal an amount of the siRNA composition in
an amount effective to stabilize or improve vision. Retinal
disorders, which are characterized by increased connective or
fibrous tissue, also may be treated using methods which comprise
the steps of removing the vitreous humor from the eye; removing the
epiretinal membrane, if present, from the eye; and administering a
composition comprising the siRNA compositions of the invention by
cannula to place the therapeutic composition immediately over the
portion of the retina requiring treatment.
[0011] In certain other embodiments, the siRNA composition may be
administered by intraocular injection or by application to the
cornea. Such corneal application may be achieved using eye drops or
a timed release capsule placed in the cul de sac.
[0012] In another embodiment, there is provided a method for
treating a mammal for ocular neovascularization, said method
comprising administering to a mammal an effective amount of the
siRNA compositions of the present invention.
[0013] Other non-ocular disorders that may be treated using the
siRNA-based methods of the present invention include but are not
limited to fibroproliferative disorders such as those selected from
the group consisting of diabetic nephropathy, glomerulonephritis,
proliferative vitreoretinopathy, liver cirrhosis, biliary fibrosis,
and myelofibrosis, post-radiation fibrosis. Connective tissue
disorders such as rheumatoid arthritis, scleroderma, myelofibrosis,
and hepatic, and pulmonary fibrosis also may be treated. Disorders
involving defective T-cell response, such as trypanosomal infection
or viral infections such as human immunosuppression virus, human T
cell lymphotropic virus, lymphocytic choroiomeningitis virus and
hepatitis may be treated. siRNA methods may be used to treat
patients with cancer, including patients with prostate cancer,
ovarian cancer, plasmacytoma and glioblastoma. siRNA may be used to
treat patient with collagen vascular diseases such as progressive
systemic sclerosis (PSS), polymyositis, dermatomyosistis and
systemic lupus erythamaosus.
[0014] In addition, siRNA-based methods may be used to treat wounds
other than those induce by ocular trauma, disorders or surgery.
Surgical incisions in general, trauma-induced lacerations, fibrosis
due to radiation therapy and wounds involving the peritoneum may be
treated. Scarring resulting from restenosis of blood vessels,
hypertrophic scars and keloids may also be treated with siRNA
methods.
[0015] Particularly preferred siRNA molecules include 21-23 bases.
Four specific sequences for the TGF.beta.RII siRNA were derived
from the human TGF.beta.RII sequence (Genbank Accession Number:
M85079) and were designated as NK1, NK2, SS1 and SS2. The target
sequences (5' to 3') are set out as below, with the position of the
first nucleotide in the human TGF.beta.II receptor sequence (from
M85079) shown in parenthesis. The corresponding commercially
synthesized siRNA duplexes are also set out below: TABLE-US-00001
Target Sequence 5' to 3' Nucleotide number in parenthesis siRNA
duplex NK1 (529) UCCUGCAUGAGCAACUGCAdTdT AATCCTGCATGAGCAACTGCA
dTdTAGGACGUACUCGUUGACGU (SEQ ID NO: 1) (SEQ ID NOS: 5-6) NK2 (1113)
GGCCAAGCUGAAGCAGAACdTdT AAGGCCAAGCTGAAGCAGAAC
dTdTCCGGUUCGACUUCGUCUUG (SEQ ID NO: 2) (SEQ ID NOS: 7-8) SS1 (1253)
GCAUGAGAACAUACUCCAGdTdT AGCATGAGAACATACTCCAG
dTdTCGUACUCUUGUAUGAGGUC (SEQ ID NO: 3) (SEQ ID NO: 9-10) SS2 (948)
GACGCGGAAGCUCAUGGAGdTdT AAGACGCGGAAGCTCATGGAG
dTdTCUGCGCCUUCGAGUACCUC (SEQ ID NO: 4) (SEQ ID NO: 11-12)
[0016] It should be understood that those of skill in the art will
be able to produce additional siRNA molecules surrounding positions
529, 1113, 1253 and 948 of the human TGF.beta.RII gene sequence at
Genbank Accession Number: M85079. It should be understood that the
siRNA molecules of the invention may be conveniently formulated
into pharmaceutical formulations using methods known to those of
skill in the art. Such pharmaceutical compositions also may
comprise other non-siRNA based therapeutic agents for the
therapeutic intervention of the particular disorder being treated.
Other wound healing compositions include anti-cancer drugs
Mitomycin and 5-fluorouracil, agaricus bisporus lectin,
metallocomplexes such as zinc-desferrioxaminde or
gallium-desferrioxamine, methyl xanthine derivatives such as
pentoxifylline, collagen-based sealants such as GE Amidon Oxyde,
agents that inhibit fibroblast growth factors and connective tissue
growth factor, and matrix metalloproteinase inhibitors such as
ilomastat. Other anti-angiogenic agents include inhibitors of
vascular endothelial growth factor (VEGF) and antiangiogenic
steroids. Inhibitors of VEGF include siRNA molecules targeting VEGF
or its receptor.
[0017] Other features and advantages of the present invention will
become apparent from the following detailed description. It should
be understood, however, that the detailed description and the
specific examples, while indicating preferred embodiments of the
invention, are given by way of illustration only, because various
changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from
this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] The following drawings form part of the present
specification and are included to further illustrate aspects of the
present invention. The invention may be better understood by
reference to the drawings in combination with the detailed
description of the specific embodiments presented herein.
[0019] FIG. 1. Inhibition of TGF.beta. type II receptor expression
by siRNA. Immunofluorescence analysis of human corneal fibroblasts
untreated (1.sup.st row), or treated with scrambled siRNA (2.sup.nd
row) or 100 nM NK1 (3.sup.rd and 4.sup.th rows) was performed to
visualize TGF.beta.RII receptor expression (left column). Staining
of nuclei using DAPI stain is shown in the right column. Note the
large reduction in staining of cells treated with NK1 siRNA at 48
(3.sup.rd row, left column) and 72 h (4.sup.th row, left column)
compared to control cells (1.sup.st and 2.sup.nd rows, left
column).
[0020] FIG. 2. Suppression of TGF.beta.I type II receptor protein
expression by siRNA in corneal fibroblasts. Lysates from human
corneal fibroblasts treated with different concentrations of
TGF.beta.RII receptor siRNA or control, scrambled siRNA for 16 (top
panel) or 48 hours (bottom panel) were separated on 10%
SDS-polyacrylamide gels and immunoblotted with a TGF.beta.RII
receptor antibody. Lane 1 contains lysate from cells incubated only
with TransIT-TKO reagent (no siRNA). Lanes 2 and 8 contain lysates
from cells treated with 100 nM scrambled siRNA. Lanes 3, 4, 9 and
10 contain lysates of cells treated with NK1 siRNA at a final
concentration of 50 (lanes 3 and 9) or 100 nM (lanes 4 and 10).
Lanes 5, 6, 11 and 12 contain lysates of cells treated with SS1
siRNA at 50 (lanes 5 and 11) or 100 nM (lanes 6 and 12). In lane 7,
the TGF.beta.RII receptor antibody was preincubated with antigenic
peptide before probing the normal cell lysate. Similar amounts of
total protein were loaded in each lane.
[0021] FIG. 3A-3G provides target sequences in the TGF.beta. type
II receptor sequence and the corresponding siRNA molecule
sequences. The nucleotide numbers refer to the location in the type
II TGF-.beta. receptor sequence (Genbank Accession Number: M85079).
The GC content refers to the content of guanine and cytosines (GC)
within the target sequence.
[0022] FIG. 4. Inhibition of TGF.beta.RII using siRNA on HUVEC
cells. Human umbical vein endothelial cells (HUVEC) were plated at
3.times.10.sup.-5 and allowed to grow into confluent monolayers.
Following day the cells were treated with (a) control (TKO reagent
only), (b) scrambled (c) NK1 siRNA oligonucleotides, (d) SS1 siRNA
oligonucleotides, all in the TKO reagent. Images were taken 48
hours post RNAi treatment. e, f, g, and h are corresponding DAPI
nuclear staining of the cells in panels a, b. c, and d
respectively. Scale bar is 10 microns.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE
INVENTION
[0023] There is a need to develop new therapies for reducing
scarring that result during wound healing. TGF.beta. is known to be
involved in the fibrogenic response in wound healing, and
inhibition of TGF.beta.-induced activities may be therapeutically
effective for reducing fibrosis and scarring. The present invention
provides specific siRNA compositions for use in methods of
promoting wound healing and for reducing scarring as a result of
wound healing. In addition, the present invention provides specific
siRNA compounds for use in methods of inhibiting angiogenesis.
These compositions are described in further detail herein.
Definitions
[0024] The term RNA interference (RNAi) refers to
post-transcriptional gene silencing induced by the introduction of
double stranded RNA.
[0025] The term small interfering RNAs (siRNAs) refers to
nucleotides of 19-23 bases in length which incorporate into an
RNA-induced silencing complex in order to guide the complex to
homologous endogenous mRNA for cleavage and degradation of
TGF.beta.RII and that mRNA.
[0026] The term transforming growth factor (TGF.beta.) refers to a
family of peptide growth factors including five member, numbered 1
through 5.
[0027] The term TGF.beta. receptors refers to cell surface
proteins, of which three (Type I, Type II and Type III) are known
in mammals. The TGF.beta. type II receptor (TGF.beta.RII) is a
membrane bound protein with an intracellular domain, a
transmembrane domain and extracellular domain that binds to
TGF.beta.. As reviewed in Massague et al., Annu. Rev. Biochem. 67:
753-791, (1998) incorporated herein by reference.
[0028] The term therapeutically effective amount refers the amount
of a siRNA molecule which effectively suppresses expression of the
TGF.beta.RII protein in a mammal in need.
Role of TGF.beta. Family in Wound Healing
[0029] Transforming growth factor-.beta. (TGF.beta.) family of
cytokines is an important mediator in the wound healing process in
various tissues. In the eye, TGF.beta. has been implicated in the
corneal haze and scarring at the wound site following glaucoma
surgery. TGF.beta. has also been associated with diabetic
retinopathy, proliferative vitreoretinopathy and macular
degeneration. The inventors designed small interfering RNAs
(siRNAs) targeting the type II receptor of TGF.beta. and found that
these RNA fragments were effective in abrogating the receptor
protein and transcript in cultured human corneal fibroblasts.
TGF.beta.-mediated processes such as fibronectin assembly and cell
migration were inhibited. The siRNAs, when introduced
subconjunctivally into mouse eyes, were also efficacious in
reducing the inflammatory response and matrix deposition. These
findings indicate that siRNAs can be successfully delivered both in
vitro and in vivo to regulate the TGF.beta. type II receptor level
and modulate wound response. The RNA interference technology may
have a wide-ranging application, extending from the eye to other
organs and tissues throughout the body.
[0030] In addition to wound healing, TGF.beta. is known to play an
important role in the regulation of growth and differentation of
many cell types. As TGF.beta. is also known to control the
accumulation of matrix proteins such as collagen, fibronectin,
thrombospondin, osteopotin, proteoglycans and glycosamineoglycans,
it is thought to contribute to carcinogenic changes within many
organ systems. Therefore, suppression of TGF.beta.RII gene
expression may be a method of treating fibroproliferative
disorders, and connective tissue disorders.
[0031] TGF.beta. is also known to induce endothelial tube formation
in vitro and is thought to affect the organizational process of
capillary tube that formation in vivo. TGF.beta. levels are known
to be elevated in some cancers such as prostate cancer, ovarian
cancer, plasmacytoma and gliablastoma. Furthermore, it is
associated with angiogenesis in part by its association with CTGF.
Thus, suppression of TGF.beta.RII receptor gene expression may be a
method of treating these and other types of cancers, as well as
abnormal blood vessel growth.
[0032] TGF.beta. is also known to inhibit the growth to both T- and
B-lymphocytes, natural killer cells and lymphokine-activated killer
cells. Therefore, in addition to cancers, suppression of
TGF.beta.RII gene expression may be a method of treating immune
disorders such as AIDS, other viral infections and trypanosomal
infections.
[0033] In addition, siRNA-based methods may be used to treat wounds
other than those induce by ocular trauma, disorders or surgery.
Surgical incisions in general, trauma-induced lacerations, fibrosis
due to radiation therapy and wounds involving the peritoneum may be
treated. Scarring resulting from restenosis of blood vessels,
hypertrophic scars and keloids may be treated with siRNA
methods.
