U.S. patent application number 15/208424 was filed with the patent office on 2017-01-05 for ptd-smad7 therapeutics.
This patent application is currently assigned to The Regents of the University of Colorado, a body corporate. The applicant listed for this patent is The Regents of the University of Colorado, a body corporate. Invention is credited to Yosef REFAELI, Xiao-Jing WANG, Qinghong ZHANG.
Application Number | 20170000851 15/208424 |
Document ID | / |
Family ID | 51492023 |
Filed Date | 2017-01-05 |
United States Patent
Application |
20170000851 |
Kind Code |
A1 |
WANG; Xiao-Jing ; et
al. |
January 5, 2017 |
PTD-SMAD7 THERAPEUTICS
Abstract
The present technology provides methods and compositions for the
treatment of inflammatory and/or tissue damage conditions. In
particular, the use of Smad7 compositions delivered locally or
systemically to a site of inflammation and/or tissue damage is
described. Other specific embodiments concern treatment or
prevention of side effects caused by radiation and/or chemotherapy,
including but not limited to oral and gastric mucositis. Also
provided are codon-optimized nucleic acids encoding for Smad7
fusion proteins.
Inventors: |
WANG; Xiao-Jing; (Greenwood
Village, CO) ; ZHANG; Qinghong; (Englewood, CO)
; REFAELI; Yosef; (Denver, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Regents of the University of Colorado, a body
corporate |
Denver |
CO |
US |
|
|
Assignee: |
The Regents of the University of
Colorado, a body corporate
|
Family ID: |
51492023 |
Appl. No.: |
15/208424 |
Filed: |
July 12, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14201488 |
Mar 7, 2014 |
9422352 |
|
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15208424 |
|
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61775252 |
Mar 8, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 14/475 20130101;
A61P 37/02 20180101; A61K 38/162 20130101; C07K 2319/20 20130101;
C12N 7/00 20130101; A61K 48/005 20130101; A61P 17/06 20180101; C12N
2740/16322 20130101; C12N 2740/16311 20130101; A61K 38/00 20130101;
C07K 2319/23 20130101; C12N 2800/22 20130101; C07K 14/4703
20130101; A61P 29/00 20180101; A61K 38/18 20130101; C07K 14/4702
20130101; A61P 1/02 20180101; C07K 14/005 20130101; A61P 17/02
20180101; C07K 2319/10 20130101 |
International
Class: |
A61K 38/18 20060101
A61K038/18; C12N 7/00 20060101 C12N007/00; C07K 14/475 20060101
C07K014/475; A61K 38/16 20060101 A61K038/16; C07K 14/005 20060101
C07K014/005 |
Goverment Interests
STATEMENT OF GOVERNMENT INTEREST
[0002] This invention was made with government support under grant
number AR061796 awarded by the National Institutes of Health. The
government has certain rights in the invention.
Claims
1.-20. (canceled)
21. A fusion protein comprising a protein transduction domain and a
functional Smad7 domain, wherein the functional domain is selected
from the group consisting of amino acids 2-258 of the amino acid
sequence as set forth in SEQ ID NO: 12 and amino acids 259-426 of
the amino acid sequence as set forth in SEQ ID NO: 12.
22. The fusion protein of claim 21, wherein the protein
transduction domain is a variant of a Tat protein from HIV.
23. The fusion protein of claim 22, wherein the protein
transduction domain is selected from the group consisting of SEQ ID
NO: 2, SEQ ID NO: 4, and SEQ ID NO: 6.
24. The fusion protein of claim 21, wherein the fusion protein
further comprises one or more of an epitope tag or a purification
tag.
25. The fusion protein of claim 21, wherein the amino acid sequence
of the fusion protein is set forth in SEQ ID NO: 27.
26. The fusion protein of claim 25, wherein the functional Smad7
domain has one or more biological activities selected from the
group consisting of mediating cell migration, promoting cell
proliferation, reducing radiation-induced DNA damage, and blocking
fibrotic response.
27. The fusion protein of claim 21, wherein the amino acid sequence
of the fusion protein is set forth in SEQ ID NO: 25.
28. The fusion protein of claim 27, wherein the functional Smad7
domain has one or more biological activities selected from the
group consisting of reducing or eliminating phosphorylation of
Smad2, reducing or eliminating nuclear translocation of NF.kappa.B
p50 subunit, increasing cell proliferation, increasing epithelial
migration, reducing apoptosis, reducing radiation-induced DNA
damage, reducing inflammation, reducing angiogenesis, promoting
healing in oral mucositis, promoting wound healing, and treating
auto-immune disease.
29. A nucleic acid molecule encoding a fusion protein comprising a
protein transduction domain and a functional Smad7 domain, wherein
the functional domain is selected from the group consisting of
amino acids 2-258 of the amino acid sequence as set forth in SEQ ID
NO: 12 and amino acids 259-426 of the amino acid sequence as set
forth in SEQ ID NO: 12.
30. The nucleic acid molecule of claim 29, wherein the nucleic acid
molecule is codon-optimized for expression in one or more of
bacteria or yeast.
31. The nucleic acid molecule of claim 29, wherein the nucleic acid
molecule is selected from the group consisting of SEQ ID NOs: 26,
32.
32. The nucleic acid molecule of claim 29, wherein the nucleic acid
molecule is selected from the group consisting of SEQ ID NOs: 24,
33, 34.
33. The nucleic acid molecule of claim 29, wherein the protein
transduction domain is a variant of a Tat protein from HIV.
34. The nucleic acid molecule of claim 33, wherein the nucleic acid
sequence encoding the Tat protein from HIV is selected from the
group consisting of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, and
SEQ ID NO: 7.
35. A method for preventing or treating an inflammatory disease or
disorder in a subject, comprising providing to a subject in need
thereof a therapeutically effective amount of a pharmaceutical
composition comprising a fusion protein comprising a protein
transduction domain and a functional Smad7 domain, wherein the
functional domain is selected from the group consisting of amino
acids 2-258 of the amino acid sequence as set forth in SEQ ID NO:
12 and amino acids 259-426 of the amino acid sequence as set forth
in SEQ ID NO: 12.
36. The method of claim 35, wherein the inflammatory disease or
disorder is selected from the group consisting of wound healing,
auto-immune disease, psoriasis, oral mucositis, fibrosis, and
radiation-induced tissue damage.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 14/201,488, filed Mar. 7, 2014, which claims
priority to U.S. provisional patent application U.S. Ser. No.
61/775,252, filed Mar. 8, 2013, which are incorporated by reference
herein in their entireties.
SEQUENCE LISTING
[0003] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on May 28, 2014, is named 089491-0302_SL.txt and is 92,533 bytes in
size.
BACKGROUND
[0004] Oral mucositis, a severe oral ulceration, is a common
adverse effect of a large dose of radiation for bone marrow
transplant or craniofacial radiotherapy for cancer. Severe oral
mucositis could require feeding tubes, management of severe pain,
and prematurely halting radiotherapy. Excessive inflammation and
epithelial ablation are key features of oral mucositis.
[0005] Palifermin, a KGF (human keratinocyte growth factor)
recombinant protein, is approved for preventing oral mucositis in
bone-marrow transplant patients. Two Palifermin clinical trials in
head and neck cancer patients showed that Palifermin reduced severe
oral mucositis incidence from 67% and 69% to 51% and 54%,
respectively. Other oral mucositis drugs in clinical trials or
pre-clinical studies include growth factors, agents for
radioprotection, anti-inflammatory agents or immune modulators.
[0006] The modest effects of Palifermin and drugs being developed
in the above mentioned categories highlight the need for
identification of biomarkers for novel therapies. However, the lack
of routine diagnostic biopsies or discarded tissues from oral
mucositis patients has hindered this effort.
[0007] Cutaneous wound healing progresses through three overlapping
phases: inflammation, tissue formation, and tissue remodeling.
These are dynamic processes that involve interactions among the
epidermis, leukocytes, extracellular matrix (ECM), and dermal
fibroblasts. In response to skin injury, blood clots, infiltrated
inflammatory cells and other cell types in the wound release
multiple cytokines and chemokines. These cytokines initiate
fibroblast proliferation and synthesis of ECM that fill the wound
deficit and lead to wound closure.
[0008] Meanwhile, keratinocytes at the wound edge begin to
proliferate and migrate to cover the wound surface. Underneath the
re-epithelialized epidermis, new stroma, called granulation tissue,
begins to fill the wound space, which contains provisional ECM,
inflammatory cells, fibroblasts, and blood vessels. Once the wound
area is filled with the granulation tissue and covered by newly
re-epithelialized epidermis, the process of wound closure is
completed. Later on, the wound gradually returns to normal strength
and texture through tissue remodeling.
[0009] Among the many molecules known to influence wound healing,
transforming growth factor .beta. (TGF-.beta.) has the broadest
spectrum of action, affecting all cell types that are involved in
all stages of wound healing (Feng et al., Annu Rev Cell Dev Biol
21:659-693, 2005). The various functions of TGF-.beta. are mediated
by a number of signaling molecules, including the Smad family
members. When a ligand binds to TGF-.beta. type I and type II
receptors (TGF.beta.RI and TGF-.beta.RII), TGF-.beta.RI
phosphorylates Smad2 and Smad3. Phosphorylated Smad2 and Smad3 bind
a co-Smad, Smad4, to form heteromeric Smad complexes and
translocate into the nucleus to regulate transcription of
TGF-.beta. target genes.
[0010] TGF-.beta. signaling has been reported to exert both
positive and negative effects on wound healing (Wang et al., J
Investig Dermatol Symp Proc 11: 112-117, 2006). For instance, Smad3
deficient mice, in which TGF-.beta. signaling is partially
abrogated, exhibit accelerated wound healing (Ashcroft et al., Nat
Cell Biol 1:260-266, 1999). In contrast, the introduction of
exogenous Smad3 to wound sites to enhance TGF-.beta. signaling also
accelerated wound healing in a rabbit dermal ulcer model (Sumiyoshi
et al., J Invest Dermatol 123:229-236, 2004). Skin wounds in
Smad4-deficient mice have a dramatic increase in inflammation and
angiogenesis causing a delay in wound closure and formed an
excessive scar (Owens et al., Am J Pathol 176:122-133, 2010).
Transient adenoviral gene transfer of Smad7, an antagonist of
TGF-.beta. signaling, in corneal epithelium and stroma resulted in
accelerated corneal wound healing with reduced inflammation (Saika
et al., Am J Pathol 166:1405-1418, 2005). Further, Smad7 gene
transfer to the lens epithelium and stroma prevented injury-induced
epithelial-mesenchymal transition of lens epithelial cells and
suggests a potential role of Smad7 in prevention of capsular
fibrosis (Saika et al., Lab Invest 84:1259-1270, 2004). However,
adenoviral vector delivery of Smad7 to balloon injury in rat
carotid arteries resulted in reduced vascular healing
(Mallawaarachchi et al., Arterioscler Thromb Vasc Biol 25:
1383-1387, 2005). These studies suggest that the effects of
TGF-.beta. signaling components, such as Smad7, on wound healing
are complex and highly context-specific. Additionally, the effect
of Smad7 may not always be explained by its role in TGF-.beta.
signaling. For instance, Smad7 has also been shown to interact with
components of the Wnt/.beta.-catenin (Han et al., Dev Cell Biol
11:301-312, 2006) and the TNF.beta./NF-.kappa.B (Hong et al., Nat
Immunol 8:504-513, 2007) families.
SUMMARY
[0011] The present technology provides a nucleic acid molecule
comprising a codon-optimized human Smad7 cDNA nucleotide sequence.
In some embodiments, the codon-optimized human Smad7 nucleotide
sequence may include one or more codons for arginine optimized for
expression in one or more of bacteria or yeast, including one or
more codons for serine optimized for expression in one or more of
bacteria or yeast, and/or including one or more codons for
histidine optimized for expression in one or more of bacteria or
yeast. In some embodiments, the codon-optimized human Smad7
nucleotide sequence may include 28 serine codons, 6 histidine
codons, and 9 arginine codons optimized for expression in one or
more of bacteria or yeast. In some embodiments, the codon-optimized
human Smad7 nucleotide sequence may be selected from the group
consisting of SEQ ID NOs: 9, 21, 23, 24, 26, 28, 30, 32-34, 36, 38,
and 39. In some embodiments, the codon-optimized human Smad7
nucleotide sequence may have about 65 to 75 percent homology to
human Smad7 cDNA, may comprise a nucleotide sequence encoding an
N-terminal fragment SMAD7, may comprise a nucleotide sequence
encoding a C-terminal fragment of SMAD7, may comprise nucleotides
encoding amino acids 2-258 of the human Smad7 protein, may comprise
nucleotides encoding amino acids 259-426 of the human Smad7
protein, or may comprise nucleotides encoding amino acids 204-258
of the human Smad7 protein. In some embodiments, any of the
foregoing may further comprise a nucleotide sequence encoding a
protein transduction domain, such as Tat. In some embodiments, any
of the foregoing may also further comprise a nucleotide sequence
encoding one or more of an epitope tag or a purification tag, such
as V5, glutathione-S-transferase, or 6-Histidine (6H) (SEQ ID NO:
40).
[0012] In some embodiments, any of the foregoing may be isolated
and/or purified. In some embodiments, any one of the foregoing may
also encode a polypeptide having one or more biological activities
selected from the group consisting of reducing or eliminating
phosphorylation of Smad2, reducing or eliminating nuclear
translocation of the NF-.kappa.B p50 subunit, increasing cell
proliferation, reducing apoptosis, reducing radiation-induced DNA
damage, reducing inflammation, reducing angiogenesis, promoting
healing in oral mucositis, promoting wound healing, and treating
auto-immune disease. In some embodiments, pharmaceutical
compositions comprising the nucleic acid molecules above and one or
more pharmaceutically acceptable excipients are provided. In some
embodiments, expression vectors comprising the nucleic acid
molecules above operably linked to a promoter are provided, as are
host cells comprising such expression vectors, and pharmaceutical
compositions comprising such vectors and host cells with one or
more pharmaceutically acceptable excipients.
[0013] In one aspect, a protein molecule comprising a human Smad7
protein having leucine at position 216 is provided. In some
embodiments, the human Smad7 protein may be truncated at the
C-terminal, or truncated at the N-terminal. In some embodiments,
the truncated human Smad7 protein may include about 50% of the
full-length Smad7 sequence, or may include about 13% of the
full-length Smad7 sequence. In some embodiments, the human Smad7
protein may comprise or consist of amino acids 2-258, amino acids
204-258, or amino acids 259-426 of the human Smad7 protein. In some
embodiments, the protein molecule may have one or more biological
activities selected from the group consisting of reducing or
eliminating phosphorylation of Smad2, reducing or eliminating
nuclear translocation of the NF-.kappa.B p50 subunit, increasing
cell proliferation, reducing apoptosis, reducing radiation-induced
DNA damage, reducing inflammation, reducing angiogenesis, promoting
healing in oral mucositis, promoting wound healing, and treating
auto-immune disease. In some embodiments, any of the foregoing may
further comprise a protein transduction domain, such as Tat. In
some embodiments, any of the foregoing may also further comprise
one or more of an epitope tag or a purification tag, such as V5,
glutathione-S-transferase or 6-histidine (6H) (SEQ ID NO: 40). In
some embodiments, a pharmaceutical composition comprising any of
the foregoing, a protein molecule, and one or more pharmaceutically
acceptable excipients is provided.
[0014] In another aspect, a method is provided for treating or
preventing an inflammatory condition in a subject comprising
providing to the subject a therapeutically effective amount of the
pharmaceutical composition described above. In some embodiments,
the inflammatory condition may be one or more of a chronic wound,
skin inflammation, psoriasis, or an autoimmune disease. In some
embodiments, the composition may reduce inflammation through
inhibition of TGF-.beta. and NF-.kappa.B signaling.
[0015] In another aspect, a method is provided for preventing or
treating a disease or disorder in a subject comprising one or more
of increasing one or more of cell proliferation or cell migration,
or preventing one or more of apoptosis or DNA damage in the subject
comprising providing to the subject a therapeutically effective
amount of the pharmaceutical composition as described above,
wherein one or more of increasing one or more of cell proliferation
or cell migration, or preventing one or more of apoptosis or DNA
damage is useful in preventing or treating the disease or disorder.
In some embodiments, the disease or disorder may include one or
more of chronic wounds, acute wounds, or mucositis. In some
embodiments, the chronic wounds may include one or more of diabetic
ulcers, pressure ulcers, venous ulcers, or oral ulcers, the acute
wounds may include one or more of trauma-induced wounds, surgical
wounds, or scarring, the mucositis may include one or more of
radiation-induced mucositis or chemotherapy-induced mucositis and
the mucositis may include one or more of oral mucositis or gut
mucositis.
[0016] It is contemplated that any method or composition described
herein can be implemented with respect to any other method or
composition described herein
[0017] The use of the word "a" or "an" when used in conjunction
with the term "comprising" in the claims and/or the specification
may mean "one," but it is also consistent with the meaning of "one
or more," "at least one," and "one or more than one." The word
"about" means plus or minus 5% of the stated number.
[0018] Other objects, features and advantages of the present
technology will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating specific
embodiments of the present technology, are given by way of
illustration only, since various changes and modifications within
the spirit and scope of the present technology will become apparent
to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The following drawings form part of the present
specification and are included to further demonstrate certain
embodiments of the present technology. The embodiments may be
better understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0020] FIGS. 1A-G provide an illustrative embodiment of data
showing that K5.Smad7 mice were resistant to radiation-induced oral
mucositis. FIG. 1A provides an illustrative embodiment of H&E
staining in non-irradiated and irradiated (day 9 after initiation
of radiation) wild-type (WT) and K5.Smad7 tongues. The vertical
lines in the images of tongues from WT mice highlight the ulcer
boundary and dotted lines in the images indicate the
epithelial-stromal boundary (scale bar, 50 .mu.m). FIG. 1B provides
a graphical representation of the quantification of sizes of tongue
ulcers (mean.+-.s.e.m); n=8 for WT mice and n=7 for K5.Smad7 mice
in 8 Gy.times.3 radiation; n=5 for WT mice and n=4 for K5.Smad7
mice in 18-Gy radiation; n=5 per group for WT and K5.Smad7 mice in
22-Gy radiation. FIG. 1C provides an illustrative embodiment of
human ventricular posterior of the tongue (top) and
radiation-induced tongue mucositis (bottom) visualized using
H&E (left) and CD45 staining (right). The solid line indicates
the ulcer boundary, and dotted lines indicate the basement membrane
(scale bar, 25 .mu.m). FIG. 1D provides an illustrative embodiment
of immunostaining of CD45, proliferating cell nuclear antigen
(PCNA), and TUNEL assay in irradiated sections adjacent to an ulcer
from WT mice and in damaged areas from K5.Smad7 mice (PI, propidium
iodide). Dotted lines indicate the basement membrane (scale bar, 25
.mu.m). FIGS. 1E-1G provide graphical representations of the
quantification of staining in FIG. 1D (n=3 or 4 per group). Data
are expressed as mean.+-.s.e.m (FIG. 1B) or mean.+-.s.d (FIGS.
1E-1G), and two-tail Student t-test is used to calculate P values.
*P<0.05, **P<0.01, ***P<0.001, NS, no significance
determined by two-tailed Student's t-test. Dotted lines in (FIG.
1A), (FIG. 1C) and (FIG. 1D) highlight the basement membrane. Scale
bar: 50 .mu.m for all panels in (FIG. 1A) and (FIG. 1C), 25 .mu.m
for all panels in (FIG. 1D).
[0021] FIGS. 2A-G provide an illustrative embodiment of data
showing molecular alterations attenuated by Smad7. FIG. 2A provides
an illustrative embodiment of immune-staining of NF-.kappa.B p50,
TGF-.beta.1 and pSmad2. Irradiated tongue sections of wild-type
(WT) were adjacent to ulcer and sections of K5.Smad7 were from the
damaged area. Human samples were from non-irradiated oral mucosa
and radiation-induced mucositis. Dotted lines delineate
epithelial-stromal boundary. Scale bar, 25 .mu.m for all panels.
FIG. 2B provides a graphical representation of the quantification
of immunostaining of NF-.kappa.B p50 and pSmad2 shown in (FIG. 2A).
FIG. 2C provides an illustrative embodiment of qRT-PCR of
TGF-.beta.1 (normalized by Keratin 5, n=6 per group for day 0, n=4
for day 7 and day 9, and n=7 for day 10). FIG. 2D provides a
graphical representation of the quantification of human oral
keratinocyte migration (see images in FIG. 8). Scrambled, scrambled
siRNA; n=3 per group. FIG. 2E provides an illustrative embodiment
of a western analysis of knockdown efficiency of siSmad7-1 and
siSmad7-2 for Smad7 and for Rac1, 72 hours after Smad7 knockdown.
M, molecular markers. FIG. 2F provides an illustrative embodiment
of western analysis of total and activated (GTP-bound) Rac1
protein. M: molecular markers. FIG. 2G provides a graphical
representation of the quantification of the effect of Rac1
knockdown on Smad7-mediated keratinocyte migration (see knockdown
efficiency in FIG. 9A and images in FIG. 9D). n=3 per group. Data
are presented as mean.+-.s.d. and two-tail Student's t-test was
used to calculate P values for (FIGS. 2B-2D) and (FIG. 2G).
*P<0.05, **P<0.01, ***P<0.001. NS, no significance.
[0022] FIGS. 3A-H provide an illustrative embodiment of data
showing Smad7 increased Rac1 expression by repressing individual
Smad and CtBP1 binding to the SBE of the Rac1 promoter. FIG. 3A
provides a graphical representation of the quantification of Rac1
mRNA in wild-type (WT) and Smad7 transgenic keratinocytes. n=4 per
group. FIG. 3B provides an illustrative embodiment of western
analysis of GTP-Rac1 and total Rac1 in WT and Smad7 keratinocytes.
Smad7 protein levels in WT and Smad7 keratinocytes were determined
by reprobing the tubulin western blot with an antibody to Smad7
(see an additional western blot and quantification in FIGS. 10A-B).
FIG. 3C provides an illustrative embodiment of western analysis of
Rac1 protein level after knocking down individual Smad2, Smad3 or
Smad4 in human keratinocytes (see FIG. 10C-10E for Smad knockdown
efficiencies). FIG. 3D provides an illustrative embodiment of a
ChIP assay for Smad-2, -3, -4, and -7 binding to the -1.5 Kb SBE
site of the Rac1 promoter in WT and Smad7 transgenic keratinocytes.
FIG. 3E provides a graphical representation of the quantification
of Rac1 luciferase reporter assay in mouse keratinocytes.
Scrambled: scrambled siRNA. n=6. FIG. 3F provides a graphical
representation of the quantification of the activities of Rac1
luciferase reporter containing SBE or mutant (mut) SBE in WT or
Smad7 transgenic keratinocytes. n=6. FIG. 3G provides an
illustrative embodiment of images of ChIP assays of CtBP1 binding
to the SBE-1.5 Kb site of the Rac1 promoter in WT or K5.Smad7
keratinocytes. FIG. 3H provides a graphical representation of
ChIP-qPCR quantification of CtBP1 binding to the SBE shown in FIG.
3G in WT and Smad7 transgenic keratinocytes. n=4. Data are
presented as mean.+-.s.d. and two-tail Student's t-test is used to
calculate P values for FIGS. 3A, 3E, 3F and 3H. *P<0.05,
**P<0.01, ***P<0.001.
[0023] FIGS. 4A-G provide an illustrative embodiment of data
showing CtBP1-associated Rac1 repression contributed to inhibition
of keratinocyte migration. FIG. 4A provides an illustrative
embodiment of western analysis of Rac1 protein after knockdown of
CtBP1 in human oral keratinocytes. FIG. 4B provides a graphical
representation of the quantification of SBE-containing Rac1 luc
reporter activity. n=6. FIG. 4C provides a graphical representation
of the quantification of the effect of CtBP1 knockdown on human
oral keratinocyte migration. n=3 per group. FIG. 4D provides an
illustrative embodiment of immunostaining of CtBP1. Irradiated
sections were adjacent to the ulcer (WT) or the damaged area
(K5.Smad7). Dotted lines denote the basement membrane. Scale bar,
50 .mu.m for all panels. FIG. 4E provides an illustrative
embodiment of immunostaining of CtBP1 in non-irradiated oral mucosa
and radiation-induced oral mucositis in human specimens. Dotted
lines denote the basement membrane. Scale bar, 50 .mu.m for both
panels. FIG. 4F provides a graphical representation of the
quantification of CtBP1 nuclear positive cells in FIGS. 4D-E. n=3
or 4 per group. FIG. 4G provides a graphical representation of the
quantification of qRT-PCR for CtBP1 (normalized with Keratin K5).
n=6 per group for day 0, n=4 for day 7 and day 9, and n=7 for day
10. Data are presented as mean.+-.s.d. and two-tail Student's
t-test is used to calculate P values for FIGS. 4B, 4C, 4F and 4G.
*P<0.05, **P<0.01, ***P<0.001.
[0024] FIGS. 5A-G provide an illustrative embodiment of data
showing oral Tat-Smad7 application prevented radiation-induced oral
mucositis in mice. FIG. 5A provides a graphical representation of
the quantification of oral mucositis ulcer sizes on day 9 after
initiation of 8 Gy.times.3 radiation. Vehicle=saline or 50%
glycerol/PBS. FIG. 5B provides an illustrative embodiment of
pathological alterations on day 9 of initiation of 8 Gy.times.3
radiation. Vehicle=saline or 50% glycerol/PBS. Scale bar, 50 .mu.m
for H&E panels and 25 .mu.m for remaining panels. Dotted lines
delineate epithelial-stromal boundary; the solid line highlights
the ulcer boundary. FIGS. 5C, 5D, 5E, 5F, and 5G provide a
graphical representation of the quantification of immunostaining
shown in FIG. 5B. n=3 or 4 per group. Data are presented as
mean.+-.s.e.m (FIG. 5A) or mean.+-.s.d. (FIGS. 5C-5G) and two-tail
Student's t-test is used to calculate P values. *P<0.05,
**P<0.01, ***P<0.001. NS, no significance.
[0025] FIGS. 6A-G provide an illustrative embodiment of data
showing Tat-Smad7 treatment on oral mucositis. FIG. 6A provides a
graphical representation of the quantification of ulcer sizes
measured on day 10 after initiation of 8 Gy.times.3 radiation.
Glycerol=50% glycerol/PBS. FIG. 6B provides an illustrative
embodiment of H&E staining of oral mucosa. Upper panels: open
ulcer in Palifermin treated but not Tat-Smad7 treated mucosa. Lower
panels: comparison of epithelial thickness between Palifermin
treated and Tat-Smad7 treated mucosa. Dotted lines delineate the
basement membrane. The vertical line highlights the ulcer boundary.
