U.S. patent application number 09/875223 was filed with the patent office on 2001-12-06 for methods and compositions for inhibiting angiogenesis.
Invention is credited to Bouck, Noel P., Dawson, David W., Gillis, Paul R..
Application Number | 20010049355 09/875223 |
Document ID | / |
Family ID | 25410758 |
Filed Date | 2001-12-06 |
United States Patent
Application |
20010049355 |
Kind Code |
A1 |
Bouck, Noel P. ; et
al. |
December 6, 2001 |
Methods and compositions for inhibiting angiogenesis
Abstract
The present invention provides a method of inhibiting
angiogenesis within a tissue by providing exogenous SLED to cells
associated with the tissue. The presence of exogenous SLED inhibits
angiogenesis within the tissue, in part by interfering with the
ability of vascular endothelia to expand within the tissue. The
invention also provides a method for determining the severity of a
tumor be assaying for the presence of SLED within the tumor. To
facilitate the inventive methods, the present invention provides
pharmaceutical compositions including sources of SLED.
Inventors: |
Bouck, Noel P.; (Oak Park,
IL) ; Dawson, David W.; (Chicago, IL) ;
Gillis, Paul R.; (Chicago, IL) |
Correspondence
Address: |
KATHRYN DOYLE, PH.D., J.D.
MORGAN, LEWIS & BOCKIUS, L.L.P.
1701 Market Street
Philadelphia
PA
19103-2921
US
|
Family ID: |
25410758 |
Appl. No.: |
09/875223 |
Filed: |
June 6, 2001 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
09875223 |
Jun 6, 2001 |
|
|
|
09122079 |
Jul 23, 1998 |
|
|
|
6288024 |
|
|
|
|
09122079 |
Jul 23, 1998 |
|
|
|
08899304 |
Jul 23, 1997 |
|
|
|
Current U.S.
Class: |
514/20.7 |
Current CPC
Class: |
C07K 14/811 20130101;
A61K 38/57 20130101; A61P 35/00 20180101; A61P 17/14 20180101 |
Class at
Publication: |
514/2 |
International
Class: |
A61K 038/16 |
Goverment Interests
[0001] This invention was made with Government support under Grant
Number CA52750 and CA64239 awarded by the National Institutes of
Health. The United States Government may have certain rights in
this invention.
Claims
What is claimed is:
1. A method of inhibiting angiogenesis within a tissue, said method
comprising providing exogenous SLED to endothelial cells associated
with said tissue under conditions sufficient for said SLED to
inhibit angiogenesis within said tissue.
2. The method of claim 1, wherein said tissue is eye tissue.
3. The method of claim 1, wherein said tissue is skin tissue.
4. The method of claim 1, wherein said tissue is a tumor.
5. The method of claim 1, wherein said tissue is within a
joint.
6. The method of claim 1, wherein said tissue is ovarian or
endometrial tissue.
7. The method of claim 1, which further comprises supplying another
antiangiogenic factor to said cells in conjunction with SLED.
8. The method of claim 1, wherein said SLED is provided to said
cells by exposing a composition comprising SLED polypeptide to said
cells.
9. The method of claim 1, wherein said SLED is provided to said
cells by transferring to said cells a vector, said vector
comprising a SLED expression cassette, whereby said cells produce
and secrete SLED polypeptide.
10. The method of claim 1, wherein said SLED comprises SEQ ID
NO:1.
11. The method of claim 1, wherein said SLED comprises an active
fragment of SEQ ID NO:1.
12. The method of claim 1, wherein said SLED is supplied to said
cells via the systemic circulation.
13. The method of claim 1, wherein said SLED is supplied to said
cells topically.
14. A method of inhibiting endothelial cell migration, said method
comprising providing exogenous SLED to said cells under conditions
sufficient for said SLED to inhibit endothelial cell migration.
15. A method for stimulating the growth of hair in a mammal, said
method comprising providing exogenous SLED to cells associated with
the skin of said mammal under conditions sufficient for said SLED
to stimulate the growth of hair in said mammal.
16. A method for inhibiting the growth of a tumor, said method
comprising providing exogenous SLED to endothelial cells associated
with said tumor under conditions sufficient for said SLED to
inhibit the migration of said endothelial cells within and to said
tumor such that the growth of said tumor is inhibited.
17. The method of claim 16, which further comprises supplying
another antiangiogenic factor to said cells in conjunction with
SLED.
18. The method of claim 16, wherein said SLED is provided to said
cells by exposing a composition comprising SLED polypeptide to said
cells.
19. The method of claim 16, wherein said SLED is provided to said
cells by transferring to said cells a vector, said vector
comprising an SLED expression cassette, whereby said cells produce
and secrete SLED polypeptide.
20. The method of claim 16, wherein said SLED is supplied to said
cells via the systemic circulation.
21. The method of claim 16, wherein said SLED is supplied to said
cells topically.
22. A pharmacological composition comprising a source of SLED and a
suitable diluent.
23. The pharmacological composition of claim 17, wherein said
source of SLED is SLED polypeptide.
24. The pharmacological composition of claim 17, wherein said
source of SLED is gene transfer vector including a SLED expression
cassette.
25. A method for determining the severity of a tumor by assaying
for the presence of SLED within the tumor, wherein the absence of
SLED within the tumor indicates an advanced state and the presence
of SLED within the tumor indicates an early state of said tumor.
Description
TECHNICAL FIELD OF THE INVENTION
[0002] The present invention relates to methods and compositions
for inhibiting angiogenesis.
BACKGROUND OF THE INVENTION
[0003] Angiogenesis is the fundamental process by which new blood
vessels are formed. The process involves the migration of vascular
endothelial cells into tissue, followed by the condensation of such
endothelial cells into vessels. Angiogenesis may be induced by an
angiogenic agent or be the result of a natural condition. The
process is essential to a variety of normal body activities, such
as reproduction, development and wound repair. Although the process
is not completely understood, it involves a complex interplay of
molecules that stimulate and molecules that inhibit the growth and
migration of endothelial cells, the primary cells of the capillary
blood vessels. Under normal conditions, these molecules appear to
maintain the microvasculature in a quiescent state (i.e., without
capillary growth) for prolonged periods which can last for several
years or even decades. The turnover time for an endothelial cell is
about 1,000 days. Under appropriate conditions, however (e.g.,
during wound repair), these same cells can undergo rapid
proliferation and turnover within a much shorter period, and five
days is typical under these circumstances. (Folkman and Shing, J.
Biol. Chem., 267(16), 10931-34 (1989); Folkman and Klagsbrun,
Science, 235, 442-47 (1987)).
[0004] Although angiogenesis is a highly regulated process under
normal conditions, many diseases (characterized as "angiogenic
diseases") are driven by persistent unregulated angiogenesis. In
such disease state, unregulated angiogenesis can either cause a
particular disease directly or exacerbate an existing pathological
condition. For example, ocular neovascularization has been
implicated as the most common cause of blindness and underlies the
pathology of approximately 20 eye diseases. In certain previously
existing conditions such as arthritis, newly formed capillary blood
vessels invade the joints and destroy cartilage. In diabetes, new
capillaries formed in the retina invade the vitreous humor, causing
bleeding and blindness.
[0005] Both the growth and metastasis of solid tumors are also
angiogenesis-dependent (Folkman, J. Cancer Res., 46, 467-73 (1986);
Folkman, J. Nat. Cancer Inst., 82, 4-6 (1989); Folkman et al.,
"Tumor Angiogenesis," Chapter 10, pp. 206-32, in The Molecular
Basis of Cancer, Mendelsohn et al., eds. (W. B. Saunders, 1995)).
It has been shown, for example, that tumors which enlarge to
greater than 2 mm. in diameter must obtain their own blood supply
and do so by inducing the growth of new capillary blood vessels.
After these new blood vessels become embedded in the tumor, they
provide nutrients and growth factors essential for tumor growth as
well as a means for tumor cells to enter the circulation and
metastasize to distant sites, such as liver, lung or bone (Weidner,
New Eng. J. Med., 324(1), 1-8 (1991)). When used as drugs in
tumor-bearing animals, natural inhibitors of angiogenesis can
prevent the growth of small tumors (O'Reilly et al., O'Reilly et
al., Cell, 79, 315-28 (1994)). Indeed, in some protocols, the
application of such inhibitors leads to tumor regression and
dormancy even after cessation of treatment (O'Reilly et al., Cell,
88, 277-85 (1997)). Moreover, supplying inhibitors of angiogenesis
to certain tumors can potentiate their response to other
therapeutic regimens (e.g., chemotherapy) (see, e.g., Teischer et
al., Int. J. Cancer, 57, 920-25 (1994)).
[0006] Although several angiogenesis inhibitors are currently under
development for use in treating angiogenic diseases (Gasparini,
Eur. J. Cancer, 32A(14), 2379-85 (1996)), there are disadvantages
associated with several of these proposed inhibitory compounds. For
example, suramin is a potent angiogenesis inhibitor, but, at doses
required to reach antitumor activity, causes severe systemic
toxicity in humans. Other compounds, such as retinoids, interferons
and antiestrogens appear safe for human use but have only a weak
anti-angiogenic effect. Still other compounds may be difficult or
costly to make. In view of these problems, there exists a need for
methods and compositions for inhibiting angiogenesis.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention provides a method of inhibiting
angiogenesis within a tissue by providing exogenous SLED (an
antiangiogenic protein) to endothelial cells associated with the
tissue. The presence of exogenous SLED will inhibit angiogenesis
within the tissue, in part by interfering with the ability of
vascular endothelia to expand within the tissue. The invention also
provides a method for determining the prognosis of a tumor by
assaying for the presence of SLED within the tumor. To facilitate
the inventive method, the present invention provides pharmaceutical
compositions including sources of SLED.
[0008] The methods and compositions of the present invention are
clinically useful for treating a host of diseases associated with
angiogenesis, and for interfering with angiogenesis associated with
reproductive functions. The methods and compositions are also
diagnostically useful for assessing the prognosis of tumors and
other disorders associated with angiogenesis. Furthermore, the
methods and compositions are useful reagents for investigation of
angiogenesis in the laboratory setting. These and other advantages
of the present invention, as well as additional inventive features,
will be apparent from the accompanying drawings and in the
following detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 graphically illustrates the ability of SLED to
inhibit the migration of endothelial cells.
[0010] FIGS. 2A-2D demonstrates the specificity of SLED for
vascular endothelia by graphically representing the inability of
various doses of SLED to inhibit the migration of cells other than
vascular endothelial cells. FIG. 2A depicts data concerning WI-38
cells. FIG. 2B depicts data concerning human foreskin fibroblasts.
FIG. 2C depicts data concerning vascular smooth muscle cells. FIG.
2D depicts data concerning neutrophils.
[0011] FIG. 3 is a dose-response curve representing the
antiangiogenic activity of one type of SLED polypeptide (full
length PEDF).