[0034] An ocular fibrotic wound healing response represents a
significant pathophysiological issue especially as a consequence of
the surgical treatment for glaucoma. (Migdal et al. Ophthalmology
101, 1651-1656 (1994), Addicks et al., Arch. Ophthalmol. 101,
795-798 (1983)) Excessive post-operative scarring often leads to
failure of the filtration surgery. While the use of antimetabolites
such as mitomycin-C and 5-fluorouracil as conjunctival
anti-scarring treatments have benefited a number of patients, these
agents are associated with potentially blinding complications, such
as hypotony maclopathy and infection. (Khaw et al., Arch.
Ophthalmol. 111, 263-267 (1993), Cordeiro et al., Invest.
Ophthalmol. Vis. Sci. 40, 1975-1982 (1999), Crowston et al.,
Invest. Ophthalmol. Vis. Sci. 39, 449-454 (1998), Stamper, Am. J.
Ophthalmol. 114, 544-553 (1992)).
[0035] Sequestering of mature TGF.beta. has been a primary target
for the development of antifibrotic approaches. Antibodies to
TGF.beta.2 have been demonstrated to significantly reduce
conjunctival scarring activity. (Cordeior et al., Invest.
Ophthalmol. Vis. Sci. 40, 2225-2234 (1999), (Mead et al., Invest.
Ophthalmol. Vis. Sci. 44, 3394-3401 (2003)) In addition, modulation
of wound healing is observed when antisense oligonucleotides
(Cordeior, et al., Gene Therapy 10, 59-70 (2003), Shen et al., Eur.
J. Bioichem. 268, 2331-2337 (2001)) or ribozymes (Su et al.
Biochem. Biophys. Res. Commun. 278, 401-407 (2000), Yamamoto et
al., Circulation 102, 1308-1314 (2000)) to TGF.beta. are applied to
animal models or cultured cells. Nevertheless, neutralizing
antibodies in general exhibit relatively weak effects as these
antibodies may not gain full access to the targeted molecule. (Shen
et al., Eur. J. Bioichem. 268, 2331-2337 (2001)). Antisense
phosphorothioate oligonucleotides and ribozymes can be effective,
but their stability and specificity are at times still in question.
The concentration needed is also generally in the .mu.M range. By
comparison, the siRNAs are efficacious at 200 nM and are highly
specific. Therefore, the present invention specifically
contemplates compositions comprising siRNAs at a concentration of
100 nM, 110 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170
nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 240 nM, 250 nM, 260 nM,
270 nM, 280 nM, 290 nM, and 300 nM or more.
[0036] Such compositions of the invention will be used in methods
of treating or preventing glaucoma. In addition, recent studies
have shown that TGF.beta. may actually be involved in the
pathogenesis of primary open angle glaucoma (Inatani et al.,
Graefes Archive for Clinical & Experimental Ophthalmology.
239(2):109-13, 2001; Ochiai et al., Japanese Journal of
Ophthalmology. 46(3):249-53, 2002; Gattanka et al., Invest
Ophthalmol Vis Sci., 45(1):153-8, 2004;). Down regulation of the
TGF.beta. receptors in the anterior chamber using siRNA against the
TGF.beta. receptor will be another treatment modality against the
actual development or progression of glaucoma. Therefore, the siRNA
compositions of the present invention may be used to in treatment
methods for glaucoma that has already developed or alternatively
may be used prophylactically to prevent glaucoma. Those of skill in
the art are aware of animal models for ophthalmologic function and
methods and routes of administering therapeutic compositions (e.g.,
shunts, perfusion, etc.) for the treatment or prevention of
glaucoma, see for example, Inatani et al., supra, and Ochiai et
al., supra, U.S. Pat. Nos. 6,713,498; 6,699,211; 6,699,210;
6,649,625; 6,595,945; 6,531,128; 6,482,854. Each of these documents
are incorporated herein by reference in their entirety.
[0037] Furthermore, the use of siRNA against TGF.beta. receptors
will be of value in preventing restenosis of coronary vessels as
well as helping to arrest the progression of pulmonary fibrosis and
pulmonary scarring from chronic pulmonary obstructive disease as
well as renal fibrosis and postoperative scarring in the abdomen
and elsewhere in the body. Thus, it is contemplated that the
siRNA-based compositions of the invention will be useful as or in
conjunction with therapeutic methods for the improvement of
circulation and hemostasis in stenotic vessels. Thus, these siRAN
compositions may be used alone or in combination with (e.g.,
during, before or after) by-pass surgery and revascularization
procedures (e.g., balloon angioplasty, atherectomy, rotorary
ablation (rotoblation)) which serve to improve blood flow by
reducing or removing the stenosis. These methods will be useful in
reducing the thickness or presence of neointima within the vessel
wall which reduces the luminal area of the vessel (i.e.,
restenosis). For further details of methods and compositions for
treating restenosis and stenosis see e.g., U.S. Pat. Nos.
6,663,863; 6,648,881; 6,596,698; 6,520,957; 6,519,488; 6,458,590;
6,491,720; 6,241,718. Each of these documents are incorporated
herein by reference in their entirety. These patents are listed to
show exemplary teachings in the art for the preparation of stents
and medicaments for the treatment of restenosis. The compositions
described herein may be used in like manner to the medicaments
described therein and also may be used to supplement the treatment
methods described in those exemplary patents.
RNA Interference (RNAi) Technology
[0038] Variations on RNA interference (RNAi) technology is
revolutionizing many approaches to experimental biology,
complementing traditional genetic technologies, mimicking the
effects of mutations in both cell cultures and in living animals.
(McManus & Sharp, Nat. Rev. Genet. 3, 737-747 (2002)) The
present invention demonstrates that the RNAi technology can be
successfully used to regulate wound healing response by targeting
the TGF.beta.II receptor gene. The effect is specific and potent.
This technology may be applied not only to the conjunctiva, cornea,
retina and choroid of the eye, but also in other tissues throughout
the body to modulate wound responses in disorders including
vascular diseases, hypertension and atherosclerosis. (Yamamoto et
al., Circulation 102, 1308-1314 (2000))
[0039] In the current study, RNAi was used to target the TGF.beta.
pathway. RNAi, known to occur in animals and eukaryotes, is a
process in which double stranded RNA (dsRNA; typically >200
nucleotides in length) triggers the destruction of mRNAs sharing
the same sequence. RNAi is initiated by the conversion of dsRNA
into 21-23 nucleotide fragments and these small interfering RNAs
(siRNAs) direct the degradation of target RNAs. (Elbashir et al.,
Nature 411, 494-498 (2001), Fire et al., Nature 391, 199-213
(1998), Hannon, G. J., Nature 418,244-251 (2002)). It has been
rapidly adopted to use for silencing genes in a variety of
biological systems. (Reich et al., Mol. Vis. 9, 210-216 (2003),
Song et al., Nat. Med. 9, 347-351 (2003))
[0040] RNAi technology may be carried out in mammalian cells by
transfection of siRNA molecules. The siRNA molecules may be
chemically synthesized, generated by in vitro transcription, or
expressed by a vector or PCR product. Commercial providers such as
Ambion Inc. (Austin, Tex.), Darmacon Inc. (Lafayette, Colo.),
InvivoGen (San Diego, Calif.), and Molecula Research Laboratories,
LLC (Herndon, VA) generate custom siRNA molecules. In addition,
commercial kits are available to produce custom siRNA molecules,
such as SILENCER.TM. siRNA Construction Kit (Ambion Inc., Austin,
Tex.) or psiRNA System (InvivoGen, San Diego, Calif.). These siRNA
molecules may be introduced into cells through transient
transfection or by introduction of expression vectors that
continually express the siRNA in transient or stably transfected
mammalian cells. Transfection may be accomplished by well known
methods including methods such as infection, calcium chloride,
electroporation, microinjection, lipofection or the DEAE-dextran
method or other known techniques. These techniques are well known
to those of skill in the art.
[0041] The siRNA molecules may be introduced into a cell in vivo by
local injection of or by other appropriate viral or non-viral
delivery vectors. Hefti, Neurobiology, 25:1418-1435 (1994). For
example, the siRNA molecule may be contained in an adeno-associated
virus (AAV) vector for delivery to the targeted cells (e.g.,
Johnson, International Publication No. WO95/34670; International
Application No. PCT/US95/07178). The recombinant AAV genome
typically contains AAV inverted terminal repeats flanking the siRNA
sequence operably linked to functional promoter and polyadenylation
sequences. Alternative suitable viral vectors include, but are not
limited to, retrovirus, adenovirus, herpes simplex virus,
lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus,
alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papilloma
virus vectors.
[0042] Nonviral delivery methods include, but are not limited to,
liposome-mediated transfer, naked DNA delivery (direct injection),
receptor-mediated transfer (ligand-DNA complex), electroporation,
calcium phosphate precipitation, and microparticle bombardment
(e.g., gene gun). Methods of introducing the siRNA molecules may
also include the use of inducible promoters, tissue-specific
enhancer-promoters, DNA sequences designed for site-specific
integration, DNA sequences capable of providing a selective
advantage over the parent cell, labels to identify transformed
cells, negative selection systems and expression control systems
(safety measures), cell-specific binding agents (for cell
targeting), cell-specific internalization factors, and
transcription factors to enhance expression by a vector as well as
methods of vector manufacture.
[0043] The preferred siRNA molecule is 19-25 base pairs in length,
most preferably 21-23 base pairs, and is complementary to the
target gene sequence. The siRNA molecule preferably has two
adenines at its 5' end, but may not be an absolute requirement. The
siRNA sequences that contain 30-50% guanine-cytosine content are
known to be more effective than sequences with a higher
guanine-cytosine content. Therefore, siRNA sequence with 30-50% are
preferable, while sequences with 40-50% are more preferable. The
preferred siRNA sequence also should not contain stretches of 4 or
more thymidines or adenines.
[0044] The present specification provides details of studies
performed with siRNAs designed to target the TGF.beta. type II
receptor (TGF.beta.II) gene. The target sequence selected should
not be highly structured or bound by regulatory proteins.
Preferably, the siRNA molecules of the invention should be directed
to different positions within the target gene sequences. For
example, siRNA target sequences NK1, NK2, SS1 and SS2 (SEQ ID NO:
1-4) are directed to different portions of the TGF.beta.RII gene.
In particular nucleotides NK1 spans nucleotides 529-612, NK2 spans
nucleotides 1113-1133, SS1 spans nucleotides 1253-1273 and SS2
spans nucleotides 948-969 of the TGF.beta.RII gene. Additional
siRNA target sequences that may be effective for suppressing
TGF.beta.RII gene expression are set out in Table 1 below. These
sequences were derived by analyzing the human TGF.beta.RII sequence
(M85079) using the publicly available siRNA Target Finder program
at the Ambion, Inc. web site. The sequences were screened by BLAST
searching the Genbank database for homologous sequences. Any
sequence containing more than 16 nucleotides match to a
non-TGF.beta.RII sequence were eliminated from further
consideration.
[0045] Sequences with a GC content between 30-50% were further
analyzed. Those sequences containing four consecutive A, C, G or T
bases were eliminated. This analysis identified an additional 49
siRNA molecules that are contemplated to be effective in inhibiting
TGF.beta.RII gene. These sequences are shown in FIG. 3. The siRNA
molecules that contain up to 2 mismatches are effective in
inhibiting TGF.beta.RII expression. The effectiveness of the siRNA
containing mismatches may be dependent on their position in the
sequence. Thus, it is likely that other siRNA sequences may be
derived from the 4 already tested (NK1, NK2, SS1 and SS2) and those
indicated in FIG. 3.
[0046] The present specification provides details of studies
performed with siRNAs designed to target the TGF.beta. type II
receptor (TGF.beta.RII) gene. In cultured human corneal
fibroblasts, the siRNAs effectively suppressed gene expression of
the receptor, reduced TGF.beta.-mediated matrix deposition and
retarded cell migration. In addition, the data presented herein
shows in an in vivo model that siRNAs specific for TGF.beta.RII can
reduce inflammation and regulate wound repair in the conjunctiva of
mouse eyes. The siRNA molecules of the present invention also
effectively suppress TGF.beta.RII gene expression in human
umbilical vein endothelial cells.
[0047] siRNAs specific to human TGF.beta.RII can inhibit the
receptor expression in cultured human corneal fibroblasts as shown
by immunofluorescence, Western blotting and real time PCR analyses.