Scale bar, 50 .mu.m for all panels. FIG. 6C provides an
illustrative embodiment of immune-staining of Tat-Smad7 treatment
in 20 Gy-induced oral mucositis after ulcers healed. V5
immunostaining visualizes Tat-Smad7 in oral epithelia (sections are
away from the damaged regions). K14 immunostaining was used as
counterstain. Dotted lines delineate basement membrane. Scale bar,
25 .mu.m for all panels. FIG. 6D provides an illustrative
embodiment of Rac1 western analysis of Tat-Smad7 treated mouse
tongues, day 10 after initiation of 8 Gy.times.3 radiation. FIG. 6E
provides an illustrative embodiment of Rac1 western analysis on
Tat-Smad7 treated normal human oral keratinocytes 48 hours after
treatment. FIG. 6F provides an illustrative embodiment of the
effect of Tat-Smad7 treatment on oral human keratinocyte migration
(NOK-SI, see images in FIG. 13A). n=4 per group. FIG. 6G provides a
graphical representation of the quantification of survival curves
of NOK-SI keratinocytes and SCC lines (Cal27 and MSK921) with or
without Tat-Smad7 treatment. n=4 per group for each radiation dose.
Data are presented as mean.+-.s.e.m (FIG. 6A) or mean.+-.s.d.
(FIGS. 6F, 6G) and two-tail Student's t-test is used to calculate P
values. *P<0.05, **P<0.01,***P<0.001. NS, no
significance.
[0026] FIGS. 7A-E provide an illustrative embodiment of data
showing K5.Smad7 oral mucosal tissues were resistant to
radiation-induced oral mucositis. FIG. 7A provides an illustrative
embodiment of Smad7 western blots: undetectable in non-irradiated
wild-type (WT) tongue and barely detectable after radiation.
K5.Smad7 tongues have comparable Smad7 protein levels before and
after radiation. M: molecular marker. FIG. 7B provides an
illustrative embodiment of Smad7 immunostaining. Note that nuclei
in some irradiated epithelial cells are hypertrophic. Dotted lines
delineate epithelial-stromal boundary. FIG. 7C provides a graphical
representation of the quantification of reduced incidence of oral
mucositis-induced morbidity in K5.Smad7 mice. Fisher's exact test
is used to calculate the p value. **P=0.007. FIG. 7D provides an
illustrative embodiment of immune-staining of K5. Smad7 tongue
showing reduced infiltration of neutrophils (Ly-6G), macrophages
(BM8) and activated T cells (CD4) compared to WT oral mucositis.
Dotted lines delineate epithelial-stromal boundary. FIG. 7E
provides an illustrative embodiment of immune-staining showing no
significant difference in pSmad1/5/8-nuclear positive cells (green)
between WT and K5.Smad7 oral mucosa before or after radiation.
Keratin (K14) immunostaining (red) highlights the epithelial
compartment. Note that nuclei of irradiated epithelial cells are
hypertrophic. The scale bar is 50 .mu.m for all panels.
[0027] FIGS. 8A-D provide an illustrative embodiment of data
showing migration in spontaneously immortalized human oral
epithelial cells (NOK-SI) was delayed by knocking down Smad7 but
accelerated by knocking down TGF-.beta.1. FIGS. 8A and 8B provide
an illustrative embodiment of representative images of cell
migration. Pairs of dotted lines delineate the scratch wound.
Quantification of cell migration and efficiency of Smad7 knockdown
are presented in FIG. 2D and FIG. 2E (above). Scrambled, scrambled
siRNA. FIG. 8C provides a graphical representation of the
quantification of cell migration after TGF-.beta.1 knockdown from 3
separate experiments. FIG. 8D provides a graphical representation
of qRT-PCR showing TGF-.beta.1 knockdown efficiency. Data are
presented as mean.+-.s.d. and two-tail Student's t-test was used to
calculate P values. *P<0.05, **P<0.01. NS, no
significance.
[0028] FIGS. 9A-D provide an illustrative embodiment of data
showing knocking down Rac1 reduced proliferation and migration of
wild-type (WT) and Smad7 transgenic keratinocytes. FIG. 9A provides
an illustrative embodiment of western blot analysis for Rac1 48
hours after Rac1 siRNA (siRac1-1, siRac1-2) transfection. Control,
scrambled siRNA. FIG. 9B provides a graphical representation of the
percentage of BrdU labeled cells in WT and Smad7 cultured cells in
BrdU incorporation assay with or without Rac1 knockdown. Data from
3 separate experiments were presented as mean.+-.s.d.
***P<0.001. FIG. 9C provides an illustrative embodiment of
representative immunofluorescence of BrdU positive cells presented
in (FIG. 9B). An antibody against keratin 14 (K14, red) was used
for counterstain. FIG. 9D provides an illustrative embodiment of in
vitro cell migration assay for Smad7 transgenic and WT
keratinocytes after Rac1 knockdown. Pairs of dotted lines delineate
the scratch wound. Quantification of cell migration is presented in
FIG. 2G.
[0029] FIGS. 10A-F provide an illustrative embodiment of data
showing Smad7 increased Rac1 expression by repressing Smad and
CtBP1 binding to the SBE of the Rac1 promoter. FIG. 10A provides an
illustrative embodiment of western blot analysis for GTP-Rac1 and
total Rac1 in Smad7 transgenic keratinocytes. Additional samples
are shown in FIG. 3B. M, molecular marker. FIG. 10B provides a
graphical representation of the quantification of GTP-Rac1, total
Rac1 and Smad7 in WT and K5.Smad7 keratinocytes shown in FIG. 10A
and in FIG. 3B. The protein level in WT keratinocytes of each blot
was normalized as "1". Data is presented as mean.+-.s.d. and
two-tail Student's t-test was used to calculate P values.
**P<0.01,***P<0.001. FIGS. 10C and 10D provide an
illustrative embodiment of western blot analysis for Smad2, Smad3,
and Smad4 knockdown in NOK-SI cells. Their effects on Rac1
expression are shown in FIG. 3C. M, molecular marker. GAPDH,
internal protein control by reprobing same blot. FIG. 10F provides
an illustrative embodiment showing CtBP1 knockdown promotes NOK-SI
cell migration. Pairs of dotted lines delineate the scratch wound.
Quantification of cell migration and efficiency of CtBP1 knockdown
are shown in FIG. 4A and FIG. 4C.
[0030] FIGS. 11A-G provide an illustrative embodiment of data
showing the purification and characterization of Tat-Smad7 and
Tat-Cre proteins. FIG. 11A shows an illustrative embodiment of a
schematic representation of Tat-Smad7 protein. FIG. 11A discloses
SEQ ID NOs: 49 and 87, respectively, in order of appearance. FIG.
11B provides an illustrative embodiment of a western blot of
purified Tat-Smad7 protein. FIG. 11C provides an illustrative
embodiment of immune-staining of Tat-Smad7 protein transduction in
keratinocytes. Left and middle panels: Tat-Smad7 staining (green)
using a V5 antibody, counterstained with a K14 antibody (red).
Cells showed Tat-Smad7 in the nucleus 5 min after transduction and
in both nucleus and cytoplasm 12 hours after transduction. Right
panels: Tat-Smad7 abrogated Smad2 phosphorylation (pSmad2, green).
V5 (red) counterstain visualizes Tat-Smad7 transduced cells. FIG.
11D provides an illustrative embodiment of immune-staining showing
that V5 antibody staining detects Tat-Smad7 transduction in buccal
mucosa 12 hours after Tat-Smad7 topical application. A K14 antibody
was used for counterstain. Scale bar, 50 .mu.m for both panels.
FIG. 11E provides an illustrative embodiment of a western blot of
purified Tat-Cre protein with the same Tat and V5 tags shown in
FIG. 11A. FIG. 11F provides an illustrative embodiment of an
agarose gel showing activity of Tat-Cre: Tat-Cre cuts out a 1,460
bp floxed fragment from the 7,650 bp vector pLL3.7. FIG. 11G
provides a graphical representation showing Tat-Smad7 protein
preventive treatment reduced 20 Gy radiation-induced oral ulcers.
Data are expressed as mean.+-.s.e.m. Two-tail Student's t-test is
used to calculate P values. *P<0.05, ***P<0.001.
[0031] FIGS. 12A-I provide an illustrative embodiment of data
showing effects of Tat-Smad7 treatment on oral mucositis. FIG. 12A
provides a graphical representation of the quantification of
reduced ulcer size in Tat-Smad7 (0.8 .mu.g daily, day 6 to day 9)
treated oral mucosa. Samples were harvested on day 10. n=8 per
group. FIG. 12B provides an illustrative embodiment of
immunostaining of molecular markers for samples from FIG. 12A.
Scale bar, 50 .mu.m for the top two panels and 25 .mu.m for other
panels. Propidium iodide (PI) and K14 were used as counterstain.
FIGS. 12C-G provide graphical representation of the quantifications
of immunostaining shown in FIG. 12C. 3-4 samples were used. FIG.
12H provides a graphical representation quantification of the
Luciferase assay. Tat-Smad7 treatment increased the activity of the
Rac1 promoter with SBE but not the mutant SBE in mouse
keratinocytes. FIG. 12I provides an illustrative embodiment of a
ChIP assay for CtBP1 binding to the SBE of mouse Rac1 promoter in
Tat-Smad7 treated mouse keratinocytes. Data are expressed as
mean.+-.s.e.m (a) or mean.+-.s.d (c-h) and two-tail Student's
t-test is used to calculate P values. *P<0.05, **P<0.01,
***P<0.001. NS, no significance.
[0032] FIGS. 13A-H provide an illustrative embodiment of data
showing effects of Tat-Smad7 treatment on migration of human
keratinocytes and tumor cell lines. FIG. 13A provides an
illustrative embodiment showing Tat-Smad7 accelerates NOK-SI cell
migration. Quantification from four separate experiments is shown
in FIG. 6F (above). Pairs of dotted lines delineate initial wounds.
FIG. 13B provides an illustrative embodiment of immunostaining of
Tat-Smad7 treatment in NOK-SI cells showing attenuated
radiation-induced pSmad2 and NF-.kappa.B p50 nuclear localization.
FIG. 13C provides an illustrative embodiment showing V5 staining of
MSK921 cells 2 hours after Tat-Smad7 treatment. K14 staining was
used as counterstain. FIG. 13D provides an illustrative embodiment
of a Rac1 western analysis in MSK921 60 hours after Tat-Smad7
treatment. M, molecular marker. FIG. 13E provides a graphical
representation of quantification of MSK921 cell migration from 3
separate experiments. FIG. 13F provides an illustrative embodiment
showing a representative MSK921 cell migration assay treated with
Tat-Smad7 and PBS. Pairs of solid lines delineate initial wounds.
Dotted lines highlight the forefront of migrated cells. FIG. 13G
provides a graphical representation of quantification of Cal27 cell
migration from 3 separate experiments. FIG. 13H provides an
illustrative embodiment showing representative images for FIG. 13G.
Pairs of solid lines delineate initial wounds. Dotted lines
highlight the forefront of migrated cells. Data are expressed as
mean.+-.s.d. and the two-tail Student's t-test is used to calculate
P values. NS, no significance.
[0033] FIGS. 14A-B show an illustrative schematic of a summary of
potential mechanisms of Smad7-mediated protection and healing of
oral mucositis. FIG. 14A shows an illustrative schematic of how
radiation activates NF-.kappa.B, increases TGF-.beta.1 and CtBP1.
NF-.kappa.B and TGF-.beta.1 induce inflammation. TGF-.beta.1
induces apoptosis, growth arrest and activates Smad-2, -3 and -4,
which recruit CtBP1 to the Rac1 promoter to repress Rac1
transcription, leading to blunted re-epithelialization. FIG. 14B
shows an illustrative schematic of how Smad7 blocks NF-.kappa.B and
TGF-.beta.1-induced inflammation and blocks TGF-.beta.1-induced
apoptosis and growth arrest. Smad7 relieves Rac1 transcriptional
repression by either preventing TGF-.beta.1-mediated Smad
activation (phosphorylation) or competing with signaling
Smads/CtBP1 transcriptional repression complex in binding to the
Rac1 promoter. Increased Rac1 induced by Smad7 contributes to
keratinocyte migration during re-epithelialization.
[0034] FIG. 15 shows an illustrative schematic of Smad 7 domains
associated with protein partners, potential target effects, and
potential physiological effects.
DETAILED DESCRIPTION
[0035] As further described herein, the disclosure provides Smad7
proteins and biologically active fragments and derivatives thereof,
nucleic acids encoding such proteins, vectors including such
nucleic acids, and cells encompassing the vectors, nucleic acids,
and/or proteins all for use in formulating medicaments and for
treating and/or preventing one or more diseases or disorders. Also
provided are methods for making and for screening Smad7 proteins
and biologically active fragments and derivatives thereof useful
for treating and/or preventing one or more diseases or disorders.
Also provided are methods for predicting and/or evaluating a
response to treatment using one or more markers associated with
exposure to Smad7. Such markers may include, but are not limited
to, Rac1 for cell migration, NF-.kappa.B for inflammation, and
TGF-.beta. for growth arrest and inflammation.
[0036] Smad7 treatable diseases and disorders may include those
including one or more of reduced cell proliferation, reduced cell
migration, increased cell death, excessive inflammation, and/or DNA
damage. Smad7 treatable diseases and disorders may include those
where treatment with a Smad7 protein and biologically active
fragments and derivatives thereof that have one or more activities
including but not limited to increasing proliferation, reducing or
inhibiting cell death, reducing excessive inflammation, preventing
DNA damage, and/or increasing cell migration. Such diseases and/or
disorders may include but are not limited to acute (e.g., through
surgery, combat, trauma) and chronic wounds (e.g., ulcers, such as
diabetic, pressure, venous), scarring, fibrosis, and aberrant
healing, mucositis (e.g., oral and/or gastro-intestinal),
stomatitis, proctitis, autoimmune disease (e.g., psoriasis,
arthritis), and cancer.
[0037] It is critical for oral mucositis prevention and treatment
to overcome epithelial ablation due to massive apoptosis and
blunted keratinocyte proliferation. The proliferative and
anti-apoptotic effects of Smad7 are more obvious in oral mucositis
than in normal oral mucosa, when TGF-.beta.1, a potent growth
inhibitor and apoptosis inducer for epithelial cells, was
increased.
[0038] Although not wishing to be bound by theory, it is believed
that increased Rac1 activation is largely responsible for
Smad7-mediated keratinocyte migration in wound closure. This
finding was unexpected, given the documented role of TGF-.beta.
signaling in Rho/Rac activation in cancer cells via a
Smad-independent mechanism (Dernyck et al., Nature 415:577-584,
2003).
[0039] It is believed that during oral mucositis, Smad-dependent
Rac1 repression overcomes Smad-independent Rac1 activation (if any)
due to increased Smad signaling (evidenced by increased pSmad2) and
Smad transcriptional co-repressor CtBP1. When this repression is
abrogated by Smad7, it permits Rac1 activation-mediated
keratinocyte migration. However, in oral cancer cells, signaling
Smads are lost or inactivated, or other mechanisms independently
activate Rac1. As a result, Smad7-mediated abrogation of Rac1
repression would no longer occur.
[0040] Although Rac1 activation also contributed to keratinocyte
proliferation, knocking down Rac1 only partially attenuated the
proliferative effect of Smad7. Therefore, Rac1's contribution to
proliferation appears to be limited, and blocking
TGF-.beta.1-induced growth arrest is also needed to overcome
radiation-induced growth inhibitory effects.
[0041] Dampening excessive inflammation creates a microenvironment
for oral mucositis healing. The antagonistic effect of Smad7 on
both TGF-.beta. and NF-.kappa.B signaling makes Smad7 a more
efficient anti-inflammatory molecule than other agents targeting
only NF-.kappa.B. Because inflammatory cells produce cytokines that
further activate TGF-.beta. and NF-.kappa.B, reduced TGF-.beta. and
NF-.kappa.B signaling, found in K5.Smad7 or Tat-Smad7 treated oral
mucosa after radiation, reflects the direct antagonistic effect of
Smad7 on these two pathways and the consequence of reduced
inflammatory cytokines from infiltrated leukocytes. However, Smad7
did not reduce NF-.kappa.B or TGF-.beta. signaling below their
normal physiological conditions. This incomplete blockade of
NF-.kappa.B or TGF-.beta. signaling may be beneficial to oral
mucositis healing, as a complete loss of either pathway could
induce excessive inflammation.
[0042] The primary obstacle to using growth factors to treat oral
mucositis in cancer patients is the potential risk of promoting
cancer cell growth. The majority of human oral cancers lose
TGF-.beta. signaling in tumor epithelial cells. Thus,
anti-Smad-associated cell proliferation and migration by Smad7
would not be effective in cancer cells. In tumors with intact
TGF-.beta. signaling, activation of other oncogenic pathways could
override TGF-.beta.-induced tumor suppressive effects. These two
scenarios could explain why there was no observation of Smad7
increasing proliferation and migration in oral cancer cells with
mutant or intact TGF-.beta. signaling components.
[0043] Additionally, TGF-.beta. signaling promotes tumor invasion
mainly through Smad-independent mechanisms after loss of
TGF-.beta.-induced tumor suppression. Thus, blocking TGF-.beta.
signaling by Smad7 in cancer cells could abrogate
TGF-.beta.-mediated tumor promotion effects, which behaves
similarly to TGF-.beta. inhibitors currently being used in clinical
trials for advanced cancers. Further, the potent anti-inflammatory
effect of Smad7 may reduce the risk of tumor progression.
Therefore, long-term Smad7 application may also be helpful in
cancer treatment.
[0044] Spontaneous tumor formation in K5.Smad7 mice has not been
observed. Because Smad7 is not a secreted protein, local and
short-term Smad7 protein delivery in oral mucositis treatment
should have few systemic effects. In bone marrow transplant
patients, whose oral epithelia do not contain cancer cells, Smad7
topical application may be suitable for both prevention and
treatment of oral mucositis.
[0045] Although not wishing to be bound by any theory,
Smad7-mediated oral mucositis healing appears to be a result of
targeting multiple pathogenic processes mediated by one or more
molecules (see, e.g., FIGS. 14A-B). It is believed that one or more
of these molecules (e.g., TGF-.beta., NF-.kappa.B, CtBP1, Rac1) may
also be helpful as predictive and therapeutic responsive markers of
oral mucositis in patients.
A. Nucleic acids, Vectors and Host Cells
[0046] The present disclosure also provides, in another embodiment,
genes encoding Smad7. In addition to the wild-type SMAD7 gene (SEQ
ID NO: 22), which encodes the wild-type Smad7 protein (SEQ ID NO:
12), as well as various codon-optimized versions (SEQ ID NOs: 9,
21, 23, 24, 26, 28, 30, 32-34, 36, 38, and 39), it should be clear
that the present technology is not limited to the specific nucleic
acids disclosed herein. As discussed below, a "Smad7 gene" may
contain a variety of different bases and yet still produce a
corresponding polypeptide that is functionally indistinguishable
from, and in some cases structurally identical to, the human gene
disclosed herein.
[0047] 1. Nucleic Acids Encoding Smad7
[0048] Nucleic acids according to the present technology may
represent an entire Smad7 gene, a truncated portion, and/or a
fragment of Smad7 that expresses a polypeptide with one or more
activity associated with Smad7 such as but not limited to
increasing proliferation, reducing or inhibiting cell death,
reducing excessive inflammation, preventing DNA damage, and/or
increasing cell migration, as well as treating or preventing one or
more disease or disorders in which such treatment would be helpful
as further discussed herein. Such activities can be assessed using
one or more assays including, but not limited to, the ability to
block phosphorylation of Smad2 and/or nuclear translocation of the
NF-.kappa.B p50 subunit, increase cell proliferation, reduce
apoptosis and/or radiation-induced DNA damage, reduce inflammation
and/or angiogenesis, promote healing in oral mucositis, surgical
wounds, diabetes wounds, and/or wounds associated with chronic
inflammation in mice. The nucleic acid may be derived from genomic
DNA, i.e., cloned directly from the genome of a particular
organism. In particular embodiments, however, the nucleic acid
would comprise complementary DNA (cDNA). Also provided is a cDNA
plus a natural intron or an intron derived from another gene; such
engineered molecules are sometime referred to as "mini-genes." At a
minimum, these and other nucleic acids of the present technology
may be used as molecular weight standards in, for example, gel
electrophoresis.
[0049] The term "cDNA" is intended to refer to DNA prepared using
messenger RNA (mRNA) as template. The advantage of using a cDNA, as
opposed to genomic DNA or DNA polymerized from a genomic, non- or
partially-processed RNA template, is that the cDNA primarily
contains coding sequences of the corresponding protein. There may
be times when the full or partial genomic sequence is preferred,
such as where the non-coding regions are required for optimal
expression or where non-coding regions such as introns are to be
targeted in an antisense strategy.
[0050] As used in this application, the term "a nucleic acid
encoding a Smad7" may refer to a nucleic acid molecule that has
been isolated free of total cellular nucleic acid and/or may refer
to a cDNA encoding a Smad7 polypeptide. As used herein, the term
"isolated free of total cellular nucleic acid" means that the
nucleic acid molecule is about or at least about 75% pure, 80%
pure, 85% pure, 90% pure, 95% pure, 96% pure, 97% pure, 98% pure,
99% pure, or 100% pure of other cellular nucleic acid molecules as
determined using standard biochemical techniques, such as but not
limited to agarose gel electrophoresis. As used herein, the term
"isolated free of total cellular protein" means that the protein
molecule is about or at least about 75% pure, 80% pure, 85% pure,
90% pure, 95% pure, 96% pure, 97% pure, 98% pure, 99% pure, or 100%
pure of other cellular nucleic acid molecules as determined using
standard biochemical techniques, such as but not limited to a
western blot. In certain embodiments, the present technology
concerns a nucleic acid sequence essentially as set forth in,
and/or including any one of SEQ ID NOs: 9, 21, 23, 24, 26, 28, 30,
32-34, 36, 38, and 39.
[0051] An isolated nucleic acid molecule may be produced using
recombinant DNA technology (e.g., polymerase chain reaction (PCR)
amplification, cloning) or chemical synthesis. Isolated nucleic
acid molecules include natural nucleic acid molecules and
homologues thereof, including, but not limited to, natural allelic
variants and modified nucleic acid molecules in which nucleotides
have been inserted, deleted, substituted, and/or inverted in such a
manner that such modifications provide the desired effect (e.g.,
production of Smad7 protein in non-human expression systems).
[0052] The term "essentially as set forth in one or more nucleic
acid sequence (e.g., SEQ ID NOs: 9, 21, 23, 24, 26, 28, 30, 32-34,
36, 38, and 39)" means that the nucleic acid sequence substantially
corresponds to at least a portion, and in some cases the entirety,
of the one or more nucleic acid sequence (e.g., SEQ ID NOs: 9, 21,
23, 24, 26, 28, 30, 32-34, 36, 38, and 39). In some embodiments,
sequences that substantially correspond to at least a portion of a
nucleic acid sequence, may correspond to about, or at least about
50 nucleic acids, 75 nucleic acids, 150 nucleic acids, 200 nucleic
acids, 250 nucleic acids, 300 nucleic acids, 350 nucleic acids, 400
nucleic acids, 450 nucleic acids, 500 nucleic acids, 550 nucleic
acids, 600 nucleic acids, 650 nucleic acids, 700 nucleic acids, 750
nucleic acids, 800 nucleic acids, 900 nucleic acids, 1000 nucleic
acids, 1100 nucleic acids, 1200 nucleic acids, or 1250 nucleic
acids of one or more of the sequences described herein. In some
embodiments, sequences that substantially correspond to at least a
portion of a nucleic acid sequence, may correspond to about a range
of about 50-1250 nucleic acids, 75-1250 nucleic acids, 150-1250
nucleic acids, 200-1250 nucleic acids, 250-1250 nucleic acids,
300-1250 nucleic acids, 350-1250 nucleic acids, 400-1250 nucleic
acids, 450-1250 nucleic acids, 500-1250 nucleic acids, 550-1250
nucleic acids, 600-1250 nucleic acids, 650-1250 nucleic acids,
700-1250 nucleic acids, 750-1250 nucleic acids, 800-1250 nucleic
acids, 900-1250 nucleic acids, 1000-1250 nucleic acids, 1100-1250
nucleic acids, 1200-1250 nucleic acids, at least about 50-75
nucleic acids, 75-150 nucleic acids, 75-200 nucleic acids, 75-250
nucleic acids, 75-300 nucleic acids, 75-350 nucleic acids, 75-400
nucleic acids, 75-450 nucleic acids, 75-500 nucleic acids, 75-550
nucleic acids, 75-600 nucleic acids, 75-650 nucleic acids, 75-700
nucleic acids, 75-750 nucleic acids, 75-800 nucleic acids, 75-900
nucleic acids, 75-1000 nucleic acids, 75-1100 nucleic acids,
75-1200 nucleic acids, or 75-1250 nucleic acids or 1250 nucleic
acids of one or more of the sequences described herein.
[0053] In some embodiments, sequences that substantially correspond
to at least a portion of a nucleic acid sequence include identical
sequences to that portion of the nucleic acid sequence. In some
embodiments, sequences that substantially correspond to at least a
portion of a nucleic acid sequence or the entirety of a nucleic
acid sequence may include one or more functionally equivalent
codons. The term "functionally equivalent codon" is used herein to
refer to one or more codons that encode the same amino acid, such
as the six codons for arginine or serine, and in some embodiments
refers to codons that encode biologically equivalent amino acids,
as discussed in the following pages. The term "biologically
equivalent" amino acid is used herein to refer to one or more amino
acids that when changed from the amino acid present in the amino
acid sequence of human Smad7 wild-type protein, do not change one
or more (or in some embodiments any) of the biological activities
of Smad7 described herein, such as but not limited to, increasing
proliferation, reducing or inhibiting cell death, reducing
excessive inflammation, preventing DNA damage, and/or increasing
cell migration, as well as treating or preventing one or more
disease or disorders in which such treatment would be helpful as
further discussed herein.
[0054] In some embodiments, allowing for the degeneracy of the
genetic code, sequences that have about or at least about 60%, 70%,
80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and/or 99% of
nucleotides that are identical to the nucleotides of any one of the
codon-optimized nucleic acid sequences (e.g., SEQ ID NOs: 9, 21,
23, 24, 26, 28, 30, 32-34, 36, 38, and 39) may be considered
substantially corresponding nucleic acid sequences. Sequences that
are essentially the same as those set forth in any one of the
nucleic acid sequences (e.g., SEQ ID NOs: 9, 21, 23, 24, 26, 28,
30, 32-34, 36, 38, and 39) also may be functionally defined as
sequences that are capable of hybridizing to a nucleic acid segment
containing the complement of SEQ ID NOs: 9, 21, 23, 24, 26, 28, 30,
32-34, 36, 38, and 39 under various standard conditions.