[0012] FIG. 4 is a dose response curve representing the
antiangiogenic activity of one type of SLED polypeptide (a fragment
of PEDF).
DETAILED DESCRIPTION OF THE INVENTION
[0013] Within the context of the inventive method, SLED is a
protein having potent antiangiogenic properties, and it includes
any antiangiogenic derivative of pigment epithelium derived factor
(PEDF, Steele et al., Proc. Nat. Acad. Sci. (USA), 90(4), 1526-30
(1993)), such as those described herein. One form of SLED
polypeptide (Full length PEDF) is set forth at SEQ ID NO:1;
however, the invention is not limited to the use of this exemplary
sequence. Indeed, other PEDF sequences are known in the art (see,
e.g., published international patent applications WO 95/33480 and
WO 93/24529), and genetic sequences can vary between different
species and individuals, and this natural scope of allelic
variation is included within the scope of the invention.
Additionally and alternatively, a SLED polypeptide can include one
or more point mutations from the exemplary sequence or another
naturally occurring SLED polypeptide. Thus, a SLED polypeptide is
typically at least about 75% homologous to all or a portion of SEQ
ID NO:1 and preferably is at least about 80% homologous to all or a
portion of SEQ ID NO:1 (e.g., at least about 85% homologous to SEQ
ID NO:1); more preferably the SLED polypeptide is at least about
90% homologous to all or a portion of SEQ ID NO:1 (such as at least
about 95% homologous to all or a portion of SEQ ID NO:1), and most
preferably the SLED polypeptide is at least about 97% homologous to
all or a portion of SEQ ID NO:1. Indeed, the SLED polypeptide can
also include other domains, such as epitope tags and His tags
(e.g., the protein can be a fusion protein).
[0014] Within the context of the present invention, a SLED
polypeptide can be or comprise insertion, deletion, or substitution
mutants of a known PEDF sequence or derivative thereof. Preferably,
any substitution is conservative in that it minimally disrupts the
biochemical properties of the SLED polypeptide. Thus, where
mutations are introduced to substitute amino acid residues,
positively-charged residues (H, K, and R) preferably are
substituted with positively-charged residues; negatively-charged
residues (D and E) preferably are substituted with
negatively-charged residues; neutral polar residues (C, G, N, Q, S,
T, and Y) preferably are substituted with neutral polar residues;
and neutral non-polar residues (A, F, I, L, M, P, V, and W)
preferably are substituted with neutral non-polar residues.
Moreover, the SLED polypeptide can be an active fragment of a known
PEDF protein or fragment thereof. Indeed, it has been found that
truncated fragments derived from SEQ ID NO:1 are active SLED
polypeptides. For example, it is believed that residues 1 through
20 of SEQ ID NO:1 are cleaved during secretion and thus are
dispensable for SLED activity. Moreover, other active SLED
polypeptides comprise sequences derived from residues 21 through
382 of SEQ ID NO:1, such as residues 44 through 157 of SEQ ID NO:1
(e.g., residues 45 through 121 of SEQ ID NO:1). Of course, while
insertion, deletion, or substitution mutations can affect
glycosylation of the protein, a SLED polypeptide need not be
glycosylated to possess the requisite antiangiogenic properties for
use in the inventive method.
[0015] SLED polypeptides inhibit angiogenesis, in part, by
attenuating the migration of endothelial cells, thus reducing the
ability of endothelia to expand within the tissue. Thus, the
invention provides a method of inhibiting endothelial cell
migration by providing exogenous SLED to such cells. Aside from
attenuating angiogenesis, the method is useful for treating
disorders associated with stimulation of endothelial cell migration
such as intestinal adhesions, Crohn's disease, atherosclerosis,
scleroderma and hypertrophic scars (e.g., keloids).
[0016] In accordance with the inventive method, SLED is provided to
endothelial cells associated with the tissue of interest. Such
cells can be cells comprising the tissue of interest, exogenous
cells introduced into the tissue, or neighboring cells not within
the tissue. Thus, for example, the cells can be cells of the
tissue, and SLED is provided to them in situ such that the SLED
contacts the cells. Alternatively, the cells can be cells
introduced into the tissue, in which case the SLED can be
transferred to the cells before they are so introduced into the
tissue (e.g., in vitro), as well as being transferred in situ after
introduction into the tissue.
[0017] The tissue with which the endothelial cells are associated
is any tissue in which it is desired to inhibit the migration or
expansion of endothelia, (e.g., for inhibiting angiogenesis). In
one application, the tissue can be eye tissue, in which case the
presence of exogenous SLED will inhibit novel angiogenesis
associated with a variety of disorders of the eye. For example, the
inventive method is useful for treating eye injury, hypoxia,
infection, surgery, laser surgery, diabeties, retinoblastoma or
other diseases or disorders of the eye. In this respect, the method
is useful for preventing blindness or retarding loss of vision
associated with a variety of eye diseases.
[0018] In another application, the tissue is skin tissue, in which
case the presence of exogenous SLED prevents neovascularization
associated with several skin diseases. For example, the inventive
method is useful for treating diseases and disorders such as
psoriasis, scleroderma, tumors of the skin, neovascularization as a
consequence of infection (e.g., cat scratch disease, bacterial
ulceration, etc.) or other skin disorders. Where SLED is provided
to the skin, it can be provided to the surface of the skin or to
skin tissue beneath the skin's surface. Furthermore, transfer of
SLED to skin of a mammal can also stimulate the growth of hair in
the skin. Without being bound by any particular theory, it is
believed that SLED affects hair growth by mediating angiogenesis
within the hair follicle.
[0019] In other embodiments, the tissue is a tumor (e.g., a
cancerous tumor), in which case the inventive method will inhibit
the growth of blood vessels within and to the tumor. Inhibiting the
growth of blood vessels within tumors prevents sufficient nutrients
and oxygen from being supplied to the tumor to support growth
beyond a given size. Thus, the inventive method can prevent the
nucleation of tumors from cancerous cells already present due to
genetic predisposition (e.g., BRCA-1 mutation carriers, Li Fraumeni
patients with p53 mutations, etc.) or the presence of external
carcinogens (e.g., tobacco, alcohol, industrial solvents, etc.).
Aside from preventing tumerogenesis, the inventive method can
retard the growth of existing tumors, thus rendering them more
easily contained and excised. This application is highly
advantageous for treating tumors that are difficult to operate on
(e.g., brain or prostate tumors). Moreover, minimizing the number
of blood vessels within existing tumors lessens the probability
that the tumor will metastasize. In treating tumors, the method can
be used alone or in conjunction with other treatments, to control
the growth of tumors. Indeed, employing the inventive method can
potentiate the response of some tumors to other therapies. For
example, the inventive method optionally can be employed as a
pretreatment for (e.g., for about a week in advance of), and
continued during, a chemotherapeutic or radiation regimen.
[0020] Where the inventive method is applied to other tissues, the
prevention of neovascularization effectively treats a host of
disorders. Thus, for example, the inventive method can be used as
part of a treatment for disorders of blood vessels (e.g.,
hemangiomas and capillary proliferation within atherosclerotic
plaques), muscle diseases (e.g., myocardial angiogenesis or
angiogenesis within smooth muscles), joints (e.g., arthritis,
hemophiliac joints, etc.), and other disorders associated with
angiogenesis (e.g., Osler-Webber Syndrome, plaque
neovascularization, telangiectasia, angiofibroma, wound
granularization, etc.).
[0021] Aside from treating disorders and symptoms associated with
neovascularization, the inhibition of angiogenesis can be used to
modulate or prevent the occurrence of normal physiological
conditions associated with neovascularization. Thus, for example
the inventive method can be used as a birth control. In accordance
with the inventive method, the presence of SLED within the ovaries
or endometrium can attenuate neovascularization associated with
ovulation, implantation of an embryo, placenta formation, etc.
[0022] Within the context of the inventive method, SLED can be
supplied alone or in conjunction with other known antiangiogenic
factors. For example, SLED can be used in conjunction with
antibodies and peptides that block integrin engagement, proteins
and small molecules that inhibit metalloproteinases (e.g.,
marmistat), agents that block phosphorylation cascades within
endothelial cells (e.g., herbamycin), dominant negative receptors
for known inducers of angiogenesis, antibodies against inducers of
angiogenesis or other compounds that block their activity (e.g.,
suramin), or other compounds (e.g., retinoids, IL-4, interferons,
etc.) acting by other means. Indeed, as such factors modulate
angiogenesis by different mechanisms, employing SLED in combination
with other antiangiogenic agents can potentiate a more potent (and
potentially synergistic) inhibition of angiogenesis within the
desired tissue.
[0023] As discussed herein, SLED is a proteinatious factor. Thus,
in one protocol, the method involves providing SLED by supplying a
SLED polypeptide to the cells (e.g., within a suitable
composition). Any suitable method can be employed to obtain a SLED
polypeptide for use in the present invention. Many suitable SLED
polypeptides can be purified from tissues which naturally produce
SLED or from media conditioned by a variety of SLED-producing cells
(e.g., retinoblastoma cell line WER127). For example, it is known
that SLED is produced by all types of muscle, megakaryocytes of the
spleen, fibroblasts, kidney tubules, cerebellar Purkinje cells,
piliosebaceous glands of hair follicles, and retinal cells. A
particularly good source of naturally occurring SLED is vitreous
and aqueous humors extracted from the eye. One protocol for
purifying SLED from protein extracts of these (or other sources) is
by concentration/dialysis using a 30 kDa ultrafiltration membrane
followed by protein precipitation in a range of about 65% to about
95% ammonium sulfate, followed by a lentil lectin sepharose column
at 0.5 M methyl-.alpha.-D-mannopytanoside, followed by
gradient/isocratic elution at 0.5 M NaCl from a PHARMACIA HiTrap
heparin column. Other protocols for purifying SLED polypeptides are
known in the art (see, e.g., published international patent
applications WO 95/33480 and WO 93/24529). The native SLED
polypeptide represented by SEQ ID NO:1 is identified via SDS-PAGE
as a protein of about 45 kDa. Other SLED polypeptides can be
synthesized using standard direct peptide synthesizing techniques
(e.g., as summarized in Bodanszky, Principles of Peptide Synthesis
(Springer-Verlag, Heidelberg: 1984)), such as via solid-phase
synthesis (see, e.g., Merrifield, J. Am. Chem. Soc., 85, 2149-54
(1963); Barany et al., Int. J. Peptide Protein Res., 30, 705-739
(1987); and U.S. Pat. No. 5,424,398). Of course, as genes for SLED
polypeptides are known (see, e.g., published international patent
applications WO 95/33480 and WO 93/24529); see also GenBanc
accession no. U29953), or can be deduced from the polypeptide
sequences discussed herein, a SLED polypeptide can be produced by
standard recombinant methods.