Four concentrations of siRNAs ranging from 25 to 200 nM and four
time points from 16 to 72 hours were tested. The inhibitory
response is both dose and time dependent. Specificity of the siRNAs
for the TGF.beta.RII has also been established. All four siRNAs
tested were found to be efficacious, although two of them showed
greater effect. Given the teachings provided herein, one of skill
in the art would expect that other siRNAs deduced from the cDNA
sequence of human TGF.beta.RII also will be as effective.
Assays to Test Efficacy of siRNA Specific to Human TGF.beta. Type
II Receptor In Vitro Models
[0048] Corneal fibroblasts constitutively express TGF.beta.. (Song
et al., J. Cell. Biochem. 77, 186-199 (2000), Imanishi et al.,
Prog. Retin. Eye Res. 19, 113-129 (2000)) The effects of siRNAs in
blocking autocrine TGF.beta. signaling in corneal fibroblasts was
examined and are described herein. The functional roles of the
siRNAs are thus well established in this in vitro culture
model.
[0049] TGF.beta. has been shown to enhance the expression of matrix
molecules such as fibronectin and collagen type I (Song et al., J.
Cell. Biochem. 77, 186-199 (2000), Massague, Annu. Rev. Cell Biol.
6, 597-641 (1990)) and to facilitate migration of corneal
fibroblasts, (Imanishi et al., Prog. Retin. Eye Res. 19, 113-129
(2000), Andersen et al, Curr. Eye Res. 16, 605-613 (1997)), and the
steps involved in the complex wound repair process. (Clark,
Physiology, Biochemistry and Molecular Biology of the Skin, Oxford
University Press. P. 576-601, 1997) As has been demonstrated in
hepatic stellate cells with antisense RNA complementary to
TGF.beta.1, (Arias et al., Cell Growth Differ. 13, 265-273 (2002))
diminished receptor level and blockade of receptor binding for
TGF.beta. caused a reduction in the secreted fibronectin level and
its incorporation into the matrix. Corneal fibroblast migration is
also markedly retarded.
[0050] Given the teachings of the present invention, those of skill
in the art are instructed to produce siRNA molecules discussed
herein and employ such molecules in in vitro assays to assess the
effects of such siRNA molecules on migration of corneal
fibroblasts, the expression of fibronectin, and/or the expression
of collagen type I. Any decrease or diminution of the level of
migration of corneal fibroblasts, the level of expression and/or
secretion of either fibronectin or collagen type I will be
indicative of the given siRNA molecule being effective for use as a
therapeutic agent in accordance with the present invention.
Mouse Models
[0051] The therapeutic effects of the TGF.beta. specific siRNA
molecules are also demonstrated in a conjunctival scarring mouse
model. The model was similar to that described previously by
Reichel et al. (Br. J. Ophthalmol. 82, 1072-1077 (1998)). However,
instead of injecting only PBS into the subconjunctival space, the
injected PBS was mixed with latex beads to have an improved mouse
model with augmented inflammatory and scarring response. siRNA at
200 nM clearly showed its effectiveness in reducing the
inflammatory and fibrotic response in this new mouse model. Those
of skill in the art could repeat these model studies with any other
TGF.beta. specific siRNA molecule. Any other molecule that reduces
the inflammatory or fibrotic response in this mouse model is
contemplated to be a useful siRNA molecule of the invention.
Cell Growth Assays
[0052] TGF.beta. is known to stimulate fibroblast proliferation and
inhibit proliferation of epithelial cells, in particular tumor
cells. Therefore, measuring the effect of siRNA on
TGF.beta.-induced fibroblast proliferation or epithelial cell
growth inhibition is a method for evaluating the effectiveness of
the siRNA molecules.
[0053] Cell growth may be monitored by measuring DNA synthesis. DNA
synthesis may be measured using [.sup.3H]-thymidine incorporation
in cells as described in Lee et al., (Endocrinology 136:796-803,
(1995)). Cells are seeded at approximately 2.times.10.sup.4 per
well (24-well plate) and are incubated for 22 hours in 1 ml culture
medium with or without 1% PBS and containing TGF.beta. at selected
concentrations. Then 2 mCi per well [.sup.3H]-thymidine is added,
subsequently incubation continues for 4 hours, and radioactivity is
counted with a scintillation counter.
[0054] Cell proliferation can be measured by cell counting. Cells
are seeded (24-well plates) in culture medium with or without 1%
FBS and medium is changed every other day. At the end of a 4-day
culture, cells are trypsinized and counted in a Coulter
counter.
TGF.beta.RII Activation Assays
[0055] The use of the p3TP-lux construct allows for evaluation of
activation of the TGF.beta. type II receptor. Cells are seeded at
1.times.10.sup.5 cells per well in 6-well plates and are
transiently transfected with the plasmid p3TP-Lux using lipofection
according to manufacturer's instructions (Life technologies,
Gaithersburg, Md.). p3TP-Lux contains three
12-O-tetradecanoylphorbol-13-acetate-responsive elements from the
human collagen gene and one TGF.beta.-responsive element from the
human plasminogen activator inhibitor-1 (PAI-1) promoter linked to
the luciferase reporter gene (Wrana et al., Cell 71: 1003-14,
(1996)). Cells are incubated with 1 .mu.g/ml p3TP-Lux and 12
.mu.g/ml Lipofectamine for 24 hours. Subsequently, cells are
treated with 5 ng/ml TGF.beta. in RPMI for 24 hours and lysed with
extraction buffer (100 mM potassium phosphate, pH 7.5, 1% Triton
X-100, 100 mg/ml bovine serum albumin, 2.5 mM
phenylmethylsulfonylfluoride, 1 mM dithiothreitol). Lysates are
diluted into reaction buffer (75 mM MgCl.sub.2, 1 M glycylglycine,
pH 7.8, 100 mg/ml bovine serum albumin, 60 mg/ml ATP) and are
assayed for luciferase activity using a luminometer.
[0056] Use of this assay allows one to evaluate the effectiveness
of the siRNA on TGF.beta.RII activity. An effective siRNA molecule
of the present invention will inhibit the amount of signaling
through the TGF.beta.RII receptor as it will reduce the number of
receptors available for signaling. Preferably, the effective siRNA
molecule will inhibit signaling through TGF.beta.RII by at least
20%, or more preferably by at least 25%, 30%, 35%, 40% or 45%. It
is highly preferable that the effective siRNA molecule inhibit
signaling through the TGF.beta.RII by at least 50%, 55%, 60%, 65%,
70, 75% or more.
Chemotaxis Assays.
[0057] TGF.beta. is a cytokine and those of skill in the art
monitor the activity of such agents through well known chemotaxis
assays. Exemplary chemotaxis assays that may be performed are
described in Martinet et al., J. Immunol. Meth., 174:209, 1994 and
Keller et al., J. Immunol. Meth., 1:165, 1972. Briefly, 20 ml of
peripheral blood is collected from health volunteers in 10 ml
heparinized tubes. Blood is diluted 1:1 and then under laid with 10
ml of Histopaque (Sigma). After centrifugation at 400 g for 25
minutes, cells at the interface are collected and washed twice in
PBS. Cells are resuspended in DMEM (Life Technologies,
Gaithersburg, Md.) with 100 U/ml penicillin and 100 .mu.g/ml
streptomycin (tissue culture antibiotics, Life Technologies) at
106/ml. Sterile bovine serum albumin (Sigma) is added to final
concentration of 0.2 mg/ml.
[0058] 100 .mu.l of this cell suspension is added to each transwell
insert (Costar). DMEM with antibiotics and 0.2% BSA with or without
siRNA molecules is added to the lower wells in the 24 well plate.
Transwell inserts are placed into the lower walls, and incubated at
37N C for 90 minutes. At the completion of the incubation period
inserts are removed and the adherent cells are removed. The entire
insert is then stained with Wright-Giemsa. Cells adherent to the
lower surface of the insert and those that migrated to the lower
well are counted under microscope, and added together to obtain a
total number of migrating cells.
Assay of Chemoattractant and Cell-Activation Properties.
[0059] The effects of siRNA directed to TGF.beta.RII upon human
monocytes/macrophages or human neutrophils may be evaluated, e.g.,
by methods described by Devi et al., J. Immunol., 153:5376-5383
(1995) for evaluating murine TCA3-induced activation of neutrophils
and macrophages. Indices of activation measured in such studies
include increased adhesion to fibrinogen due to integrin
activation, chemotaxis, induction of reactive nitrogen
intermediates, respiratory burst (superoxide and hydrogen peroxide
production), and exocytosis of lysozyme and elastase in the
presence of cytochalasin B.
[0060] As discussed by Devi et al., these activities correlate to
several stages of the leukocyte response to inflammation. This
leukocyte response, reviewed by Springer, Cell, 76:301-314 (1994),
involves adherence of leukocytes to endothelial cells of blood
vessels, migration through the endothelial layer, chemotaxis toward
a source of chemokines, and site-specific release of inflammatory
mediators.
Assays of Effect on Myeloid Progenitor Cells.
[0061] The inhibition of TGF.beta.-induced suppression of
hematopoiesis may be tested in assays of stem/progenitor cell
function and number, including LTC-IC, CFU-GEMM, CFU-GM, BFU-E.
These assays are well known to those of skill in the art and are
relatively straightforward to set up as described in for example
Broxmeyer et al., Blood, 76:1110 (1990). Briefly, bone marrow cells
are collected from human donors after obtaining informed consent.
Low density human bone marrow cells at 5.times.104/ml are plated in
1% methylcellulose in Iscove's Modified Essential Medium
(Biowhitaker, Walkersville, Md.) supplemented with 30% FCS
(Hyclone), recombinant human erythropoietin (EPO, 1 U/ml, Amgen,
Thousand Oaks, Calif.), recombinant human interleukin-3 (IL-3, 100
U/ml, Immunex, Seattle, Wash.), and recombinant human stem cell
factor (SCF, 50 ng/ml, Amgen) for colony forming unit
granulocyte/macrophage (CFU-GM), colony forming unit
granulocyte/erythrocyte/macrophage/megakaryocyte (CFU-GEMM) or
blast forming unit-erythrocyte (BFU-E) analysis. Cultures are
incubated at 5% CO2 and low oxygen tension (5%) for 14 days, and
then scored for colony formation using an inverted microscope in a
blinded fashion.
Assays for Effects on Myeloid Cell Lines.
[0062] The effect of siRNA on TGF.beta.-induced inhibition of
myeloid cell proliferation also may be a useful test of functional
activity of the siRNA molecules. Such a functional assay may be
assessed using the human myeloid cell lines TF-1 and MO7E (Avanzi
et al., Brit. J Haematol., 69:359; 1988), which require GM-CSF and
SCF for maximal proliferation. The cytokine-dependent primitive
acute myeloid leukemia cell lines TF-1 and MO7E may be cultured in
RPMI 1640 (Life Technologies, Gaithersburg, Md.) plus 10% FCS
(Hyclone) and 100 U/ml penicillin and 100 .mu.g/ml streptomycin
(tissue culture antibiotics, Life Technologies, Gaithersburg, Md.).
This media is supplemented with granulocyte-macrophage colony
stimulating factor (GM-CSF, 100 U/ml, Immunex, Seattle, Wash.) and
stem cell factor (SCF, 50 ng/ml, Amgen, Thousand Oaks, Calif.) to
promote normal log phase growth.
Assays for Effect on Chronic Myelogenous Leukemia Progenitors.
[0063] The effect of siRNA on TGF.beta.-induced inhibition of
progenitor proliferation in chronic myelogenous leukemia (CML) may
be evaluated using colony formation assays as described in Hromas
et al., Blood, 89:3315-3322 (1997). Briefly, bone marrow cells are
collected from six CML patients in chronic phase. Low density
marrow cells at for example, 5.times.10.sup.4 cells/mL are plated
in 1% methylcellulose in Iscove's modified Dulbecco's medium
supplemented with 30% fetal calf serum, 1 U/mL human
erythropoietin. (Epogen.RTM., Amgen), 100 U/mL human interleukin-3
(Genetics Institute) and 50 ng/mL human stem cell factor (Amgen),
in the presence or absence of an appropriate concentration of
TGF.beta. (e.g. 100 ng/ml) alone or in combination with other
chemokines such as EXODUS, MIP-1.alpha. and the like.
[0064] Cultures are incubated at 5% CO.sub.2 and low (5%) oxygen
tension for 14 days, and then scored using an inverted microscope
for CFU-GM, CFU-GEMM and BFU-E. Colony counts for cultures treated
with chemokines are compared to colony counts of the control
cultures and were expressed as a percentage of control CFU or
FU.