[0055] For applications requiring high selectivity, one will
typically desire to employ relatively high stringency conditions to
form the hybrids. For example, relatively low salt and/or high
temperature conditions, such as provided by about 0.02 M to about
0.10 M NaCl at temperatures of about 50.degree. C. to about
70.degree. C. Such high stringency conditions tolerate little, if
any, mismatch between the probe or primers and the template or
target strand and would be particularly suitable for isolating
specific genes or for detecting specific mRNA transcripts. It is
generally appreciated that conditions can be rendered more
stringent by the addition of increasing amounts of formamide.
[0056] For certain applications it is appreciated that lower
stringency conditions are preferred. Under these conditions,
hybridization may occur even though the sequences of the
hybridizing strands are not perfectly complementary, but are
mismatched at one or more positions. Conditions may be rendered
less stringent by increasing salt concentration and/or decreasing
temperature. For example, a medium stringency condition could be
provided by about 0.1 to 0.25 M NaCl at temperatures of about
37.degree. C. to about 55.degree. C., while a low stringency
condition could be provided by about 0.15 M to about 0.9 M salt, at
temperatures ranging from about 20.degree. C. to about 55.degree.
C. Hybridization conditions can be readily manipulated depending on
the desired results.
[0057] In other embodiments, hybridization may be achieved under
conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3
mM MgCl.sub.2, 1.0 mM dithiothreitol, at temperatures between
approximately 20.degree. C. to about 37.degree. C. Other
hybridization conditions utilized could include approximately 10 mM
Tris-HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl.sub.2, at temperatures
ranging from approximately 40.degree. C. to about 72.degree. C.
[0058] To determine the percent homology of two amino acid
sequences or of two nucleic acids, the sequences are aligned for
optimal comparison purposes (e.g., gaps are introduced in the
sequence of a first amino acid or nucleic acid sequence for optimal
alignment with a second amino acid or nucleic acid sequence). The
amino acid residues or nucleotides at corresponding amino acid
positions or nucleotide positions can then be compared. When a
position in the first sequence is occupied by the same amino acid
residue or nucleotide as the corresponding position in the second
sequence, then the molecules are identical at that position. The
percent homology between the two sequences is a function of the
number of identical positions shared by the sequences (% identity=#
of identical positions/total # of positions (e.g., overlapping
positions).times.100). In some embodiments the two sequences are
the same length.
[0059] To determine percent homology between two sequences, the
algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA
87:2264-2268, modified as in Karlin and Altschul (1993) Proc. Natl.
Acad. Sci. USA 90:5873-5877 can be used. Such an algorithm is
incorporated into the NBLAST and XBLAST programs of Altschul et al.
(1990) J. Mol Biol. 215:403-410. BLAST nucleotide searches is
performed with the NBLAST program, score=100, wordlength=12 to
obtain nucleotide sequences homologous to a nucleic acid molecules
described or disclose herein. BLAST protein searches is performed
with the (BLAST program, score=50, wordlength=3. To obtain gapped
alignments for comparison purposes, Gapped BLAST may be utilized as
described in Altschul et al. (1997) Nucleic Acids Res.
25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the
default parameters of the respective programs (for example, XBLAST
and NBLAST) are used. See the website of the National Center for
Biotechnology Information for further details (on the World Wide
Web at ncbi.nlm.nih.gov). Proteins suitable for use in the methods
described herein also includes proteins having between 1 to 15
amino acid changes, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,
12, 13, 14, or 15 amino acid substitutions, deletions, or
additions, compared to the amino acid sequence of any protein
described herein. In other embodiments, the altered amino acid
sequence is at least 75% identical, for example, 77%, 80%, 82%,
85%, 88%, 90%, 92%, 95%, 97%, 98%, 99%, or 100% identical to the
amino acid sequence of any protein inhibitor described herein. Such
sequence-variant proteins are suitable for the methods described
herein as long as the altered amino acid sequence retains
sufficient biological activity to be functional in the compositions
and methods described herein. In certain instances conservative
amino acid substitutions are utilized. Illustrative conservative
substitution among amino acids are within each of the following
groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2)
phenylalanine, tyrosine, and tryptophan, (3) serine and threonine,
(4) aspartate and glutamate, (5) glutamine and asparagine, and (6)
lysine, arginine and histidine. The BLOSUM62 table is an amino acid
substitution matrix derived from about 2,000 local multiple
alignments of protein sequence segments, representing highly
conserved regions of more than 500 groups of related proteins
(Henikoff et al. (1992), Proc. Natl Acad. Sci. USA,
89:10915-10919). The BLOSUM62 substitution frequencies can be used
to define conservative amino acid substitutions that, in some
embodiments, are introduced into the amino acid sequences described
or disclosed herein. Although it is possible to design amino acid
substitutions based solely upon chemical properties (as discussed
above), the language "conservative amino acid substitution"
preferably refers to a substitution represented by a BLOSUM62 value
of greater than -1. For example, an amino acid substitution is
conservative if the substitution is characterized by a BLOSUM62
value of 0, 1, 2, or 3. According to this system, preferred
conservative amino acid substitutions are characterized by a
BLOSUM62 value of at least 1 (e.g., 1, 2 or 3), while more
preferred conservative amino acid substitutions are characterized
by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
[0060] The DNA segments of the present technology include those
encoding biologically functional equivalent Smad7 proteins and
peptides, as described above. Such sequences may arise as a
consequence of codon redundancy and amino acid functional
equivalency that are known to occur naturally within nucleic acid
sequences and the proteins thus encoded. Alternatively,
functionally equivalent proteins or peptides may be created via the
application of recombinant DNA technology, in which changes in the
protein structure may be engineered, based on considerations of the
properties of the amino acids being exchanged. Changes designed by
man may be introduced through the application of site-directed
mutagenesis techniques or may be introduced randomly and screened
later for the desired function, as described elsewhere.
[0061] As described in greater detail below, the Smad7 nucleic acid
sequence has been optimized for expression in alternative host
organisms (e.g., non-human). Although as described above, the
genetic code is degenerate, so frequently one amino acid may be
coded for by two or more nucleotide codons. Thus, multiple nucleic
acid sequences may encode one amino acid sequence. Although this
creates identical proteins, the nucleic acids themselves are
distinct, and can have distinct properties. As described herein,
one aspect of the choice of codon usage can be (but is not limited
to) the ability to express a protein in a non-native cells (e.g., a
human protein in bacteria or yeast), or the level of expression in
such cells. In order to obtain enough protein for purification,
testing, and use in in vitro assays, in animal models, and
eventually in clinical development, efficient protein expression in
non-human systems is needed.
[0062] A series of 23 arginine amino acids in the human Smad7
protein sequence coded for by one or more of AGG (1.7% codon
utilization; 9 residues), AGA (2.8% codon utilization; 2 residues),
CGA (3.5% codon utilization; 4 residues), or CGG (5.4% codon
utilization; 8 residues) has been identified, and it has been
determined that in order to have efficient protein expression from
non-human sources, such as, but not limited to, bacteria and/or
yeast that one or more, and potentially all the arginine codons
should be modified to CGT (20.6% codon utilization). Therefore, in
some embodiments, the Smad7 codon-optimized nucleic acid sequence
includes at least 1, at least 2, at least 3, at least 4, at least
5, at least 6, at least 7, at least 8, at least 9, at least 10, at
least 11, at least 12, at least 13, at least 14, at least 15, at
least 16, at least 17, at least 18, at least 19, at least 20, at
least 21, at least 22, or 23 codons for arginine that have been
changed to CGT. In some embodiments, the Smad7 codon-optimized
nucleic acid sequence includes one or more or all of the arginine
codons at nucleic acid sequence positions 7-9, 43-45, 169-171,
403-405, 490-492, 526-528, 526-528, 823-825, 1057-1059, 16-18,
136-138, 199-201, 598-600, 31-33, 112-114, 316-318, 772-774,
940-942, 973-975, 1135-1137, 1276-1278, 637-639, or 814-816 be
changed to CGT.
[0063] A series of 33 serine residues in the human Smad7 protein
sequence coded for by TCC or TCG (9%) has been identified, and it
has been determined that it may be beneficial to efficient protein
expression and purification from non-human sources, such as, but
not limited to, bacteria and/or yeast, that one or more, and
potentially all the serine codons be modified to AGC (15% codon
utilization). Therefore, in some embodiments, the Smad7
codon-optimized nucleic acid sequence includes at least 1, at least
2, at least 3, at least 4, at least 5, at least 6, at least 7, at
least 8, at least 9, at least 10, at least 11, at least 12, at
least 13, at least 14, at least 15, at least 16, at least 17, at
least 18, at least 19, at least 20, at least 21, at least 22, at
least 23, at least 24, at least 25, at least 26, at least 27, at
least 28, at least 29, at least 30, at least 31, at least 32 or 33
codons for serine that have been changed to (AGC). In some
embodiments, the Smad7 codon-optimized nucleic acid sequence
includes one or more or all of the serine codons at nucleic acid
sequence positions 19-21, 46-48, 133-135, 292-294, 349-351,
451-453, 454-456, 460-462, 511-513, 514-516, 544-546, 595-597,
616-618, 634-636, 691-693, 694-696, 739-741, 745-747, 775-777,
847-849, 907-909, 919-921, 943-945, 1006-1008, 1009-1101,
1030-1032, 1054-1056, 1093-1095, 1126-1128, 1192-1194, 1237-1239,
1240-1242, 1273-1275. Of these, 23 codons (19-21, 292-294, 349-351,
451-453, 454-456, 460-462, 511-513, 514-516, 544-546, 616-618,
634-636, 691-693, 694-696, 739-741, 745-747, 775-777, 847-849,
907-909, 919-921, 1009-1101, 1030-1032, 1054-1056, 1093-1095) can
be changed without introducing potential alternative open reading
frames.
[0064] A series of 12 histidine residues in the human Smad7 protein
sequence coded for by CAC (9.6% codon usage) has also been
identified, and it has been determined that it may be beneficial to
efficient protein expression and purification from non-human
sources, such as but not limited to bacteria and/or yeast, that one
or more, and potentially all the serine codons be modified to CAT
(optionally to 12.6% usage). Therefore, in some embodiments, the
Smad7 codon-optimized nucleic acid sequence includes at least 1, at
least 2, at least 3, at least 4, at least 5, at least 6, at least
7, at least 8, at least 9, at least 10, at least 11, or 12 codons
for histidine that have been changed to (CAT). In some embodiments,
the Smad7 codon-optimized nucleic acid sequence includes one or
more or all of the serine codons at nucleic acid sequence positions
142-144, 214-216, 217-219, 220-222, 226-228, 289-291, 589-591,
778-780, 1072-1074, 1147-1149. Of these, 4 codons (nucleotides
217-219, 220-222, 589-591, 778-780) can be changed without
introducing potential alternative open reading frames.
[0065] In some embodiments, one or more codon-optimized nucleic
acids may include one or more of at least one and any integer up to
22 of its arginine codons modified to CGT, at least one and any
integer up to 28 of its serine codons (optionally that are able to
be modified with introducing open reading frames) modified to AGC,
or at least one and any integer up to 12 of its histidine codons
(optionally that are able to be modified with introducing open
reading frames) modified to CAT. In some embodiments, one or more
codon-optimized nucleic acid may include at least one and any
integer up to 22 of its arginine codons modified to CGT, at least
one and any integer up to 28 of its serine codons (optionally that
are able to be modified with introducing open reading frames)
modified to AGC, and at least one and any integer up to 12
(optionally that are able to be modified with introducing open
reading frames) of its histidine codons modified to CAT. In some
embodiments, one or more codon-optimized nucleic acid may include
22 of its arginine codons modified to CGT, 28 of its serine codons
(optionally that are able to be modified with introducing open
reading frames) modified to AGC, and 12 of its histidine codons
(optionally that are able to be modified with introducing open
reading frames) modified to CAT. In some embodiments, one or more
codon-optimized nucleic acid may also have a nucleotide
substitution in the codon for Met216 (ATG), to form the codon for
Leu216 (CTG).
[0066] In some embodiments, one or more codon-optimized nucleic
acids may have about 65% to 75%, about 65% to 68%, about 68% to
75%, or about 68% to 71% homology to human Smad7 wild-type cDNA
(SEQ ID NO: 22), which encodes the amino acid sequence as set forth
in SEQ ID NO: 12. In some embodiments, one or more codon-optimized
nucleic acid may have about 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
73%, 74%, or 75%, homology to human Smad7 wild-type cDNA (SEQ ID
NO: 22). In some embodiments, one or more codon-optimized nucleic
acid may also have a nucleotide substitution in the codon for
Met216 (ATG), to form the codon for Leu216 (CTG).
[0067] A methionine codon (Met216; ATG) that has the potential for
being perceived by translation machinery (e.g., such as but not
limited bacteria or yeast) as an alternative open reading frame has
been identified. Although not intending to be bound by theory, it
is believed that the presence of the second potential open reading
frame may decrease expression of the Smad7 protein. In some
embodiments, one or more Smad7 nucleic acid sequences are modified
at nucleotide position (646-648) to encode a human Smad7 protein
where Met216 (ATG) is modified to Leu216 (CTG).
[0068] It has also been discovered that various truncated forms and
fragments of Smad7 protein retain one or more of the activities of
full-length human Smad7, such as, but not limited to, increasing
proliferation, reducing or inhibiting cell death, reducing
excessive inflammation, preventing DNA damage, and/or increasing
cell migration, as well as treating or preventing one or more
disease or disorders in which such treatment would be helpful as
further discussed herein. Such activities can be assessed using one
or more assays including, but not limited to, the ability to block
phosphorylation of Smad2 and/or nuclear translocation of the
NF-.kappa.B p50 subunit, increase cell proliferation, reduce
apoptosis and/or radiation-induced DNA damage, reduce inflammation
and/or angiogenesis, promote healing in oral mucositis, surgical
wounds, diabetes wounds, and/or wounds associated with chronic
inflammation in mice.
[0069] Further, in some embodiments, various truncated forms and
fragments of Smad7 protein retain only a subset of the one or more
of the activities of full-length human Smad7. For example, the
C-terminal MH2 domain of Smad7 may primarily mediate the
anti-inflammatory effect of Smad7. Smad7 peptides having this
anti-inflammatory function may be sufficient and optionally an
improvement for treating chronic inflammation associated
conditions, such as but not limited to, oral mucositis, stomatitis,
arthritis, and psoriasis, among others. The N-terminal MH1 domain
may primarily mediate cell migration and/or blocking
TGF-.beta.-induced growth arrest and/or fibrotic response. Smad7
peptides having this cell migration and proliferation function may
be sufficient, and optionally an improvement, for enhancing healing
that is not associated with excessive inflammation. Types of wounds
that might benefit from this form of treatment include, but are not
limited to, surgical wounds, fibrotic scarring, and diabetes
wounds, defective healing and/or scarring among others.
[0070] In some embodiments, nucleic acid molecules (optionally
codon-optimized nucleic acid molecules as described above and
herein) encode fragments or truncated forms of Smad7 protein
(optionally including Leu216). In some embodiments, these fragments
and/or truncated forms of Smad7 protein retain one or more or all
of the activities of full-length human Smad7 protein. In some
embodiments, such truncated nucleic acid sequences encode the
N-terminal portion of the Smad7 protein. In some embodiments, such
truncated nucleic acid sequences encode the C-terminal portion of
the Smad7 protein. In some embodiments, such truncated nucleic acid
sequences (nucleotide positions 4-774) encode amino acids 2-258 of
the human Smad7 protein. In some embodiments, such truncated
nucleic acid sequences (nucleotide positions 775-1278) encode amino
acids 259-426 of the human Smad7 protein. In some embodiments, such
fragments of the nucleic acid sequences (nucleotide positions
610-774) encode amino acids 204-258 of the human Smad7 protein.
[0071] The term "truncated" as used herein in reference to nucleic
acid molecules refers to a molecule that contains nucleotide
sequences encoding the natural N-terminus of a corresponding
protein (with or without a cleaved leader sequence), but lacks one
or more nucleotides starting from the C-terminus-encoding portion
of the molecule, or a molecule that contains nucleotide sequences
encoding the natural C-terminus of a corresponding protein (with or
without a cleaved leader sequence), but lacks one or more
nucleotides starting from the N-terminus-encoding portion of the
molecule. In some embodiments, molecules lacking nucleotides
encoding at least about 25, at least about 50, at least about 75,
at least about 100, at least about 125, at least about 150, at
least about 200, at least about 250, at least about 300, or at
least about 350, or at least about 400 amino acids from one or the
other terminus are specifically provided. Similarly, the term
"truncated" may also be used in reference to protein molecules
encoded by truncated nucleic acid molecules. In some embodiments, a
"truncated" molecule is biologically active, having (or encoding a
polypeptide having) one or more of the Smad7 activities described
herein.
[0072] The term "fragment" as used herein in reference to nucleic
acid molecules refers to a molecule containing contiguous residues
of a full length sequence but lacking some 5' and/or 3' sequences
of the full length sequence. In some embodiments, a "fragment"
includes a portion of one or more of the full length sequences
described herein. In some embodiments, the "fragment" does not
include sequences encoding either the N-terminal or the C-terminal,
but only internal fragments. In some embodiments, a "fragment"
encodes a polypeptide that is biologically active, having one or
more of the Smad7 activities described herein. In some embodiments,
nucleic acid fragments may encode proteins having at least about
25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100,
150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750,
800, 850, 900, 950, 1000, 1050, 1100, 1150 amino acids. Similarly,
"fragment" may also be used in reference to protein molecules
encoded by Smad7 nucleic acid fragments.
[0073] The term "N-terminal portion" as used herein in refers to a
fragment of a corresponding protein that contains the protein's
N-terminus but lacks all sequences C-terminal to an internal
residue.
[0074] The term "C-terminal portion" as used herein in refers to a
fragment of a corresponding protein that contains the protein's
C-terminus but lacks all sequences N-terminal to an internal
residue.
[0075] Although not intending to be bound by theory, the Smad7
protein activity is generally believed to be the result of
interactions in both the cytoplasm and nucleus of a cell. For that
reason among others, there existed a general belief that Smad7
protein was not a candidate for a therapeutic role. However, it was
decided to pursue development of Smad7 as a protein therapeutic,
and modify the Smad7 nucleic acid sequence to encode a protein
transduction domain (PTD) in frame with the Smad7 nucleic acid
sequence (e.g., optionally any nucleic acid sequence described
herein encoding Smad7 protein, including human wild-type and
codon-optimized sequences, both full-length and biologically active
fragments or truncated portions). In some embodiments, the PTD is
located at the 3' end of the Smad7 nucleic acid sequence, and in
some embodiments the PTD is located at the 5' end of the Smad7
nucleic acid sequence. In some embodiments, there is a linker
sequence encoding 1, 2, 3, 4, 5, or 6 amino acids that connects the
PTD and the Smad7 nucleic acid sequence.
[0076] In some embodiments, the PTD nucleic acid sequence is a Tat
nucleic acid sequence. ggccgtaaaaaacgccgtcaacgccgccgt (SEQ ID NO:
1) encoding GRKKRRQRRR (SEQ ID NO: 2),
tatggccgtaaaaaacgccgtcaacgccgccgt (SEQ ID NO: 3) encoding
YGRKKRRQRRR (SEQ ID NO: 4), or ggccgtaaaaaacgccgtcaa (SEQ ID NO: 5)
encoding GRKKRRQ (SEQ ID NO: 6).
[0077] In some embodiments, the nucleic acid sequence further
includes a nucleotide sequence encoding one or more of an epitope
tag or a purification tag. In some embodiments, the epitope tag is
V5. In some embodiments, the purification tag is one or more of
glutathione-S-Transferase (GST) or 6-histidine (6H) (SEQ ID NO:
40).
[0078] The term "epitope tag" as used herein in reference to
nucleic acid molecules refers to nucleotides encoding peptide
sequences that are recognized and bound by the variable region of
an antibody or fragment. In some embodiments, the epitope tag is
not part of the native protein. In some embodiments, the epitope
tag is removable. In some embodiments, the epitope tag is not
intrinsic to the protein's native biological activity. Examples of
epitope tags include, but are not limited to V5.
[0079] The term "purification tag" as used herein in reference to
nucleic acid molecules refers to nucleotides encoding peptide
sequences that facilitate the purification of the protein, but are
generally not necessary for the protein's biological activity. In
some embodiments, purification tags may be removed following
protein purification. Examples of purification tags include, but
are not limited to GST and 6H (SEQ ID NO: 40).
[0080] 2. Vectors for Cloning, Gene Transfer and Expression
[0081] Within certain embodiments, expression vectors are employed
to express the Smad7 polypeptide product, which can then be
purified for various uses. In other embodiments, the expression
vectors are used in gene therapy. Expression requires that
appropriate signals be provided in the vectors, and which include
various regulatory elements, such as enhancers/promoters from both
viral and mammalian sources that drive expression of the genes of
interest in host cells. Elements designed to optimize messenger RNA
stability and translatability in host cells also are defined. The
conditions for the use of a number of dominant drug selection
markers for establishing permanent, stable cell clones expressing
the products are also provided, as is an element that links
expression of the drug selection markers to expression of the
polypeptide.
[0082] Throughout this application, the term "expression construct"
is meant to include any type of genetic construct containing a
nucleic acid coding for a gene product in which part or all of the
nucleic acid encoding sequence is capable of being transcribed. The
transcript may be translated into a protein, but it need not be. In
certain embodiments, expression includes both transcription of a
gene and translation of mRNA into a gene product. In other
embodiments, expression only includes transcription of the nucleic
acid encoding a gene of interest.
[0083] The term "vector" is used to refer to a carrier nucleic acid
molecule into which a nucleic acid sequence can be inserted for
introduction into a cell where it can be replicated. A nucleic acid
sequence can be "exogenous," which means that it is foreign to the
cell into which the vector is being introduced or that the sequence
is homologous to a sequence in the cell but in a position within
the host cell nucleic acid in which the sequence is ordinarily not
found. Vectors include plasmids, cosmids, viruses (bacteriophage,
animal viruses, and plant viruses), and artificial chromosomes
(e.g., YACs). One of skill in the art would be well-equipped to
construct a vector through standard recombinant techniques, which
are described, e.g., in Sambrook, et al., Molecular Cloning (Cold
Spring Harbor Lab Press, 1989), and Ausubel, et al., Current
Protocols in Molecular Biology (Wiley, 1994), both incorporated
herein by reference.
[0084] The term "expression vector" refers to a vector containing a
nucleic acid sequence coding for at least part of a gene product
capable of being transcribed. In some cases, RNA molecules are then
translated into a protein, polypeptide, or peptide. In other cases,
these sequences are not translated, for example, in the production
of antisense molecules or ribozymes. Expression vectors can contain
a variety of "control sequences," which refer to nucleic acid
sequences necessary for the transcription and possibly translation
of an operably linked coding sequence in a particular host
organism, including promoters and enhancers. In addition to control
sequences that govern transcription and translation, vectors and
expression vectors may contain nucleic acid sequences that serve
other functions, such as transcription termination signals and
poly-adenylation sites.
[0085] The capacity of certain viral vectors to efficiently infect
or enter cells, to integrate into a host cell genome and stably
express viral genes, have led to the development and application of
a number of different viral vector systems. Robbins, et al.,
Pharmacol. Ther. 80:35-47 (1998). Viral systems are currently used
as vectors for ex vivo and in vivo gene transfer. For example,
adenovirus, herpes-simplex virus, lentiviruses, retrovirus and
adeno-associated virus vectors are being evaluated currently for
treatment of diseases such as cancer, cystic fibrosis, Gaucher
disease, renal disease and arthritis. Robbins, et al., Pharmacol.
Ther. 80:35-47 (1998); Imai, et al., Nephrologie 19:379-402 (1998);
U.S. Pat. No. 5,670,488. The various viral vectors present specific
advantages and disadvantages, depending on the particular
gene-therapeutic application.
[0086] Suitable non-viral methods for nucleic acid delivery for
transformation of an organelle, a cell, a tissue or an organism for
use with the present technology are believed to include virtually
any method by which a nucleic acid (e.g., DNA) can be introduced
into an organelle, a cell, a tissue or an organism, as described
herein or as would be known to one of ordinary skill in the art.
Such methods include, but are not limited to, direct delivery of
DNA such as by injection (U.S. Pat. Nos. 5,994,624, 5,981,274,
5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466
and 5,580,859, each incorporated herein by reference), including
microinjection (Harland and Weintraub, 1985; U.S. Pat. No.
5,789,215, incorporated herein by reference); by electroporation
(U.S. Pat. No. 5,384,253, incorporated herein by reference); by
calcium phosphate precipitation (Graham, et al., Virology
52:456-467 (1973); Chen, et al., Mol. Cell Biol. 7:2745-2752
(1987); Rippe, et al., Mol. Cell Biol. 10:689-695 (1990)); by using
DEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell
Biol. 5:1188-1190 (1985)); by direct sonic loading (Fechheimer, et
al., PNAS 84:8463-8467 (1987)); by liposome mediated transfection
(Nicolau, et al., Biochim. Biophys. Acta 721:185-190 (1982);
Fraley, et al., PNAS 76:3348-3352 (1979); Nicolau, et al., Methods
Enzymol. 149: 157-176 (1987); Wong, et al., Gene 10:87-94 (1980);
Kaneda, et al., J. Biol. Chem. 264:12126-12129 (1989); Kato, et
al., J. Biol. Chem. 266:3361-3364 (1991)); by microprojectile
bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S.
Pat. Nos. 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and
5,538,880, and each incorporated herein by reference); by agitation
with silicon carbide fibers (Kaeppler, et al., Plant Cell Rep.
9:415-418 (1990); U.S. Pat. Nos. 5,302,523 and 5,464,765, each
incorporated herein by reference); or by PEG-mediated
transformation of protoplasts (Omirulleh, et al., Plant Mol. Biol.
21:415-428 (1993); U.S. Pat. Nos. 4,684,611 and 4,952,500, each
incorporated herein by reference); by
desiccation/inhibition-mediated DNA uptake (Potrykus, et al., Mol.
Gen. Genet. 199:169-177 (1985)). Through the application of
techniques such as these, organelle(s), cell(s), tissue(s) or
organism(s) may be stably or transiently transformed.
[0087] 3. Expression Systems
[0088] Numerous expression systems exist that comprise at least a
part or all of the compositions discussed above. Prokaryote- and/or
eukaryote-based systems can be employed for use with the present
technology to produce nucleic acid sequences, or their cognate
polypeptides, proteins and peptides. Many such systems are
commercially and widely available.
[0089] The insect cell/baculovirus system can produce a high level
of protein expression of a heterologous nucleic acid segment, such
as described in U.S. Pat. Nos. 5,871,986 and 4,879,236, both herein
incorporated by reference, and which can be bought, for example,
under the name MAXBAC.RTM. 2.0 from INVITROGEN.RTM. and BACPACK.TM.
BACULOVIRUS EXPRESSION SYSTEM FROM CLONTECH.RTM..
[0090] Other examples of expression systems include
STRATAGENE.RTM.'s COMPLETE CONTROL.TM. Inducible Mammalian
Expression System, which involves a synthetic ecdysone-inducible
receptor, or its pET Expression System, an E. coli expression
system. Another example of an inducible expression system is
available from INVITROGEN.RTM., which carries the T-REX.TM.