[0024] In other protocols, SLED polypeptide can be provided to the
tissue of interest by transferring an expression cassette including
a nucleic acid disclosing SLED to cells associated with the tissue
of interest. The cells produce and secrete the SLED polypeptide
such that it is suitably provided to endothelial cells within the
tissue to inhibit their migration and, thus, to attenuate
angiogenesis within the tissue of interest. Coding sequences for
SLED polypeptides are known (see, e.g., published international
patent applications WO 95/33480 and WO 93/24529); see also GenBanc
accession no. U29953), and others can be deduced from the
polypeptide sequences discussed herein. Thus, SLED expression
cassettes typically employ coding sequences homologous to these
known sequences, e.g., they will hybridize to at least a fragment
of the known sequences under at least mild stringency conditions,
more preferably under moderate stringency conditions, most
preferably under high stringency conditions (employing the
definitions of mild, moderate, and high stringency as set forth in
Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d
edition, Cold Spring Harbor Press (1989)).
[0025] In addition to the SLED coding sequence, an expression
cassette includes a promoter, and, in the context of the present
invention, the promoter must be able to drive the expression of the
SLED gene within the cells. Many viral promoters are appropriate
for use in such an expression cassette (e.g., retroviral ITRs,
LTRs, immediate early viral promoters (IEp) (such as herpesvirus
IEp (e.g., ICP4-IEp and ICP0-IEp) and cytomegalovirus (CMV) IEp),
and other viral promoters (e.g., late viral promoters,
latency-active promoters (LAPs), Rous Sarcoma Virus (RSV)
promoters, and Murine Leukemia Virus (MLV) promoters)). Other
suitable promoters are eukaryotic promoters, such as enhancers
(e.g., the rabbit .beta.-globin regulatory elements),
constitutively active promoters (e.g., the .beta.-actin promoter,
etc.), signal specific promoters (e.g., inducible and/or
repressible promoters, such as a promoter responsive to TNF or
RU486, the metallothionine promoter, etc.), and tumor-specific
promoters.
[0026] Within the expression cassette, the SLED gene and the
promoter are operably linked such that the promoter is able to
drive the expression of the SLED gene. As long as this operable
linkage is maintained, the expression cassette can include more
than one gene, such as multiple genes separated by ribosome entry
sites. Furthermore, the expression cassette can optionally include
other elements, such as polyadenylation sequences, transcriptional
regulatory elements (e.g., enhancers, silencers, etc.), or other
sequences.
[0027] The expression cassette must be introduced into the cells in
a manner suitable for them to express the SLED gene contained
therein. Any suitable vector can be so employed, many of which are
known in the art. Examples of such vectors include naked DNA
vectors (such as oligonucleotides or plasmids), viral vectors such
as adeno-associated viral vectors (Bems et al., Ann. N.Y Acad.
Sci., 772, 95-104 (1995)), adenoviral vectors (Bain et al., Gene
Therapy, 1, S68 (1994)), herpesvirus vectors (Fink et al., Ann.
Rev. Neurosci., 19, 265-87 (1996)), packaged amplicons (Federoff et
al., Proc. Nat. Acad. Sci. USA, 89, 1636-40 (1992)), pappiloma
virus vectors, picornavirus vectors, polyoma virus vectors,
retroviral vectors, SV40 viral vectors, vaccinia virus vectors, and
other vectors. In addition to the expression cassette of interest,
the vector can also include other genetic elements, such as, for
example, genes encoding a selectable marker (e.g., .beta.-gal or a
marker conferring resistance to a toxin), a pharmacologically
active protein, a transcription factor, or other biologically
active substance.
[0028] Once a given type of vector is selected, its genome must be
manipulated for use as a background vector, after which it must be
engineered to incorporate exogenous polynucleotides. Methods for
manipulating the genomes of vectors are well known in the art (see,
e.g., Sambrook et al., supra) and include direct cloning, site
specific recombination using recombinases, homologous
recombination, and other suitable methods of constructing a
recombinant vector. In this manner, the expression cassette can be
inserted into any desirable position of the vector.
[0029] The vector harboring the SLED expression cassette is
introduced into the cells by any means appropriate for the vector
employed. Many such methods are well-known in the art (Sambrook et
al., supra; see also Watson et al., Recombinant DNA, Chapter 12, 2d
edition, Scientific American Books (1992)). Thus, plasmids are
transferred by methods such as calcium phosphate precipitation,
electroporation, liposome-mediated transfection, gene gun,
microinjection, viral capsid-mediated transfer, polybrene-mediated
transfer, protoplast fusion, etc. Viral vectors are best
transferred into the cells by infecting them; however, the mode of
infection can vary depending on the virus.
[0030] Cells into which the SLED gene has been transferred can be
used in the inventive method as transient transformants.
Alternatively, where the cells are cells in vitro, they can be
subjected to several rounds of clonal selection (if the vector also
contains a gene encoding a selectable marker, such as a gene
conferring resistance to a toxin) to select for stable
transformants.
[0031] Within the cells, the SLED gene is expressed such that the
cells express and secrete the SLED polypeptide. Successful
expression of the gene can be assessed via standard molecular
biological techniques (e.g., Northern hybridization, Western
blotting, immunoprecipitation, enzyme immunoassay, etc.). Reagents
for detecting the expression of SLED genes and the secretion of
SLED from transfected cells are known in the art (see, e.g.,
published international patent applications WO 95/33480 and WO
93/24529); Steele et al., supra).
[0032] Depending on the location of the tissue of interest, SLED
can be supplied in any manner suitable to provide it to endothelial
cells within the tissue of interest. Thus, for example, a
composition containing a source of SLED (i.e., a SLED polypeptide
or a SLED expression cassette, as described herein) can be
introduced into the systemic circulation, which will distribute the
source of SLED to the tissue of interest. Alternatively, a
composition containing a source of SLED can be applied topically to
the tissue of interest (e.g., injected as a bolus within a tumor or
intercutaneous or subcutaneous site, applied to all or a portion of
the surface of the skin, dropped onto the surface of the eye,
etc.).
[0033] Where the source of SLED is a SLED polypeptide (e.g., within
a suitable composition), it is provided in a concentration and for
a time sufficient to inhibit angiogenesis within the tissue. Where
SLED is produced naturally, it can be present in concentrations as
high as about 250 nM. Because SLED is non-toxic, it can be supplied
to tissues in a far more concentrated form. However, given SLED's
potency, it can be employed in the inventive method at far reduced
concentrations, such as about 50 nM or less (e.g., about 10 nM or
less). Indeed, in some protocols, about 2 nM SLED or less
effectively inhibits angiogenesis and endothelial cell migration.
Depending on the formulation of a composition comprising the
protein, it is supplied over a time course sufficient to retard
angiogenesis within the desired tissue. In some protocols (e.g.,
where the SLED is supplied to the surface of skin or to the eye),
repeated application enhances the antiangiogenic effect and may be
required in some applications. Where the source of SLED is a SLED
expression cassette, the cells expressing the cassette produce an
effective amount of the protein (i.e., sufficient to inhibit
angiogenesis in the tissue).
[0034] To facilitate the inventive method, the invention provides a
pharmacological composition comprising a source of SLED and a
suitable diluent. In addition to the source of SLED, the
composition includes a diluent, which includes one or more
pharmacologically-acceptable carriers. Pharmaceutical compositions
for use in accordance with the present invention can be formulated
in a conventional manner using one or more pharmacologically or
physiologically acceptable carriers comprising excipients, as well
as optional auxiliaries which facilitate processing of the active
compounds into preparations which can be used pharmaceutically.
Proper formulation is dependent upon the route of administration
chosen. Thus, for systemic injection, the source of SLED can be
formulated in aqueous solutions, preferably in physiologically
compatible buffers. For transmucosal administration, penetrants
appropriate to the barrier to be permeated are used in the
formulation. Such penetrants are generally known in the art. For
oral administration, the source of SLED can be combined with
carriers suitable for inclusion into tablets, pills, dragees,
capsules, liquids, gels, syrups, slurries, diposomes, suspensions
and the like. For administration by inhalation, the source of SLED
is conveniently delivered in the form of an aerosol spray
presentation from pressurized packs or a nebuliser, with the use of
a suitable propellant. The source of SLED can be formulated for
parenteral administration by injection, e.g., by bolus injection or
continuous infusion. Such compositions can take such forms as
suspensions, solutions or emulsions in oily or aqueous vehicles,
and can contain formulatory agents such as suspending, stabilizing
and/or dispersing agents. For application to the skin, the source
of SLED can be formulated into a suitable gel, magma, creme,
ointment, or other carrier. For application to the eyes, the source
of SLED can be formulated in aqueous solutions, preferably in
physiologically compatible buffers. The source of SLED can also be
formulated into other pharmaceutical compositions such as those
known in the art.
[0035] Because it is known that SLED is absent from some tumors,
the invention also provides a method for determining the prognosis
of a tumor by assaying for the presence of SLED within the tumor.
The method involves obtaining tissue or fluid from the tumor and
detecting the presence or absence of SLED within the tissue or
fluid. Greater the SLED concentration within the tumor correlates
with a lesser likelihood that the tumor is undergoing angiogenesis.
Thus, higher SLED concentration within the tumor is indicative of a
relatively early stage of tumerogenesis and is, thus, an optimistic
indication. Conversely, the absence of SLED within a given tumor is
indicative of a more advanced stage of tumerogenesis. The method
can employ an assay for the presence of PEDF gene expression (e.g.,
via rtPCR, Northern hybridization, in situ hybridization etc.).
Alternatively, the method can employ an assay for the presence of
secreted SLED (e.g., immunological assays, SLED purification and
PAGE analysis, etc.). Reagents for detecting the presence of SLED
within such tumors are known in the art (see, e.g., published
international patent applications WO 95/33480 and WO 93/24529).
[0036] While it is believed that one of skill in the art is fully
able to practice the invention after reading the foregoing detailed
description, the following examples further illustrate some of its
features. As these examples are included for purely illustrative
purposes, they should not be construed to limit the scope of the
invention in any respect.
[0037] The procedures employed in these examples, such as cell
culture, manipulation of protein and DNA, etc. are well known in
the art (see generally Sambrook et al., Molecular Cloning, A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring
Harbor, N.Y. (1989)). Accordingly, in the interest of brevity, the
experimental protocols are not discussed in detail.
EXAMPLE 1
[0038] This example demonstrates that SLED prevents endothelial
cell migration.
[0039] The migration of different vascular endothelial cell types
was determined by adding SLED to cultured endothelial cells.
Specifically, endothelial cells isolated from bovine adrenal
capillaries, human umbilical chords, and human dermal microvascular
tissue.