[0065] As stated earlier, the assays described above are intended
to exemplify the types of assays that may be conducted to determine
the in vitro and in vivo effects of the siRNA molecules of the
present invention. These are by no means the only assays known to
be used for determine TGF.beta. activity. Those of skill in the art
will know of other assays that may be substituted for these
described above but nonetheless measure similar parameters of
function and activity.
Angiogenesis Assays
[0066] The effect of siRNA molecules on angiogenesis may be
monitored using the following assays. Angiogenesis is the multistep
process of new capillary formation originating from sprouting of
endothelial cells from the wall of an existing small blood vessel.
In order for new capillary tubes to form, endothelial cells must
elongate and migrate.
[0067] A tube formation assay may be utilized to determine if the
siRNA molecules targeting TGF.beta.RII inhibit tube formation in
endothelial cells such as HUVEC cells. For example endothelial tube
formation assays may be carried out in vitro using Matrigel. When
endothelial cells are plated on BD Matrigel.TM. (BD Biosciences),
the cells stop proliferating, and display high motility and
cell-cell communication. Furthermore, within 24 hours, the cells
align and form a three-dimensional network of capillary tubes that
has been proposed as a model of endothelial cell differentiation as
well as one of the final steps of the angiogenic cascade.
[0068] A 24-well tissue culture plate is coated with 500 .mu.l of
the Matrigel Matrix with reduced growth factors and allowed to gel
thoroughly by incubating at 37.degree. C. for at least 30 minutes.
After the Matrigel forms a gel, endothelial cells such as bovine
aortic endothelial cells (BAEC) or human umbilical vein endothelial
cells (HUVEC), are washed and seeded on Matrigel coated wells. The
cells are treated with TGF.beta. in the presence and absence of
siRNA molecules targeted to TGF.beta.RII, To view tube formation,
cells are treated with 1 mM Calcein AM (Molecular Probes) diluted
at 1:2000 in media, incubated in the dark for at least 15 minutes,
and subsequently washed with media+10% FBS.
[0069] Other assays to evaluate the effect of siRNA molecules on
TGF.beta.-induced angiogeneis include endothelial cell
proliferation assays and endothelial cell migration assays. In
addition, alterations in endothelial cells occur during
angiogenesis as vessels invade tumors, and have effects on
endothelial cell morphology and function. Endothelial cell
morphology may be evaluated using immunohistochemistry or electron
microscopy to view endothelial cell sprouting, migration, and
proliferation.
[0070] The Chicken Chorioallantoic Membrane (CAM) assay is also a
well known method of evaluating angiogenesis. The developing
chicken embryo is surrounded by a chorioallantoicmembrane, which
becomes vascularized as the embryo develops. Tissue grafts are
placed on the CAM through a window made in the eggshell. This
causes a typical radial rearrangement of vessels towards, and a
clear increase of vessels around the graft within four days after
implantation. Blood vessels entering the graft are counted under a
stereomicroscope. To assess the anti-angiogenic or angiogenic
activity of the siRNA molecules, the compounds are either prepared
in slow release polymer pellets, absorbed by gelatin sponges or
air-dried on plastic discs and then implanted onto the CAM. In the
CAM assay, siRNA of the present invention that lead to the
regression of newly developed CAM vasculature are determined to be
effective inhibitors of TGF.beta.-induced angiogenesis.
[0071] The effect of the siRNA molecules of the present invention
on TGF.beta.-induced angiogeneis may also be measured in the mouse
cornea using the micropocket assay. The mouse cornea presents an in
vivo avascular site. This makes it a very good model for studying
angiogenesis, as the growth of new blood vessels easily can be
studied under microscope. Any vessels penetrating from the limbus
into the corneal stroma can be identified as newly formed. To
induce an angiogenic response, slow release polymer pellets (i.e.
poly-2-hydroxyethyl-methacrylate (hydron) or ethylene-vinyl acetate
copolymer (ELVAX)), containing an TGF.beta. is implanted in
"pockets" created in the corneal stroma of a mouse. After 4-6 days,
new vessel growth occurs. The vascular response can be quantified
by computer image analysis after perfusion of the cornea with India
ink. The blood vessels in this model can also be studied
ultrastructurally by electron microscope, or by the use of
immunohistochemistry.
Pharmaceutical Compositions.
[0072] Where clinical applications are contemplated, it will be
necessary to prepare the viral expression vectors, nucleic acids
and other compositions identified by the present invention as
pharmaceutical compositions, i.e., in a form appropriate for in
vivo applications. Generally, this will entail preparing
compositions that are essentially free of pyrogens, as well as
other impurities that could be harmful to humans or animals. In
preferred embodiments, the present invention contemplates
pharmaceutical compositions containing siRNA molecules described as
the present invention.
[0073] The active compositions of the present invention include
classic pharmaceutical preparations. Administration of these
compositions according to the present invention will be via any
common route so long as the target tissue is available via that
route. The pharmaceutical compositions may be introduced into the
subject by any conventional method, e.g., by intravenous,
intradermal, intramusclar, intramammary, intraperitoneal,
intrathecal, intraocular, retrobulbar, intrapulmonary (e.g., term
release); by oral, sublingual, nasal, anal, vaginal, or transdermal
delivery, or by surgical implantation at a particular site, e.g.,
embedded under the splenic capsule, brain, or in the cornea. The
treatment may consist of a single dose or a plurality of doses over
a period of time.
[0074] The active compounds may be prepared for administration as
solutions of free base or pharmacologically acceptable salts in
water suitably mixed with a surfactant, such as
hydroxypropylcellulose. Dispersions also can be prepared in
glycerol, liquid polyethylene glycols, and mixtures thereof and in
oils. Under ordinary conditions of storage and use, these
preparations contain a preservative to prevent the growth of
microorganisms.
[0075] The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms, such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (for
example, glycerol, propylene glycol, and liquid polyethylene
glycol, and the like), suitable mixtures thereof, and vegetable
oils. The proper fluidity can be maintained, for example, by the
use of a coating, such as lecithin, by the maintenance of the
required particle size in the case of dispersion and by the use of
surfactants. The prevention of the action of microorganisms can be
brought about by various antibacterial an antifungal agents, for
example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal,
and the like. In many cases, it will be preferable to include
isotonic agents, for example, sugars or sodium chloride. Prolonged
absorption of the injectable compositions can be brought about by
the use in the compositions of agents delaying absorption, for
example, aluminum monostearate and gelatin.
[0076] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0077] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
antibacterial and antifungal agents, isotonic and absorption
delaying agents and the like that do not produce adverse, allergic,
or other untoward reactions when administered to an animal or
human. The use of such media and agents for pharmaceutical active
substances is well known in the art. Except insofar as any
conventional media or agent is incompatible with the active
ingredient, its use in the therapeutic compositions is
contemplated. Supplementary active ingredients also can be
incorporated into the compositions.
[0078] For oral administration the polypeptides of the present
invention may be incorporated with excipients and used in the form
of non-ingestible mouthwashes and dentifrices. A mouthwash may be
prepared incorporating the active ingredient in the required amount
in an appropriate solvent, such as a sodium borate solution
(Dobell's Solution). Alternatively, the active ingredient may be
incorporated into an antiseptic wash containing sodium borate,
glycerin and potassium bicarbonate. The active ingredient may also
be dispersed in dentifrices, including: gels, pastes, powders and
slurries. The active ingredient may be added in a therapeutically
effective amount to a paste dentifrice that may include water,
binders, abrasives, flavoring agents, foaming agents, and
humectants.
[0079] The compositions of the present invention may be formulated
in a neutral or salt form. Pharmaceutically-acceptable salts
include the acid addition salts (formed with the free amino groups
of the protein) and which are formed with inorganic acids such as,
for example, hydrochloric or phosphoric acids, or such organic
acids as acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups also can be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0080] The compositions of the present invention may be formulated
in a neutral or salt form. Pharmaceutically-acceptable salts
include the acid addition salts (formed with the free amino groups
of the protein) and which are formed with inorganic acids such as,
for example, hydrochloric or phosphoric acids, or such organic
acids as acetic, oxalic, tartaric, mandelic, and the like. Salts
formed with the free carboxyl groups also can be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0081] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as injectable solutions, drug
release capsules and the like. For parenteral administration in an
aqueous solution, for example, the solution should be suitably
buffered if necessary and the liquid diluent first rendered
isotonic with sufficient saline or glucose. These particular
aqueous solutions are especially suitable for intravenous,
intramuscular, subcutaneous and intraperitoneal administration.
[0082] "Unit dose" is defined as a discrete amount of a therapeutic
composition dispersed in a suitable carrier. For example, where
siRNA molecules are being administered parenterally, siRNA
compositions are generally injected in doses ranging from 1 mg/kg
to 100 mg/kg body weight/day, preferably at doses ranging from 0.1
mg/kg to about 50 mg/kg body weight/day. Parenteral administration
may be carried out with an initial bolus followed by continuous
infusion to maintain therapeutic circulating levels of drug
product. Those of ordinary skill in the art will readily optimize
effective dosages and administration regimens as determined by good
medical practice and the clinical condition of the individual
patient.
[0083] The frequency of dosing will depend on the pharmacokinetic
parameters of the agents and the routes of administration. The
optimal pharmaceutical formulation will be determined by one of
skill in the art depending on the route of administration and the
desired dosage. See for example Remington's Pharmaceutical
Sciences, 18th Ed. (1990, Mack Publ. Co, Easton Pa. 18042) pp
1435-1712, incorporated herein by reference. Such formulations may
influence the physical state, stability, rate of in vivo release
and rate of in vivo clearance of the administered agents. Depending
on the route of administration, a suitable dose may be calculated
according to body weight, body surface areas or organ size. Further
refinement of the calculations necessary to determine the
appropriate treatment dose is routinely made by those of ordinary
skill in the art without undue experimentation, especially in light
of the dosage information and assays disclosed herein as well as
the pharmacokinetic data observed in animals or human clinical
trials.
[0084] Appropriate dosages may be ascertained through the use of
established assays for determining blood levels in conjunction with
relevant dose-response data. The final dosage regimen will be
determined by the attending physician, considering factors which
modify the action of drugs, e.g., the drug's specific activity,
severity of the damage and the responsiveness of the patient, the
age, condition, body weight, sex and diet of the patient, the
severity of any infection, time of administration and other
clinical factors. As studies are conducted, further information
will emerge regarding appropriate dosage levels and duration of
treatment for specific diseases and conditions.
[0085] In gene therapy embodiments employing viral delivery, the
unit dose may be calculated in terms of the dose of viral particles
being administered. Viral doses include a particular number of
virus particles or plaque forming units (pfu). For embodiments
involving adenovirus, particular unit doses include 10.sup.3,
10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7, 10.sup.8, 10.sup.9,
10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13 or 10.sup.14 pfu.
Particle doses may be somewhat higher (10 to 100-fold) due to the
presence of infection defective particles.
[0086] It will be appreciated that the pharmaceutical compositions
and treatment methods of the invention may be useful in fields of
human medicine and veterinary medicine. Thus the subject to be
treated may be a mammal, preferably human or other animal. For
veterinary purposes, subjects include for example, farm animals
including cows, sheep, pigs, horses and goats, companion animals
such as dogs and cats, exotic and/or zoo animals, laboratory
animals including mice rats, rabbits, guinea pigs and hamsters; and
poultry such as chickens, turkey ducks and geese.
Combined Therapy.
[0087] In addition to therapies based solely on the delivery of
siRNA molecules and related composition, combination therapy is
specifically contemplated. In the context of the present invention,
it is contemplated that siRNA methods could be used similarly in
conjunction with other agents for promoting wound-healing, reducing
scarring, inhibiting angiogenesis, or those used in the therapy of
the disorders enumerated herein. It is also contemplated that the
siRNA molecules directed to TGF.beta.RII could be used in
conjunction with other siRNA molecules that promote wound healing,
reducing scarring, inhibiting angiogenesis or those used in the
therapy of the disorders described herein.
[0088] To achieve the appropriate therapeutic outcome, be it a
decrease in scarring, decrease in fibrogen accumulation, reduction
in angiogenesis or any other use for the siRNA molecules discussed
herein, using the methods and compositions of the present
invention, one would generally contact a "target" cell with a siRNA
expression construct and at least one other therapeutic agent
(second therapeutic agent). These compositions would be provided in
a combined amount effective to produce the desired therapeutic
outcome. This process may involve contacting the cells with the
expression construct and the agent(s) or factor(s) at the same
time. This may be achieved by contacting the cell with a single
composition or pharmacological formulation that includes both
agents, or by contacting the cell with two distinct compositions or
formulations, at the same time, wherein one composition includes
the expression construct and the other includes the second
therapeutic agent.