(tetracycline-regulated expression) System, an inducible mammalian
expression system that uses the full-length CMV promoter.
INVITROGEN.RTM. also provides a yeast expression system called the
Pichia methanolica Expression System, which is designed for
high-level production of recombinant proteins in the methylotrophic
yeast Pichia methanolica. One of skill in the art would know how to
express a vector, such as an expression construct, to produce a
nucleic acid sequence or its cognate polypeptide, protein, or
peptide.
[0091] Primary mammalian cell cultures may be prepared in various
ways. In order for the cells to be kept viable while in vitro and
in contact with the expression construct, it is necessary to ensure
that the cells maintain contact with the correct ratio of oxygen
and carbon dioxide and nutrients but are protected from microbial
contamination. Cell culture techniques are well documented.
[0092] One embodiment of the foregoing involves the use of gene
transfer to immortalize cells for the production of proteins. The
gene for the protein of interest may be transferred as described
above into appropriate host cells followed by culture of cells
under the appropriate conditions. The gene for virtually any
polypeptide may be employed in this manner. The generation of
recombinant expression vectors, and the elements included therein,
are discussed above. Alternatively, the protein to be produced may
be an endogenous protein normally synthesized by the cell in
question.
[0093] Examples of useful mammalian host cell lines are Vero and
HeLa cells and cell lines of Chinese hamster ovary, W138, BHK,
COS-7, 293, HepG2, NIH3T3, RIN and MDCK cells. In addition, a host
cell strain may be chosen that modulates the expression of the
inserted sequences, or modifies and process the gene product in the
manner desired. Such modifications (e.g., glycosylation) and
processing (e.g., cleavage) of protein products may be important
for the function of the protein. Different host cells have
characteristic and specific mechanisms for the post-translational
processing and modification of proteins. Appropriate cell lines or
host systems can be chosen to insure the correct modification and
processing of the foreign protein expressed.
[0094] A number of selection systems may be used including, but not
limited to, HSV thymidine kinase, hypoxanthine-guanine
phosphoribosyltransferase and adenine phosphoribosyltransferase
genes, in tk-, hgprt- or aprt-cells, respectively. Also,
anti-metabolite resistance can be used as the basis of selection
for dhfr, that confers resistance to; gpt, that confers resistance
to mycophenolic acid; neo, that confers resistance to the
aminoglycoside G418; and hygro, that confers resistance to
hygromycin.
[0095] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. All of these terms also
include their progeny, which are any and all subsequent
generations. It is understood that all progeny may not be identical
due to deliberate or inadvertent mutations. In the context of
expressing a heterologous nucleic acid sequence, "host cell" refers
to a prokaryotic or eukaryotic cell, and it includes any
transformable organism that is capable of replicating a vector
and/or expressing a heterologous gene encoded by a vector. A host
cell can, and has been, used as a recipient for vectors. A host
cell may be "transfected" or "transformed," which refers to a
process by which exogenous nucleic acid is transferred or
introduced into the host cell. A transformed cell includes the
primary subject cell and its progeny.
[0096] Host cells may be derived from prokaryotes or eukaryotes
(e.g., bacteria or yeast), depending upon whether the desired
result is replication of the vector or expression of part or all of
the vector-encoded nucleic acid sequences. Numerous cell lines and
cultures are available for use as a host cell, and they can be
obtained through the American Type Culture Collection (ATCC), which
is an organization that serves as an archive for living cultures
and genetic materials (atcc.org). An appropriate host can be
determined by one of skill in the art based on the vector backbone
and the desired result. A plasmid or cosmid, for example, can be
introduced into a prokaryote host cell for replication of many
vectors. Bacterial cells used as host cells for vector replication
and/or expression include DH5.alpha., JM109, and KC8, as well as a
number of commercially available bacterial hosts such as SURE.RTM.
Competent Cells and SOLOPACK.TM. Gold Cells (STRATAGENE.RTM., La
Jolla). Alternatively, bacterial cells such as E. coli LE392 could
be used as host cells for phage viruses.
[0097] Examples of eukaryotic host cells for replication and/or
expression of a vector include HeLa, NIH3T3, Jurkat, 293, Cos, CHO,
Saos, and PC12. Many host cells from various cell types and
organisms are available and would be known to one of skill in the
art. Similarly, a viral vector may be used in conjunction with
either a eukaryotic or prokaryotic host cell, particularly one that
is permissive for replication or expression of the vector.
[0098] Some vectors may employ control sequences that allow it to
be replicated and/or expressed in both prokaryotic and eukaryotic
cells. One of skill in the art would further understand the
conditions under which to incubate all of the above described host
cells to maintain them and to permit replication of a vector. Also
understood and known are techniques and conditions that would allow
large-scale production of vectors, as well as production of the
nucleic acids encoded by vectors and their cognate polypeptides,
proteins, or peptides.
B. Smad7 Proteins and Protein Fragments
[0099] Mothers against decapentaplegic homolog 7 (Smad7) was
previously identified as an antagonist of TGF-.beta. signaling by
several mechanisms including: (a) blockade of TGF-.beta.
receptor-mediated phosphorylation and nuclear translocation of
signaling Smads; (b) increased degradation of TGF-.beta. receptors
and signaling Smads through specific ubiquitin-proteasome pathways
and (c) inhibition of signaling Smads for their binding to Smad
binding elements (SBEs). Smad7 also antagonizes other signaling
pathways, like the NF-.kappa.B pathway.
[0100] Smad7 protein is encoded by the SMAD7 gene, discussed above.
Like many other TGF-.beta. family members, Smad7 is involved in
cell signaling. It is a TGF-.beta. type 1 receptor antagonist. It
blocks TGF-.beta.1 and activin associating with the receptor,
blocking access to Smad2. It is an inhibitory Smad (I-SMAD) and is
enhanced by SMURF2. Smad7 also enhances muscle differentiation.
[0101] The terms "polypeptide," "peptide," and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues. The terms apply to naturally occurring amino acid
polymers as well as amino acid polymers in which one or more amino
acid residues is a non-naturally occurring amino acid, for example,
an amino acid analog. As used herein, the terms encompass amino
acid chains of any length, including full length proteins, wherein
the amino acid residues are linked by covalent peptide bonds.
[0102] In one embodiment, the present technology relates to Smad7
protein compositions. In addition to the entire Smad7 molecule, the
present technology also relates to truncated portions and fragments
of the polypeptide that retain one or more activity associated with
Smad7, such as, but not limited to, increasing proliferation,
reducing or inhibiting cell death, reducing excessive inflammation,
preventing DNA damage, and/or increasing cell migration, as well as
treating or preventing one or more disease or disorders in which
such treatment would be helpful as further discussed herein. Such
activities can be assessed using one or more assays including, but
not limited to, the ability to block phosphorylation of Smad2
and/or nuclear translocation of the NF-.kappa.B p50 subunit,
increase cell proliferation, reduce apoptosis and/or
radiation-induced DNA damage, reduce inflammation and/or
angiogenesis, promote healing in oral mucositis, surgical wounds,
diabetes wounds, and/or wounds associated with chronic inflammation
in mice.
[0103] Protein fragments may be generated by genetic engineering of
translation stop sites within the coding region (discussed below).
Alternatively, treatment of the Smad7 molecule with proteolytic
enzymes, known as proteases, can produces a variety of N-terminal,
C-terminal and internal fragments. These fragments may be purified
according to known methods, such as precipitation (e.g., ammonium
sulfate), HPLC, ion exchange chromatography, affinity
chromatography (including immunoaffinity chromatography) or various
size separations (sedimentation, gel electrophoresis, gel
filtration).
[0104] As used herein, reference to an isolated protein or
polypeptide in the present embodiments include full-length
proteins, fusion proteins, chimeric proteins, or any fragment
(truncated form, portion) or homologue of such a protein. More
specifically, an isolated protein can be a protein (including a
polypeptide or peptide) that has been removed from its natural
milieu (i.e., that has been subject to human manipulation), and can
include, but is not limited to, purified proteins, partially
purified proteins, recombinantly produced proteins, proteins
complexed with lipids, soluble proteins, synthetically produced
proteins, and isolated proteins associated with other proteins. As
such, "isolated" does not reflect the extent to which the protein
has been purified. Preferably, an isolated protein is produced
recombinantly.
[0105] Variants of Smad7 are also provided--these can be
substitutional, insertional or deletion variants. Deletion variants
lack one or more residues of the native protein that are not
essential for activity, including the truncation mutants described
above and herein. Substitutional variants typically contain the
exchange of one amino acid for another at one or more sites within
the protein, and may be designed to modulate one or more properties
of the polypeptide, such as stability against proteolytic cleavage
and/or translation and/or transcription (protein expression),
without the loss of other functions or properties. Substitutions of
this kind preferably are conservative, that is, one amino acid is
replaced with one of similar shape and charge. Conservative
substitutions are well known in the art and include, for example,
each amino acid can be changed or substituted with a different
amino acid. In making substitutional variants, the hydropathic
index, hydrophilicity, charge and size are normally considered.
[0106] Specifically contemplated deletion variants of Smad7 include
truncations and fragments, for example, including polypeptide
molecules having N-terminal sequences, but not C-terminal
sequences, having C-terminal sequences but not N-terminal
sequences, or having internal sequences, but not N-terminal or
c-terminal sequences. Specifically contemplated Smad7 polypeptide
truncations or fragments include, but are not limited to, molecules
including amino acid residues 2-258, 259-426, 204-258 corresponding
to the native human Smad7 protein sequence.
[0107] The term "truncated" as used herein in reference to protein
sequences refers to a molecule that contains the natural N-terminus
of a corresponding protein (with or without a cleaved leader
sequence), but lacks one or more amino acids starting from the
C-terminus of the molecule, or a molecule that contains the natural
C-terminus of a corresponding protein (with or without a cleaved
leader sequence), but lacks one or more amino acids starting from
the N-terminus of the molecule. In some embodiments, molecules
lacking at least about 25, at least about 50, at least about 75, at
least about 100, at least about 125, at least about 150, at least
about 200, at least about 250, at least about 300, or at least
about 350, or at least about 400 amino acids from one or the other
terminus are specifically provided. In some embodiments, a
"truncated" molecule is biologically active, having one or more of
the Smad7 activities described herein.
[0108] The term "fragment" as used herein in reference to
polypeptide sequences refers to a molecule containing contiguous
residues of a full length sequence but lacking some N-terminal
and/or C-terminal residues of the full length sequence. In some
embodiments, a "fragment" includes a portion of one or more of the
full length sequences described herein. In some embodiments, the
"fragment" does not include sequences encoding either the
N-terminal or the C-terminal, but only internal fragments. In some
embodiments, a "fragment" encodes a polypeptide that is
biologically active, having one or more of the Smad7 activities
described herein. In some embodiments, polypeptide fragments have
at least about 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,
90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650,
700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 amino
acids.
[0109] A specialized kind of variant is the fusion protein. This
molecule generally has all or a substantial portion of the native
molecule, linked at the N- or C-terminus, to all or a portion of a
second polypeptide. However, in some embodiments, the fusion
protein may include any one of the fragments and/or truncated
(N-terminal, C-terminal) Smad7 proteins described throughout the
disclosure. For example, fusions may employ leader sequences from
other species to permit the recombinant expression of a protein in
a heterologous host. Another useful fusion includes the addition of
an optional functionally active domain, such as but not limited to
an antibody epitope and/or a purification tag (e.g., V5:
GKPIPNPLLGLDST (SEQ ID NO: 41); Flag: KYKDDDDK (SEQ ID NO: 42); HA:
YPYDVPDYA (SEQ ID NO: 43)). Another type of fusion includes
attaching a domain that can act as the target for an activating or
inactivating ligand, thereby permitting control of the fusion
protein's function once delivered to a subject. Such domains
include, for example, steroid ligand binding (e.g., ER, PR, GR),
which can be activated by small molecules, e.g., 4-hydroxyl
tamoxifen or RU486 that are either uniquely able to activate those
steroid ligand binding domains and/or do not exist in nature and
will therefore enable full control of the Smad7 function by the
presence of these small molecules.
[0110] Another specific form of a fusion protein finding particular
utility in the present technology is a fusion including a protein
transduction domain (PTD), also called a cell delivery domain or
cell transduction domain. Such domains have been described in the
art and are generally characterized as short amphipathic or
cationic peptides and peptide derivatives, often containing
multiple lysine and arginine resides (Fischer, Med. Res. Rev.
27:755-795 (2007)). In some embodiments, the PTD is one or more
variant of TAT protein from HIV (GRKKRRQRRR (SEQ ID NO: 2),
YGRKKRRQRRR (SEQ ID NO: 4), or GRKKRRQ (SEQ ID NO: 6)) or
alternatively, HSV VP 16. Alternate forms of Tat may be used. In
some embodiments, a linker may be used to connect one or more PTDs
and SMad7. In some embodiments, the PTD (optionally Tat) is fused
or linked in frame to the N-terminal and/or C-terminal end of any
one of the Smad7 full-length, fragments, and/or truncated
(N-terminal, C-terminal) proteins described throughout the
disclosure. Other examples of PTDs provided by the present
technology are shown in Table 1.
TABLE-US-00001 TABLE 1 PROTEIN TRANSDUCTION DOMAINS SEQ SEQ ID NO:
ID NO: GALFLGWLGAAGSTMGAKK 44 QAATATRGRS 65 KRKV AASRPTERPR
APARSASRPR RPVE RQIKIWFQNRRMKWKK 45 MGLGLHLLVL 66 AAALQGAKSK RKV
RRMKWKK 46 AAVALLPAVL 67 LALLAPAAAN YKKPKL RRWRRWWRRWWRRWRR 47
MANLGYWLLA 68 LFVTMWTDVG LCKKRPKP RGGRLSYSRRRFSTSTGR 48 LGTYTQDFNK
69 FHTFPQTAIG VGAP YGRKKRRQRRR 4 DPKGDPKGVT 70 VTVTVTVTGK GDPXPD
RKKRRQRRR 49 PPPPPPPPPP 71 PPPP YARAAARQARA 50 VRLPPPVRLP 72
PPVRLPPP RRRRRRRR 51 PRPLPPPRPG 73 KKKKKKKK 52 SVRRRPRPPY 74
LPRPRPPPFF PPRLPPRIPP GWTLNSAGYLLGKINLKALA 53 TRSSRAGLQF 75 ALAKXIL
PVGRVHRLLR K LLILLRRRIRKQANAHSK 54 GIGKFLHSAK 76 KFGKAFVGEI MNS
SRRHHCRSKAKRSRHH 55 KWKLFKKIEK 77 VGQNIRDGII KAGPAVAVVG QATQIAK
NRARRNRRRVR 56 ALWMTLLKKV 78 LKAAAKAALN AVLVGANA RQLRIAGRRLRGRSR 57
GIGAVLKVLT 79 TGLPALISWI KRKRQQ KLIKGRTPIKFGK 58 INLKALAALA 80 KKIL
RRIPNRRPRR 59 GFFALIPKII 81 SSPLPKTLLS AVGSALGGSG GQE
KLALKLALKALKAALKLA 60 LAKWALKQGF 82 AKLKS KLAKLAKKLAKLAK 61
SMAQDIISTI 83 GDLVKWIIQT VNXFTKK GALFLGFLGAAGSTNGAWSQ 62 LLGDFFRKSK
84 PKKKRKV EKIGKEFKRI VQRIKQRIKD FLANLVPRTE S KETWWETWWTEWSQPKKKR
63 PAWRKAFRWA 85 KV WRMLKKAA LKKLLKKLLKKLLKKLLKKL 64 KLKLKLKLKL 86
KLKLKLKL
[0111] In particular embodiments, the present technology provides
for sequence variants of Smad7 in which one or more residues have
been altered. For example, in one embodiment, the methionine
residue found at position 216 of the human Smad7 sequence is
modified to a leucine residue (ATG to CTG).
C. Methods of Treatment
[0112] Smad7-treatable diseases and disorders may include those
including one or more of reduced cell proliferation, reduced cell
migration, increased cell death, excessive inflammation, and/or DNA
damage. Smad7-related diseases and disorders may include those
where treatment with a Smad7 protein and biologically active
fragments and derivatives thereof that have one or more activities
including but not limited to increasing proliferation, reducing or
inhibiting cell death, reducing excessive inflammation, preventing
DNA damage, and/or increasing cell migration is helpful. Such
diseases and/or disorders may include but are not limited to acute
(e.g., through surgery, combat, trauma) and chronic wounds (e.g.,
ulcers, such as diabetic, pressure, venous), scarring, fibrosis,
and aberrant healing, mucositis (e.g., oral and/or
gastro-intestinal), stomatitis, proctitis, autoimmune disease
(e.g., psoriasis, arthritis), and cancer.
[0113] In some embodiments, one or more of the diseases and or
disorders described herein may be prevented, treated, and/or
ameliorated by providing to a subject in need of such treatment a
therapeutically effective amount of one or more of the Smad7
proteins (e.g., full-length or biologically active truncated (e.g.,
N-terminal or C-terminal) or fragment thereof) described in the
disclosure. In some embodiments, the one or more Smad7 proteins are
fusion proteins including a PTD domain. In some embodiments, the
one or more Smad7 proteins includes Leu216. In some embodiments,
the Smad7 proteins make part of a pharmaceutical composition
including one or more pharmaceutically acceptable excipients.
[0114] In some embodiments, one or more of the diseases and or
disorders described herein may be prevented, treated, and/or
ameliorated by providing to a subject in need of such treatment a
therapeutically effective amount of one or more of the nucleic acid
molecules encoding one or more Smad7 proteins (e.g., full-length or
biologically active truncated (e.g., N-terminal or C-terminal) or
fragment thereof) described in the disclosure. In some embodiments,
the one or more nucleic acid molecules include codon-optimized
nucleotide sequences and/or sequences that encode Leu216. In some
embodiments, the one or more Smad7 nucleic acid molecules are
provided to the subject in a construct including an expression
vector. In some embodiments, the Smad7 nucleic acid molecules
(optionally part of an expression vector) make part of a
pharmaceutical composition including one or more pharmaceutically
acceptable excipients.
[0115] The term "subject" or "patient" as used herein refers to
persons or non-human animals in need of treatment and or prevention
using one or more of the treatments described herein. In some
embodiments, non-human animals include laboratory animals such as
monkeys, mice, rats, and rabbits, domestic pets such as dogs and
cats, and livestock such as cattle, horses, pigs, goats and
sheep.
[0116] 1. Chronic Wounds
[0117] A chronic wound is a wound that does not heal in an orderly
set of stages and in a predictable amount of time the way most
wounds do; wounds that do not heal within three months are often
considered chronic. Chronic wounds seem to be detained in one or
more of the phases of wound healing. For example, chronic wounds
often remain in the inflammatory stage for too long. In acute
wounds, there is a precise balance between production and
degradation of molecules such as collagen; in chronic wounds this
balance is lost and degradation plays too large a role.
[0118] As described in more detail elsewhere herein, PTD-Smad7 has
been shown to enhance wound healing in a mouse skin model and a
mucosal model. Application of PTD-Smad7 was effective through a
topical route, which is desirable for wound treatment. Although not
intending to be bound by theory, it is believed that PTD-Smad7 may
act to treat or ameliorate chronic wounds through multiple routes,
which may include one or more of reducing inflammation, increasing
cell proliferation (e.g., keratinocytes), increasing cell migration
(e.g., keratinocytes), or reducing fibrosis (e.g., through
modulation of collagen), among others.
[0119] Chronic wounds may never heal or may take years to do so.
These wounds cause patients severe emotional and physical stress as
well as creating a significant financial burden on patients and the
whole healthcare system. Acute and chronic wounds are at opposite
ends of a spectrum of wound healing types that progress toward
being healed at different rates. The vast majority of chronic
wounds can be classified into three categories: venous ulcers,
diabetic, and pressure ulcers. A small number of wounds that do not
fall into these categories may be due to causes such as radiation
poisoning or ischemia.
[0120] Venous and Arterial Ulcers.
[0121] Venous ulcers, which usually occur in the legs, account for
about 70% to 90% of chronic wounds and mostly affect the elderly.
They are thought to be due to venous hypertension caused by
improper function of valves that exist in the veins to prevent
blood from flowing backward. Ischemia results from the dysfunction
and, combined with reperfusion injury, causes the tissue damage
that leads to the wounds.
[0122] Diabetic Ulcers.
[0123] Another major cause of chronic wounds, diabetes, is
increasing in prevalence. Diabetics have a 15% higher risk for
amputation than the general population due to chronic ulcers.
Diabetes causes neuropathy, which inhibits nociception and the
perception of pain. Thus patients may not initially notice small
wounds to legs and feet, and may therefore fail to prevent
infection or repeated injury. Further, diabetes causes immune
compromise and damage to small blood vessels, preventing adequate
oxygenation of tissue, which can cause chronic wounds. Pressure
also plays a role in the formation of diabetic ulcers.
[0124] Pressure Ulcers.
[0125] Another leading type of chronic wounds is pressure ulcers,
which usually occur in people with conditions such as paralysis
that inhibit movement of body parts that are commonly subjected to
pressure such as the heels, shoulder blades, and sacrum. Pressure
ulcers are caused by ischemia that occurs when pressure on the
tissue is greater than the pressure in capillaries, and thus
restricts blood flow into the area. Muscle tissue, which needs more
oxygen and nutrients than skin does, shows the worst effects from
prolonged pressure. As in other chronic ulcers, reperfusion injury
damages tissue.
[0126] Chronic wounds may affect only the epidermis and dermis, or
they may affect tissues all the way to the fascia. They may be
formed originally by the same things that cause acute wounds, such
as surgery or accidental trauma, or they may form as the result of
systemic infection, vascular, immune, or nerve insufficiency, or
comorbidities such as neoplasias or metabolic disorders. Although
not intending to be bound by theory, the reason a wound becomes
chronic is that the body's ability to deal with the damage is
overwhelmed by factors such as repeated trauma, continued pressure,
ischemia, or illness. Some of the major factors that lead to
chronic wounds include, but are not limited to, ischemia,
reperfusion injury, and bacterial colonization.
[0127] Ischemia.
[0128] Ischemia is an important factor in the formation and
persistence of wounds, especially when it occurs repetitively (as
it usually does) or when combined with a patient's old age.
Ischemia causes tissue to become inflamed and cells to release
factors that attract neutrophils such as interleukins, chemokines,
leukotrienes, and complement factors.
[0129] While they fight pathogens, neutrophils also release
inflammatory cytokines and enzymes that damage cells. One of their
important functions is to produce Reactive Oxygen Species (ROS) to
kill bacteria, for which they use an enzyme called myeloperoxidase.
The enzymes and ROS produced by neutrophils and other leukocytes
damage cells and prevent cell proliferation and wound closure by
damaging DNA, lipids, proteins, the ECM, and cytokines that speed
healing. Neutrophils remain in chronic wounds for longer than they
do in acute wounds, and contribute to the fact that chronic wounds
have higher levels of inflammatory cytokines and ROS. Because wound
fluid from chronic wounds has an excess of proteases and ROS, the
fluid itself can inhibit healing by inhibiting cell growth and
breaking down growth factors and proteins in the ECM.
[0130] Bacterial Colonization.
[0131] Since more oxygen in the wound environment allows white
blood cells to produce ROS to kill bacteria, patients with
inadequate tissue oxygenation, for example, those who suffered
hypothermia during surgery, are at higher risk for infection. The
host's immune response to the presence of bacteria prolongs
inflammation, delays healing, and damages tissue. Infection can
lead not only to chronic wounds but also to gangrene, loss of the
infected limb, and death of the patient.
[0132] Like ischemia, bacterial colonization and infection damage
tissue by causing a greater number of neutrophils to enter the
wound site. In patients with chronic wounds, bacteria with
resistance to antibiotics may have time to develop. In addition,
patients carrying drug resistant bacterial strains, such as
methicillin-resistant Staphylococcus aureus (MRSA), have more
chronic wounds.
[0133] Growth Factors and Proteolytic Enzymes.
[0134] Chronic wounds also differ in makeup from acute wounds in
that their levels of proteolytic enzymes such as elastase and
matrix metalloproteinases (MMPs) are higher, while their
concentrations of growth factors such as Platelet-derived growth
factor and Keratinocyte Growth Factor are lower.
[0135] Since growth factors (GFs) are imperative in timely wound
healing, inadequate GF levels may be an important factor in chronic
wound formation. In chronic wounds, the formation and release of
growth factors may be prevented, the factors may be sequestered and
unable to perform their metabolic roles, or degraded in excess by
cellular or bacterial proteases.
[0136] Chronic wounds such as diabetic and venous ulcers are also
caused by a failure of fibroblasts to produce adequate ECM proteins
and by keratinocytes to epithelialize the wound. Fibroblast gene
expression is different in chronic wounds than in acute wounds.
[0137] Although all wounds require a certain level of elastase and
proteases for proper healing, too high a concentration is damaging.
Leukocytes in the wound area release elastase, which increases
inflammation, destroys tissue, proteoglycans, and collagen, and
damages growth factors, fibronectin, and factors that inhibit
proteases. The activity of elastase is increased by human serum
albumin, which is the most abundant protein found in chronic
wounds. However, chronic wounds with inadequate albumin are
especially unlikely to heal, so regulating the wound's levels of
that protein may in the future prove helpful in healing chronic
wounds.
[0138] Excess matrix metalloproteinases, which are released by
leukocytes, may also cause wounds to become chronic. MMPs break
down ECM molecules, growth factors, and protease inhibitors, and
thus increase degradation while reducing construction, throwing the
delicate compromise between production and degradation out of
balance.
[0139] Oral Ulcers.
[0140] A mouth ulcer (also termed an oral ulcer, or a mucosal
ulcer) is an ulcer that occurs on the mucous membrane of the oral
cavity. More plainly, a mouth ulcer is a sore or open lesion in the
mouth. Mouth ulcers are very common, occurring in association with
many diseases and by many different mechanisms, but usually there
is no serious underlying cause. The two most common causes of oral
ulceration are local trauma (e.g., rubbing from a sharp edge on a
filling) and aphthous stomatitis ("canker sores"), a condition
characterized by recurrent formation of oral ulcers for largely
unknown reasons. Some consider ulcers on the lips or on the skin
around the mouth to be included under the general term oral
ulceration (e.g., an ulcer left by rupture of a blister caused by
herpes labialis, i.e., a cold sore). Mouth ulcers often cause pain
and discomfort, and may alter the person's choice of food while
healing occurs (e.g., avoiding acidic or spicy foods and
beverages). They may occur singly or multiple ulcers may occur at
the same time (a "crop" of ulcers). Once formed, the ulcer may be
maintained by inflammation and/or secondary infection. Rarely, a
mouth ulcer that does not heal for many weeks may be a sign of oral
cancer. Other causes include burns, chemical injury, or
infection.