[0040] The cells were plated on gelatinized Nucleopore membranes (5
.mu.m pores for bovine capillary cells and 8 .mu.m pores for other
cells) in an inverted modified Boyden chamber. After two hours, the
chamber was reinverted and test substances added to the top wells
of each. Specifically, populations were exposed to either culture
medium alone (control), 10 ng/ml bFGF, 2 nM SLED (full length
PEDF), or both 10 ng/ml bFGF (Fibroblast Growth Factor) and 10 nM
SLED. The cells were then permitted to migrate for 3-4 hours.
Following this, the membranes were fixed and stained, and the
number of cells that had migrated were counted.
[0041] The results of the assay are presented in FIG. 1 as a
percentage of maximal migration (error bars represent standard
error measurement, n=4). As is depicted, all three types of
vascular endothelial cells exhibited nearly 100% migration in the
presence of bFGF. However, in the presence of SLED, considerably
less migration was observed. These results demonstrate that SLED
inhibits endothelial cell migration. These results are surprising,
given that the PEDF protein is known to induce neural
differentiation of cultured retinoblastoma tumor cells, to be a
neurotrophic factor for cerebellar granular cells and a cytostatic
factor for glial cells (Taniwaki et al., J. Neurochem., 68, 26-32
(1997); Sugita et al., J. Neurosci. Res., 49, 710-18 (1997);
Tombran-Tink et al., Exp. Eye Res., 53, 411-14 (1991); Becerra,
"Structure-Function Studies on PEDF," Chapter 21, in Chemistry and
Biology of Serpins, Church et al., eds. (Plenum Press, 1997)).
EXAMPLE 2
[0042] This example demonstrates that the prevention of cell
migration by SLED is specific for endothelial cells.
[0043] The ability of SLED to prevent migration of fibroblasts or
smooth muscle was tested using cells obtained from human diploid
fibroblast cell line WI-38, human foreskin fibroblasts, vascular
smooth muscle, and normal human neutrophils.
[0044] The assay was performed as indicated in Example 1, except
that the dose of SLED varied from 0.01 nM to about 50 nM and that
the migration assay was performed without inverting the chambers.
Moreover, the inducer of migration varied with the cell type (IL-8
was used at 1 .mu.g/ml and PDGF was used at 250 pg/ml).
[0045] The results of this experiment are presented in FIGS. 2A-2D.
As indicated in these figures, SLED did not inhibit migration of
any of the cell lines. This result indicates that the antimigratory
activity of SLED is specific for vascular endothelial cells.
EXAMPLE 3
[0046] This example demonstrates that SLED is among the most potent
inhibitors of endothelial cell migration.
[0047] Using a protocol similar to that outlined in Example 1,
bovine adrenal capillary endothelial cells were exposed to bFGF,
SLED, and several known antiangiogenic factors. The amount of a
given factor necessary to achieve 50% of migration was determined
and is reported here as ED.sub.50. A smaller ED.sub.50 measurement
indicates a more potent antiangiogenic factor. The results of this
experiment, presented in Table 1, indicate that SLED is a highly
potent antiangiogenic factor.
1 TABLE 1 Agent ED.sub.50 (nM) SLED 0.1-0.5 Thrombospondin 0.5
Endostatin 3.0 Angiostatin 3.5 Retinoic Acid 15 Tissue Inhibitor of
Metalloproteinase-1 3500 Captopril 10,000
EXAMPLE 4
[0048] This example demonstrates that SLED inhibits the angiogenic
activity of known angiogenic agents.
[0049] Using a protocol similar to that outlined in Example 1,
bovine adrenal capillary endothelial cells were exposed to five
known angiogenic agents alone or in combination with 0.1 .mu.g/ml
SLED. In particular, aFGF was employed at a concentration of 50
ng/ml, bFGF was employed at a concentration of 10 ng/ml, IL-8 was
employed at a concentration of 40 ng/ml, PDGF was employed at a
concentration of 250 pg/ml, and VEGF was employed at a
concentration of 100 pg/ml.
[0050] The results of the assay are presented in FIG. 3. As is
depicted, the migration of the cells was considerably inhibited by
SLED, regardless of the angiogenic agent. These results demonstrate
that SLED-mediated inhibition of vascular endothelial migration is
not specific for bFGF induction, but that SLED acts generally to
inhibit migration of these cells.
EXAMPLE 5
[0051] This example demonstrates that SLED inhibits
neovascularization in vivo.
[0052] Pellets containing various proteins were implanted in the
avascular corneas of rats. Pellets either contained or lacked bFGF,
and the pellets also contained either SLED or bovine serum albumin
(BSA) as a control. After seven days, the corneas of the rats were
examined to note whether angiogenesis had occurred.
[0053] The results of this assay are presented in Table 2. As
indicated, no vascularization was observed from injecting pellets
lacking bFGF. However, vascularization was observed in all eyes
implanted with bFGF and BSA. Co-injection of bFGF and SLEF,
however, resulted in no neovascularization in any cornea. These
results indicate that SLED is a potent inhibitor of angiogenesis in
vivo.
2 TABLE 2* Treatment Without bFGF With bFGF SLED (8 nM) 0/3 0/3 BSA
0/2 4/4 *results expressed as number of corneas with
angiogenesis/number of test animals.
EXAMPLE 6
[0054] This example demonstrates that SLED polypeptides other than
the full PEDF protein are active antiangiogenic agents.
[0055] Trypsin digestion of the complete PEDF protein cleaves the
protein at amino acid 352 of SEQ ID NO:1, removing the
approximately 3-5 kDa carboxy-terminal portion of the protein
(Becerra et al., J. Biol. Chem., 270, 25992-99 (1995)). This
procedure was employed to generate the fragments, and the truncated
N-PEDF fragment (representing amino acids 21-382 of SEQ ID NO:1)
was purified from trypsin by heparin affinity chromatography.
[0056] Using a protocol similar to that outlined above, various
concentrations of either full length PEDF or the truncated peptide
were assayed for their respective abilities to affect endothelial
cell migration. Data generated for the truncated peptide are
indicated in FIG. 4. Comparison of these data with the activity of
the full length PEDF (see FIG. 3) reveals both proteins to be
similarly potent at inhibiting endothelial cell migration. These
results indicate that peptides other than full length PEDF are
active SLED polypeptides.
EXAMPLE 7
[0057] This example demonstrates that exogenous SLED applied to the
skin promotes the growth of hair therein.
[0058] In this example, the ability of SLED to induce hair growth
is investigated using an established mouse model. In particular, in
the C57BL/6 mouse strain, a change in skin pigmentation correlates
with the physiology of the skin being in anagen (growing), catagen
(transitional), or telogen (resting) phase (see, e.g., Jiang et
al., J. Invest. Dermatol., 104, 523-25 (1995); Slominski et al., J.
Invest. Dermatol., 102, 862-69 (1994); Paus et al., Lab. Invest.,
71, 134-40 (1994); and Paus et al., Lab. Invest., 60, 365-69
(1989)). Six or seven week old C57BL/6 mice have pink skin,
indicating telogen phase. Depilation of these mice triggers hair
growth and skin darkening.
[0059] To test for the effect of SLED on hair growth, six or
seven-week-old C57BL/6 mice are inspected for the absence of skin
pigmentation to verify that their skin is in telogen (resting)
stage. Following this, they are anesthetized and their hair is
removed by clipping (rather than shaving) so as not to trigger
anagen. A plasmid having a SLED expression cassette is precipitated
onto gold particles. Similarly, a control plasmid having a
.beta.-galactosidase expression cassette is also precipitated onto
gold particles. To avoid significant trauma to the skin (which
could trigger anagen), the plasmids are transferred to the prepared
skin of the mice at 250 psi using a gene gun (see, e.g.,
Rakmilevich et al., Proc. Nat. Acad. Sci. (USA), 93, 6291-96
(1996)).
[0060] At weekly intervals following gene transfer, animals are
sacrificed and the presence of circulating levels of administered
gene product are assessed by Western blotting. Additionally, skin
from the prepared and transfected skin is assayed for the presence
of gene product within the skin by immunohistochemistry. The mice
are also visually observed weekly for the presence of coloration
within the treated areas of skin.
[0061] Results indicate that transfer of the .beta.-galactosidase
or SLED expression cassette leads to detectable product in the
animals. However, skin transfected with the .beta.-gal expression
cassette does not progress to anagen any more rapidly than
untreated skin. Conversely, skin treated with the SLED expression
cassette becomes pigmented more rapidly than untreated skin. The
results indicate that transfer of exogenous SLED to the skin
promotes the growth of hair.
[0062] All of the references cited herein, including patents,
patent applications, and publications, are hereby incorporated in
their entireties by reference.
[0063] While this invention has been described with an emphasis
upon preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations of the preferred embodiments may
be used and that it is intended that the invention may be practiced
otherwise than as specifically described herein. Accordingly, this
invention includes all modifications encompassed within the spirit
and scope of the invention as defined by the following claims.