[0089] Alternatively, the siRNA treatment may precede or follow the
other agent treatment by intervals ranging from minutes to weeks.
In embodiments where the second therapeutic agent and expression
construct are applied separately to the cell, one would generally
ensure that a significant period of time did not expire between the
time of each delivery, such that the agent and expression construct
would still be able to exert an advantageously combined effect on
the cell. In such instances, it is contemplated that one would
contact the cell with both modalities within about 12-24 hours of
each other and, more preferably, within about 6-12 hours of each
other, with a delay time of only about 12 hours being most
preferred. In some situations, it may be desirable to extend the
time period for treatment significantly, however, where several
days (2, 3, 4, 5, 6 or 7) to several weeks (1, 2, 3, 4, 5, 6, 7 or
8) lapse between the respective administrations.
[0090] Local delivery of siRNA expression constructs or sequences
to patients may be a very efficient method for delivering the siRNA
molecules to counteract a clinical disease. Similarly, the second
therapeutic agent may be directed to a particular, affected region
of the subject's body. Alternatively, systemic delivery of
expression construct and/or the second therapeutic agent may be
appropriate in certain circumstances.
[0091] Other antiproliferative and anti-angiogenic compositions
which may be effective include in combination treatments with the
siRNA molecules of the present invention include anti-cancer drugs
mitomycin-C and 5-fluorouracil, agaricus bisporus lectin,
metallocomplexes such as zinc-desferrioxaminde or
gallium-desferrioxamine, methyl xanthine derivatives such as
pentoxifylline, collagen-based sealants such as GE Amidon Oxyde. In
addition, agents that inhibit VEGF, fibroblast growth factors,
connective tissue growth factors and matrix metalloproteinase
inhibitors such as ilomastat are contemplated as second therapeutic
agents for use with the siRNA molecules of the present invention.
Such inhibitors include siRNA molecules that target VEGF,
fibroblast growth factors, connective tissue growth factors or the
respective receptors for these growth factors and matrix
metalloproteinases.
EXAMPLES
[0092] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Materials & Methods
Human Corneal Fibroblast Cultures.
[0093] Normal human corneas from donors aged 13, 29, 34, 45, and 47
years were obtained from either the Illinois Eye Bank (Chicago,
Ill.) or the National Disease Research Interchange Philadelphia,
Pa.). The procurement of tissue was approved by the IRB Committee
at the University of Illinois at Chicago in compliance with the
declaration of Helsinki. The endothelial and epithelial layers were
removed from the corneas and the stroma was used as explants to
initiate corneal fibroblast cultures. The cells were maintained in
Dulbecco's modified Eagle's minimum essential medium (MEM)
supplemented with glutamine, 10% fetal calf serum, 5% calf serum,
nonessential and essential amino acids and antibiotics as
previously described in Yue and Blum. (Vision Res. 21, 41-43
(1981)) Third- to fifth-passaged cells were used for the study.
TGF.beta.II Receptor siRNA Sequences.
[0094] Four sequences for the TGF.beta.II receptor siRNA were
derived from the human TGF.beta.II receptor sequence (Genbank
Accession Number: M85079). The siRNAs were custom synthesized and
purified by Dharmacon Research (Lafayette, Colo.). The target
sequences (5' to 3') were as follows, with the position of the
first nucleotide in the human TGF.beta.II receptor sequence shown
in brackets: TABLE-US-00002 NK1: (529) AATCCTGCATGAGCAACTGCA (SEQ
ID NO: 1) NK2: (1113) AAGGCCAAGCTGAAGCAGAAC (SEQ ID NO: 2) SS1:
(1253) AAGCATGAGAACATACTCCAG (SEQ ID NO: 3) SS2: (948)
AAGACGCGGAAGCTCATGGAG (SEQ ID NO: 4)
[0095] RNA of a scrambled sequence was used as a control.
Transfection of siRNA Duplexes
[0096] Normal human corneal fibroblasts were plated at 50-70%
confluence onto Lab-Tek 4- or 8-well chamber slides, coverslips, or
6-well plates the day prior to the transfection. Transfection
complexes were prepared by adding 2 .mu.l of TransIT-TKO reagent
(Takara Mirus Corporation, Madison, Wis.) to 50 .mu.l of serum-free
media, vortexing and incubating the mixture at room temperature for
10 min. To the mixture, anti-TGF.beta.II receptor siRNA duplex (25,
50, 100, or 200 nM final concentration) was added. The solution was
further mixed by gently pipeting and was incubated for another 20
minutes. The final mixture was then added dropwise to the cells in
complete media. After gentle rocking, the cells were incubated at
37.degree. C. for 16, 24, 48, or 72 hours before assaying for gene
expression. As controls, corneal fibroblasts were either untreated
or treated only with the transfection reagent. Non-specific
scrambled siRNA duplex (Dharmacon; 100 and 200 mM) was also used in
place of the TGF.beta.RII specific siRNAs.
Immunofluorescence.
[0097] At selected time points after siRNA transfection, cells in
coverslips or 8-well chamber slides (Nalge Nunc International,
Naperville, Ill.) were fixed with 2% formaldehyde solution and
permeabilized with 0.1% Triton-X100 in PBS. Cells were blocked for
45 minutes at room temperature in 10% heat-inactivated normal goat
serum (Colorado Serum Company, Denver, Colo.), and incubated with a
rabbit anti-TGF.beta.II receptor antibody (1:100, Santa Cruz
Biologicals, Santa Cruz, Calif., SC1700) for 60 min. Following
washes, a goat FITC-anti-rabbit (Southern Biotechnology) at 1:200
was applied for a 60-minutes incubation. The nuclei of the cells
were counterstained with DAPI (4',6'-diamidino-2-phenylindole
dihydrochloride). The slides were examined by epifluorescence under
a Zeiss Axiovert fluorescence microscope (Carl Zeiss, Jena,
Germany).
[0098] For fibronectin staining, cells on Lab-Tek 4-well glass
chamber slides were fixed 48 hours after transfection in ice cold
methanol. Immunofluorescence was performed using a rabbit
anti-human fibronectin (1:100, BD Science, Lexington, Ky.) as the
primary antibody and FITC-conjugated goat anti-rabbit IgG (1:100,
Jackson ImmunoResearch, West Grove, Pa.) as the secondary antibody.
The slides were mounted in Vectashield (Vector Laboratories,
Burlingame, Calif.) with DAPI. The staining was examined under a
Zeiss 100M microscope.
Western Blotting.
[0099] After siRNA transfection, the media were removed and corneal
fibroblasts in 6-well plates were harvested. Cells were lysed in a
Triton buffer, followed by addition of sodium dodecyl sulfate (SDS)
sample buffer. Protein samples were separated on a 10%
SDS-polyacrylamide gel, transferred to nitrocellulose membranes and
blocked with BLOTTO. Subsequently, blot was incubated with rabbit
anti-TGF.beta.II receptor at 1:200 dilution (of course, other
dilutions e.g., 1:2000, and dilutions in between these figures also
could be used) and horseradish peroxidase (HRP)-conjugated goat
anti-rabbit IgG (Jackson ImmunoResearch). Signals were detected by
chemiluminescence.
[0100] For fibronectin study, corneal fibroblasts after
transfection for 48 hours were incubated with serum-free MEM for 24
hours. The media were collected and the cells were lysed on ice in
10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.5% NP40, 2 mM
phenylmethylsulfonyl fluoride, and 1.times. cocktail protease
inhibitors (Roche). Cellular debris was pelleted, and the proteins
in the lysate were quantified by Bradford protein assay. After
adjusting the protein amounts, equal aliquots of media samples were
resolved on 10% SDS-polyacrylamide gels under reducing conditions.
The proteins were electroblotted onto nitrocellulose membranes.
After blocking with 5% nonfat dry milk, the membranes were
incubated with rabbit anti-human fibronectin (1:5000) and HRP-goat
anti-rabbit IgG (1:10,000). Protein bands were detected using
SuperSignal Substrate from Pierce (Rockford, Ill.). Densitometric
analysis was performed to measure the intensity of the fibronectin
bands with the use of 1D Image Analysis software (Kodak Digital
Imaging, Eastman Kodak Company, New Haven, Conn.).
Real Time PCR.
[0101] Total RNA was extracted with Trizol from cells treated for
24, 48, and 72 hours with scrambled, NK1, or SS1 siRNA. Real time
PCR was performed according to methods known to those of skill in
the art.
Cell Migration Assay.
[0102] A wound scratch assay was used to assess cell migration.
Forty eight hours after transfection, corneal fibroblasts in
24-well plates were scratched with a sterile P20 pipette tip as
previously described in Mostafavi-Pour et al., J. Cell Biol. 161:
155-167 (2003). The ability of cells to migrate into the wound was
examined under phase contrast microscopy 7 hours after wounding. To
quantify the extent of migration, total area of the wound in each
10.times. field and the areas devoid of cells within the wound were
measured with the use of the Image Processing Tool Kit version 3.0
(an Adobe Photoshop 7:1 plugin software, Reindeer Graphics, Inc.,
Asheville, N.C.). A total of 10 fields were analyzed and the mean
percentage of areas covered by the migratory cells in each specimen
was calculated. Student's t tests were used for statistical
evaluation. All experiments were repeated at least 3 times.
Mouse Model of Conjunctival Scarring.
[0103] All experiments were performed using 6 week old C57BL6 mice.
Treatment of the animals was conformed to the ARVO Statement for
the Use of Animals in Ophthalmic and Vision Research. Mice
underwent general anesthesia with intraperitoneal injections
(pentobarbital, 0.1 ml/10 g body weight). Surgery was performed as
reported previously with modifications. (Reichel et al. Br. J.
Ophthalmol. 82, 1072-1077 (1998)) A blunt dissection of the
temporal subconjunctival space was performed using 1 ml syringe and
30 gauge needle by injecting of sterile PBS (pH 7.4) containing
latex beads (1.053 .mu.m diameter, 300 .mu.g/ml, Polysciences,
Warrington, Pa.) with transfection reagent mixed with 200 nM NK1,
SS1, or scrambled missense oligonucleotide. One eye of each mouse
was treated with HK1 or SS1, and the contralateral eye was treated
with the scrambled siRNA in a double masked manner. Eyes in other
mice were either left untreated or injected with PBS and latex
beads alone to serve as controls. Mice were sacrificed by cervical
dislocation 2, 7, and 14 days after surgery. For each
treatment/time point, three mice were used.
[0104] Eyes enucleated eyes were fixed at room temperature with 10%
buffered formalin for 24 hours, and were processed for paraffin
sections. Five-.mu.m-thick paraffin-embedded sections were
deparaffinized, rehydrated, and stained with hematoxylin and eosin
(H & E) to assess the inflammatory reaction and picrocirius red
to demonstrate collagen deposition.
Example 2
Suppression of TGF.beta.II Receptor Protein and mRNA Expression
[0105] Human corneal fibroblasts were transfected with all four
exemplary siRNAs designed using the TransIT-TKO reagent. The
cellular uptake of oligonucleotides was demonstrated by
fluorescence microscopy using the Cy3-labeled luciferase. The
transfection seemed to be extremely efficient, with more than 90%
of the cells displaying red fluorescence. Little cytotoxicity of
the transfection reagent or the siRNAs was observed.
[0106] Immunofluorescence analyses showed that TGF.beta.RII was
distributed diffusely in the cytoplasm of untreated control corneal
fibroblasts (FIG. 1, row 1). When treated with 100 nM of SS1 siRNA
for 48 h, the TGF.beta.RII staining intensity was dramatically
reduced (FIG. 1, rows 3 and 4). At 100 nM, NK1, NK2 and SS2 siRNAs
also suppressed the TGF.beta.RII intensity. While not evident at
the lowest concentration (25 nM) and the shortest time point (16 h)
tested, the inhibiting effects, to varying degrees, were also
observed for all four siRNAs tested with other concentrations (50
and 200 nM) and time points (24 and 72 h). Overall, NK1 and SS1
appeared to result in a greater inhibition than the others. Cells
treated with scrambled siRNA (FIG. 1, row 2) showed a similar
intensity and pattern as the untreated control cells, demonstrating
the specificity of NK1 and SS1 effects.