[0141] A mucosal ulcer is an ulcer which specifically occurs on a
mucous membrane. An ulcer is a tissue defect which has penetrated
the epithelial-connective tissue border, with its base at a deep
level in the submucosa, or even within muscle or periosteum. An
ulcer is a deeper breech of the epithelium than an erosion or an
excoriation, and involves damage to both epithelium and lamina
propria. An erosion is a superficial breach of the epithelium, with
little damage to the underlying lamina propria. A mucosal erosion
is an erosion which specifically occurs on a mucous membrane. Only
the superficial epithelial cells of the epidermis or of the mucosa
are lost, and the lesion can reach the depth of the basement
membrane. Erosions heal without scar formation. Excoriation is a
term sometimes used to describe a breach of the epithelium which is
deeper than an erosion but shallower than an ulcer. This type of
lesion is tangential to the rete pegs and shows punctiform (small
pinhead spots) bleeding, caused by exposed capillary loops.
[0142] 2. Acute Wounds/Trauma
[0143] Physical trauma is a serious and body-altering physical
injury, such as the removal of a limb. Blunt force trauma, a type
of physical trauma caused by impact or other force applied from or
with a blunt object, whereas penetrating trauma is a type of
physical trauma in which the skin or tissues are pierced by an
object. Trauma can also be described as both unplanned, such as an
accident, or planned, in the case of surgery. Both can be
characterized by mild to severe tissue damage, blood loss and/or
shock, and both may lead to subsequent infection, including sepsis.
The present technology provides for treatment of trauma, including
both pre-treatment (in the case of a medical procedure) and
treatment after trauma injury has occurred.
[0144] As described in more detail elsewhere herein (and briefly
mentioned above), PTD-Smad7 has been shown to enhance wound healing
in a mouse skin model and a mucosal model. Application of PTD-Smad7
was effective through a topical route, which is desirable for wound
treatment. Although not intending to be bound by theory, it is
believed that PTD-Smad7 may act to treat or ameliorate wounds
through multiple routes, which may include one or more of reducing
inflammation, increasing cell proliferation (e.g., keratinocytes),
increasing cell migration (e.g., keratinocytes), or reducing
fibrosis (e.g., through modulation of collagen), among others. As
described briefly below, reduced inflammation could significantly
contribute to accelerated wound healing, optionally through reduced
angiogenesis and collagen production and/or reduced leukocyte
infiltration leading to reduction of cytokines and chemokines
normally released by leukocytes, which are angiogenic and
fibrogenic. Temporal treatment with Smad7 may allow early stage
angiogenesis and collagen production required for wound repair,
while preventing prolonged angiogenesis and collagen production.
These changes could potentially accelerate wound stromal remodeling
and prevent excessive scarring due to unresolved inflammation or
collagen overproduction. For surgical procedures (as well as
everyday injuries), particularly where the potential for scarring
is an issue, treatment with Smad7 may be beneficial.
[0145] Surgery.
[0146] Surgery uses operative manual and instrumental techniques on
a patient to investigate and/or treat a pathological condition such
as disease or injury, to help improve bodily function or
appearance, or sometimes for some other reason. The present
technology can address trauma resulting from surgeries, as defined
further below.
[0147] As a general rule, a procedure is considered surgical when
it involves cutting of a patient's tissues or closure of a
previously sustained wound. Other procedures that do not
necessarily fall under this rubric, such as angioplasty or
endoscopy, may be considered surgery if they involve common
surgical procedure or settings, such as use of a sterile
environment, anesthesia, antiseptic conditions, typical surgical
instruments, and suturing or stapling. All forms of surgery are
considered invasive procedures; so-called noninvasive surgery
usually refers to an excision that does not penetrate the structure
being addressed (e.g., laser ablation of the cornea) or to a
radiosurgical procedure (e.g., irradiation of a tumor). Surgery can
last from minutes to hours.
[0148] Surgical procedures are commonly categorized by urgency,
type of procedure, body system involved, degree of invasiveness,
and special instrumentation. Elective surgery is done to correct a
non-life-threatening condition, and is carried out at the patient's
request, subject to the surgeon's and the surgical facility's
availability. Emergency surgery is surgery which must be done
quickly to save life, limb, or functional capacity. Exploratory
surgery is performed to aid or confirm a diagnosis. Therapeutic
surgery treats a previously diagnosed condition.
[0149] Amputation involves cutting off a body part, usually a limb
or digit. Replantation involves reattaching a severed body part.
Reconstructive surgery involves reconstruction of an injured,
mutilated, or deformed part of the body. Cosmetic surgery is done
to improve the appearance of an otherwise normal structure.
Excision is the cutting out of an organ, tissue, or other body part
from the patient. Transplant surgery is the replacement of an organ
or body part by insertion of another from different human (or
animal) into the patient. Removing an organ or body part from a
live human or animal for use in transplant is also a type of
surgery.
[0150] When surgery is performed on one organ system or structure,
it may be classified by the organ, organ system or tissue involved.
Examples include cardiac surgery (performed on the heart),
gastrointestinal surgery (performed within the digestive tract and
its accessory organs), and orthopedic surgery (performed on bones
and/or muscles).
[0151] Minimally invasive surgery involves smaller outer
incision(s) to insert miniaturized instruments within a body cavity
or structure, as in laparoscopic surgery or angioplasty. By
contrast, an open surgical procedure requires a large incision to
access the area of interest. Laser surgery involves use of a laser
for cutting tissue instead of a scalpel or similar surgical
instruments. Microsurgery involves the use of an operating
microscope for the surgeon to see small structures. Robotic surgery
makes use of a surgical robot, such as Da Vinci or Zeus surgical
systems, to control the instrumentation under the direction of the
surgeon.
[0152] 3. Autoimmune/Inflammatory Disease
[0153] The present technology contemplates the treatment of a
variety of autoimmune and/or inflammatory disease states such as
spondyloarthropathy, ankylosing spondylitis, psoriatic arthritis,
reactive arthritis, enteropathic arthritis, ulcerative colitis,
Crohn's disease, irritable bowel disease, inflammatory bowel
disease, rheumatoid arthritis, juvenile rheumatoid arthritis,
familial Mediterranean fever, amyotrophic lateral sclerosis,
Sjogren's syndrome, early arthritis, viral arthritis, multiple
sclerosis, or psoriasis. The diagnosis and treatment of these
diseases are well documented in the literature.
[0154] In general, autoimmune diseases are associated with an
overactive immune response of a body against substances and tissues
normally present in the body, and not normally the focus of an
immune response. There are more than 80 types of autoimmune
diseases, some of which have similar symptoms, and they may arise
from a similar underlying cause. The classic sign of an autoimmune
disease is inflammation, which as disclosed herein is amenable to
treatment with Smad7 (optionally PTD-Smad7) compositions.
[0155] 4. Chemotherapy, Radiotherapy and Cytokine Therapy
Toxicity
[0156] Various forms of cancer therapy, including chemotherapy,
radiation, and cytokines, are associated with toxicity, sometimes
severe, in the cancer patient. The present technology seeks to
reduce this toxicity using the pharmaceutical compositions of the
present technology, thereby reducing or alleviating discomfort on
the part of the patient, as well as permitting higher doses of the
therapy.
[0157] As described at length throughout this disclosure, it has
been found that PTD-Smad7 acts to heal as well as to prevent oral
mucositis in a mouse model. PTD-Smad7 was shown to be more
effective than palifermin, the existing approved drug for
preventing oral mucositis, in direct comparisons.
[0158] Oral cancer, the 6.sup.th most common cancer worldwide, is a
subtype of head and neck cancer, and includes any cancerous tissue
growth located in the oral cavity. It may arise as a primary lesion
originating in any of the oral tissues, by metastasis from a
distant site of origin, or by extension from a neighboring anatomic
structure, such as the nasal cavity or the oral cancers may
originate in any of the tissues of the mouth, and may be of varied
histologic types: teratoma, adenocarcinoma derived from a major or
minor salivary gland, lymphoma from tonsillar or other lymphoid
tissue, or melanoma from the pigment-producing cells of the oral
mucosa. There are several types of oral cancers, but around 90% are
squamous cell carcinomas, originating in the tissues that line the
mouth and lips. Oral or mouth cancer most commonly involves the
tongue. It may also occur on the floor of the mouth, cheek lining,
gingiva (gums), lips, or palate (roof of the mouth). Most oral
cancers look very similar under the microscope and are called
squamous cell carcinoma. These are malignant and tend to spread
rapidly.
[0159] Over 80% of oral cancer patients are treated with radiation
therapy and at least 75% of these individuals will develop oral
mucositis. Oral mucositis is a chronic oral ulceration. This
disease frequently occurs in radiation-treated patients of all
cancer types, including but not limited to patients who are
radiation-treated for organ transplants (to eliminate rejection of
the transplants), and patients undergoing routine chemotherapy.
Severe oral mucositis is extremely painful and impairs food/liquid
intake, hence is often the most severe complication of cancer
therapy. Oral mucositis is a major factor in determining the
maximum dose possible of radiation and chemotherapy to the head and
neck region; it can significantly complicate cancer treatment,
extend hospitalization, decrease quality of life and increase
costs.
[0160] Currently, there is no established therapy to effectively
treat severe oral mucositis. To date, palifermin (KEPIVANCE.RTM.),
a recombinant protein of human keratinocyte growth factor (KGF), is
the only FDA approved drug for intravenous (i.v.) injections for
severe oral mucositis in bone-marrow transplant patients, and its
use in cancer patients remains to be determined. It is also used
for prevention of oral mucositis. Hence, this drug is available for
only 4% of the at-risk population. It also suffers from the need
for medical service providers due to the i.v. administration route.
Other potential therapies include topical rinses, such as viscous
2% lidocaine rinses, or baking soda and saline solutions, or a
cocktail solution, for instance BAX (lidocaine, diphenhyramine,
sorbitol and MYLANTA.RTM.). Other investigative or mucoprotective
adjuvant therapies include, but are not limited to, beta carotene,
tocopherol, laser irradiation, prophylactic brushing the oral
mucosa with silver-nitrate, misoprostol, leucovorin, systemic KGF,
pentoxifylline, allopurinol mouthwash, systemic sucralfate,
chlorhexidine gluconate, and cryotherapy.
[0161] Chemotherapy- and radiation-induced gut mucositis is an
inflammatory condition that arises as a result of the acute death
of rapidly dividing intestinal epithelial cells. Most
chemotherapeutic drugs used for treatment of solid tumors, alone,
in a combination of drugs, or with radiation, will result in the
death of a large number of intestinal epithelial cells. The
clinical manifestations of the ensuing mucositis include digestive
symptoms such as nausea and vomiting, serious diarrhea, acute
weight loss and wasting. This is fast becoming one of the limiting
factors for administering chemotherapy for many cancer patients.
The ability of Tat-Smad7 to protect intestinal epithelial cells
from either chemotherapeutic agents, radiation, or a combination of
those, will significantly decrease the undesirable side effects of
cancer therapies, and enable more aggressive ways to treat the
disease with existing tools.
[0162] Bone marrow failure syndromes are a set of conditions that
develop when the hematopoietic stem cell compartment is compromised
and fails to give rise to normal cell types. Bone marrow failure
occurs as a result of inherited genetic abnormalities, exposure to
a noxious substance, such as toxins, chemicals or viruses. Although
the nature and identity of environmental factors that can lead to
the development of acquired bone marrow failure is still not
completely understood, a few factors have been linked to the
development of acquired bone marrow failure among military
personnel including exposure to mustard gas, ionizing radiation,
and infectious agents such as visceral leishmaniasis or African
trypanosomiasis. The best approach for management of bone marrow
failure syndromes is still the transplantation of hematopoietic
stem cells (HSCs), unless a sufficient number of the remaining
resident bone marrow HSCs can be spared from these stresses and
encouraged to repopulate the hematopoietic compartment. The
modulation of Smad 7, as described here, should enable for the
deliberate protection of the remaining resident HSCs in patients
that exhibit clinical signs consistent with bone marrow
failure.
[0163] 5. Cancer
[0164] TGF-.beta. and NF-.kappa.B activations are known to promote
cancer invasion and metastasis. Currently, TGF-.beta. inhibitors
are in clinical trials for treating metastatic cancer and
NF-.kappa.B inhibitors are used in cancer prevention. The
demonstrated effect of Smad7 on blocking both TGF-.beta. and
NF-.kappa.B signaling present the possibility that it is an even
stronger anti-cancer/anti-metastasis agent than other inhibitors
that inhibit only one of these two pathways. Smad7 has been shown
to prevent angiogenesis and fibrogenesis, and may therefore be
particularly useful in situations where the tumor needs to develop
a blood supply and/or stroma.
[0165] The cancer may be selected from the group consisting of
brain, lung, liver, spleen, kidney, lymph node, small intestine,
pancreas, blood cells, colon, stomach, breast, endometrium,
prostate, testicle, cervix, uterus, ovary, skin, head & neck,
esophagus, bone marrow and blood cancer. The cancers may be
metastatic or primary, recurrent or multi-drug resistant. In some
embodiments, the cancer is a solid tumor (organ tumor). Solid
tumors refer to a mass of cells that grow in organ systems and can
occur anywhere in the body. Two types of solid tumors include
epithelial tumors (carcinomas) that occur in the epithelial tissue
inside or outside an organ, and sarcomas (connective tissue tumors)
that occur in connective tissue such as, but not limited to,
muscles, tendons, fat, nerves and other connective tissues that
support, surround, or connect structures and organs in the body. In
some embodiments the cancer is a liquid tumor or cancer of the
blood, bone marrow, or lymph nodes. These tumors include, but are
not limited to, leukemia, lymphoma, and myeloma.
[0166] 6. Scarring, Fibrosis, and Aberrant Healing
[0167] In addition to accelerated re-epithelialization (e.g.,
through increasing cell proliferation and/or increasing cell
migration), Smad7 effects on wound stroma include one or more of
reducing inflammation, angiogenesis, or collagen production, among
others. Although not intending to be bound by theory these effects
may be mediated through reduction of NF-.kappa.B signaling
(evidenced by reduced p50), and blocking TGF-.beta. signaling
(evidenced by reduced pSmad2). As a result, reduced inflammation
could significantly contribute to accelerated wound healing,
optionally through reduced angiogenesis and collagen production
and/or reduced leukocyte infiltration leading to reduction of
cytokines and chemokines normally released by leukocytes, which are
angiogenic and fibrogenic. Temporal treatment with Smad7 may allow
early stage angiogenesis and collagen production required for wound
repair, while preventing prolonged angiogenesis and collagen
production. These changes could potentially accelerate wound
stromal remodeling and prevent excessive scarring due to unresolved
inflammation or collagen overproduction.
[0168] 7. Stomatitis
[0169] Stomatitis is an inflammation of the mucous lining of any of
the structures in the mouth, which may involve the cheeks, gums,
tongue, lips, throat, and roof or floor of the mouth. The
inflammation can be caused by conditions in the mouth itself, such
as poor oral hygiene, dietary protein deficiency, poorly fitted
dentures, or from mouth burns from hot food or drinks, toxic
plants, or by conditions that affect the entire body, such as
medications, allergic reactions, radiation therapy, or infections.
Severe iron deficiency anemia can lead to stomatitis. Iron is
necessary for the upregulation of transcriptional elements for cell
replication and repair. Lack of iron can cause the genetic
downregulation of these elements, leading to ineffective repair and
regeneration of epithelial cells, especially in the mouth and lips.
This condition is also prevalent in people who have a deficiency in
vitamin B.sub.2 (Riboflavin), B.sub.3 (Niacin), B.sub.6
(Pyridoxine), B.sub.9 (folic acid) or B.sub.12 (cobalamine). When
it also involves an inflammation of the gingiva (gums), it is
called gingivostomatitis. It may also be seen in ariboflavinosis
(riboflavin deficiency) or neutropenia.
[0170] Irritation and fissuring in the corners of the lips is
termed angular stomatitis or angular cheilitis. In children,
angular stomatitis is a frequent cause is repeated lip-licking and
in adults it may be a sign of underlying iron deficiency anemia, or
vitamin B deficiencies (e.g., B.sub.2-riboflavin, B.sub.9-folate or
B.sub.12-cobalamin), which in turn may be evidence of poor diets or
malnutrition (e.g., celiac disease). Also, angular cheilitis can be
caused by a patient's jaws at rest being "overclosed" due to
edentulousness or tooth wear, causing the jaws to come to rest
closer together than if the complete/unaffected dentition were
present. This causes skin folds around the angle of the mouth which
are kept moist by saliva which in turn favours infection; mostly by
Candida albicans or similar species. Treatment usually involves the
administration of topical nystatin or similar antifungal agents.
Another treatment can be to correct the jaw relationship with
dental treatment (e.g., dentures or occlusal adjustment).
[0171] Migratory stomatitis is a condition in which extensive areas
in the oral cavity mucosa are affected by annular atrophic red
lesions that are surrounded by a thin white rim. This is a
relatively uncommon form of the geographic tongue condition, that,
as opposed to migratory stomatitis, is confined to the dorsal and
lateral aspects of the tongue mucosa only.
[0172] 8. Proctitis
[0173] Proctitis is inflammation of the lining of the rectum, the
lower end of the large intestine leading to the anus. With
proctitis, inflammation of the rectal lining--called the rectal
mucosa--is uncomfortable and sometimes painful. The condition may
lead to bleeding or mucous discharge from the rectum, among other
symptoms. Some causes of proctitis include, but are not limited to:
sexually transmitted diseases (STDs), such as those that can be
transmitted during anal sex (e.g., gonorrhea, chlamydia, syphilis,
and herpes); non-STD infections from, for example, food borne
bacteria (e.g., Salmonella and Shigella); anorectal trauma from,
for example, anal sex or the insertion of objects or substances
into the rectum (e.g., chemicals from enemas); ulcerative colitis
and Crohn's disease or other inflammatory bowel diseases, may cause
ulcers (e.g., sores) in the inner lining of the colon and rectum;
radiation therapy, particularly of the pelvic area (e.g., rectal,
ovarian, or prostate cancer) which may lead to rectal bleeding;
antibiotics which lead to a loss of commensal bacteria allowing
harmful bacteria (e.g., Clostridium difficile) to cause
disease.
[0174] 9. Formulations and Routes of Administration
[0175] Where clinical applications are contemplated, it will be
necessary to prepare pharmaceutical compositions--proteins,
expression vectors, virus stocks, proteins and drugs--in a form
appropriate for the intended application. 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.
[0176] PTD-Smad7 (and truncated variants) were purified extensively
prior to use in animal models. PTD-Smad7 (and truncated versions)
were prepared for topical and trans-mucosal application using a
mixture of glycerol and PBS.
[0177] One will generally desire to employ appropriate salts and
buffers to render delivery vectors stable and allow for uptake by
target cells. Buffers also will be employed when recombinant cells
are introduced into a patient. Aqueous compositions of the present
technology comprise an effective amount of the vector to cells,
dissolved or dispersed in a pharmaceutically acceptable carrier or
aqueous medium. Such compositions also are referred to as inocula.
The phrase "pharmaceutically or pharmacologically acceptable" refer
to molecular entities and compositions that do not produce adverse,
allergic, or other untoward reactions when administered to an
animal or a human. 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. The use of such media and agents for
pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the
vectors or cells of the present technology, its use in therapeutic
compositions is contemplated. Supplementary active ingredients also
can be incorporated into the compositions.
[0178] The active compositions of the present technology may
include classic pharmaceutical preparations. Administration of
these compositions according to the present technology will be via
any common route so long as the target tissue is available via that
route. Such routes of administration may include oral parenteral
(including intravenous, intramuscular, subcutaneous, intradermal,
intra-articular, intra-synovial, intrathecal, intra-arterial,
intracardiac, subcutaneous, intraorbital, intracapsular,
intraspinal, intrastemal, and transdermal), nasal, buccal,
urethral, rectal, vaginal, mucosal, dermal, or topical (including
dermal, buccal, and sublingual). Alternatively, administration may
be by orthotopic, intradermal, subcutaneous, intramuscular,
intraperitoneal or intravenous injection. Such compositions would
normally be administered as pharmaceutically acceptable
compositions, described supra. Of particular interest is direct
intratumoral administration, perfusion of a tumor, or
administration local or regional to a tumor, for example, in the
local or regional vasculature or lymphatic system, or in a resected
tumor bed. Administration can also be via nasal spray, surgical
implant, internal surgical paint, infusion pump, or via catheter,
stent, balloon or other delivery device. The most useful and/or
beneficial mode of administration can vary, especially depending
upon the condition of the recipient and the disorder being
treated.
[0179] Solutions of the active compounds as free base or
pharmacologically acceptable salts can be prepared in water
suitably mixed with a surfactant, such as hydroxypropylcellulose.
Dispersions can also 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.
[0180] 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 (e.g.,
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 and 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.
[0181] 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.
[0182] 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. 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 can also be
incorporated into the compositions.
[0183] The compositions of the present technology 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 can also be derived from
inorganic bases such as, for example, sodium, potassium, ammonium,
calcium, or ferric hydroxides, and such organic bases as
isopropylamine, trimethylamine, histidine, procaine and the
like.
[0184] The formulations are easily administered in a variety of
dosage forms. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject. Moreover, for human
administration, preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biologics standards.
[0185] For oral administration the polypeptides of the present
technology may be incorporated with excipients and used in the form
of non-ingestible mouthwashes and dentifrices. It is anticipated
that virtually any pill or capsule type known to one of skill in
the art including, e.g., coated, and time delay, slow release,
etc., may be used with the present technology. 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, creams,
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.
[0186] Pharmaceutical compositions suitable for oral dosage may
take various forms, such as tablets, capsules, caplets, and wafers
(including rapidly dissolving or effervescing), each containing a
predetermined amount of the active agent. The compositions may also
be in the form of a powder or granules, a solution or suspension in
an aqueous or non-aqueous liquid, and as a liquid emulsion
(oil-in-water and water-in-oil). The active agents may also be
delivered as a bolus, electuary, or paste. It is generally
understood that methods of preparations of the above dosage forms
are generally known in the art, and any such method would be
suitable for the preparation of the respective dosage forms for use
in delivery of the compositions.
[0187] In one embodiment, an active agent compound may be
administered orally in combination with a pharmaceutically
acceptable vehicle such as an inert diluent or an edible carrier.
Oral compositions may be enclosed in hard or soft shell gelatin
capsules, may be compressed into tablets or may be incorporated
directly with the food of the patient's diet. The percentage of the
composition and preparations may be varied; however, the amount of
substance in such therapeutically useful compositions is preferably
such that an effective dosage level will be obtained.
[0188] Hard capsules containing the active agent compounds may be
made using a physiologically degradable composition, such as
gelatin. Such hard capsules comprise the compound, and may further
comprise additional ingredients including, for example, an inert
solid diluent such as calcium carbonate, calcium phosphate, or
kaolin. Soft gelatin capsules containing the compound may be made
using a physiologically degradable composition, such as gelatin.
Such soft capsules comprise the compound, which may be mixed with
water or an oil medium such as peanut oil, liquid paraffin, or
olive oil.
[0189] Sublingual tablets are designed to dissolve very rapidly.
Examples of such compositions include ergotamine tartrate,
isosorbide dinitrate, and isoproterenol HCL. The compositions of
these tablets contain, in addition to the drug, various soluble
excipients, such as lactose, powdered sucrose, dextrose, and
mannitol. The solid dosage forms of the present technology may
optionally be coated, and examples of suitable coating materials
include, but are not limited to, cellulose polymers (such as
cellulose acetate phthalate, hydroxypropyl cellulose, hydroxypropyl
methylcellulose, hydroxypropyl methylcellulose phthalate, and
hydroxypropyl methylcellulose acetate succinate), polyvinyl acetate
phthalate, acrylic acid polymers and copolymers, and methacrylic
resins (such as those commercially available under the trade name
EUDRAGIT.RTM.), zein, shellac, and polysaccharides.
[0190] Powdered and granular compositions of a pharmaceutical
preparation may be prepared using known methods. Such compositions
may be administered directly to a patient or used in the
preparation of further dosage forms, such as to form tablets, fill
capsules, or prepare an aqueous or oily suspension or solution by
addition of an aqueous or oily vehicle thereto. Each of these
compositions may further comprise one or more additives, such as
dispersing or wetting agents, suspending agents, and preservatives.
Additional excipients (e.g., fillers, sweeteners, flavoring, or
coloring agents) may also be included in these compositions.
[0191] Liquid compositions of pharmaceutical compositions which are
suitable for oral administration may be prepared, packaged, and
sold either in liquid form or in the form of a dry product intended
for reconstitution with water or another suitable vehicle prior to
use.
[0192] A tablet containing one or more active agent compounds
described herein may be manufactured by any standard process
readily known to one of skill in the art, such as, for example, by
compression or molding, optionally with one or more adjuvant or
accessory ingredient. The tablets may optionally be coated or
scored and may be formulated so as to provide slow or controlled
release of the active agents.
[0193] Solid dosage forms may be formulated so as to provide a
delayed release of the active agents, such as by application of a
coating. Delayed release coatings are known in the art, and dosage
forms containing such may be prepared by any known suitable method.
Such methods generally include that, after preparation of the solid
dosage form (e.g., a tablet or caplet), a delayed release coating
composition is applied. Application can be by methods, such as
airless spraying, fluidized bed coating, use of a coating pan, or
the like. Materials for use as a delayed release coating can be
polymeric in nature, such as cellulosic material (e.g., cellulose
butyrate phthalate, hydroxypropyl methylcellulose phthalate, and
carboxymethyl ethylcellulose), and polymers and copolymers of
acrylic acid, methacrylic acid, and esters thereof.
[0194] Solid dosage forms according to the present technology may
also be sustained release (i.e., releasing the active agents over a
prolonged period of time), and may or may not also be delayed
release. Sustained release compositions are known in the art and
are generally prepared by dispersing a drug within a matrix of a
gradually degradable or hydrolyzable material, such as an insoluble
plastic, a hydrophilic polymer, or a fatty compound. Alternatively,
a solid dosage form may be coated with such a material.
[0195] Compositions for parenteral administration include aqueous
and non-aqueous sterile injection solutions, which may further
contain additional agents, such as antioxidants, buffers,
bacteriostats, and solutes, which render the compositions isotonic
with the blood of the intended recipient. The compositions may
include aqueous and non-aqueous sterile suspensions, which contain
suspending agents and thickening agents. Such compositions for
parenteral administration may be presented in unit-dose or
multi-dose containers, such as, for example, sealed ampoules and
vials, and may be stores in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carrier, for
example, water (for injection), immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared
from sterile powders, granules, and tablets of the kind previously
described.
[0196] Compositions for rectal delivery include rectal
suppositories, creams, ointments, and liquids. Suppositories may be
presented as the active agents in combination with a carrier
generally known in the art, such as polyethylene glycol. Such
dosage forms may be designed to disintegrate rapidly or over an
extended period of time, and the time to complete disintegration
can range from a short time, such as about 10 minutes, to an
extended period of time, such as about 6 hours.