Sequence CWU 1
1
2 1 362 PRT Homo sapiens 1 Met Gln Ala Leu Val Leu Leu Leu Cys Ile
Gly Ala Leu Leu Gly His 1 5 10 15 Ser Ser Cys Gln Asn Pro Ala Ser
Pro Pro Glu Glu Gly Ser Pro Asp 20 25 30 Pro Asp Ser Thr Gly Ala
Leu Val Glu Glu Glu Asp Pro Phe Phe Lys 35 40 45 Val Pro Val Asn
Lys Leu Ala Ala Ala Val Ser Asn Phe Gly Tyr Asp 50 55 60 Leu Tyr
Arg Val Arg Ser Ser Met Ser Pro Thr Thr Asn Val Leu Leu 65 70 75 80
Ser Pro Leu Ser Val Ala Thr Ala Leu Ser Ala Leu Ser Leu Gly Ala 85
90 95 Glu Gln Arg Thr Glu Ser Ile Ile His Arg Ala Leu Tyr Tyr Asp
Leu 100 105 110 Ile Ser Ser Pro Asp Ile His Gly Thr Tyr Lys Glu Leu
Leu Asp Thr 115 120 125 Val Thr Ala Pro Gln Lys Asn Leu Lys Ser Ala
Ser Arg Ile Val Phe 130 135 140 Glu Lys Lys Leu Arg Ile Lys Ser Ser
Phe Val Ala Pro Leu Glu Lys 145 150 155 160 Ser Tyr Gly Thr Arg Pro
Arg Val Leu Thr Gly Asn Pro Arg Leu Asp 165 170 175 Leu Gln Glu Ile
Asn Asn Trp Val Gln Ala Gln Met Lys Gly Lys Leu 180 185 190 Ala Arg
Ser Thr Lys Glu Ile Pro Asp Glu Ile Ser Ile Leu Leu Leu 195 200 205
Gly Val Ala His Phe Lys Gly Gln Trp Val Thr Lys Phe Asp Ser Arg 210
215 220 Lys Thr Ser Leu Glu Asp Phe Tyr Leu Asp Glu Glu Arg Thr Val
Arg 225 230 235 240 Val Pro Met Met Ser Asp Pro Lys Ala Val Leu Arg
Tyr Gly Leu Asp 245 250 255 Ser Asp Leu Ser Cys Lys Ile Ala Gln Leu
Pro Leu Thr Gly Ser Met 260 265 270 Ser Ile Ile Phe Phe Leu Pro Leu
Lys Val Thr Gln Asn Leu Thr Leu 275 280 285 Ile Glu Glu Ser Leu Thr
Ser Glu Phe Ile His Asp Ile Asp Arg Glu 290 295 300 Leu Lys Thr Val
Gln Ala Val Leu Thr Val Pro Lys Leu Lys Leu Ser 305 310 315 320 Tyr
Glu Gly Glu Val Thr Lys Ser Leu Gln Glu Met Lys Leu Gln Ser 325 330
335 Leu Phe Asp Ser Pro Asp Phe Ser Lys Ile Thr Gly Lys Pro Ile Lys
340 345 350 Leu Thr Gln Gly Gly Thr Pro Gly Trp Leu 355 360 2 22484
DNA Homo sapiens Unsure 1...22484 "n" means either a, c, t, or g 2
gcggccgcag ggtggactgt gctgaggaac cctgggccca gcaggggtgg cagcccgcgc
60 agtgccacgt ttggcctctg gccgctcgcc aggcatcctc caccccgtgg
tcccctctga 120 cctcgccagc cctcccccgg gacacctcca cgccagcctg
gctctgctcc tggcttcttc 180 ttctctctat gcctcaggca gccggcaaca
gggcggctca gaacagcgcc agcctcctgg 240 tttgggagaa gaactggcaa
ttagggagtt tgtggagctt ctaattacac accagcccct 300 ctgccaggag
ctggtgcccg ccagccgggg gcaggctgcc gggagtaccc agctccagct 360
ggagacagtc agtgcctgag gatttggggg aagcaggtgg ggaaaccttg gcacagggct
420 gacaccttcc tctgtgccag agcccaggag ctggggcagc gtgggtgacc
atgtgggtgg 480 gcacgcttcc ctgctggggg tgcagggggt ccacgtggca
gcggccacct ggagccctaa 540 tgtgcagcgg ttaagagcaa gcccctggaa
gtcagagagg cctggcatgg agtcttgctt 600 cttgcaaacg agccgtgtgg
agagagagat agtaaatcaa caaagggaaa tacatggtct 660 gtccgaggat
gagctgccgg agagcaatgg tgaaagtgaa gtgggggagg gggcggggct 720
gggaggaaaa gccttgtgag aaggtgacac gagagcacgg ccttgaaggg gaagaaggag
780 ggcactatgg aggtcccggc gaagcgtggc ctggccgagg aacggcatgt
gcagaggtcc 840 tgccgaggag ctcaagacaa gtaggggacg gtggggctgg
agtggagaga gtgagtggga 900 ggaggagtag gagtcagaga ggagctcagg
acagatcctt taggctctag ggacacgata 960 aacacagtgt tttttgtctt
gtcaagtgtg tcctttttat ttttttgaaa gagtctcgct 1020 ctgtagccca
ggctggagtg cagcggtgcg acctcggctc actgcaacct ctgcctcccg 1080
ggtccaagca attctcctgc ctcagcctcc cgagtagctg ggattacagg cacccgccac
1140 cacgcactgc taatttttgt attttagtag agaccgggtt ttgccatgtt
ggtcaggctg 1200 gtctcgaact cctgacctca ggtgatccgc ccgcctcggc
ctcccagagt ggtgtgagcc 1260 actatgccct gcagcacttg tcaagtcttt
ctcagcgttc ccctcctctc cactgcagct 1320 cccagtgccc cagtctgggc
ctcgtcttca cttcctggga tccctgacat tgcctgctag 1380 gctctccctg
tctctggtct ggctgccttc actgtaacct ccacccagca ggtacctctt 1440
cagcacctcc catgaaccca gcagaatacc aagccctggg gatgcagcaa cgaacaggta
1500 gacgctgcac tccagcctgg gcgacagagc aagactccgc ctgaagaaaa
aaaaaaggac 1560 caggccgggc gcggtggctc acgcctgtaa tcccagcact
ttgggaggcc gaggtgggtg 1620 gatcatgagg tcaggagttc aagaccagcc
tggccaaaat ggtgaaaccc cgtctctact 1680 gaaaaataca aaaattagct
gggtgcagtg gcgggcgcct gtagtctcag ctactcagga 1740 ggctgaggca
ggataattgc ttgaccccag gaggcagagg ttgcagtgaa ccgagatcac 1800
gccactgcac tccagcctgg gcgacagagc aagactctgc ctcaaaaaaa agaataaaaa
1860 taaaaaaaag gaccagatac agaaaacaga aggagacgta ctatgaagga
aattggagag 1920 cttttgggat actgagtaac tcagggtggc ctttcccagg
ggacatttag ctgagagata 1980 gacggtatga agacctgacc gttcagaaac
aggggaagag gcagcagccc gggcaaaggc 2040 ctttggggca ggaaagggct
tggatcactg gagaagcaga aagatggcca gtgtgaccag 2100 agtgtgacaa
agtcagagaa aaccaggaag atggagctgg agacacaggc ggggccagat 2160
cacgagggtc ctcgcagacc agagcaaggg tttggatttt attccaagta tgaagggaag
2220 ctgctgaagt gtgttttcct ttacaatttg tagttgaaat ataatatgca
aagtacacaa 2280 gtcttaacta tatgtaagct taatgaatgt ttccatgaac
caaataccgc tgtgcaacca 2340 tcaccagctc aagagacgaa cccttctccc
tcctcctgac tgccagtaac atagtggttc 2400 agctcaagaa acagaactct
tctgacttcc cctaacatag cgggttttct tttttgtttt 2460 gttttttgtt
gttttttaag agacaatgtc tttattattt ttattttttt ttatttttga 2520
gacggagtct tgctgtcgcc caggctggag tgcagtggtg cgatctcggc tcactgcagg
2580 ctctgccccc cggggttcat gccattctcc tgcctcagcc tccctagcag
ctgggactac 2640 aggtgcccgc cacctcgccc ggctattttt ttgtattttt
agtggagacg gggtttcacc 2700 gtgttagcca ggatggtctc gatctcctga
cctcgtgatc cgcccacctc ggcctcccaa 2760 agtgctggga ttacaggcat
gagccaccgc gcccagccaa gagacacggt cttgctctgt 2820 cgcccaggct
ggatggagtg ccgtggtgcg atcacagctc gcggcagcct tgacatcctg 2880
ggctcaagca accttcctgc cttggcctcc caaatgttgg gattataggc atgagccact
2940 gtgcttggca tctattcatc tttaatgtca agcaggcaat tgaatatttg
atcagggata 3000 gaattgtcta tttgggggta tgcagatgtg cttcatgtca
tggaactggg ccgggcgcgg 3060 tggctcatgc ctataatccc agcactttgg
gaggccgagg caggcggatc ataaggtcag 3120 gagatcgaga ccatccgggc
caacacggtg aaaccccgtc tctactaaaa atacaaaaat 3180 taggcaggtg
tggtggtgcg tgcctgtagt cccagctact cagggaggct gagacaggag 3240
aattgattga acctgggagg cagaggttgt agtgagccaa gatcgcgcca ctgcactcca
3300 gcctgggcga catgagcgag actccgtctc aaaaataaac aaaaaaaagt
catggaattg 3360 atggaaattg cctaagggga gatgtagaag aaaaggggtc
tcaggatcaa gccagcagag 3420 aaggcagaaa aggtaaggtg tgtgaggtgg
cagaaaaagg gaagagtgtg gacagtgagg 3480 gtttcaagga ggaggaactg
tctactgcct cctgccaagg acggaggtgt ccactgccag 3540 ttgacataag
gtcacccatg aacttggtga caggaatttc agtggagaag tggccacaga 3600
cacaagtcta gaattgaaat gggagccgag gcagcgtaga caaaagagga aactgctcct
3660 tccagagcgg ctctgagcga gcaccgagaa atgggcagtg gctttagggg
atgtagcgtc 3720 aaggaagtgt cttttaaaga agtcgggggc cgggcacggt
ggctcacgcc tgtagtccca 3780 gcactttggg aggccgaggc aggcagatca
cttgaggtca ggagttcgag accagcctgg 3840 ctaacacgat gaaaccccgt
ctctactaaa aatacaaaaa attagctggg cacggtggct 3900 cgtgcctgta
atcccagcac tttgggaggc agaggtgggc agatcacttg aggtcaggag 3960
tttgagacca gcctagccaa catggtgaaa ccccatctct actaaaacta caaaaattag
4020 ccgggagtgg tggcacgtgc ctgtaatccc agccagtcag gaggctgagg
caggagaatc 4080 actggaatcc tggaggtgga ggtggcagtg agccgagatg
gtacctctgt actccagcct 4140 gggggacaga gtgagactcc gtctcaaaaa
aaaaagaagg tggggaagga tctttgaggg 4200 ccggacacgc tgaccctgca
ggagaggaca cattcttcta acaggggtcg gacaaaagag 4260 aactcttctg
tataatttat gattttaaga tttttattta ttattatttt ttatagaggc 4320
aagcattttt caccacgtca cccaggctgg tctccaactc ctgggctcaa gtgtgctggg
4380 attatagcca tgagtcacca cacctggccc agaaacttta ctaaggactt
atttaaatga 4440 tttgcttatt tgtgaatagg tattttgttc acgtggttca
caactcaaaa gcaacaaaaa 4500 gcacccagtg aaaagccttc ctctcattct