[0107] Western blotting (FIG. 2) yielded a 73-75 kDa band (a
diffuse band as the receptor is a glycoprotein) immunoreactive to
anti-TGF.beta.RII in the vehicle-treated control and scrambled
siRNA-transfected samples. There was no discernible difference in
the TGF.beta.RII protein level at the 16 hours time point except
for the cells treated with SS1 (lane 6) where a reduction was seen.
At 48 h, both NK1 (lanes 9 and 10) and SS1 (lanes 11 and 12) siRNAs
showed a marked decrease in signal intensity for TGF.beta.II
receptor compared to control cells (lane 8). A densitometric
analysis suggests a 70-85% reduction of the TGF.beta.RII in the
siRNA treated immunoblots. NK1 siRNA appeared to be more effective
than SS1 in reducing the TGF.beta.RII expression at this time point
at both 50 and 100 nM siRNA concentrations. When the TGF.beta.II
receptor antibody was preincubated with the antigenic peptide
before probing, the immunoreactive band disappeared (FIG. 2, lane
7). The lack of a signal in this lane demonstrates the specificity
of the antibody. The turnover rate varies with the presence of
ligand binding and with the cell type used. The half life of
TGF.beta.RII receptor varied from 2-6 hours. TGF.beta.BII receptor
transcript was examined by real time PCR and it was seen that the
siRNA compositions significantly changed the level of receptor
mRNA.
Example 3
Reduction of Fibronectin Assembly and Secreted Fibronectin by
siRNAs
[0108] Using immunofluorescence, it was demonstrated that untreated
control corneal fibroblasts exhibited robust fibronectin deposition
and a dense fibrillar network over cells. A similar pattern was
also observed in cells treated with scrambled siRNA. In these
analyses immunofluorescence of untreated fibroblasts or fibroblasts
treated for 48 hours with scrambled siRNA, 100 or 200 nM NK1, or
100 or 200 nM SS1 was performed to visualize fibronectin matrix.
Staining of nuclei was performed using DAPI stain. These studies
showed that fibronectin deposition was markedly reduced in corneal
fibroblasts 48 hours after transfection with both 100 and 200 nM of
NK1 and SS1 siRNAs. The nuclei were counterstained by DAPI. The
cell density was similar in the various specimens and thus the
decreased fibronectin assembly was not related to a decrease in
cell number.
[0109] The effects of the siRNAs on the fibronectin fibrillogenesis
also was examined through observing changes in fibronectin
secretion. Corneal fibroblasts, 48 hours after transfection, were
incubated in serum-free medium for 24 hours. Proteins collected in
the media were subjected to Western blotting. A 220-Kda fibronectin
band was observed in all samples. Consistent with the
immunofluorescence data, treatment with 100 and 200 nM NK1 and SS1
resulted in a decreased level of fibronectin secreted into the
culture media. The two siRNAs were equally effective, eliciting
greater effect with 200 nM than 100 nM.
Example 4
Retardation of Cell Migration by siRNAs
[0110] Wound scratch assays indicated that corneal fibroblasts were
able to move into the wounded area. Within 7 hours, untreated
control and scrambled RNA-transfected cells filled most of the
pipette tip-generated wound, covering 83.0.+-.2.2% and 80.4.+-.2.6%
of the area, respectively. By contrast, the wound area covered by
100 and 200 nM NK1 and SS1 transfected cells was significantly
smaller (P<0.0001) varying from 37 to 57%. The blockage of cell
migration was more dramatic with the higher concentration of
siRNAs. Experiments were repeated 3 times yielding similar
results.
Example 5
Reduction of Inflammatory Response and Fibrosis in a Mouse
Model
[0111] A conjunctival scarring mouse model was generated by
injecting phosphate buffered saline (PBS) and latex beads into
subconjunctival space. Inflammation response, as judged by the
number of inflammatory cells in tissue sections, was more severe on
post-injection day 2 compared to those obtained from eyes injected
with PBS alone. The inflammatory response observed on day 2
subsided on days 4 and 7.
[0112] NK1, SS1, and scrambled siRNAs were introduced into mouse
eyes together with phosphate buffered saline (PBS) and latex beads
in a double masked manner. One eye of each mouse was treated with
NK1 or SS1, and the contralateral eye was treated with the
scrambled RNA. Eyes in other mice were either left untreated or
injected with PBS and latex beads alone to serve as controls. Two
days following the injection, numerous inflammatory cells were
observed underneath the conjunctival epithelium in the scrambled
RNA-treated and PBS/beads-injected control eyes. The inflammatory
cells were less in NK1 and SS1-treated eyes.
[0113] On post-injection days 7 and 14, the number of inflammatory
cells was reduced in all treated or injected eyes. The
subconjunctival space in the scrambled RNA-treated and
PBS/beads-injected control eyes was filled with fibroblasts. The
density of conjunctival fibroblasts was higher than that seen in
eyes treated with NK1 or SS1. Picrocirius red staining to
demonstrate collagen deposition further showed that the fibrotic
response on day 14 was repressed by NK1 and SS1 siRNAs.
Example 6
Inhibition of TGF.beta.RII Using siRNA on Endothelial Cells
[0114] Human umbical vein endothelial cells (HUVEC) were plated at
3.times.10.sup.-5 cells/well and allowed to grow into confluent
monolayers. The following day, the cells were treated with
TransIT-TKO reagent only (negative control), scrambled siRNA
oligonucleotides, NK1 siRNA oligonucleotides and SS1 siRNA
oligonucleotides, all in TransIT-TKO reagent. 200 nM concentrations
of the oligonucleotides was used, however, greater or lesser
concentrations may be used. The cellular uptake of the
oligonucleotides was demonstrated by fluorescence microscopy using
the Cy3-labeled luciferase. Images were taken 48 hours post RNAi
treatment.
[0115] Immunofluorescence analyses showed that TGF.beta.RII was
distributed diffusely in the cytoplasm of untreated control corneal
fibroblasts (FIG. 4a). In the presence of NK1 and SS1 siRNAs, the
TGF.beta.RII staining intensity was dramatically reduced (FIG. 4 c
and d; respectively). These results are consistent with the
experiments carried out in corneal fibroblasts described in Example
2. Cells treated with scrambled siRNA (FIG. 4, b) showed a similar
intensity and pattern as the untreated control cells, demonstrating
the specificity of NK1 and SS1 effects.
Sequence CWU 1
1
161 1 21 DNA Homo sapiens 1 aatcctgcat gagcaactgc a 21 2 21 DNA
Homo sapiens 2 aaggccaagc tgaagcagaa c 21 3 20 DNA Homo sapiens 3
agcatgagaa catactccag 20 4 21 DNA Homo sapiens 4 aagacgcgga
agctcatgga g 21 5 21 DNA Artificial Sequence Synthetic
oligonucleotide 5 uccugcauga gcaacugcat t 21 6 21 DNA Artificial
Sequence Synthetic oligonucleotide 6 ttaggacgua cucguugacg u 21 7
21 DNA Artificial Sequence Synthetic oligonucleotide 7 ggccaagcug
aagcagaact t 21 8 21 DNA Artificial Sequence Synthetic
oligonucleotide 8 ttccgguucg acuucgucuu g 21 9 21 DNA Artificial
Sequence Synthetic oligonucleotide 9 gcaugagaac auacuccagt t 21 10
21 DNA Artificial Sequence Synthetic oligonucleotide 10 ttcguacucu
uguaugaggu c 21 11 21 DNA Artificial Sequence Synthetic
oligonucleotide 11 gacgcggaag cucauggagt t 21 12 21 DNA Artificial
Sequence Synthetic oligonucleotide 12 ttcugcgccu ucgaguaccu c 21 13
21 DNA Homo sapiens 13 aagtcggtta ataacgacat g 21 14 20 DNA
Artificial Sequence Synthetic oligonucleotide 14 gucguuaaua
acgacaugtt 20 15 21 DNA Artificial Sequence Synthetic
oligonucleotide 15 caugucguua uuaaccgact t 21 16 21 DNA Homo
sapiens 16 aacgacatga tagtcactga c 21 17 21 DNA Artificial Sequence
Synthetic oligonucleotide 17 cgacaugaua gucacugact t 21 18 21 DNA
Artificial Sequence Synthetic oligonucleotide 18 gucagugacu
aucaugucgt t 21 19 21 DNA Homo sapiens 19 aacaacggtg cagtcaagtt t
21 20 21 DNA Artificial Sequence Synthetic oligonucleotide 20
caacggugca gucaaguuut t 21 21 21 DNA Artificial Sequence Synthetic
oligonucleotide 21 aaacuugacu gcaccguugt t 21 22 21 DNA Homo
sapiens 22 aacggtgcag tcaagtttcc a 21 23 21 DNA Artificial Sequence
Synthetic oligonucleotide 23 cggugcaguc aaguuuccat t 21 24 21 DNA
Artificial Sequence Synthetic oligonucleotide 24 uggaaacuug
acugcaccgt t 21 25 21 DNA Homo sapiens 25 aagtttccac aactgtgtaa a
21 26 21 DNA Artificial Sequence Synthetic oligonucleotide 26
guuuccacaa cuguguaaat t 21 27 21 DNA Artificial Sequence Synthetic
oligonucleotide 27 uuuacacagu uguggaaact t 21 28 21 DNA Homo
sapiens 28 aaatcctgca tgagcaactg c 21 29 21 DNA Artificial Sequence
Synthetic oligonucleotide 29 auccugcaug agcaacugct t 21 30 21 DNA
Artificial Sequence Synthetic oligonucleotide 30 gcaguugcuc
augcaggaut t 21 31 21 DNA Homo sapiens 31 aagtctgtgt ggctgtatgg a
21 32 21 DNA Artificial Sequence Synthetic oligonucleotide 32
gucugugugg cuguauggat t 21 33 21 DNA Artificial Sequence Synthetic
oligonucleotide 33 uccauacagc cacacagact t 21 34 21 DNA Homo
sapiens 34 aaagaatgac gagaacataa c 21 35 21 DNA Artificial Sequence