[0197] Topical compositions may be in any form suitable and readily
known in the art for delivery of active agents to the body surface,
including dermally, buccally, and sublingually. Typical examples of
topical compositions include ointments, creams, gels, pastes, and
solutions. Compositions for administration in the mouth include
lozenges.
[0198] In accordance with these embodiments, oral (topical,
mucosal, and/or dermal) delivery materials can also include creams,
salves, ointments, patches, liposomes, nanoparticles,
microparticles, timed-release formulations and other materials
known in the art for delivery to the oral cavity, mucosa, and/or to
the skin of a subject for treatment and/or prevention of a
condition disclosed herein. Certain embodiments concern the use of
a biodegradable oral (topical, mucosal, and/or dermal) patch
delivery system or gelatinous material. These compositions can be a
liquid formulation or a pharmaceutically acceptable delivery system
treated with a formulation of these compositions, and may also
include activator/inducers.
[0199] The compositions for use in the methods of the present
technology may also be administered transdermally, wherein the
active agents are incorporated into a laminated structure
(generally referred to as a "patch") that is adapted to remain in
intimate contact with the epidermis of the recipient for a
prolonged period of time. Typically, such patches are available as
single layer "drug-in-adhesive" patches or as multi-layer patches
where the active agents are contained in a layer separate from the
adhesive layer. Both types of patches also generally contain a
backing layer and a liner that is removed prior to attachment to
the recipient's skin. Transdermal drug delivery patches may also be
comprised of a reservoir underlying the backing layer that is
separated from the skin of the recipient by a semi-permeable
membrane and adhesive layer. Transdermal drug delivery may occur
through passive diffusion, electrotransport, or iontophoresis.
[0200] In certain embodiments, a patch contemplated herein may be a
slowly dissolving or a time-released patch. In accordance with
these embodiments, a slowly dissolving patch can be an alginate
patch. In certain examples, a patch may contain a detectible
indicator dye or agent such as a fluorescent agent. In other
embodiments, a tag (e.g., detectible tag such as a biotin or
fluorescently tagged agent) can be associated with a treatment
molecule in order to detect the molecule after delivery to the
subject. In certain embodiments, one or more oral delivery patches
or other treatment contemplated herein may be administered to a
subject three times daily, twice daily, once a day, every other
day, weekly, and the like, depending on the need of the subject as
assessed by a health professional. Patches contemplated herein may
be oral-biodegradable patches or patches for exterior use that may
or may not degrade. Patches contemplated herein may be 1 mm, 2 mm,
3 mm, 4 mm to 5 mm in size or more depending on need. In addition,
skin patches are contemplated herein for use for example in a
subject suffering from psoriasis. In treating psoriasis and chronic
wounds, Smad7 can be delivered topically using vehicles such as
glycerol, carboxymethycellulose. It can also use transdermal system
(e.g., commercially available from 3M) for delivery. Subcutaneous
injection into the lesion (in normal saline or PBS) can also be
used.
[0201] In some embodiments, compositions may include short-term,
rapid-onset, rapid-offset, controlled release, sustained release,
delayed release, and pulsatile release compositions, providing the
compositions achieve administration of a compound as described
herein. See Remington's Pharmaceutical Sciences (18th ed.; Mack
Publishing Company, Eaton, Pa., 1990), herein incorporated by
reference in its entirety.
[0202] In certain embodiments, the compounds and compositions
disclosed herein can be delivered via a medical device. Such
delivery can generally be via any insertable or implantable medical
device, including, but not limited to stents, catheters, balloon
catheters, shunts, or coils. In one embodiment, the present
technology provides medical devices, such as stents, the surface of
which is coated with a compound or composition as described herein.
The medical device of this technology can be used, for example, in
any application for treating, preventing, or otherwise affecting
the course of a disease or condition, such as those disclosed
herein.
[0203] It is contemplated that any molecular biology, cellular
biology or biochemical technique known in the art may be used to
generate and/or test treatments provided herein. In addition,
protein chemistry techniques are contemplated to assess utility of
treatments in model systems developed herein (e.g., mouse model
system).
[0204] 10. Combination Therapies
[0205] It is common in many fields of medicine to treat a disease
with multiple therapeutic modalities, often called "combination
therapies." Many of the diseases described herein (e.g.,
inflammatory disease and cancer) are no exception. In some
embodiments, to treat inflammatory disorders using the methods and
compositions of the present technology, one would contact a target
cell, organ or subject with a Smad7 protein, expression construct
or activator and at least one other therapy. These therapies would
be provided in a combined amount effective to achieve a reduction
in one or more disease parameter. This process may involve
contacting the cells/subjects with the both agents/therapies at the
same time, e.g., using a single composition or pharmacological
formulation that includes both agents, or by contacting the
cell/subject with two distinct compositions or formulations, at the
same time, wherein one composition includes the Smad7 agent and the
other includes the other agent.
[0206] Alternatively, the Smad7 agent may precede or follow the
other treatment by intervals ranging from minutes to weeks. One
would generally ensure that a significant period of time did not
expire between the time of each delivery, such that the therapies
would still be able to exert an advantageously combined effect on
the cell/subject. In such instances, it is contemplated that one
would contact the cell with both modalities within about 12-24
hours of each other, within about 6-12 hours of each other, or with
a delay time of only about 12 hours. 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.
[0207] It also is conceivable that more than one administration of
either the Smad7 agent or the other therapy will be desired.
Various combinations may be employed, where the Smad7 agent is "A,"
and the other therapy is "B," as exemplified below:
TABLE-US-00002 A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A A/A/A/B
B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B
[0208] Other combinations are provided. Other agents suitable for
use in a combined therapy against an inflammatory disorder include
steroids, glucocorticoids, non-steriodal anti-inflammatory drugs
(NSAIDS; including COX-1 and COX-2 inhibitors), aspirin, ibuprofen,
and naproxen. Analgesics are commonly associated with
anti-inflammatory drugs but which have no anti-inflammatory
effects. An example is paracetamol, called acetaminophen in the
U.S. and sold under the brand name of Tylenol. As opposed to
NSAIDS, which reduce pain and inflammation by inhibiting COX
enzymes, paracetamol has recently been shown to block the reuptake
of endocannabinoids, which only reduces pain, likely explaining why
it has minimal effect on inflammation. A particular agent for
combination use is an anti-TGF-.beta. antibody.
[0209] The skilled artisan is directed to Remington's
Pharmaceutical Sciences, 15th Edition, chapter 33, in particular,
pages 624-652, 1990. Some variation in dosage will necessarily
occur depending on the condition of the subject being treated. The
person responsible for administration will, in any event, determine
the appropriate dose for the individual subject. Moreover, for
human administration, preparations should meet sterility,
pyrogenicity, general safety and purity standards as required by
FDA Office of Biologics standards.
[0210] It also should be pointed out that any of the foregoing
therapies may prove useful by themselves in treating
inflammation.
[0211] As discussed above, the present technology has particular
relevance to the treatment of DNA damage and/or inflammation
resulting from certain anti-cancer therapies, and for the treatment
of cancer. Thus, in particular, the present technology may be
applied as a combination with cancer therapies. This process may
involve contacting the cells, organ, or patient with the
agents/therapies at the same time, including by contacting the
cells, organ or patient with a single composition or
pharmacological formulation that includes both agents, or with two
distinct compositions or formulations at the same time, wherein one
composition includes the Smad7 agent and the other includes the
other agent. Alternatively, analogous to the chart set forth above,
the compositions can be delivered at different times, including
repeated doses of one or both agents.
[0212] Agents or factors suitable for use in a combined therapy
include any chemical compound or treatment method that induces DNA
damage when applied to a cell. Such agents and factors include
radiation and waves that induce DNA damage such as, irradiation,
microwaves, electronic emissions, and the like. A variety of
chemical compounds, also described as "chemotherapeutic" or
"genotoxic agents," are intended to be of use in the combined
treatment methods disclosed herein. In treating cancer according to
the present technology, one would contact the tumor cells with an
agent in addition to the expression construct. This may be achieved
by irradiating the localized tumor site; alternatively, the tumor
cells may be contacted with the agent by administering to the
subject a therapeutically effective amount of a pharmaceutical
composition.
[0213] Various classes of chemotherapeutic agents are provided for
use with in combination with peptides of the present technology.
For example, selective estrogen receptor antagonists ("SERMs"),
such as Tamoxifen, 4-hydroxy Tamoxifen (Afimoxfene), Falsodex,
Raloxifene, Bazedoxifene, Clomifene, Femarelle, Lasofoxifene,
Ormeloxifene, and Toremifene.
[0214] Chemotherapeutic agents contemplated to be of use, include,
e.g., camptothecin, actinomycin-D, mitomycin C. The present
technology also encompasses the use of a combination of one or more
DNA damaging agents, whether radiation-based or actual compounds,
such as the use of X-rays with cisplatin or the use of cisplatin
with etoposide. The agent may be prepared and used as a combined
therapeutic composition, or kit, by combining it with a MUC1
peptide, as described above.
[0215] Heat shock protein 90 is a regulatory protein found in many
eukaryotic cells. HSP90 inhibitors have been shown to be useful in
the treatment of cancer. Such inhibitors include Geldanamycin,
17-(Allylamino)-17-demethoxygeldanamycin, PU-H71 and Rifabutin.
[0216] Agents that directly cross-link DNA or form adducts are also
envisaged. Agents such as cisplatin, and other DNA alkylating
agents may be used. Cisplatin has been widely used to treat cancer,
with efficacious doses used in clinical applications of 20
mg/m.sup.2 for 5 days every three weeks for a total of three
courses. Cisplatin is not absorbed orally and must therefore be
delivered via injection intravenously, subcutaneously,
intratumorally or intraperitoneally.
[0217] Agents that damage DNA also include compounds that interfere
with DNA replication, mitosis and chromosomal segregation. Such
chemotherapeutic compounds include adriamycin, also known as
doxorubicin, etoposide, verapamil, podophyllotoxin, and the like.
Widely used in a clinical setting for the treatment of neoplasms,
these compounds are administered through bolus injections
intravenously at doses ranging from 25-75 mg/m.sup.2 at 21 day
intervals for doxorubicin, to 35-50 mg/m.sup.2 for etoposide
intravenously or double the intravenous dose orally. Microtubule
inhibitors, such as taxanes, also are contemplated. These molecules
are diterpenes produced by the plants of the genus Taxus, and
include paclitaxel and docetaxel.
[0218] Epidermal growth factor receptor inhibitors, such as Iressa,
mTOR, the mammalian target of rapamycin, also known as
FK506-binding protein 12-rapamycin associated protein 1 (FRAP1) is
a serine/threonine protein kinase that regulates cell growth, cell
proliferation, cell motility, cell survival, protein synthesis, and
transcription. Rapamycin and analogs thereof ("rapalogs") are
therefore provided for use in combination cancer therapy in
accordance with the present technology.
[0219] Another possible combination therapy with the peptides
claimed herein is TNF-.alpha. (tumor necrosis factor-alpha), a
cytokine involved in systemic inflammation and a member of a group
of cytokines that stimulate the acute phase reaction. The primary
role of TNF is in the regulation of immune cells. TNF is also able
to induce apoptotic cell death, to induce inflammation, and to
inhibit tumorigenesis and viral replication.
[0220] Agents that disrupt the synthesis and fidelity of nucleic
acid precursors and subunits also lead to DNA damage. As such a
number of nucleic acid precursors have been developed. Particularly
useful are agents that have undergone extensive testing and are
readily available. As such, agents such as 5-fluorouracil (5-FU),
are preferentially used by neoplastic tissue, making this agent
particularly useful for targeting to neoplastic cells. Although
quite toxic, 5-FU, is applicable in a wide range of carriers,
including topical, however intravenous administration with doses
ranging from 3 to 15 mg/kg/day being commonly used.
[0221] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
x-rays, and/or the directed delivery of radioisotopes to tumor
cells. Other forms of DNA damaging factors are also contemplated
such as microwaves and UV-irradiation. It is most likely that all
of these factors effect a broad range of damage DNA, on the
precursors of DNA, the replication and repair of DNA, and the
assembly and maintenance of chromosomes. Dosage ranges for x-rays
range from daily doses of 50 to 200 roentgens for prolonged periods
of time (3 to 4 weeks), to single doses of 2000 to 6000 roentgens.
Dosage ranges for radioisotopes vary widely, and depend on the
half-life of the isotope, the strength and type of radiation
emitted, and the uptake by the neoplastic cells.
[0222] The skilled artisan is directed to Remington's
Pharmaceutical Sciences, 15th Edition, chapter 33, in particular
pages 624-652. Some variation in dosage will necessarily occur
depending on the condition of the subject being treated. The person
responsible for administration will, in any event, determine the
appropriate dose for the individual subject. Moreover, for human
administration, preparations should meet sterility, pyrogenicity,
general safety and purity standards as required by FDA Office of
Biologics standards.
[0223] In addition to combining Smad7 therapies with chemo- and
radiotherapies, it also is contemplated that combination with
immunotherapy, hormone therapy, toxin therapy and surgery. In
particular, one may employ targeted therapies such as AVASTIN.RTM.,
ERBITUX.RTM., GLEEVEC.RTM., HERCEPTIN.RTM., and RITUXAN.RTM..
[0224] In other embodiments, to assess the roles and mechanisms of
Smad7 within the context of oral mucositis, "gene-switch"
transgenic mouse models were developed to allow control of the
level and duration of Smad7 transgene expression specifically in
oral epithelia. In accordance with these embodiments, these models
may be used to test other genes or downstream molecules for their
effects on oral epithelia and oral mucosa. Thus, these models can
be used for, but are not limited to, further analysis of oral wound
healing biology and testing therapeutic approaches to oral wound
healing. Molecular Smad7 targets identified in these studies can
provide additional therapeutic targets for subjects suffering from
oral mucositis. Models and resources developed herein can provide
unique tools for analytical studies to identify biomarkers and
therapeutic targets related to Smad7 overexpression and control,
for example, downstream molecules turned on or bound by Smad7 can
be identified as additional therapeutic targets for example, to
treat oral mucositis, psoriasis and other conditions aggravated by
TGF-.beta. activities and NF-.kappa.B activities.
D. Kits
[0225] In certain embodiments, a kit provided herein may include
compositions discussed above for treating a subject having a
condition provided herein, such as but not limited to oral
mucositis, psoriasis, or wound healing. The kits can include one or
more containers containing the therapeutic Smad7 compositions of
the present technology. Any of the kits will generally include at
least one vial, test tube, flask, bottle, syringe or other
container, into which compositions may be preferably and/or
suitably aliquoted. Kits herein may also include a kit for
assessing biological targets that contribute to a condition
provided herein.
E. Methods of Predicting or Evaluating Responses
[0226] Also provided are methods for predicting and/or evaluating a
response to treatment with Smad7 using by assessing the level of
expression of one or more markers associated with exposure to
Smad7. Such markers may include, but are not limited to, Rac1 for
cell migration, NF-.kappa.B for inflammation, and TGF-.beta. for
growth arrest and inflammation. As is discussed in the Examples,
methods for detection of and/or changes in the levels of one or
more markers associated with Smad7 activity are provided and/or
known in the art. In some embodiments, the level of expression of
one or more of the Smad7 markers in a subject may be assessed, and
based on the level detected, a decision may be made to treat (or to
continue or discontinue treatment) with Smad7, or to employ an
alternate treatment.
[0227] The term "detection of" as used herein refers to the ability
to measure the presence or absence of a marker at some repeatable
and controlled level. Typically, detection is performed over
background values, which may include the noise (or detection
limits) inherent in the testing system. As such, there is typically
a "lower limit" of detection associated with an assay, and in order
to be detected, a change may need to be above a certain cut-off
level, for example. Determination of such limits is well-known in
the art.
[0228] In some embodiments, detection is performed as compared to
controls, which may include, but are not limited to, a comparison
with data from normal subjects and/or comparable normal tissue (in
the same or different subjects) absent the disease or disorder
present in the subject (or the specific tissue of the subject
tested). In some embodiments, the comparison may be between levels
detected at a variety of time intervals (and/or locations) in a
patient. In some embodiments, the detection needs to be
statistically significant as compared to background or control
levels; the ability to assess significance is well-known in the
art, and exemplified in the Examples.
[0229] The term "changes in the levels" as used herein refers to a
detectable change from a control or background level, and or a
previously detected level. In some embodiments, the change is an
increase as compared to another level, and in some embodiments the
change is a decrease as compared to another level. In some
embodiments, the detectable change (increase or decrease) is
statistically significant. In some embodiments, such changes can be
assessed quantitatively as at least about a 5%, 10%, 25%, 50%,
100%, 200%, 500% or greater change, and/or about a 5-10%, 10-25%,
10-50%, 25-50%, 50-75%, 50-100%, 100-150%, 100-200%, 200-300%,
300-500%, or 500-1000% change.
F. Method of Screening for Additional Biologically Active
Fragments
[0230] In another aspect, methods of screening for additional
biologically active fragments (including, but not limited to
truncations) of Smad7 are contemplated. In some embodiments,
biological activity may be assessed using one of the methods
described herein, including those described below in Examples 5 and
8. Some of the biological activities that can be assessed include,
but are not limited to, increasing cell proliferation, reducing or
inhibiting cell death, reducing excessive inflammation, preventing
DNA damage, and/or increasing cell migration, as well as animal
models treating or preventing one or more disease or disorders in
which such treatment would be helpful as further discussed herein.
Such activities can be assessed using one or more assays including,
but not limited to, the ability to block phosphorylation of Smad2
and/or nuclear translocation of the NF-.kappa.B p50 subunit,
increase cell proliferation, reduce apoptosis and/or
radiation-induced DNA damage, reduce inflammation and/or
angiogenesis, promote healing in oral mucositis, surgical wounds,
diabetes wounds, and/or wounds associated with chronic inflammation
in mice and other laboratory models. Some specific examples
include, but are not limited to, immunofluorescence (IF),
immunohistochemistry (IHC), and TUNEL assay for apoptosis.
[0231] In some embodiments, biologically active fragments are those
that are selected to include one or more or all of the activities
described herein. In some embodiments, biologically active
fragments selected to include only or primarily 1, only or
primarily 2, only or primarily 3, only or primarily 4, or only or
primarily 5 of the activities described herein. In some
embodiments, biologically active fragments selected to exclude only
or primarily 1, only or primarily 2, only or primarily 3, only or
primarily 4, or only or primarily 5 of the activities described
herein. In some embodiments, the biologically active fragments are
selected to include or to exclude a specific subset of the
activities described herein. For instance, increased proliferation
and migration may be sufficient for treating diabetic wounds,
whereas anti-inflammation is needed in chronic inflammatory wounds.
Reduced apoptosis and DNA damage activities are needed for treating
oral mucositis but not for treating surgical wounds
[0232] The term "primarily includes" as used herein refers to
fragments in which although some level of other biological activity
may remain, that activity is reduced as compared with full-length
fragments, whereas the activity that is considered "primary"
remains at about the same or an increased level as that observed in
the full-length native protein. Similarly, the term "primarily
excludes" as used herein refers to fragments in which although some
level of a particular biological activity may remain, the level of
that particular activity is reduced (optionally significantly
and/or statistically significantly reduced) as compared with
full-length fragments, whereas one or more other biological
activities remains at about the same or increased level as that
observed in the full-length native protein.
[0233] In some embodiments involving selection of biologically
active fragments, the methods include assessing changes in the
level of expression of one or more biological activities, including
increases and decreases of one or more activities in a selected
fragment are assessed as changes in reference to the activities
observed in the full-length protein. In some embodiments, one or
more biological activities are being selected to remain the same as
that observed in the full-length fragments while other activities
may be increased or decreased or even eliminated (e.g., such
fragments would lack one or more of the activities discussed). In
some embodiments, the change is an increase as compared to another
level, and in some embodiments the change is a decrease as compared
to another level. In some embodiments, the detectable change
(increase or decrease) is statistically significant. In some
embodiments, such changes can be assessed quantitatively as at
least about a 5%, 10%, 25%, 50%, 100%, 200%, 500% or greater
change, and/or about a 5-10%, 10-25%, 10-50%, 25-50%, 50-75%,
50-100%, 100-150%, 100-200%, 200-300%, 300-500%, or 500-1000%
change. In some embodiments, an activity that "remains the same"
can still be observed to have some change from the activity of the
full-length protein, but such change might be limited to, for
example, about a 1%, 2%, 5%, 10%, or 20% change or less.
[0234] In a non-limiting example, fragments of interest may include
those that primarily mediate the anti-inflammatory effect of Smad7.
Smad7 peptides having this anti-inflammatory function may be
sufficient and optionally an improvement for treating chronic
inflammation associated conditions, such as but not limited to,
oral mucositis, stomatitis and psoriasis, among others. In another
non-limiting example, fragments of interest may include those that
primarily mediate cell migration and/or blocking TGF-.beta.-induced
growth arrest and/or fibrotic response. Smad7 peptides having this
cell migration and proliferation function may be sufficient, and
optionally an improvement, for enhancing healing that is not
associated with excessive inflammation. Types of wounds that might
benefit from this form of treatment include, but are not limited
to, surgical wounds, fibrotic scarring, and diabetes wounds,
defective healing and/or scarring among others.
G. Methods of Producing Smad7 Protein
[0235] In another aspect, methods for producing Smad7 protein,
including any of the Smad7 variants, fragments, truncations, fusion
proteins (e.g., PTD-Smad7) described herein are contemplated. The
inventors have discovered methods of producing Smad7 protein at
levels and purity sufficient for research, development, or
commercialization that include nucleic acid codon optimization. As
a result, methods for producing Smad7 including the use of one or
more of the codon-optimized Smad7 nucleic acid molecules described
herein (e.g., within the Examples) are expressly contemplated.
EXAMPLES
[0236] The following examples are included to illustrate various
embodiments. It should be appreciated by those of skill in the art
that the techniques disclosed in the examples that follow represent
techniques discovered to function well in the practice of the
claimed methods, compositions and apparatus. However, those of
skill in the art should, in light of the present disclosure,
appreciate that many changes may 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 present
technology.
Example 1
K5.Smad7 Mice are Resistant to Oral Mucositis
[0237] A transgenic mouse model expressing a human Smad7 protein in
keratinocytes (K5.Smad7) was generated as previously described (Han
et al., Dev. Cell, 11:301-312, 2006). Transgene expression in oral
epithelia was confirmed (FIGS. 7A-B). The mice were bred into in
the C57BL/6 background, and 8-10 weeks old male and female
transgenic mice and wild-type littermates were used in studies.
These mice showed improved healing of excisional skin wounds (Han
et al., Am. J. Pathol., 179:1768-1779, 2011) and radiation-induced
oral mucositis.
[0238] K5.Smad7 mice and wild-type littermates were exposed to
cranial radiation to determine the biological equivalent dose (BED)
required to induce oral mucositis in mice. It was determined that 8
Gy.times.3 (BED=43.2), a regimen relevant to hypo-fractionated
radiotherapy in clinic, was the minimal dose needed to induce oral
mucositis (FIGS. 1A-B). To evaluate the potency of Smad7 effects,
they also tested single doses of cranial radiation and found that
oral mucositis severity correlated with BED values between 18 Gy
(BED=50.4) and 22 Gy (BED=70.4) (FIGS. 1A-B, FIG. 7C). By day 9
after initiation of radiation, wild-type mice developed oral ulcers
(FIGS. 1A-B).
[0239] K5.Smad7 oral mucosa prior to irradiation had morphology
similar to wild-type mice, but exhibited resistance to
radiation-induced oral mucositis (FIGS. 1A-B). Histological
analyses revealed that wild-type mice developed oral mucositis
(FIG. 1A) similar to that in humans (FIG. 1C). The First Affiliated
Hospital of Kunming Medical University, China provided
de-identified archived human tissue paraffin sections and approved
the study as an exempt for human subjects. Oral mucositis lesions
were from the tongue, buccal or oropharyngeal mucosa adjacent to
recurrent oral cancers that had undergone radiotherapy.
Non-irradiated oral mucosa sections were from surgically removed
sleep apnea oral tissues and a tongue biopsy adjacent to a cyst
(mucocele).
[0240] K5.Smad7 oral epithelia typically showed radiation
dose-dependent damage, i.e., thinning epithelium and flattened
tongue papillae after 8 Gy.times.3 radiation, and more damaged
(hypo- or hypertrophic) epithelial cells after 18 Gy and 22 Gy
radiation (FIG. 1A). Consistent with increased leukocyte
infiltration in human oral mucositis lesions (FIG. 1C), lesions in
wild-type mice harbored numerous infiltrated leukocytes (FIGS.
1D-E) consisting of neutrophils, macrophages, and lymphocytes (FIG.
7D); all were substantially reduced in K5.Smad7 oral mucosa (FIGS.
1D-E and 7D).
[0241] Because it is difficult to capture human oral mucositis
pathology at the acute phase, a mouse model was utilized to assess
proliferation and apoptosis when ulcers are just formed. Similar to
previous reports, proliferative cells were sparse in irradiated
wild-type oral epithelium, but were seen more in irradiated
K5.Smad7 oral epithelium (FIGS. 1D and 1F). Conversely, apoptotic
cells were significantly reduced in irradiated K5.Smad7 oral mucosa
compared to wild-type mice (FIGS. 1D and 1G).
[0242] As expected, cells with nuclear NF-.kappa.B p50 subunit were
significantly increased in oral mucositis compared to
non-irradiated wild-type oral mucosa (FIGS. 2A-B). Interestingly,
TGF-.beta.1, an immune suppressant in internal organ, but
pro-inflammatory in oral mucosa, together with its activated
signaling mediator, phosphorylated (p) Smad2, were also increased
in oral mucositis compared to non-irradiated oral mucosa in
wild-type mice (FIGS. 2A-B). Similar changes were also detected in
human oral mucositis lesions (FIGS. 2A-B).
[0243] Irradiated K5.Smad7 oral epithelia significantly reduced
cells positive for nuclear NF-.kappa.B p50 and pSmad2, even though
they still had abundant TGF-.beta.1 protein (FIGS. 2A-B).
TGF-.beta.1 mRNA in irradiated wild-type oral mucosa was
significantly increased on day 9 and day 10 (FIG. 2C). TGF-.beta.1
mRNA level in K5.Smad7 mucosa was similar to wild-type mucosa at
earlier time points, but was back to normal by day 10 (FIG. 2C).
Although not wishing to be bound by any theory, these data suggest
that TGF-.beta.1 transcription is not inhibited by Smad7, but its
more rapid decline in K5.Smad7 mucosa could be a consequence of
accelerated healing.
[0244] Phospho-Smad1/5/8, markers for activated BMP signaling, were
not affected by Smad7 before and after radiation (FIG. 7E). This
result is consistent with the ability of Smad7 to preferentially
inhibit TGF-.beta. signaling.
Example 2
Rac1 Contributes to Smad7-Mediated Keratinocyte Migration
[0245] To determine if Smad7 contributes to healing in human oral
keratinocytes, Smad7 was knocked down in spontaneously immortalized
human oral keratinocytes (NOK-SI) Smad7 knockdown blunted
keratinocyte migration after wounding (FIG. 2D and FIG. 8A).