gatttccagt cactggattc tactcttggg 4560 atgcagtgtt tttcatctct
tttttgtatc cttttggaaa tagtattctg ctttaaaaag 4620 caaatacagg
ccaggtatgg tggctcactc ctgtaatccc agcactttgg gaggccgagg 4680
caggtgatca cctaaggtca ggagttcaag accagcctgg ccaatatggt gaaaccctgt
4740 ctgtaccaaa acacaaaaac aaaaacaaaa acaaaaatta gccgggcgtg
gtggcgtgct 4800 cctgtaatcc cagctactca ggaggctgag gcaggagaat
cgcttgaacc tgggaggcag 4860 aggttgcagt gagccgagat tgtgccactg
tactccagcc tgggccacag agcaaggttc 4920 catctcaaac aaaacaaaac
aaaacaaaca aaaaaacaaa acaaaagcta atacaaacac 4980 atatacaata
gacaaaactg taaatatttt attattttta ttttttttag tagagacagg 5040
gtttcaccat gttggccagg atggtctcaa actcctgacc tcaggtgatc cacccacctc
5100 agcctcccga tagttaggat tacaggcatg agccaccaca cccggcctaa
aattgtaaac 5160 gttttagaag aaagtataga tgaatccctt cgtgatctcg
gggaagaaga gattttttaa 5220 aaaagatacc aaaagaagca caaattataa
aagaaaagat tgaaaatgtt ggtgttaaaa 5280 ttaaaaactt gttttaaaac
aagcttgtgt aacccatgac ccacaggctg catgtggccc 5340 agaaaagctt
tgactgcagc ccaacacaaa ttcgtaaact ttcctaaaac attatgagat 5400
tttttttgag attttgtttt gttttgtttt ttgttttttt agctcattcg gtatcattaa
5460 tgttagcata ttttacgtgg ggcccaagac aattcttctt ccaatgtgtc
tcaggggagc 5520 caaaagattg gacacccctg ccataaacat gaaaagacaa
tggccgggca cggtggctca 5580 cgcctgtaat cccagcactt tgggaggctg
aggggggcgg gatcacctga ggtcaggagt 5640 ttgagacaag cgtgaccaat
gtggtgaaac cctgtctcta ctaaaaatac aaaaattagc 5700 cgggcatgct
cgtgcacacc tatagtccca actactcagc agggtgaggc aggagaacct 5760
cttgaacccg ggaagcggag gttgcagtga gccgacattg cacccctgca ctccagcctg
5820 ggtgacagag tgagtctcca ctggaaaaaa aaaaaaaaga acagtgtgat
acattgacct 5880 aaggtttaag aacatgcaaa ctgatactat atatcactta
gggacaaaaa cttacatggt 5940 aaaagtaaaa agaaatgtac gaaaataata
aaaatcaaat tcaagatggt ggttatggtg 6000 acgggaaaga actgaggcgg
aaatataagg ttgtcactat attgagaaat ttttctatct 6060 ttttttcttt
tttctttttt tgagacgggg tctcgctctg tcgcccagga tggagtgcag 6120
tggtgtgatc tcagctcact gcaacctccg cctcccaggt ttaagtgatt ctcctgcctc
6180 agactcccaa gtagctggga ctacaggtgc gcgccaacac acctgggtaa
ttttgtttgt 6240 atttttagta gagatggggt ttcaccgtgt tgactaggct
ggtctcgaac tcctgacctc 6300 aggtgatccc ccggcctcgg tctcccaaag
tgctgggata acaagcgtga gccactgcgc 6360 ccagctttgt ttgcattttt
aggtgagatg gggtttcacc acgttggcca ggctggtctt 6420 gaactcctga
cctcaggtga tgcacctgcc tcagtctccc aaagtgctgg attacaggcg 6480
ttagcccctg cgcccggccc ctgaaggaaa atctaaagga agaggaaggt gtgcaaatgt
6540 gtgcgcctta ggcgtaatgg atggtggtgc agcagtgggt taaagttaac
acgagacagt 6600 gatgcaatca cagaatccaa attgagtgca ggtcgcttta
agaaaggagt agctgtaatc 6660 tgaagcctgc tggacgctgg attagaaggc
agcaaaaaaa gctctgtgct ggctggagcc 6720 ccctcagtgt gcaggcttag
agggactagg ctgggtgtgg agctgcagcg tatccacagg 6780 taaagcagct
ccctggctgc tctgatgcca gggacggcgg gagaggctcc cctgggctgg 6840
ggggacaggg gagaggcagg ggcactccag ggagcagaaa agaggggtgc aagggagagg
6900 aaatgcggag acagcagccc ctgcaatttg ggcaaaaggg tgagtggatg
agagagggca 6960 gagggagctg gggggacaag gccgaaggcc aggacccagt
gatccccaaa tcccactgca 7020 ccgacggaag aggctggaaa ggcttttgaa
tgaagtgagt gggaaacagc ggaggggcgg 7080 tcatggggag gaaaggggag
ctaagctgct gggtcgggtc tgagcagcac cccaagactg 7140 gagcccgagg
caaggaggct cacgggagct gcttccacca agggcagtca ggaaggcggc 7200
cgccctgcag cccagccctg gcccctgctc cctcggctcc ctgctacttt ttcaaaatca
7260 gctggtgctg actgttaagg caatttccca gcaccaccaa accgctggcc
tcggcgccct 7320 ggctgagggc tgggatggag gacagctggg tccttctagc
cagcccccac ccactctctt 7380 tggctacatg agtcaaggct gggcgaccaa
tgaggttgtg gcctccggca aacaatgacc 7440 actatttagg ccggcaggtg
tatagggcgt gggggcccag ctgccagtgc tggagacaag 7500 ggctgtccga
gatgaaccct ttctgctgcc tgccaagcca ctgggagggg taggtctcag 7560
caggattccc agaaaccccg cccctgtcca gcctaggccc cccacccggt gttagctaac
7620 ccaacgttag cccccaggtt ccgtggggtt ggggggcagg gagtcctatt
cttggggctg 7680 ctgcttctgg ggtgtgggga agtgcaactc cacggcaccc
tgggctgact cattcagctt 7740 ctaaagcttc aggaaacatt gtttggggct
gggtcaccat gggtgggcca gagaggaccc 7800 ctcaatcccc tccggagagc
caggggaggg ggaggtgccc ttccccatgc tatctccgag 7860 gcccactgcc
atgtggctga aggctgtgcg gttctgggaa gagggggagg tggcggtgga 7920
ggctgtttgt ctcctaactg ggcttaatct gaaacacatg tattggcttg agttgatccg
7980 cctcacgtgg aggcaagatc acaaaagctt ctgtgtttct tgatgtgggc
aattgtcaga 8040 aaataaggcc tgaccttggc ccagcaggga gggtatctac
ctctccctga gccctccccc 8100 gcctgctagg acgagagcgg ggcttggata
ctgccctttg gacaggatgg catcattgtc 8160 tgtggctgca gccagccagc
ggtcgcctgc tcagcccatg agcaaccact gtggacaggg 8220 tattgcgtgt
gtgctgaggg gcgtccatgc agacccccac gcttgccctc tcactgccct 8280
tgtagggttt tcaatcatct ctcctcttcc cttatccaga tggcttgaag tggaggattc
8340 agacttgccg ttaatactct gggtccctgt gtctagctcg gggccacctt
tggacccatg 8400 tcccttccct gccaggctcc ctcacctcac ctcagcctac
ccacattgtg acaatcatct 8460 accacctgat ctggggtttg ggcttagatt
ctgtaggcac caagactaaa gtcgctcctt 8520 caagtccatt tgaattgtga
ctttagtttc cttaaatact atgccaggat aatggccagg 8580 gatggtggct
cacgcctgta ctcctggcac tttgggatgc tggtggatca cctgagatca 8640
ggattccagg ccagcctggc caacacggtg aaaccccatc tctactaaaa cataaaaatt
8700 aaccaggtgt ggtggcgggc acctgtaatc ccagctactc aggagactga
ggcaggagaa 8760 ttgcttgaac ccgggaggtg gaagttgcac tgagctgaga
tcgcgccact gcactttagc 8820 ctgggcgaca agagtgaaac tctgtctcaa
aaacaaaaaa aactatgccg ggatgagcct 8880 gtctcctccc ttaatttctt
acttgggcca gaggaactag aactaacaac ttctcttcta 8940 gccttgcctc
ctgtgtacct cactgaattt ttggtctcta ataaaccagt ctgcagaggc 9000
tcaggggagg caggctcctg gcagctgggt ggggctggcc ccagccgggt ggagaccagc
9060 tgtaggcctg gatggtggtg aggcctctgt cttgcactgc agaaagcttt
tcctgttgtc 9120 tacacgaaag ttttctccct gcatgtcagg gcagccacgt
gcaagagcag ctggctggga 9180 acgcagaggt ctgcggctcg aggcggggtt
tagaaagaaa accaggctgc ttcctgctgc 9240 ccgtcctgcc ttaagctgag
taaactcaaa ggcaatcttc tttcatgcct cacgatattg 9300 tccagtggat
tatctgattt aatttgaagg acgagagcca acaatcacac aacgtcctcc 9360
caaattttct gatccacttt gttctgggaa gtcaaaaagt gcgtgtgctg tgtgggtgga
9420 tgtttgtgta tataaatgga taatgaagga tgatgtgttg ggggccaggg
caggggagac 9480 aacgctgttc agattctaca tttttttttc cttttttttt
tttttttgag atggagtctt 9540 gctctgttgc ccagcctgga gtgcagtggc
gcgatctcag ctcactgcaa cctccacttc 9600 ctggattcaa gtgattctcc
tgccttagcc tcccaagtag ctgggattac aggcatgcgc 9660 caccacaccc
ggctaatttt tgtattttta gtagagatgg ggtttctcca tgttggccag 9720
gatggtctca aactcctgac ctcaggtgat ctacccgcct cggcctctca aagtgctggg
9780 attacaggtt tgagccactg cgcctggcct tttttttttt ttttgagatg
gagttttcac 9840 tcttgttgcc caggctggag tgcagtggtg cgatcttggc
tcactgcaac ctccacctcc 9900 caagttcaag tgattctcca gccttagccc
tccaagtagc tgggactaca ggtgtgtgcc 9960 accatgcctg gctattttat
tttattttat tttatttatt tatttttgag actaagtctt 10020 gctctgttgc
ccaggctgga gtgcagtggc ataatcggct cactgcaacc tctgcctccc 10080
aggttcaagt gattctcctg cctcagcctc ctgagtaact gggattacag gggcctgcca
10140 ccacgcctgg ctactttttg tatttttagt atagatgggg tttcaccatg
ttggccaggc 10200 tggtctcgaa ctcctgacct caggctatcc gcctgcctca
gcctcccaaa gtgctgggat 10260 tacaggcatg agccactgtg ctcggtagtt
gttttatttt aatagtaggt tattttattt 10320 ccattttaca agagaaaaaa
tggtgattta aagagctact aagacacagc actgagacca 10380 tgtgtgatgg
catgcgcctg cagtcccagc tactcacgag gctgaggcag gaggatcaca 10440
tgaggtcagg agttccaggc tgtggagtgc tatggttgtg tagtgaatag ccactacact
10500 ccagcctggg cagcacagca agatcttgtc tcccaaaaaa aaaaaaaaaa