Synthetic oligonucleotide 35 agaaugacga gaacauaact t 21 36 21 DNA
Artificial Sequence Synthetic oligonucleotide 36 guuauguucu
cgucauucut t 21 37 21 DNA Homo sapiens 37 aatgacgaga acataacact a
21 38 21 DNA Artificial Sequence Synthetic oligonucleotide 38
ugacgagaac auaacacuat t 21 39 21 DNA Artificial Sequence Synthetic
oligonucleotide 39 uaguguuaug uucucgucat t 21 40 21 DNA Homo
sapiens 40 aacataacac tagagacagt t 21 41 21 DNA Artificial Sequence
Synthetic oligonucleotide 41 cauaacacua gagacaguut t 21 42 21 DNA
Artificial Sequence Synthetic oligonucleotide 42 aacugucucu
aguguuaugt t 21 43 21 DNA Homo sapiens 43 aacactagag acagtttgcc a
21 44 21 DNA Artificial Sequence Synthetic oligonucleotide 44
cacuagagac aguuugccat t 21 45 21 DNA Artificial Sequence Synthetic
oligonucleotide 45 uggcaaacug ucucuacugt t 21 46 21 DNA Homo
sapiens 46 aagatgctgc ttctccaaag t 21 47 21 DNA Artificial Sequence
Synthetic oligonucleotide 47 gaugcugcuu cuccaaagut t 21 48 21 DNA
Artificial Sequence Synthetic oligonucleotide 48 acuuuggaga
agcagcauct t 21 49 21 DNA Homo sapiens 49 aagcctggtg agactttctt c
21 50 21 DNA Artificial Sequence Synthetic oligonucleotide 50
gccuggugag acuuucuuct t 21 51 21 DNA Artificial Sequence Synthetic
oligonucleotide 51 gaagaaaguc ucaccaggct t 21 52 21 DNA Homo
sapiens 52 aatgacaaca tcatcttctc a 21 53 21 DNA Artificial Sequence
Synthetic oligonucleotide 53 ugacaacauc aucuucucat t 21 54 21 DNA
Artificial Sequence Synthetic oligonucleotide 54 ugagaagaug
auguugucat t 21 55 21 DNA Homo sapiens 55 aacatcatct tctcagaaga a
21 56 21 DNA Artificial Sequence Synthetic oligonucleotide 56
caucaucuuc ucagaagaat t 21 57 21 DNA Artificial Sequence Synthetic
oligonucleotide 57 uucuucugag aagaugaugt t 21 58 21 DNA Artificial
Sequence Synthetic oligonucleotide 58 gaauauaaca ccagcaauct t 21 59
21 DNA Artificial Sequence Synthetic oligonucleotide 59 gauugcuggu
guuauauuct t 21 60 21 DNA Homo sapiens 60 aatataacac cagcaatcct g
21 61 21 DNA Artificial Sequence Synthetic oligonucleotide 61
uauaacacca gcaauccugt t 21 62 21 DNA Artificial Sequence Synthetic
oligonucleotide 62 caggauugcu gguguuauat t 21 63 21 DNA Homo
sapiens 63 aacaccagca atcctgactt g 21 64 21 DNA Artificial Sequence
Synthetic oligonucleotide 64 caccagcaau ccugacuugt t 21 65 21 DNA
Artificial Sequence Synthetic oligonucleotide 65 caagucagga
uugcuggugt t 21 66 21 DNA Homo sapiens 66 aatcctgact tgttgctagt c
21 67 21 DNA Artificial Sequence Synthetic oligonucleotide 67
uccugacuug uugcuaguct t 21 68 21 DNA Artificial Sequence Synthetic
oligonucleotide 68 gacuagcaac aagucaggat t 21 69 21 DNA Homo
sapiens 69 aagctgagtt caacctggga a 21 70 21 DNA Artificial Sequence
Synthetic oligonucleotide 70 gcugaguuca accugggaat t 21 71 21 DNA
Artificial Sequence Synthetic oligonucleotide 71 uucccagguu
gaacucagct t 21 72 21 DNA Homo sapiens 72 aagatcaccg ctctgacatc a
21 73 21 DNA Artificial Sequence Synthetic oligonucleotide 73
gaugaccgcu cugacaucat t 21 74 21 DNA Artificial Sequence Synthetic
oligonucleotide 74 ugaugucaga gcggucauct t 21 75 21 DNA Homo
sapiens 75 aacaacatca accacaacac a 21 76 21 DNA Artificial Sequence
Synthetic oligonucleotide 76 caacaucaac cacaacacat t 21 77 21 DNA
Artificial Sequence Synthetic oligonucleotide 77 uguguugugg
uugauguugt t 21 78 21 DNA Homo sapiens 78 aacatcaacc acaacacaga g
21 79 21 DNA Artificial Sequence Synthetic oligonucleotide 79
caucaaccac aacacagagt t 21 80 21 DNA Artificial Sequence Synthetic
oligonucleotide 80 cucuguguug ugguugaugt t 21 81 21 DNA Homo
sapiens 81 aagctgaagc agaacacttc a 21 82 21 DNA Artificial Sequence
Synthetic oligonucleotide 82 ugaaguguuc ugcuucagct t 21 83 21 DNA
Homo sapiens 83 aagcagaaca cttcagagca g 21 84 21 DNA Artificial
Sequence Synthetic oligonucleotide 84 gcagaacacu ucagagcagt t 21 85
21 DNA Artificial Sequence Synthetic oligonucleotide 85 cugcucugaa
guguucugct t 21 86 21 DNA Homo sapiens 86 aacacttcag agcagtttga g
21 87 21 DNA Artificial Sequence Synthetic oligonucleotide 87
cacuucagag cacuuugagt t 21 88 21 DNA Artificial Sequence Synthetic
oligonucleotide 88 cucaaacugc ucugaagugt t 21 89 21 DNA Homo
sapiens 89 aagatctttc cctatgagga g 21 90 21 DNA Artificial Sequence
Synthetic oligonucleotide 90 gaucuuuccc uaugaggagt t 21 91 21 DNA
Artificial Sequence Synthetic oligonucleotide 91 cuccucauag
ggaaagauct t 21 92 21 DNA Homo sapiens 92 aagacagaga aggacatctt c
21 93 21 DNA Artificial Sequence Synthetic oligonucleotide 93
gacagagaag gacaucuuct t 21 94 21 DNA Artificial Sequence Synthetic
oligonucleotide 94 gaagaugucc uucucuguct t 21 95 21 DNA Homo
sapiens 95 aaggacatct tctcagacat c 21 96 21 DNA Artificial Sequence
Synthetic oligonucleotide 96 ggacaucuuc ucagacauct t 21 97 21 DNA
Artificial Sequence Synthetic oligonucleotide 97 gaugucugag
aagaugucct t 21 98 21 DNA Homo sapiens 98 attctgaagc atgagaacat a
21 99 21 DNA Artificial Sequence Synthetic oligonucleotide 99
ucugaagcau gagaacauat t 21 100 21 DNA Artificial Sequence Synthetic
oligonucleotide 100 uauguucuca ugcuucagat t 21 101 21 DNA
Artificial Sequence Synthetic oligonucleotide 101 gcaugagaac
auacuccagt t 21 102 21 DNA Artificial Sequence Synthetic
oligonucleotide 102 cuggaguaug uucucaugct t 21 103 21 DNA Homo
sapiens 103 aacatactcc agttcctgac g 21 104 21 DNA Artificial
Sequence Synthetic oligonucleotide 104 cauacuccag uuccugacgt t 21
105 21 DNA Artificial Sequence Synthetic oligonucleotide 105
cgucaggaac uggaguaugt t 21 106 21 DNA Homo sapiens 106 aagacggagt
tggggaaaca a 21 107 21 DNA Artificial Sequence Synthetic
oligonucleotide 107 gacggaguug gggaaacaat t 21 108 21 DNA
Artificial Sequence Synthetic oligonucleotide 108 uuguuucccc
aacuccguct t 21 109 21 DNA Homo sapiens 109 aaacaatact ggctgatcac c
21 110 21 DNA Artificial Sequence Synthetic oligonucleotide 110
acaauacugg cugaucacct t 21 111 21 DNA Artificial Sequence Synthetic
oligonucleotide 111 ggugaucagc caguauugut t 21 112 21 DNA Homo
sapiens 112 aagagctcca atatcctcgt g 21 113 21 DNA Artificial
Sequence Synthetic oligonucleotide 113 gagcuccaau auccucgugt t 21
114 21 DNA Artificial Sequence Synthetic oligonucleotide 114
cacgaggaua uuggagcuct t 21 115 21 DNA Homo sapiens 115 aatatcctcg
tgaagaacga c 21 116 21 DNA Artificial Sequence Synthetic
oligonucleotide 116 uauccucgug aagaacgact t 21 117 21 DNA
Artificial Sequence Synthetic oligonucleotide 117 gucguucuuc
acgaggauat t 21 118 21 DNA Homo sapiens 118 aactgcaaga tacatggctc c
21 119 21 DNA Artificial Sequence Synthetic oligonucleotide 119
cugcaagaua cauggcucct t 21 120 21 DNA Artificial Sequence Synthetic
oligonucleotide 120 ggagccaugu aucuugcagt t 21 121 21 DNA Homo
sapiens 121 aagatacatg gctccagaag t 21 122 21 DNA Artificial
Sequence Synthetic oligonucleotide 122 gauacauggc uccagaagut t 21
123 21 DNA Artificial Sequence Synthetic oligonucleotide 123
acuucuggag ccauguauct t 21 124 21 DNA Homo sapiens 124 aagtcctaga
ttccaggatg a 21 125 21 DNA Artificial Sequence Synthetic
oligonucleotide 125 guccuagaau ccaggaugat t 21 126 21 DNA
Artificial Sequence Synthetic oligonucleotide 126 ucauccugga
uucuaggact t 21 127 21 DNA Homo sapiens 127 aatccaggat gaatttggag a
21 128 21 DNA Artificial Sequence Synthetic oligonucleotide 128
uccaggauga auuuggagat t 21 129 21 DNA Artificial Sequence Synthetic
oligonucleotide 129 ucuccaaauu cauccuggat t 21 130 21 DNA Homo
sapiens 130 aatttggaga atgctgagtc c 21 131 21 DNA Artificial
Sequence Synthetic oligonucleotide 131 uuuggagaau gcugagucct t 21
132 21 DNA Artificial Sequence Synthetic oligonucleotide 132
ggacucagca uucuccaaat t 21 133 21 DNA Homo sapiens 133 aatgctgagt
ccttcaagca g 21 134 21 DNA Artificial Sequence Synthetic
oligonucleotide 134 ugcugagucc uucaagcagt t 21 135 21 DNA
Artificial Sequence Synthetic oligonucleotide 135 cugcuugaag
gacucagcat t 21 136 21 DNA Homo sapiens 136 aaatgacatc tcgctgtaat g
21 137 21 DNA Artificial Sequence Synthetic oligonucleotide 137
augacaucuc gcuguaaugt t 21 138 21 DNA Artificial Sequence Synthetic
oligonucleotide 138 cauuacagcg agaugucaut t 21 139 21 DNA Homo
sapiens 139 aatgcagtgg gagaagtaaa a 21 140 21 DNA Artificial
Sequence Synthetic oligonucleotide 140 ugcaguggga gaaguaaaat t 21
141 21 DNA Artificial Sequence Synthetic oligonucleotide 141
uuuuacuucu cccacugcat t 21 142 21 DNA Homo sapiens 142 aagattatga
gcctccattt g 21 143 21 DNA Artificial Sequence Synthetic
oligonucleotide 143 gauuaugagc cuccauuugt t 21 144 21 DNA
Artificial Sequence Synthetic oligonucleotide 144 caaauggagg
cucauaauct t 21 145 21 DNA Homo sapiens 145 aaagcatgaa ggacaacgtg t
21 146 21 DNA Artificial Sequence Synthetic oligonucleotide 146
agcaugaagg acaacgugut t 21 147 21 DNA Artificial Sequence Synthetic
oligonucleotide 147 acacguuguc cuucaugcut t 21 148 21 DNA Homo
sapiens 148 aaggacaacg tgttgagaga t 21 149 21 DNA Artificial
Sequence Synthetic oligonucleotide 149 ggacaacgug uugagagaut t 21
150 21 DNA Artificial Sequence Synthetic oligonucleotide 150
aucucucaac acguugucct t 21 151 21 DNA Homo sapiens 151 aaattcccag
cttctggctc a 21 152 21 DNA Artificial Sequence Synthetic