Conversely, knocking down TGF-.beta.1 accelerated keratinocyte
migration (FIGS. 8B-8D), consistent with accelerated wound healing
seen in mice null for TGF-.beta.1 or Smad3.
[0246] To search for molecular mechanisms associated with
Smad7-mediated keratinocyte migration, Rac1, a protein
indispensable for oral wound healing was examined. Rac1 was reduced
after Smad7 knockdown (FIG. 2E). It was expected that TGF-.beta.1
overexpression in oral mucositis would activate Rac1 through a
Smad-independent mechanism. However, although total Rac1 protein
increased by 2-fold after irradiation, activated Rac1 protein did
not change considerably in wild-type tongues (FIG. 2F).
[0247] In K5.Smad7 oral mucosa, both total and activated Rac1 were
significantly increased by 4-fold and 8-fold, respectively,
compared to wild-type oral mucosa (FIG. 2F). To determine the
functional significance of Smad7-induced Rac1 activation, Rac1 was
knocked down in primary keratinocytes isolated from wild-type and
Smad7 transgenic neonatal skin, and assays for cell proliferation
and migration were performed. Rac1 knockdown showed modestly
reduced proliferation in wild-type and Smad7 keratinocytes (FIGS.
9A-9C), but almost completely abrogated Smad7-induced migration
(FIG. 2G and FIG. 9D), suggesting that increased Rac1 contributes
to Smad7-mediated cell migration.
[0248] It was observed that increased Rac1 mRNA levels in Smad7
transgenic keratinocytes correlated with total and active Rac1
protein levels (FIGS. 3A-B and FIGS. 10A-B), suggesting that
increased Rac1 activation in Smad7 keratinocytes is, at least in
part, a consequence of increased Rac1 transcripts. Further, Rac1
protein increased by .about.3-fold (FIG. 3C) after knockdown of
individual Smads in NOK-SI cells (FIGS. 10C-10E). These data
suggest that normal Smad signaling represses Rac1
transcription.
[0249] Among the two putative Smad binding elements (SBEs) in the
mouse Rac1 promoter (-2.1 Kb and -1.5 Kb upstream of the coding
sequence), which are in similar regions of the human Rac1 promoter,
chromatin immunoprecipitation (ChIP) identified Smad-2, -3, -4, and
-7 binding to the -1.5 Kb site (FIG. 3D), but not the -2.1 Kb site
in wild-type keratinocytes; binding of Smad-2, -3 and -4 was
significantly reduced in Smad7 transgenic keratinocytes (FIG.
3D).
[0250] Luciferase reporter assays using a SBE-containing Rac1-Luc
construct show that knockdown of Smad7 in wild-type keratinocytes
significantly reduced luciferase activity (FIG. 3E). Conversely,
Smad7 transgenic cells had increased luciferase activity compared
to wild-type cells, and mutating the SBE attenuated this increase
(FIG. 3F). Thus, Smad7 binding to SBE appears necessary to expel
signaling Smads to abrogate Rac1 repression.
[0251] Among known Smad transcriptional co-repressors, it was found
that CtBP1 bound to the Rac1 promoter SBE-1.5 Kb site in wild-type
keratinocytes (FIG. 3G), and Smad7 transgene expression
significantly reduced CtBP1 binding to the SBE (FIGS. 3G-H). When
CtBP1 was knocked down in NOK-SI cells, Rac1 protein and Rac1-Luc
activity were increased compared to keratinocytes transfected with
scrambled siRNA (FIGS. 4A-B), suggesting that CtBP1 binding to
SBE-1.5 Kb represses Rac1 expression. Further, knocking down CtBP1
in NOK-SI cells increased their migration (FIG. 4C and FIG.
10F).
[0252] Upon examination of CtBP1 protein in radiation-induced oral
mucositis, it was found that CtBP1 is barely detectable in
non-irradiated mouse and human oral mucosa (FIGS. 4D-4F); however,
CtBP1 positive cells were significantly increased in irradiated
oral mucosa of wild-type and K5.Smad7 mice as well as in human oral
mucositis (FIGS. 4D-4F). Additionally, CtBP1 mRNA in irradiated
wild-type oral mucosa was significantly increased on day 9 and day
10 (FIG. 4G). CtBP1 mRNA level in K5.Smad7 mucosa was similar to
wild-type mucosa at earlier time points, but declined to normal by
day 10 (FIG. 4G). These results indicate that Smad7 does not reduce
CtBP1 mRNA but instead inhibits CtBP1 binding to the Rac1 promoter
by repelling the Smad/CtBP1 complex from the SBE binding site;
further, more rapid CtBP1 reduction in K5.Smad7 mucosa serves as a
marker of healing.
Example 3
Tat-Smad7 Alleviates Radiation-Induced Oral Mucositis
[0253] Smad7 transgene's ability to block multiple pathological
processes of oral mucositis prompted us to explore if localized
Smad7 delivery can be used to prevent and treat oral mucositis.
Because Smad7 is a nuclear protein, local Smad7 delivery needs to
allow Smad7 to rapidly enter into cells before saliva washes off
the protein. Thus, a recombinant human Smad7 with an N-terminal
Tat-tag allowing proteins to rapidly permeate the cell membrane and
enter the nucleus was produced. A V5 epitope was added to the
C-terminal end of the Tat-Smad7 protein to track Tat-Smad7 cell
penetration (FIGS. 11A-11D).
[0254] Using its ability to block Smad2 phosphorylation, Tat-Smad7
bioactivity was tested (FIG. 11C). Tat-Cre recombinant protein with
the same tags as a control (FIGS. 11E-F) was produced, and cloned
into the pET101-Topo protein expression vector (Invitrogen) that
contains a sequence encoding C-terminal 6H (SEQ ID NO: 40). Tat-Cre
was transformed into BL-21 STAR.TM. E. coli (Invitrogen) to produce
Tat-Cre protein and was purified with Ni-NTA column.
[0255] The purity and size of both proteins was verified using
SDS-PAGE electrophoresis. To evaluate transduction and activity of
Tat-Smad7 protein in vitro, Tat-Smad7 was added to primary mouse
keratinocytes. Slides were fixed in cold methanol for 5 minutes and
stained for V5 and pSmad2. Tat-Cre activity was verified by
digesting a 1,460 bp floxed fragment from the 7,650 bp vector
pLL3.7 (Addgene). For in vivo treatments, 30 .mu.L 50% glycerol/PBS
as a vehicle control and Tat-Cre as a non-irrelevant protein
control were used. Tat-Smad7 or Tat-Cre (in 30 .mu.L 50%
glycerol/PBS, doses and regimens are specified in each figure) was
topically applied to mouse oral cavity and mice were restricted
from oral intake for 1 hour.
[0256] For oral mucositis prevention, both Tat-Smad7 and Tat-Cre
(in 50% glycerol/PBS) were topically applied to the oral cavity of
8-10 week old C3H females (Jackson Laboratory) or C57BL/6 mice
daily, starting 24 hours prior to radiation through day 8 after
initiation of radiation. Treated tissues were examined on day 9.
Mouse tongues were harvested, fixed in 10% formalin, embedded in
paraffin, and cut into 5 .mu.m sections. Histological changes were
analyzed and ulcers were measured using H&E stained slides. An
additional group received Palifermin treatment with a clinical
regimen, i.e., 6.25 mg kg.sup.-1 (i.p.) daily for 3 days prior to
irradiation, and daily for 3 days 24 hours after the last dose of
radiation.
[0257] Tat-Cre showed no effect compared to vehicle controls (FIGS.
5A-B). Tat-Smad7 treatments showed preventive effects on ulcer
formation similar to Palifermin (FIG. 5A). The dose-dependent
effect of Tat-Smad7 was more obvious when used on animals given a
20 Gy (BED=60) single dose of radiation that induced larger oral
ulcers than fractionated radiation (FIG. 11G). Microscopically,
both Palifermin and Tat-Smad7 treated oral mucosa prevented open
ulceration in the majority of cases (FIG. 5B). Palifermin-treated
mucosa exhibited more keratinocyte down-growth but also more
damaged keratinocytes (condensed or charcoal-like nuclei, swelled
mono- or multi-nucleated cells and shattered nuclear fragments in
conified layers) than Tat-Smad7-treated mucosa (FIG. 5B).
Immunostaining revealed that Palifermin increased proliferation
more significantly than Tat-Smad7. Tat-Smad7 reduced apoptosis,
leukocyte infiltration, nuclear pSmad2 and NF-.kappa.B p50, but
Palifermin did not (FIGS. 5B-5G).
[0258] To test whether Tat-Smad7 can be used to treat existing oral
mucositis, mice were exposed to fractionated (8 Gy.times.3) cranial
radiation and Tat-Smad7 (topically) or Palifermin (6.25 mg
kg.sup.-1, i.p.) was applied daily from day 6 after initiation of
radiation (when mucosal damage was obvious) till day 9. Treated
tissues were examined on day 10. Although beginning post-radiation
administration of Palifermin at earlier time points than the
current protocol reduced oral mucositis in mice, Palifermin
administration with the current protocol did not accelerate ulcer
closure (FIG. 6A), regardless of its hyperproliferative effect on
the entire oral mucosa (FIG. 6B). This is not surprising, as
Palifermin is approved to prevent but not treat oral mucositis.
[0259] Tat-Smad7 treated oral mucositis reduced ulcer sizes and
pathological alterations after both fractionated and single dose
radiation (FIGS. 6A-B and FIGS. 12A-12G). Away from ulcers,
Tat-Smad7 treated oral mucosa exhibited less hyperplasia and more
differentiated epithelia than Palifermin-treated oral mucosa (FIG.
6B). With a 20 Gy single dose radiation that caused slower healing
than fractionated radiation, the effect of Tat-Smad7 on recovery
after wound closure was more obvious. When vehicle treated ulcer
was just re-epithelialized, Tat-Smad7 treated mucosa had almost
recovered to normal morphology (FIG. 6C).
[0260] Consistent with observations in K5.Smad7 mice, Tat-Smad7
increased Rac1 promoter activity and reduced CtBP1 binding to the
SBE of the mouse Rac1 promoter (FIGS. 12G and 12I), and increased
Rac1 protein in mouse oral mucositis and human oral keratinocytes
(FIGS. 6D-E).
[0261] Tat-Smad7-treated human oral keratinocytes after wound
scratch had accelerated wound closure (FIG. 6F and FIG. 13A).
Further, irradiated human oral keratinocytes increased nuclear
pSmad2 and NF-.kappa.B p50, which were attenuated by Tat-Smad7
treatment (FIG. 13B). In contrast, although Tat-Smad7 penetrated
oral cancer cells efficiently (FIG. 13C), it did not further
elevate Rac1 protein level that is already abundant in cancer cells
(FIG. 13D). This result could account for faster migration of
cancer cells than normal keratinocytes (FIG. 6F and FIGS. 13A,
13E-13H), and the lack of an effect of Tat-Smad7 on migration in
two oral cancer cell lines: MSK921 which does not contain genetic
loss of TGF-.beta. signaling components; and Cal27 which has a
mutated Smad4 (FIGS. 13E-13H).
[0262] Colony assays show that survival of human oral keratinocytes
was slightly increased by Tat-Smad7 treatment with or without
radiation (FIG. 6G). Consistent with the notion that reduced
survival after irradiation is more prominent in cancer cells than
in normal cells, SCC cells showed substantial reductions in cell
survival after radiation. Treatment with Tat-Smad7 did not affect
survival in SCC cells with or without radiation (FIG. 6G).
Example 4
Design of a Cell-Penetrating Smad7 Protein
[0263] It was hypothesized that in order to be effective as a
therapeutic, SMAD7 needed to be able to penetrate cells
efficiently. In order to achieve this, the Smad7 sequence was
modified to include a protein transduction domain.
[0264] The Tat sequence from HIV was selected to test with Smad7 as
a protein transduction domain. The nucleotide and protein sequences
of Tat that were used in fusion proteins with Smad7 and Smad7
fragments are derived from Cardarelli et al., Traffic Apr
9(4):528-39 (2008). The Tat nucleotide and amino acid sequences are
provided below:
TABLE-US-00003 (SEQ ID NO: 1) ggccgtaaaaaacgccgtcaacgccgccgt (SEQ
ID NO: 2) G R K K R R Q R R R
Fusion proteins were prepared having Tat directly linked in frame
to human Smad7 complementary DNA (cDNA) either at the 5' or 3' ends
of Smad7 as shown below:
TABLE-US-00004 5'Tat: (SEQ ID NO: 7)
Ggccgtaaaaaacgccgtcaacgccgccgt-Smad7 3'Tat: (SEQ ID NO: 8)
Smad7-Ggccgtaaaaaacgccgtcaacgccgccgt
The 5' Tat-Smad7 construct included a 3' V5 tag sequence, and was
cloned into the pGEX-6p-1 protein expression vector (New England
Biolabs) to make a GST-Tat-Smad7 fusion protein. Tat-Smad7 gene was
transformed into BL-21 Star Escherichia coli (Invitrogen) to
produce Tat-Smad7 protein. The protein was purified by glutathione
column purification and elution, using enzymatic cleavage from the
Glutathione S Transferase (GST) fusion (Precision enzyme, GE Life
Sciences).
[0265] While creating a PTD-Smad7 fusion protein, a V5 tag at the
3' end was included to monitor Tat-Smad7 penetration into cells by
immunostaining using a V5 antibody. This epitope tag can be deleted
for use in the clinic (e.g., by re-cloning the sequence in the
absence of the V5 tag), if appropriate.
[0266] A PTD-Smad7 fusion protein (Tat-Smad7-V5-6H) ("6H" disclosed
as SEQ ID NO: 40) was also created having a 6-Histidine (6H) tag
(SEQ ID NO: 40) for protein purification, and is shown below.
Tat-Smad7-V5-6H ("6H" disclosed as SEQ ID NO: 40) has the following
nucleotide sequence: 1-53 include the 5' sequence of pET-TOPO;
54-1365 include Tat-Smad7; 1366-1497 include 3' pET-TOPO containing
the V5 epitope and 6H tag (SEQ ID NO: 40) (V5 includes 1393-1434,
His tag includes 1444-1461, and the Stop includes 1462-1464).
[0267] Tat-human Smad7, codon-optimized for protein production,
cloned to pET101/D-Topo vector is shown below:
TABLE-US-00005 (SEQ ID NO: 9)
ttcccctctagaaataattttgtttaactttaagaaggaattcaggagcccttcaccatg M
cgtaaaaaacgccgtcaacgccgccgtggtttccgtacgaaacgctcggccctggtccgt R K K
R R Q R R R G F R T K R S A L V R
cgcctgtggcgctcccgtgctccgggtggtgaagatgaagaagaaggtgctggcggcggt R L W
R S R A P G G E D E E E G A G G G
ggcggtggcggtgaactgcgtggcgagggtgcaaccgatagtcgtgcacacggtgcaggc G G G
G E L R G E G A T D S R A H G A G
ggtggcggtccgggtcgtgctggttgctgtctgggtaaagctgtgcgcggcgcgaaaggt G G G
P G R A G C C L G K A V R G A K G
catcaccatccgcacccgccggcagcaggtgcaggtgcagctggcggtgcggaagccgat H H H
P H P P A A G A G A A G G A E A D
ctgaaagccctgacccatagtgtcctgaaaaaactgaaagaacgtcagctggagctgctg L K A
L T H S V L K K L K E R Q L E L L
ctgcaagcagtagaatcccgtggcggtacccgtacggcttgtctgctgctgccgggtcgt L Q A
V E S R G G T R T A C L L L P G R
ctggattgccgtctgggtccgggtgcaccggctggtgcgcagccggcacaaccgccgagc L D C
R L G P G A P A G A Q P A Q P P S
tcttacagcctgccgctgctgctgtgtaaagtgtttcgttggccggacctgcgccacagt S Y S
L P L L L C K V F R W P D L R H S
tccgaagttaaacgcctgtgctgttgcgagagctatggcaaaattaacccggaactggtt S E V
K R L C C C E S Y G K I N P E L V
tgttgcaatccgcaccatctgtctcgtctgtgtgaactggagagcccgccgccgccgtat C C N
P H H L S R L C E L E S P P P P Y
tctcgttacccgatggatttcctgaaaccgactgcagattgcccggacgcagtcccgtca S R Y
P M D F L K P T A D C P D A V P S
tcggctgagaccggcggcaccaactatctggcaccgggcggtctgagtgattcccagctg S A E
T G G T N Y L A P G G L S D S Q L
ctgctggaaccgggcgaccgttcacattggtgtgtggttgcctattgggaagagaaaacg L L E
P G D R S H W C V V A Y W E E K T
cgtgtcggtcgcctgtactgcgtacaggaaccgtcgctggatatcttttatgacctgccg R V G
R L Y C V Q E P S L D I F Y D L P
cagggcaatggtttctgtctgggccaactgaactcagataataaatcgcagctggtgcaa Q G N
G F C L G Q L N S D N K S Q L V Q
aaagttcgctcaaaaattggctgcggtatccagctgacccgtgaagttgacggtgtctgg K V R
S K I G C G I Q L T R E V D G V W
gtatataaccgcagctcttacccgatttttatcaaaagtgccaccctggataatccggac V Y N
R S S Y P I F I K S A T L D N P D
tcccgtacgctgctggtccacaaagtatttccgggcttctcaatcaaagcgttcgattac S R T
L L V H K V F P G F S I K A F D Y
gagaaagcctactcgctgcagcgcccgaacgaccatgaattcatgcagcaaccgtggacg E K A
Y S L Q R P N D H E F M Q Q P W T
ggttttactgtgcagatctctttcgttaaaggctggggtcaatgctacacccgtcagttt G F T
V Q I S F V K G W G Q C Y T R Q F
atctcgtcctgtccgtgctggctggaagtgattttcaatagccgcaagggcgagctcaat I S S
C P C W L E V I F N S R K G E L N
tcgaagcttgaaggtaagcctatccctaaccctctcctcggtctcgattctacgcgtacc S K L
E G K P I P N P L L G L D S T R T
Ggtcatcatcaccatcaccattgagtttgatccggctgctaacaaagcccgaaagga (SEQ ID
NO: 10) G H H H H H H-
[0268] A comparison of the protein sequence of Tat-Smad7-v5 and
Smad7 is provided below. The first amino acid of Smad7 in Tat-Smad7
is not M (unlike Smad7), because Tat-Smad7 is designed to be
in-frame with Tat and/or GST to form a GST fusion protein.
Tat-Smad7 is then cleaved from the GST fusion protein after
purification. Upper case nucleotides identify the V5 tag.
Underlined italics indicate amino acids from the optional
pET101-Topo backbone vector.
[0269] Below a Tat-Smad7-v5 and Smad7 comparison is presented:
TABLE-US-00006 Tat-Smad7-V5 1 gsgrkkrrqrrrgfrtkrsalvrrlwrsra
pggedeeegagggggggelr human Smad7 1 ------------mfrtkrsalvrrlwrsra
pggedeeegagggggggelr Tat-Smad7-V5 51 gegatdsrahgaggggpgragcclgkavr
gakghhhphppaagagaagga human Smad7 39 gegatdsrahgaggggpgragcclgkavr
gakghhhphppaagagaagga Tat-Smad7-V5 101 eadlkalthsvlkklkerqlelllqave
srggtrtaclllpgrldcrlgp human Smad7 89 eadlkalthsvlkklkerqlelllqaves
rggtrtaclllpgrldcrlgp Tat-Smad7-V5 151 gapagaqpaqppssyslplllckvfrwp
dlrhssevkrlcccesygkinp human Smad7 139 gapagaqpaqppssyslplllckvfrwp
dlrhssevkrlcccesygkinp Tat-Smad7-V5 201
elvccnphhlsrlcelesppppysrypm dflkptadcpdavpssaetggt human Smad7 189
elvccnphhlsrlcelesppppysrypm dflkptadcpdavpssaetggt Tat-Smad7-V5
251 nylapgglsdsqlllepgdrshwcvvay weektrvgrlycvqepsldify human Smad7
239 nylapgglsdsqlllepgdrshwcvvay weektrvgrlycvqepsldify
Tat-Smad7-V5 301 dlpqgngfclgqlnsdnksqlvqkvrsk
igcgiqltrevdgvwvynrssy human Smad7 289 dlpqgngfclgqlnsdnksqlvqkvrsk
igcgiqltrevdgvwvynrssy Tat-Smad7-V5 351
pifiksatldnpdsrtllvhkvfpgfsi kafdyekayslqrpndhefmqq human Smad7 339
pifiksatldnpdsrtllvhkvfpgfsi kafdyedayslqrpndhefmqq Tat-Smad7-V5
401 pwtgftvqisfvkgwgqcytrqfisscp cwlevifnsrkgelnskleGKP human Smad7
389 pwtgftvqisfvkgwgqcytrqfisscp cwlevifnsr------------
Tat-Smad7-V5 451 (SEQ ID NO: 11) IPNPLLGLDST human Smad7 (SEQ ID
NO: 12) 427 -----------
Example 5
Additional Assays for PTD-Smad7 Protein Activity
[0270] Immunofluorescence (IF), Immunohistochemistry (IHC), and
TUNEL Assay for Apoptosis.
[0271] IF and IHC were performed as previously described (Han, G.,
Li, F., Ten Dijke, P. & Wang, X. J. Temporal smad7 transgene
induction in mouse epidermis accelerates skin wound healing. Am J
Pathol 179, 1768-1779 (2011)). Primary antibodies used were guinea
pig antibody to K14 (1:400, Fitzgerald, 20R-CP200), rat antibody to
CD4 (1:20, BD Bioscience, 550278), Ly-6G (1:20, BD Bioscience,
550291), BM8 (antibody to F4/80, 1:20, Invitrogen, MF48000),
FITC-labeled antibody to BrdU (BD Bioscience, 347583), rat antibody
to CD45 (1:50, BD Bioscience, 550539) for mouse samples, mouse
antibody to CD45 (1:50, Abcam, Ab781) for human samples, chicken
antibody to TGF-.beta.1 (1:50, R&D, AF-101-NA), rabbit antibody
to CtBP1 (1:100, Millipore, 07-306), rabbit antibody to NF-.kappa.B
p50 (1:200, Santa Cruz Biotechnology, SC-7178), rabbit antibody to
PCNA (1:200, Santa Cruz Biotechnology, SC-7907), rabbit antibody to
pSmad2 (1:100, Cell Signaling Technology, 3101), and mouse antibody
to V5 (1:500, Invitrogen, 460705). For IF, secondary antibodies to
different species IgG were Alexa Fluor.RTM. 594 (red) or 488
(green) conjugated (1:200 for all, Invitrogen). For IHC, secondary
biotinylated antibodies to different species IgG (1:300, Vector
Labs) were used and were developed using Vectastain ABC kit (Vector
Labs). A Terminal deoxynucleotidyl transferase uridine nick
end-labeling (TUNEL, G3250) kit (Promega) was used on formalin
fixed tissue sections to detect apoptotic cells. BrdU labeling was
performed in vivo by i.p. injection of 0.125 mg g.sup.-1 BrdU 1
hour prior to euthanization. PCNA or BrdU were quantified as cells
mm.sup.-1 epithelial length including all epithelial cells, TUNEL
or CD45-positive cells as cells mm.sup.-1 epithelial length
including all epithelial layers and stroma above the muscle layer,
nuclear pSmad2 or NF-.kappa.B p50 positive cells as the number of
positive cells/existing total remaining epithelial cells (i.e.,
excluding sloughed epithelial cells induced by irradiation).
Consecutive fields of slides were used to count BrdU-labeled cells
using MetaMorph software.
[0272] Cell Culture.
[0273] Smad7 transgenic and wild-type primary keratinocytes were
prepared from neonatal mouse skin as previously described (Han, G.,
Li, F., Ten Dijke, P. & Wang, X. J. Temporal smad7 transgene
induction in mouse epidermis accelerates skin wound healing. Am J
Pathol 179, 1768-1779 (2011)), and cultured in PCT medium
(CELLnTEC). Spontaneously immortalized normal oral keratinocytes
(NOK-SI) derived from gingival tissues of healthy volunteers were
cultured and maintained in defined keratinocyte medium (Castilho,
R. M., et al. Rac1 is required for epithelial stem cell function
during dermal and oral mucosal wound healing but not for tissue
homeostasis in mice. PloS one 5, e10503 (2010)). Oral cancer cells
Cal27 (ATCC) and MSK921 were cultured (D. Raben's lab,
fingerprinted by University of Colorado Cancer Center Tissue
Culture Core) in Dulbecco Modified Eagle Medium supplemented with
10% fetal bovine serum (GIBCO.RTM.; Invitrogen). To assess the
effect of Tat-Smad7 in irradiated cells, the above human cell lines
were cultured in chamber slides (BD Bioscience, 354108), irradiated
with 3 Gy, and Tat-Smad7 (1 .mu.g mL.sup.-1) was added to the
culture medium immediately after irradiation. Cells were fixed in
100% cold methanol 4 hours after Tat-Smad7 treatment for
immunostaining of pSmad2, NF-.kappa.B p50 and V5.
[0274] Transfection with siRNA.
[0275] When cultured keratinocytes reached 70% confluency, 100 nM
of target siRNA or scrambled siRNA (Dharmacon) was transfected
using LIPOFECTAMINE.RTM. 2000 (Invitrogen). Cells were harvested
48-72 hours after transfection and subjected to western analyses to
determine knockdown efficiency. For migration assays, siRNA was
transfected when cells were plated. Target siRNAs included in this
study are: mouse siRac1-1 (Invitrogen, MSS237708) and siRac1-2
(IDT, MMC.RNAI.N009007.12.3); human siSmad2 (Dharmacon,
L-003561-00-0005), siSmad3 (Invitrogen, HSS106252), and siSmad4
(Invitrogen, HSS118066); human siCtBP1-1 and siCtBP1-2; human
siSmad7-1 and siSmad7-2; human TGF-.beta.1 (Dharmacon,
J-012562-08-0005); mouse siSmad7.
[0276] In Vitro Keratinocyte Proliferation Assay.
[0277] In vitro keratinocyte proliferation was determined by BrdU
incorporation in wild-type and Smad7 transgenic keratinocytes.
Cells at 70% confluency were transfected with Rac1 siRNAs, and
changed to regular culture medium 24 hours later. An in situ cell
proliferation kit (Roche Applied Science) was used to perform in
vitro BrdU labeling and detection, and MetaMorph software was used
to count BrdU-labeled cells.
[0278] In Vitro Cell Migration Assays.
[0279] When cells reached 100% confluency, the cells were treated
with mitomycin C (Sigma) at 10 .mu.g mL.sup.-1 for 2 hours to
inhibit cell proliferation and a scratch wound was introduced with
a Fisherbrand pipet tip. Cell migration was photographed daily.