aaaaatttca 10560 aatgtgaacc caggatctct gaccctaggc cctgcactcc
taaccatggg aggaagagct 10620 cttgaaaggg aactgtggga gaagggaatg
agctgccttg tgaggccaca gaagtccaaa 10680 gacagcttga gaatttggag
ggacagcacg tgccggactg ggtgcctcta tgcttggtat 10740 ccggtgattc
catggaggag acctgggttc tgccccattc tcctgggagg ggttgcccaa 10800
agtcttatca ccggagtggg tcagctgcct ccaggacaaa gctttagcat acacttgtgc
10860 tgggccatac tccacgtgga gaagccctgc tggggctggg gccccactgc
tctggatctt 10920 taaaagctat tggttcaggg gccaggtgta atggctcaca
cctataaccc tagcactttg 10980 ggaggctgaa gcaggtggat agcctgaggt
caggagtttg agacaagcct gatgaacgtg 11040 gtgaaacccc atcgctatta
aaatacaaaa aattagccgg gcatggtggc aggtgcctgt 11100 aattccagct
acttgggagg ctgaggcggg agaatcgctt gaacccagga ggcggaggtt 11160
gcagtgagcc aagatcgctc cactgtactc cagcctgggc gacagagcca gactctgttt
11220 caaaaaataa aatataaata aataaataaa taaataaata aataaataaa
agctttaggc 11280 ttaaaggagg gtcccctgac gcagacagtg gaacaaaagc
acaagcttat ggtatgactg 11340 tgggccctga ggcaggggga ggggcgggag
aaccttgctg ggagggatgg gccatcaagc 11400 tgagggtcca cttctggggg
cctggagggg tgaggggtgg tcgctgcagg gggtggggga 11460 aagtgactag
ccctgcccaa cccctgggtc ctggctgggg tggccaggaa ggggtagcgg 11520
ggcagtgcag tgtcggggga gagcggcttg ctgcctcgtt cttttcttgc aggccccagg
11580 atgcaggccc tggtgctact cctctgcatt ggagccctcc tcgggcacag
cagctgccag 11640 aaccctgcca gccccccgga ggaggtcagt aggcaggcgg
ggagggcgtg gtcagcattc 11700 cccgcccctc cttggcaggc agcacgggaa
acaggacagg gaacccggac ccaggttcca 11760 ggccaggctt gggcctttat
ttctctaggg ctggagtttc tccagcagca aaacagagag 11820 aaaatgtctt
gccttgcctt tcaggggatg gagtagggac atgaataaga tcccaaaaga 11880
gtaaaaatct gaagcacttt taacaagtcc agggcaattc tcctgcctca gcttcccaag
11940 cagctgggat tacaggcatg caccaccaag cccggctcat tttgtatttt
tagtagagac 12000 ggggtttctc catgttggtc aggctggtct cgaactcccg
acctcaagtg attctcctgc 12060 ctcggcctcc caaagtgccg ggatgacagg
tgtgagccac cgcacctggc caggatcttt 12120 tctcattacc ttgtcttcct
agtgggggct ccactgagca ggtcatgttc ccggacattt 12180 gttcggatac
tgaccaggct gtggcaggga gtgagggtat ggagtgacct ctctcctgcc 12240
cagaaagggc gcagctgggt tcccaaggca gatacaggca catggaggga agcctgggcc
12300 atatgagtgt tatggggtga gtgttggcgg aggcccaccc ttgagggaca
agagcagctg 12360 ggcatcttgg cgagagccct ggactttcgt gaggtcagag
tatgaattct gcgtctccct 12420 cttcctagct ttgtgaccct agacaaccct
tacctcagtc tttgcttcct tgcctatgaa 12480 atgggataaa aacacccatt
ctacagggcc atgtggccac tcatttattt ctcatctacc 12540 aaacacctac
tcgacagggg ctggcaatgg gcggaaataa aaactcagtt ctgccgggtg 12600
cggtggctca cacctgtaat cccagcagtg tgggaggcgg agcaggacga tcccttgaat
12660 ccaggagttt gagaccagca taggcaacat agtgagaccc ctgtctctac
acaaaagcaa 12720 aaattaccag gcgtggtggc
aagtgcttgt ggtactacct acttgggaag ctgaggtggg 12780 aggatcactt
gagcccagga gattaagact gcagtgaggg gccgggcgcg gtggctcacg 12840
cctgtaatcc cagcactttg ggaggtggag gtgggtggat cacgaggtca ggagatcgag
12900 accatcctgg ctaacacggt gaaaccccgt ctctactaaa aatacaaaaa
attagctggg 12960 tgtggtgggg ggcgcctgta gtcccagcta ctcgggaggc
tgaggcagga gaatggcgtg 13020 aacccgggag gtggaggttg cagtgagctg
agctcgcacc actgcactcc agcctgggcg 13080 acagagtgag actccgtctc
aaaaaaaaaa aaaaaaaaaa gaaagaaaga aaaactgagt 13140 tctttttttt
aactttcttt ttttagagac agagtctcac tccatcaccc atgctggagt 13200
acagtggtgc gatcttggct cactgcaatc ttggcctcct gagttcaacc aattctcatg
13260 cctcagcctc ccaaatagct gggaccacag gcacgtgcca ccacgcccag
ctaatttttt 13320 gggtattttt agtagagatg gggcctcacc atgttgctca
ggttggtctg aaactcctga 13380 gctcaagtga tccatcttcc tcggcctgcc
aaagtgctgg gattataggc ataagccact 13440 gcacctagct cccaattttt
atatttatat ttatttttat ttacttattt attttttgag 13500 acagggtctc
actctgtcac ccaggctgga gtacagtggc actatctcag ctcactgcaa 13560
cctctgcctc ctgggttcaa gcgaatctcg tgcctcagcc tcctgagtag ctgggattac
13620 aggcatgcac caccatgccc cgttaatttt tttgtatttt tagtagagac
gggtttcacc 13680 gtgttgccca ggatggtctc gaactcctga cctcaagtga
ttcacccacc tcagcctccc 13740 aaagtgctgg gattataggt gtgagccact
cggctgatgg tttttaaaaa gtgggtcatg 13800 gggctgggcg cggtggctca
tgcctgtaat cccagcactt tggtagaccg aggcgggtgg 13860 atcacaaggt
caggagatcg agaccatcct gcctaacacg gtgaaacccc gtctctacta 13920
aaaatacaaa aaattaccca ggcatggtgg tgggcgcctg tagtcccagc tactcgggag
13980 gctgaggcag gagaatggcg tgaacctggg aggcggagct tgcagtgagc
cgagatcacg 14040 ccaccgtact ccagcctgag cgacagagcg agactccgtc
tcaaaaaaaa aaaaaaaaag 14100 tgggtcatag gtttcggctt ataggtcaca
agtgtttaaa cctggccatg aggccaggcg 14160 cagtggcgca tgcctgtaat
cccagccatt tgggaggcta aggcaggaaa atcgcttgaa 14220 ccggggaggt
ggaggttgca gtgagctgag atcgcgccac tgaactctag cctgggtgac 14280
acagtaagac tctgtctcaa ataaaaaaaa aaacagctga tctctcttct gcgctgtctc
14340 tccacagaga gctcatgcgt gatcagggag taaaactcat tcccgtttta
ggccaaacac 14400 agaaaaatta ggaaggacag ccccaagggg ccagaaccac
caccctacac aaagccgtga 14460 ggagacagtc cctgtgcatc tctgcgagtc
cctgaactca aacccaagac ttcctgtctc 14520 ctgccagggc tccccagacc
ccgacagcac aggggcgctg gtggaggagg aggatccttt 14580 cttcaaagtc
cccgtgaaca agctggcagc ggctgtctcc aacttcggct atgacctgta 14640
ccgggtgcga tccagcatga gccccacgac caacgtgctc ctgtctcctc tcagtgtggc
14700 cacggccctc tcggccctct cgctgggtga gtgctcagat gcaggaagcc
ccaggcagac 14760 ctggagaggc cccctgtggc ctctgcgtaa acgtggctga
gtttattgac atttcagttc 14820 agcgaggggt gaagtagcac caggggcctg
gcctgggggt cccagctgtg taagcaggag 14880 ctcaggggct gcacacacac
gattccccag ctccccgaaa ggggctgggc accactgaca 14940 tggcgcttgg
cctcagggtt cgcttattga cacagtgact tcaaggcaca ttcttgcatt 15000
ccttaaccaa gctggtgcta gcctaggttc ctgggatgta actgcaaaca agcaggtgtg
15060 ggcttgccct caccgaggac acagctgggt tcacagggga actaatacca
gctcactaca 15120 gaatagtctt ttttttttnt ttttttnnnc tttctgagac
ggagtctcgc tttgtcncca 15180 aggctggagt gcagtggtgt gatctcagct
cactgcaacc tctgcctccc tggttcaagg 15240 aattctcctg cctcagcctc
cagagtagct gggattacag gcacctgcca tcatgcccag 15300 ctaatttttg
tatttttagt agagacgggg tttcaccatg ttgcctaggc tggtctcaaa 15360
ctcccgggct caagcgatcc acccgccttg gcctcccaaa gtgctgggat tacaggcgtg
15420 agccaccgcg cctggccaga ataatcttaa gggctatgat gggagaagta
cagggactgg 15480 tacctctcac tccctcactc ccaccttcca ggcctgatgc
ctttaaccta cttcaggaaa 15540 atctctaagg atgaaaattc cttggccacc
tagattgtct tgaagatcag cctacttggg 15600 ctctcagcag acaaaaaaga
tgagtatagt gtctgtgttc tgggaggggg cttgatttgg 15660 ggccctggtg
tgcagttatc aacgtccaca tccttgtctc tggcaggagc ggagcagcga 15720
acagaatcca tcattcaccg ggctctctac tatgacttga tcagcagccc agacatccat
15780 ggtacctata aggagctcct tgacacggtc actgcccccc agaagaacct
caagagtgcc 15840 tcccggatcg tctttgagaa gagtgagtcg cctttgcagc
ccaagttgcc tgaggcatgt 15900 gggctccatg ctgcaggctg ggggggtctt
tttttttttt ggggaaagac ggagtctcgc 15960 tctgttgccc aggttggagt
gaagtggcgt gatctcggtt cactgaaacc cccacctccc 16020 gggttcacac
catcctcctg cctcagcctc ccgagtagct gggactgcag gngcccagct 16080
aatctttntt gtatttttag cagagacggg gtttcaccgt gtttgccagg atagtctcga
16140 tctcctgacc tggtgttctg cccgcctcga cctcccaaag tgctgggatt
acaggtgtga 16200 gccaccgcgc tcggcccgtt tctaaacaat agatcatgtg
tgcccaggcc tggcctggca 16260 ctggtgtgga ggaagggccc gtgagcccaa
agaggctcag aaagaggaag tgggctgcag 16320 gagacggtgg gaggggcagg
gagggcagtg gcgcgatgtg gggaaatctg ctgcccccct 16380 ggccagtgcc
tggggatgcc agcagaagtc ctggcaagtc acaggaagat gctggctggg 16440
aagtcagggc ctgctgagcg ctaaaccaga acccgagcct ggcaggctct caaagacggg
16500 atgcttgtcg tcgagtctca tacgctaacc tctgctccgc ctcttctcag