oligonucleotide 152 auucccagcu ucuggcucat t 21 153 21 DNA
Artificial Sequence Synthetic oligonucleotide 153 ugagccagaa
gcugggaaut t 21 154 21 DNA Homo sapiens 154 aagacggctc cctaaacact a
21 155 21 DNA Artificial Sequence Synthetic oligonucleotide 155
gacggcuccc uaaacacuat t 21 156 21 DNA Artificial Sequence Synthetic
oligonucleotide 156 uaguguuuag ggagccguct t 21 157 21 DNA Homo
sapiens 157 aagaatataa caccagcaat c 21 158 21 DNA Homo sapiens 158
aagcatgaga acatactcca g 21 159 2090 DNA Homo sapiens CDS
(336)..(2039) 159 gttggcgagg agtttcctgt ttcccccgca gcgctgagtt
gaagttgagt gagtcactcg 60 cgcgcacgga gcgacgacac ccccgcgcgt
gcacccgctc gggacaggag ccggactcct 120 gtgcagcttc cctcggccgc
cgggggcctc cccgcgcctc gccggcctcc aggcccctcc 180 tggctggcga
gcgggcgcca catctggccc gcacatctgc gctgccggcc cggcgcgggg 240
tccggagagg gcgcggcgcg gagcgcagcc aggggtccgg gaaggcgccg tccgtgcgct
300 gggggctcgg tctatgacga gcagcggggt ctgcc atg ggt cgg ggg ctg ctc
353 Met Gly Arg Gly Leu Leu 1 5 agg ggc ctg tgg ccg ctg cac atc gtc
ctg tgg acg cgt atc gcc agc 401 Arg Gly Leu Trp Pro Leu His Ile Val
Leu Trp Thr Arg Ile Ala Ser 10 15 20 acg atc cca ccg cac gtt cag
aag tcg gtt aat aac gac atg ata gtc 449 Thr Ile Pro Pro His Val Gln
Lys Ser Val Asn Asn Asp Met Ile Val 25 30 35 act gac aac aac ggt
gca gtc aag ttt cca caa ctg tgt aaa ttt tgt 497 Thr Asp Asn Asn Gly
Ala Val Lys Phe Pro Gln Leu Cys Lys Phe Cys 40 45 50 gat gtg aga
ttt tcc acc tgt gac aac cag aaa tcc tgc atg agc aac 545 Asp Val Arg
Phe Ser Thr Cys Asp Asn Gln Lys Ser Cys Met Ser Asn 55 60 65 70 tgc
agc atc acc tcc atc tgt gag aag cca cag gaa gtc tgt gtg gct 593 Cys
Ser Ile Thr Ser Ile Cys Glu Lys Pro Gln Glu Val Cys Val Ala 75 80
85 gta tgg aga aag aat gac gag aac ata aca cta gag aca gtt tgc cat
641 Val Trp Arg Lys Asn Asp Glu Asn Ile Thr Leu Glu Thr Val Cys His
90 95 100 gac ccc aag ctc ccc tac cat gac ttt att ctg gaa gat gct
gct tct 689 Asp Pro Lys Leu Pro Tyr His Asp Phe Ile Leu Glu Asp Ala
Ala Ser 105 110 115 cca aag tgc att atg aag gaa aaa aaa aag cct ggt
gag act ttc ttc 737 Pro Lys Cys Ile Met Lys Glu Lys Lys Lys Pro Gly
Glu Thr Phe Phe 120 125 130 atg tgt tcc tgt agc tct gat gag tgc aat
gac aac atc atc ttc tca 785 Met Cys Ser Cys Ser Ser Asp Glu Cys Asn
Asp Asn Ile Ile Phe Ser 135 140 145 150 gaa gaa tat aac acc agc aat
cct gac ttg ttg cta gtc ata ttt caa 833 Glu Glu Tyr Asn Thr Ser Asn
Pro Asp Leu Leu Leu Val Ile Phe Gln 155 160 165 gtg aca ggc atc agc
ctc ctg cca cca ctg gga gtt gcc ata tct gtc 881 Val Thr Gly Ile Ser
Leu Leu Pro Pro Leu Gly Val Ala Ile Ser Val 170 175 180 atc atc atc
ttc tac tgc tac cgc gtt aac cgg cag cag aag ctg agt 929 Ile Ile Ile
Phe Tyr Cys Tyr Arg Val Asn Arg Gln Gln Lys Leu Ser 185 190 195 tca
acc tgg gaa acc ggc aag acg cgg aag ctc atg gag ttc agc gag 977 Ser
Thr Trp Glu Thr Gly Lys Thr Arg Lys Leu Met Glu Phe Ser Glu 200 205
210 cac tgt gcc atc atc ctg gaa gat gac cgc tct gac atc agc tcc acg
1025 His Cys Ala Ile Ile Leu Glu Asp Asp Arg Ser Asp Ile Ser Ser
Thr 215 220 225 230 tgt gcc aac aac atc aac cac aac aca gag ctg ctg
ccc att gag ctg 1073 Cys Ala Asn Asn Ile Asn His Asn Thr Glu Leu
Leu Pro Ile Glu Leu 235 240 245 gac acc ctg gtg ggg aaa ggt cgc ttt
gct gag gtc tat aag gcc aag 1121 Asp Thr Leu Val Gly Lys Gly Arg
Phe Ala Glu Val Tyr Lys Ala Lys 250 255 260 ctg aag cag aac act tca
gag cag ttt gag aca gtg gca gtc aag atc 1169 Leu Lys Gln Asn Thr
Ser Glu Gln Phe Glu Thr Val Ala Val Lys Ile 265 270 275 ttt ccc tat
gag gag tat gcc tct tgg aag aca gag aag gac atc ttc 1217 Phe Pro
Tyr Glu Glu Tyr Ala Ser Trp Lys Thr Glu Lys Asp Ile Phe 280 285 290
tca gac atc aat ctg aag cat gag aac ata ctc cag ttc ctg acg gct
1265 Ser Asp Ile Asn Leu Lys His Glu Asn Ile Leu Gln Phe Leu Thr
Ala 295 300 305 310 gag gag cgg aag acg gag ttg ggg aaa caa tac tgg
ctg atc acc gcc 1313 Glu Glu Arg Lys Thr Glu Leu Gly Lys Gln Tyr
Trp Leu Ile Thr Ala 315 320 325 ttc cac gcc aag ggc aac cta cag gag
tac ctg acg cgg cat gtc atc 1361 Phe His Ala Lys Gly Asn Leu Gln
Glu Tyr Leu Thr Arg His Val Ile 330 335 340 agc tgg gag gac ctg cgc
aag ctg ggc agc tcc ctc gcc cgg ggg att 1409 Ser Trp Glu Asp Leu
Arg Lys Leu Gly Ser Ser Leu Ala Arg Gly Ile 345 350 355 gct cac ctc
cac agt gat cac act cca tgt ggg agg ccc aag atg ccc 1457 Ala His
Leu His Ser Asp His Thr Pro Cys Gly Arg Pro Lys Met Pro 360 365 370
atc gtg cac agg gac ctc aag agc tcc aat atc ctc gtg aag aac gac
1505 Ile Val His Arg Asp Leu Lys Ser Ser Asn Ile Leu Val Lys Asn
Asp 375 380 385 390 cta acc tgc tgc ctg tgt gac ttt ggg ctt tcc ctg
cgt ctg gac cct 1553 Leu Thr Cys Cys Leu Cys Asp Phe Gly Leu Ser
Leu Arg Leu Asp Pro 395 400 405 act ctg tct gtg gat gac ctg gct aac
agt ggg cag gtg gga act gca 1601 Thr Leu Ser Val Asp Asp Leu Ala
Asn Ser Gly Gln Val Gly Thr Ala 410 415 420 aga tac atg gct cca gaa
gtc cta gaa tcc agg atg aat ttg gag aat 1649 Arg Tyr Met Ala Pro
Glu Val Leu Glu Ser Arg Met Asn Leu Glu Asn 425 430 435 gct gag tcc
ttc aag cag acc gat gtc tac tcc atg gct ctg gtg ctc 1697 Ala Glu
Ser Phe Lys Gln Thr Asp Val Tyr Ser Met Ala Leu Val Leu 440 445 450
tgg gaa atg aca tct cgc tgt aat gca gtg gga gaa gta aaa gat tat
1745 Trp Glu Met Thr Ser Arg Cys Asn Ala Val Gly Glu Val Lys Asp
Tyr 455 460 465 470 gag cct cca ttt ggt tcc aag gtg cgg gag cac ccc
tgt gtc gaa agc 1793 Glu Pro Pro Phe Gly Ser Lys Val Arg Glu His
Pro Cys Val Glu Ser 475 480 485 atg aag gac aac gtg ttg aga gat cga
ggg cga cca gaa att ccc agc 1841 Met Lys Asp Asn Val Leu Arg Asp
Arg Gly Arg Pro Glu Ile Pro Ser 490 495 500 ttc tgg ctc aac cac cag
ggc atc cag atg gtg tgt gag acg ttg act 1889 Phe Trp Leu Asn His
Gln Gly Ile Gln Met Val Cys Glu Thr Leu Thr 505 510 515 gag tgc tgg
gac cac gac cca gag gcc cgt ctc aca gcc cag tgt gtg 1937 Glu Cys
Trp Asp His Asp Pro Glu Ala Arg Leu Thr Ala Gln Cys Val 520 525 530
gca gaa cgc ttc agt gag ctg gag cat ctg gac agg ctc tcg ggg agg
1985 Ala Glu Arg Phe Ser Glu Leu Glu His Leu Asp Arg Leu Ser Gly
Arg 535 540 545 550 agc tgc tcg gag gag aag att cct gaa gac ggc tcc
cta aac act acc 2033 Ser Cys Ser Glu Glu Lys Ile Pro Glu Asp Gly
Ser Leu Asn Thr Thr 555 560 565 aaa tag ctcttatggg gcaggctggg
catgtccaaa gaggctgccc ctctcaccaa a 2090 Lys 160 567 PRT Homo
sapiens 160 Met Gly Arg Gly Leu Leu Arg Gly Leu Trp Pro Leu His Ile
Val Leu 1 5 10 15 Trp Thr Arg Ile Ala Ser Thr Ile Pro Pro His Val
Gln Lys Ser Val 20 25 30 Asn Asn Asp Met Ile Val Thr Asp Asn Asn
Gly Ala Val Lys Phe Pro 35 40 45 Gln Leu Cys Lys Phe Cys Asp Val
Arg Phe Ser Thr Cys Asp Asn Gln 50 55 60 Lys Ser Cys Met Ser Asn
Cys Ser Ile Thr Ser Ile Cys Glu Lys Pro 65 70 75 80 Gln Glu Val Cys
Val Ala Val Trp Arg Lys Asn Asp Glu Asn Ile Thr 85 90 95 Leu Glu
Thr Val Cys His Asp Pro Lys Leu Pro Tyr His Asp Phe Ile 100 105 110
Leu Glu Asp Ala Ala Ser Pro Lys Cys Ile Met Lys Glu Lys Lys Lys 115
120 125 Pro Gly Glu Thr Phe Phe Met Cys Ser Cys Ser Ser Asp Glu Cys
Asn 130 135 140 Asp Asn Ile Ile Phe Ser Glu Glu Tyr Asn Thr Ser Asn
Pro Asp Leu 145 150 155 160 Leu Leu Val Ile Phe Gln Val Thr Gly Ile
Ser Leu Leu Pro Pro Leu 165 170 175 Gly Val Ala Ile Ser Val Ile Ile
Ile Phe Tyr Cys Tyr Arg Val Asn 180 185 190 Arg Gln Gln Lys Leu Ser
Ser Thr Trp Glu Thr Gly Lys Thr Arg Lys 195 200 205 Leu Met Glu Phe
Ser Glu His Cys Ala Ile Ile Leu Glu Asp Asp Arg 210 215 220 Ser Asp
Ile Ser Ser Thr Cys Ala Asn Asn Ile Asn His Asn Thr Glu 225 230 235
240 Leu Leu Pro Ile Glu Leu Asp Thr Leu Val Gly Lys Gly Arg Phe Ala
245 250 255 Glu Val Tyr Lys Ala Lys Leu Lys Gln Asn Thr Ser Glu Gln
Phe Glu 260 265 270 Thr Val Ala Val Lys Ile Phe Pro Tyr Glu Glu Tyr
Ala Ser Trp Lys 275 280 285 Thr Glu Lys Asp Ile Phe Ser Asp Ile Asn
Leu Lys His Glu Asn Ile 290 295 300 Leu Gln Phe Leu Thr Ala Glu Glu
Arg Lys Thr Glu Leu Gly Lys Gln 305 310 315 320 Tyr Trp Leu Ile Thr
Ala Phe His Ala Lys Gly Asn Leu Gln Glu Tyr 325 330 335 Leu Thr Arg
His Val Ile Ser Trp Glu Asp Leu Arg Lys Leu Gly Ser 340 345 350 Ser
Leu Ala Arg Gly Ile Ala His Leu His Ser Asp His Thr Pro Cys 355 360
365 Gly Arg Pro Lys Met Pro Ile Val His Arg Asp Leu Lys Ser Ser Asn
370 375 380 Ile Leu Val Lys Asn Asp Leu Thr Cys Cys Leu Cys Asp Phe
Gly Leu 385 390 395 400 Ser Leu Arg Leu Asp Pro Thr Leu Ser Val Asp
Asp Leu Ala Asn Ser 405 410 415 Gly Gln Val Gly Thr Ala Arg Tyr Met
Ala Pro Glu Val Leu Glu Ser 420 425 430 Arg Met Asn Leu Glu Asn Ala
Glu Ser Phe Lys Gln Thr Asp Val Tyr 435 440 445 Ser Met Ala Leu Val
Leu Trp Glu Met Thr Ser Arg Cys Asn Ala Val 450 455 460 Gly Glu Val
Lys Asp Tyr Glu Pro Pro Phe Gly Ser Lys Val Arg Glu 465 470 475 480
His Pro Cys Val Glu Ser Met Lys Asp Asn Val Leu Arg Asp Arg Gly 485
490 495 Arg Pro Glu Ile Pro Ser Phe Trp Leu Asn His Gln Gly Ile Gln
Met 500 505 510 Val Cys Glu Thr Leu Thr Glu Cys Trp Asp His Asp Pro
Glu Ala Arg 515 520 525 Leu Thr Ala Gln Cys Val Ala Glu Arg Phe Ser
Glu Leu Glu His Leu 530 535 540 Asp Arg Leu Ser Gly Arg Ser Cys Ser
Glu Glu Lys Ile Pro Glu Asp 545 550 555 560 Gly Ser Leu Asn Thr Thr
Lys 565 161 21 DNA Artificial Sequence Synthetic oligonucleotide
161 gcugaagcag aacacuucat t 21
* * * * *