agctgcgcat 16560 aaaatccagc tttgtggcac ctctggaaaa gtcatatggg
accaggccca gagtcctgac 16620 gggcaaccct cgcttggacc tgcaagagat
caacaactgg gtgcaggcgc agatgaaagg 16680 gaagctcgcc aggtccacaa
aggaaattcc cgatgagatc agcattctcc ttctcggtgt 16740 ggcgcacttc
aagggtgagc gcgtctccaa ttctttttca tttattttac tgtattttaa 16800
ctaattaatt aattcgatgg agtcttactc tgtagcccta actggagtgc agtggtgcga
16860 tctcagctca atgcaacctc cgcctcccag gttcaagcaa ttcttgtgcc
tcagcctccc 16920 gagtagctgg gattacaggg atgtaccacc actcccggct
aattttttgt atttaataga 16980 catggggttt caccatgttg gccaggctgg
tctcgaactc ctgagctcag gtggtctgcc 17040 cgcctcagcc tcccaaagtg
ctaggattac aagcttgagc caccacgccc agcccttttt 17100 atttttaaat
taagagacaa ggtgttgcca tgatgcccag gctggtctcg aactcctggg 17160
ctcaagtaat cctcccacct tggcctccca aagtgctggg attacaggca tgagccaccg
17220 cgcccggccc ttttacattt atttatttat tttttgagac agagtcttgc
tctgtcaccc 17280 aggctggagt gcagtggcgc gatctcggct cactgcaagc
tctgccttcc aggttcacac 17340 cattctcctg cctcgacctc ccgagtagct
gggactacag gcgcccgcca ctgcgcccta 17400 ctaatttttt gtatttttag
tagagacggg gtttcaccgt ggtctcgatc tcctgacctc 17460 gtgatccacc
cgcctcagcc tcccaaagtg ctgggattac aggcgtgagc cactgcgccc 17520
ggccctttta catttatttt taaattaaga gacagggtgt cactatgatg ccgaggctgg
17580 tctcgaactc ctgagctgaa gtgatcctcc cacctcggcc tcccaaaatg
ctgggattac 17640 catgtccaac tttccacttc ttgtttgacc aaggatggat
ggcagacatc agaaggggct 17700 tggaaaggga ggtgtcaaag accttgccca
gcatggagtc tgggtcacag ctgggggagg 17760 atctgggaac tgtgcttgcc
tgaagcttac ctgcttgtca tcaaatccaa ggcaaggcgt 17820 gaatgtctat
agagtgagag acttgtggag acagaagagc agagagggag gaagaatgaa 17880
cactgggtct gtttggggct ttcccagctt ttgagtcaga caagatttat ttatttattt
17940 aagatggagt ctcattctgt tgcccaggct ggagtgcagt ggtgccatct
tggctcacta 18000 cagcctcccc acctcccagg ttcaagtgct tctcctgcct
cagcctcccg agtagttggg 18060 attacaggcg cccgccacca cacccagcta
atttttgtat tttcagtaga gatggggttt 18120 cgccatgctg gccaggctgt
tctcgaaaac tcctgacctc agatgatcca cccgcctcgg 18180 cctcccacag
tgctgggatt acaggcgtga gccactgcgc tggccaaatc agacaaggtt 18240
taaatcccag ctctgcctgt actagctgag gaactctgca cacatttcat aacctttctg
18300 ggcctacgtt ctcaccttta acgtgaggat aatatatcta cttcatagac
acctttttat 18360 gttgtctcca agttttctaa cagctctagt tctgtaccca
agacatggca ggtggccaac 18420 gacatccttc taggctgtgg tgatgtgttt
ggagcttgtt ccacgggtct tgtgtggggc 18480 cagccctgtt cagataaggc
cttgtggggt ggcctggggt agggggaggg gttgggcaaa 18540 ctctccctta
aaacgctttg taaccatctg aggcaccagc aagagcggcc cccgagcctg 18600
gacaaaatcc aaacggcttc ctacttcaag cactgatgtc tagtgagtga aggaacagct
18660 ctgggtccag gatattatag gtcacattaa actaaagggg cttggccatc
agctggcttc 18720 cagagcgtca gccagttact tcacctcttt ggctttggcc
tgttttcagc tacaagagga 18780 cttaatccag aggacctcag aggtccttcc
cagctcagac cttctttgac tgtctcccag 18840 agacactgct gtaggagtgc
acaccagttt acttttcttt cttttgtttt tgagatggag 18900 tttcgctctt
tttgcctagg ctggagtgct gtggtgtgat ctcagctcac tgcaacctct 18960
ggctcccagg ttcaagtgat tctcctgtct ctgcctcccg agtagctggg attacagaca
19020 cccaccactg cacccggcta gtttttgtat tttcagtaga gatggggttt
cgccatgctg 19080 gccaggctgt tctcgaaaac tcctgacctc agatgatcca
tccgccttgg cctcccaaag 19140 tgctgagatt acagatgtga ggcaccacac
ccggccattt ttgtattttt agtagagacg 19200 gggttttgcc atgttggcca
cgctggtctc aaactcctga cctcaagtga tctgcccacc 19260 ttggcctcct
gaagggctgg gactacaggc gtgagtcacc gtgcccggcc atttttgtat 19320
ttttaggaca gcgttttttc atgttggcca ggctggtctc aaactcctga cctcaagtga
19380 tccacccacc ccggcctccc aatatgctgg gattccaggt gtgagttacc
atgcccggct 19440 accactttac ttttcctgca ggctatcaca gaacgtgtac
aatctagact ctaatcaacc 19500 aaatcaacgt cttgccatcg gagtttgctg
gtgaagggca cttggggtcc tggaaataac 19560 tgtaggctcc aagccacaca
cactgagata ggcctattcc ctgaggcctc agagcccctg 19620 acagctaagc
tcccttgagt cgggcaattt tcaacaacgt gctctgggga cacagcatgg 19680
cgccactgtc tttctggtct cctggggctc agactatgtc atacacttct ttccagggca
19740 gtgggtaaca aagtttgact ccagaaagac ttccctcgag gatttctact
tggatgaaga 19800 gaggaccgtg agggtcccca tgatgtcgga ccctaaggct
gttttacgct atggcttgga 19860 ttcagatctc agctgcaagg tctgtgggga
taggggcagg gtggggggtg gatggaggga 19920 gaggatagag aagcaaaaca
gggtagtggg aataaaatga cctttgagat ccgacagctg 19980 tctacatgtc
gcctgctgtg tgactttgag caggttaata acatgtctga gctttcctcc 20040
tcttaagatg gggcagggga tcgttaccaa cacttaccct cccagggttt gttgtaagga
20100 cgaataaggt aataggaaat gggccctcag cactgggcac ccacatgttt
gttctcttga 20160 gactcctatt tctagaattt aaagccaaac tttgaaaaat
aatgacaaac tccaaatcgt 20220 tggcatcttt tttttttttt gagacagtct
cgctctgtcg gccaggctgg agtccagtgg 20280 cacgatctcg gctcaccaca
acctccgccc ccgctgggtt aaagcgattc tcttgcctca 20340 gcctcctgag
tagctgggat tacaggcgtg tgcctccatg cctggctaat tttatacaga 20400
cggggtttct ccatgttggt caggctggtc tcaaactccc aaactcaggt gatccgcctg
20460 cctcggtctc ccaaaacaca ggggattcca ggcatgagcc accacgcttg
gccaatcgtt 20520 ggcattctaa ggctttcagt gtacctgact tcttttagtt
ctaagtctgt aactgttaac 20580 ctttcttggg ccacggctat cacacggatc
tctctgggaa tctgacgaca gtgcctcaaa 20640 cccgagggag caccgccagg
tgtgcacaca cgtttctgtc aacgatttcg gaggactctt 20700 gggatccctg
aacaccatct gttccatggg accttaggtt aagagcctct gttcaaagga 20760
ggcttttgct cttggtgggt ggatggggtg aagtctccaa gccctcttrc ggscccttcg
20820 gtattcctat nccccggttc tgccctgtct tagtccagtg ctctctattt
aacaaatgag 20880 cagtaaatgt acaccgatgg actttgggag acaataaaga
cctgatattc aattctagct 20940 ccttaaacca caggagaaca ttctttcagc
agacaacttc agttggtatt aggccaaggt 21000 aagaaaggcc aacagcatcc
ttttctgaag aaacctcagg agatggctct ctgccagaaa 21060 gctataacct
ggaaggggaa ttgtaaaata gatgaggggc tggatgaagg acgagaccag 21120
ggccccgtca cgggagaggg aaggcagctc ctggctgtgt ctgtcccccg gcttttgggc
21180 tctgaaggac taaccacatg ctttctcact tgtctcagat tgcccagctg
cccttgaccg 21240 gaagcatgag tatcatcttc ttcctgcccc tgaaagtgac
ccagaatttg accttgatag 21300 aggagagcct cacctccgag ttcattcatg
acatagaccg agaactgaag accgtgcagg 21360 cggtcctcac tgtccccaag
ctgaagctga gttacgaagg cgaagtcacc aagtccctgc 21420 aggagatgag
tatgtctgaa gaccctttcg ctcttggtgg gtggatgggg tggggcaggg 21480
tctttgggcc ttccactgtg ctaagcagaa cgcaagggct ccacaggctt gtaggggggc
21540 cgtggatgag tccttaatcc tcatcgtgcc agaagggaag gctgaactgc
cttctctcat 21600 cagactcatt cctcagcctc acgagcagac ctccctgaca
ggcgctcaca acactgcctc 21660 tcaagacgag tctgtctgac ctgttttctc
atcttgacct aacttgctaa atgctcctgg 21720 gcaagtcact ccaccctcgg
tcagctcaga cctcttcagg cctcagagaa agtcaacagt 21780 gctgcgccat
cccagcttgc ttgcaaaggg atcccttggt tggggtgttg gggaaggcag 21840
ggttttaacg gaaatctctc tccatctcta cagagctgca atccttgttt gattcaccag
21900 actttagcaa gatcacaggc aaacccatca agctgactca aggtggaaca
ccgggctggc 21960 tttgagtgga acgaggatgg ggcgggaacc acccccagcc
cagggctgca gcctgcccac 22020 ctcaccttcc cgctggacta tcaccttaac
cagcctttca tcttcgtact gagggacaca 22080 gacacagggg cccttctctt
cattggcaag attctggacc ccaggggccc ctaatatccc 22140 agtttaatat
tccaataccc tagaagaaaa cccgagggac agcagattcc acaggacacg 22200
aaggctgccc ctgtaaggtt tcaatgcata caataaaaga gctttatccc taacttctgt
22260 tacttcgttc ctcctcctat tttgagctat gcgaaatatc atatgaagag
aaacagctct 22320 tgaggaattt ggtggtcctc tacttctagc ctggttttat
ctaaacactg caggaagtca 22380 ccgttcataa gaactcttag ttacctgtgt
tggataaggc acggacagct tctctgctct 22440 gggggtattt ctgtactagg
atcagtgatc ctcccgggag ggcg 22484
* * * * *