Migration assays were performed when cells reached confluency after
24 to 36 hours of siRNA transfection, and Image-J software was used
to document cell migration as the wound area occupied with
migrating cells. For Tat-Smad7 treatment, cells were exposed to
Tat-Smad7 protein at 1 .mu.g mL.sup.-1 or vehicle control (PBS) in
medium after wound scratch, and medium was changed every other day
with freshly added Tat-Smad7 until migrating cells fully covered
the scratched wound.
[0280] Cell Survival Assay.
[0281] Cell survival assays were performed as previously described
(Munshi, A., Hobbs, M. & Meyn, R. E. Clonogenic cell survival
assay. Methods in molecular medicine 110, 21-28 (2005)), with
slight modifications. Briefly, cells were plated in 12-well plates
at 500 cells well.sup.-1 for non-irradiated wells, and increased up
to 1,500 cells well.sup.-1 along with increased radiation doses.
Cells were irradiated 24 hours after they were plated. Tat-Smad7
was added at 1 .mu.g mL.sup.-1 or the same volume of PBS used to
dissolve Tat-Smad7 (control) to culture medium of irradiated and
non-irradiated cells. The medium was changed every other day with
freshly added Tat-Smad7 or PBS for 10 to 14 days. Colonies were
fixed in methanol, stained in 0.5% crystal violet solution
(containing 25% methanol), counted and the average from 4 wells in
each experiment was calculated. Two to three separate experiments
were performed for each cell line. The relative surviving fraction
was calculated as previously described, i.e., the absolute
surviving fraction (colony numbers/total plated cells) under each
radiation dose divided by the absolute surviving fraction of
non-irradiated cells.
[0282] Western Analysis.
[0283] Protein extraction and western analyses were performed as
previously described (Li, A. G., Lu, S. L., Zhang, M. X., Deng, C.
& Wang, X. J. Smad3 knockout mice exhibit a resistance to skin
chemical carcinogenesis. Cancer Res 64, 7836-7845 (2004)). The
antibodies used in this study included rabbit antibody to Smad7
(1:500), rabbit antibodies to Smad2 (1:300, Zymed, 51-1300) and
Smad4 (1:300, Epitomics, 1676-1), rabbit antibody to Smad3 (1:300,
Cell Signaling Technology, 9513), mouse antibody to Rac1 (1:500, BD
Biosciences, 610651), rabbit antibody to CtBP1 (1:500, Millipore,
07-306), mouse antibody to tubulin (1:3000, Sigma, T5168), mouse
antibody to GAPDH (1:5000, Abcam, Ab8245) and goat antibody to
actin (1:1000, Santa Cruz Biotechnology, SC1616). Gray-scale images
were obtained using the ODYSSEY.RTM. v.1.2 software (LI-COR
Biosciences).
[0284] Rac1 Activation Assay.
[0285] Active GTP-bound Rac1 was examined using a BIOCHEM.TM. Kit
for Rac1 activation (Cytoskeleton Inc, BK035). Wild-type and Smad7
transgenic keratinocytes were cultured in 15 cm diameter tissue
culture plates and prepared protein lysates using the provided
lysis buffer. To assay Rac1 activity, 1 mg of cell lysate was used.
To examine total Rac1 and Smad7 proteins, 50 .mu.g of lysate was
used. To measure GTP-bound Rac1 in mouse tongues, half of the
tongue was ground to a powder in liquid nitrogen and lysed with
lysis buffer to extract protein, GTP-bound Rac1 was assayed in 2 mg
of protein lysate per sample and 50 .mu.g of protein lysate was
loaded for total Rac1 protein western blot.
[0286] ChIP Assays.
[0287] ChIP assays were performed using the ChIP-IT express kit
(Active Motive, 53009) as previously described (Hoot, K. E., et al.
HGF upregulation contributes to angiogenesis in mice with
keratinocyte-specific Smad2 deletion. J Clin Invest 120, 3606-3616
(2010); Hoot, K. E., et al. Keratinocyte-specific Smad2 ablation
results in increased epithelial-mesenchymal transition during skin
cancer formation and progression. Owens, et al., J. Clin. Invest
118, 2722-2732 (2008). Smad4-dependent desmoglein-4 expression
contributes to hair follicle integrity. Owens, et al., Dev. Biol.
322:156-166 (2008). DNA-protein complex was isolated from primary
mouse keratinocytes. For ChIP, 6.3 .mu.g sheared chromatin was
incubated with protein-G magnetic beads and 2 .mu.g each of rabbit
antibodies to Smad2 (Cell Signaling Technology, 3122), Smad3 (Cell
Signaling Technology, 9523), Smad4 (Cell Signaling Technology,
9515), Smad7 antibody (Santa Cruz Biotechnology, SC-11392), CtBP1
(Millipore) or a negative control rabbit IgG (Santa Cruz
Biotechnology, SC-2027). Eluted DNA from the protein-DNA complex
was used for PCR analyses, and CtBP1 binding to the Rac1 promoter
was compared in wild-type and Smad7 transgenic keratinocytes by
ChIP band intensities on gel images or by quantitative PCR using
Power SYBR Green Master Mix (Applied Biosystems). Primers used to
amplify the Rac1 SBE-1.5 Kb promoter regions:
TABLE-US-00007 (SEQ ID NO: 13) 5'-TGGAATTCCTGGTCTGGTTT-3' (sense)
(SEQ ID NO: 14) 5'-GCCAAGCTGCTCTTCCAGTA-3' (antisense) (SEQ ID NO:
15) 5'-TCTCAGGGGGCCAAAGGTGTT-3' (sense) (SEQ ID NO: 16)
5'-TCCCAGCACCTGAATCACATGG-3' (antisense)
[0288] Rac1 Promoter Luciferase Reporter Construct, Site-Directed
Mutagenesis and Luciferase Assay.
[0289] The 883 bp fragment of -1671 bp to -789 bp of the Rac1
promoter, encompassing the SBE-1.5 Kb site, was amplified from
wild-type mouse DNA using 5' XhoI and 3' HindIII tagged primers,
and this Rac1 promoter fragment was cloned into pGL4.26 vector
(Promega) to make the Rac1 promoter-pGL4.26 luciferase reporter
(Rac1-Luc) construct. For site-directed mutagenesis, the SBE
sequence 5'-TGTCTGTGCT-3' (SEQ ID NO: 17) was mutated to
5'-TGATAGAGCT-3' (SEQ ID NO: 18). Rac1-Luc and pGL4.74 (1:20) were
co-transfected with Smad7 siRNA, CtBP1 siRNA or scrambled siRNA
using Lipofectamine 2000 (Invitrogen) to primary mouse
keratinocytes, or primary mouse keratinocytes with Tat-Smad7
treatment (1 .mu.g mL.sup.-1). Cell lysates were collected and
luciferase assays were performed 48 hours after transfection or
Tat-Smad7 treatment, using the DUAL-LUCIFERASE.RTM. Reporter Assay
kit (Promega) following manufacturer's instructions.
Rac1-luciferase activity was measured with the Glomax machine
(Promega) and expressed by the ratio of firefly activity to Renilla
activity. Primers used for amplification of Rac1 promoter sequence
were:
TABLE-US-00008 (SEQ ID NO: 19) 5'-ATCCTCGAG-TATCCTCCAGGTCTGGG-3'
(SEQ ID NO: 20) 5'-GCCAAGCTT-AGCGTCCAGCGTTAACCTG-3'
[0290] Statistical Analysis.
[0291] Statistical differences in molecular analyses and oral
mucositis ulcer size were analyzed using the Student's t-test and
all data was presented by mean.+-.s.d. except ulcer size, which was
presented by mean.+-.s.e.m. Oral mucositis incidences were analyzed
by Fisher's exact test.
Example 6
Codon Optimization for Smad7 Protein Production in E. coli or
Yeast
[0292] Although many mammalian proteins can be produced in bacteria
without nucleotide sequence modification, the analysis indicated
that the Smad7 nucleotide sequence would need to be modified to
allow protein expression in bacteria.
[0293] Analysis of Smad7 cDNA mammalian codon use revealed nine
arginine amino acids coded for by the following nucleotides: 7-9,
43-45, 169-171, 403-405, 490-492, 526-528, 526-528, 823-825,
1057-1059 are a rare codon (AGG, codon utilization 1.7%). Since
these codons are rare codons in bacteria, it is expected that they
could halt or reduce protein translation and/or production in
bacteria. The amino acids coded for by rare arginine codons are
indicated by bold capitals below in the illustrated human Smad7
protein, including arginines at positions 3, 15, 57, 135, 164, 169,
176, 275, and 353. Additionally, the following arginine codons also
have low frequency usages. CGA (3.5% codon utilization):
nucleotides 16-18, 136-138, 199-201, 598-600, which code for
arginine at positions 6, 46, 67, 200; CGG (5.4% codon utilization):
nucleotides 31-33, 112-114, 316-318, 772-774, 940-942, 973-975,
1135-1137, 1276-1278, which code for arginine at positions 11, 38,
106, 258, 314, 325, 379, 426; AGA (2.8% codon utilization):
nucleotides 637-639, 814-816, which code for arginine at positions
213, 272. These arginine residues are highlighted in bold upper
case R below and they are changed to CGC in at least one of the
codon-optimized nucleic acid sequences (20.6% codon
utilization):
TABLE-US-00009 (SEQ ID NO: 12) 1 mfRtkRsaly RrlwRsrapg gedeeegagg
gggggelRge gatdsRahga 51 ggggpgRagc clgkavrgak ghhhphppaa
gagaaggaea dlkalthsvl 101 kklkeRqlel llqavesrgg trtaclllpg
rldcRlgpga pagaqpaqpp 151 ssyslplllc kyfRwpdlRh sseykRlccc
esygkinpel vccnphhlsR 201 lcelespppp ysRypmdflk ptadcpdavp
ssaetggtny lapgglsdsq 251 lllepgdRsh wcvvayweek tRygRlycyq
epsldifydl pqgngfclgq 301 lnsdnksqlv qkvitskigcg iqltRevdgy
wvynrssypi fiksatldnp 351 dsRtllyhkv fpgfsikafd yekayslqRp
ndhefmqqpw tgftvgisfy 401 kgwgqcytrq fisscpcwle vifnsR
[0294] Based on this analysis, it was decided to optimize the Smad7
nucleotide sequence to codons that were believed to allow increased
Tat-Smad7 protein production in E. coli or yeast. Provided below is
the optimized nucleic acid codon sequence made by Genscript.
Briefly, the sequence has the following composition: nucleotides
1-6 include the restriction recognition site for BamHI; nucleotides
7-36 include the Tat sequence; nucleotides 37-1314 include
codon-optimized human Smad7 cDNA; nucleotides 1342-1383 include the
V5 epitope; nucleotides 1384-1386 are the stop codon; and
nucleotides 1387-1392 including the restriction recognition site
for SalI. In this sequence, ATG is removed to be used with GST. The
entire designed sequence was converted to E. coli codons based on
"Codon-Usage Database." The initial optimized Smad7 sequence (SEQ
ID NO: 23) is shown below:
TABLE-US-00010 (SEQ ID NO: 23) 1 ggatccggcc gtaaaaaacg ccgtcaacgc
cgccgtggtt tccgtacgaa acgctcggcc 61 ctggtccgtc gcctgtggcg
ctcccgtgct ccgggtggtg aagatgaaga agaaggtgct 121 ggcggcggtg
gcggtggcgg tgaactgcgt ggcgagggtg caaccgatag tcgtgcacac 181
ggtgcaggcg gtggcggtcc gggtcgtgct ggttgctgtc tgggtaaagc tgtgcgcggc
241 gcgaaaggtc atcaccatcc gcacccgccg gcagcaggtg caggtgcagc
tggcggtgcg 301 gaagccgatc tgaaagccct gacccatagt gtcctgaaaa
aactgaaaga acgtcagctg 361 gagctgctgc tgcaagcagt agaatcccgt
ggcggtaccc gtacggcttg tctgctgctg 421 ccgggtcgtc tggattgccg
tctgggtccg ggtgcaccgg ctggtgcgca gccggcacaa 481 ccgccgagct
cttacagcct gccgctgctg ctgtgtaaag tgtttcgttg gccggacctg 541
cgccacagtt ccgaagttaa acgcctgtgc tgttgcgaga gctatggcaa aattaacccg
601 gaactggttt gttgcaatcc gcaccatctg tctcgtctgt gtgaactgga
gagcccgccg 661 ccgccgtatt ctcgttaccc gatggatttc ctgaaaccga
ctgcagattg cccggacgca 721 gtcccgtcat cggctgagac cggcggcacc
aactatctgg caccgggcgg tctgagtgat 781 tcccagctgc tgctggaacc
gggcgaccgt tcacattggt gtgtggttgc ctattgggaa 841 gagaaaacgc
gtgtcggtcg cctgtactgc gtacaggaac cgtcgctgga tatcttttat 901
gacctgccgc agggcaatgg tttctgtctg ggccaactga actcagataa taaatcgcag
961 ctggtgcaaa aagttcgctc aaaaattggc tgcggtatcc agctgacccg
tgaagttgac 1021 ggtgtctggg tatataaccg cagctcttac ccgatatta
tcaaaagtgc caccctggat 1081 aatccggact cccgtacgct gctggtccac
aaagtatttc cgggcttctc aatcaaagcg 1141 ttcgattacg agaaagccta
ctcgctgcag cgcccgaacg accatgaatt catgcagcaa 1201 ccgtggacgg
gttttactgt gcagatctct ttcgttaaag gctggggtca atgctacacc 1261
cgtcagttta tctcgtcctg tccgtgctgg ctggaagtga tatcaatag ccgcaagggc
1321 gagctcaatt cgaagcttga aggtaagcct atccctaacc ctctcctcgg
tctcgattct 1381 acgtgagtcg ac
[0295] A nucleotide sequence comparison between Tat-Smad7-V5 (SEQ
ID NO: 23) and human Smad7 (SEQ ID NO: 22) cDNA is provided below.
Human Smad7 and codon-optimized Tat-Smad7-V5 share 68% codon
homology. Human Smad7 and codon-optimized Tat-Smad7 share 71% codon
homology. Human Smad7 and codon-optimized Smad7 share 73% codon
homology.
TABLE-US-00011 Alignment: Global DNA alignment against reference
molecule Parameters: Scoring matrix: Linear (Mismatch 2, OpenGap 4,
ExtGap 1) Reference molecule: human Smad7 mRNA, Region 1-1281
Number of sequences to align: 2 Settings: Similarity significance
value cutoff: >= 90% Summary of Percent Matches: Reference:
human Smad7 mRNA 1-1281 (1281 bps) Sequence 2: Tat-Smad7-V5 1-1392
(1392 bps) 68% ##STR00001##
[0296] In this optimization, Met216, which may form an alternative
open reading frame, was not altered as it was desired to preserve
the amino acid sequence of Smad7, if possible. In future codon
optimizations, Met216 will be mutated to Leu216 to improve protein
production without impacting function in vitro and in vivo.
Example 7
Production of Truncated Smad7 Proteins
[0297] It is believed that Smad7 has several activities in vivo
including, but not limited to, one or more of enhancing cell
proliferation, enhancing cell migration, reducing DNA damage,
reducing cell apoptosis, and decreasing inflammation. Smad7's
effects on these processes are due to one or more of blocking
TGF-.beta. signaling, blocking NF-.kappa.B signaling, blocking
CtBP1 activity, and/or increasing Rac1 expression and/or activity.
It is believed that a smaller functional domain of Ptd-Smad7 may be
sufficient to deliver a therapeutic effect (see, e.g., FIG. 15). In
addition, the resulting shorter protein sequence is expected to
enhance protein production. Additionally, it is believed that
different truncated Tat-Smad7 proteins that contain partial Smad7
sequences may be useful for different treatments.
[0298] For example, it is believed that the C-terminal MH2 domain
of Smad7 (about half length of Smad7 protein, e.g., 208-426aa) may
primarily mediate the anti-inflammatory effect of Smad7 (Hong et
al., Nat Immunology, 8, 504-513, 2007). Smad7 peptides having this
anti-inflammatory function may be sufficient and optionally an
improvement for treating chronic inflammation associated
conditions, such as but not limited to, oral mucositis, stomatitis
and psoriasis, among others.
[0299] The N-terminal MH1 domain plus the linker region of Smad7
(about half of the protein, e.g., 2-208aa) is known to activate
MAPK and binds to Smurf, a ubiquitin E3 ligase to degrade
TGF-.beta. receptor (Aragon, et al., Structure 20:1726-1736
(2012)). It is believed that it may primarily mediate cell
migration and/or blocking TGF-.beta.-induced growth arrest and/or
fibrotic response. Smad7 peptides having this cell migration and
proliferation function may be sufficient, and optionally an
improvement, for enhancing healing that is not associated with
excessive inflammation. Types of wounds that might benefit from
this form of treatment include, but are not limited to, surgical
wounds, fibrotic scarring, and diabetes wounds, defective healing
and/or scarring among others.
[0300] Truncated Smad7 N-terminal and C-terminal PTD-fusion
proteins were designed. One example of a Tat-Smad7-C-terminal
codon-optimized nucleotide and protein sequence is provided below.
In the nucleic acid sequence, nucleotides 1-6 include the
restriction recognition site for BamHI; nucleotides 7-36 include
the Tat PTD sequence; nucleotides 37-810 include codon-optimized
for the C terminal amino acids 258 to 426 of human Smad7;
nucleotides 568-609 include the V5 epitope sequence; nucleotides
610-612 include the stop sequence; and nucleotides 613-618 include
the restriction recognition site for SalI:
TABLE-US-00012
ggatccggccgtaaaaaacgccgtcaacgccgccgttcacattggtgtgtggttgcctat G S G
R K K R R Q R R R S H W C V V A Y
tgggaagagaaaacgcgtgtcggtcgcctgtactgcgtacaggaaccgtcgctggatatc W E E
K T R V G R L Y C V Q E P S L D I
ttttatgacctgccgcagggcaatggtttctgtctgggccaactgaactcagataataaa F Y D
L P Q G N G F C L G Q L N S D N K
tcgcagctggtgcaaaaagttcgctcaaaaattggctgcggtatccagctgacccgtgaa S Q L
V Q K V R S K I G C G I Q L T R E
gttgacggtgtctgggtatataaccgcagctcttacccgatttttatcaaaagtgccacc V D G
V W V Y N R S S Y P I F I K S A T
ctggataatccggactcccgtacgctgctggtccacaaagtatttccgggcttctcaatc L D N
P D S R T L L V H K V F P G F S I
aaagcgttcgattacgagaaagcctactcgctgcagcgcccgaacgaccatgaattcatg K A F
D Y E K A Y S L Q R P N D H E F M
cagcaaccgtggacgggttttactgtgcagatctctttcgttaaaggctggggtcaatgc Q Q P
W T G F T V Q I S F V K G W G Q C
tacacccgtcagtttatctcgtcctgtccgtgctggctggaagtgattacaatagccgc Y T R Q
F I S S C P C W L E V I F N S R
aagggcgagctcaattcgaagcttgaaggtaagcctatccctaaccctctcctcggtctc K G E
L N S K L E G K P I P N P L L G L gattctacgtgagtcgac (SEQ ID NO:
24) D S T - (SEQ ID NO: 25)
[0301] In the nucleic acid sequence, nucleotides 1-6 include the
restriction recognition site for BamHI; nucleotides 7-36 include
the Tat PTD sequence; nucleotides 37-810 include codon-optimized
for the N terminal amino acids 1-258 of human Smad7; nucleotides
811-852 include the V5 epitope sequence (corresponding amino acid
sequence in bold); nucleotides 853-855 include the stop sequence;
and nucleotides 856-861 include the restriction recognition site
for SalI. ATG is removed to allow for fusion with GST:
TABLE-US-00013
ggatccggccgtaaaaaacgccgtcaacgccgccgtggtttccgtacgaaacgctcggcc G S G
R K K R R Q R R R G F R T K R S A
ctggtccgtcgcctgtggcgctcccgtgctccgggtggtgaagatgaagaagaaggtgct L V R
R L W R S R A P G G E D E E E G A
ggcggcggtggcggtggcggtgaactgcgtggcgagggtgcaaccgatagtcgtgcacac G G G
G G G G E L R G E G A T D S R A H
ggtgcaggcggtggcggtccgggtcgtgctggttgctgtctgggtaaagctgtgcgcggc G A G
G G G P G R A G C C L G K A V R G
gcgaaaggtcatcaccatccgcacccgccggcagcaggtgcaggtgcagctggcggtgcg A K G
H H H P H P P A A G A G A A G G A
gaagccgatctgaaagccctgacccatagtgtcctgaaaaaactgaaagaacgtcagctg E A D
L K A L T H S V L K K L K E R Q L
gagctgctgctgcaagcagtagaatcccgtggcggtacccgtacggcttgtctgctgctg E L L
L Q A V E S R G G T R T A C L L L
ccgggtcgtctggattgccgtctgggtccgggtgcaccggctggtgcgcagccggcacaa P G R
L D C R L G P G A P A G A Q P A Q
ccgccgagctcttacagcctgccgctgctgctgtgtaaagtgtttcgttggccggacctg P P S
S Y S L P L L L C K V F R W P D L
cgccacagttccgaagttaaacgcctgtgctgttgcgagagctatggcaaaattaacccg R H S
S E V K R L C C C E S Y G K I N P
gaactggtttgttgcaatccgcaccatctgtctcgtctgtgtgaactggagagcccgccg E L V
C C N P H H L S R L C E L E S P P
ccgccgtattctcgttacccgatggatacctgaaaccgactgcagattgcccggacgca P P Y S
R Y P M D F L K P T A D C P D A
gtcccgtcatcggctgagaccggcggcaccaactatctggcaccgggcggtctgagtgat V P S
S A E T G G T N Y L A P G G L S D
tcccagctgctgctggaaccgggcgaccgtggtaagcctatccctaaccctctcctcggt S Q L
L L E P G D R G K P I P N P L L G ctcgattctacgtgagtcgac (SEQ ID NO:
26) L D S T - (SEQ ID NO: 27)
Example 8
Testing of Truncated Smad7 Proteins
[0302] Activity of truncated Smad7 proteins is tested using the in
vitro and in vivo assays used to test full-length Smad7 described
above, among other assays. Such assays include, but are not limited
to, the ability to block phosphorylation of Smad2 and/or nuclear
translocation of the NF-.kappa.B p50 subunit, increase cell
proliferation, reduce apoptosis and/or radiation-induced DNA
damage, reduce inflammation and/or angiogenesis, promote healing in
oral mucositis, surgical wounds, diabetes wounds, and/or wounds
associated with chronic inflammation in mice.
[0303] In a wound healing assay, 6-mm punch biopsies were performed
in wild-type mice followed by daily topical application of
C-terminal or N-terminal Tat-Smad7. By measuring gross wound
closure, both truncated Smad7 proteins described above (e.g.,
Tat-Smad7 C-terminal and N-terminal protein) were found to have
promoted wound healing similar to full length Tat-Smad7.
Example 9
Additional Codon Optimization for Smad7 Protein Production
[0304] Smad7 nucleic acid molecules are designed with additional
nucleotide changes selected to increase protein production. For
example, the utilization of codons encoding the amino acids Ser and
His will be manipulated. In codon-optimized human Smad7 in examples
above, the Ser codon (TCC or TCG) has an amino acid frequency of
approximately 9% of codon utilization. It is believed that changing
the codon of Ser to AGC will increase Smad7 protein production, at
least partly because it can optionally increase codon usage to 15%.
There are 33 Ser amino acids in Smad7 protein (nucleotides at
positions 19-21, 46-48, 133-135, 292-294, 349-351, 451-453,
454-456, 460-462, 511-513, 514-516, 544-546, 595-597, 616-618,
634-636, 691-693, 694-696, 739-741, 745-747, 775-777, 847-849,
907-909, 919-921, 943-945, 1006-1008, 1009-1101, 1030-1032,
1054-1056, 1093-1095, 1126-1128, 1192-1194, 1237-1239, 1240-1242,
1273-1275; with a corresponding serine amino acid position of 7,
16, 45, 98, 117, 151, 152, 154, 171, 172, 182, 199, 206, 212, 231,
232, 247, 249, 259, 283, 303, 307, 315, 336, 337, 344, 352, 365,
376, 398, 413, 414, 425). Of these, 23 (nucleotides 19-21, 292-294,
349-351, 451-453, 454-456, 460-462, 511-513, 514-516, 544-546,
616-618, 634-636, 691-693, 694-696, 739-741, 745-747, 775-777,
847-849, 907-909, 919-921, 1009-1101, 1030-1032, 1054-1056,
1093-1095; corresponding serine amino acid position of 7, 98, 117,
151, 152, 154, 171, 172, 182, 206, 212, 231, 232, 247, 249, 259,
283, 303, 307, 337, 344, 352, 365) can be changed without
introducing potential alternative open reading frames.
[0305] Similarly, in codon-optimized human Smad7 in the examples
above, the His codon (CAC) has 9.6% of codon usage. It is believed
that changing the His codon to CAT (optionally to 12.6% usage) will
increase Smad7 protein production. There are 12 His (nucleotides
142-144, 214-216, 217-219, 220-222, 226-228, 289-291, 589-591,
778-780, 1072-1074, 1147-1149; corresponding to histidine amino
acids at position 48, 72, 73, 74, 76, 97, 170, 196, 197, 260, 358,
383) in Smad7 protein. Of these, 4 (nucleotides 217-219. 220-222,
589-591, 778-780, histidine residues 73, 76, 197, 260) can be
changed without introducing potential alternative open reading
frames.
[0306] In addition, wild-type human Smad7 includes a Met amino acid
as amino acid 216 (Met216). This may be perceived as an alternative
open reading frame by bacterial machinery, for example, and
decrease protein production. It is believed that changing Met216 to
Leu216 (ATG to CTG), the amino acid that has the biochemical
property the closest to Met and thus not expected to change 3D
structure of the protein, will increase protein production.
[0307] The comparison between original codon-optimized Tat-Smad7-V5
and further changes is provided below. Top strand: Tat-Smad7-V5
(SEQ ID NO: 23); bottom strand: after optimized Ser, His and M216L
mutation (SEQ ID NO: 30).
TABLE-US-00014 Alignment: Local DNA homologies. Parameters: Both
strands. Method: FastScan-Max Score Mol 1
20120323113102812-S54366-164160-1-PGEX-5.seq (1-1463) Mol 2
TatSmad7 Ser-His optim Number of sequences to align: 2 Settings:
Similarity significance value cutoff: >= 60% Homology Block:
Percent Matches 94 Score 1227 Length 1392 ##STR00002##
[0308] Nucleic acid sequences and their corresponding amino acid
sequences that would include all these changes are provided below.
The amino acid sequence includes the V5 epitope indicated in bold,
and the pET101-Topo backbone indicated by italics and underlining.
The Tat-Smad7.sup.M216L fully-optimized full length nucleotide and
protein sequence is shown below:
TABLE-US-00015 (SEQ ID NO: 30)
ggatccggccgtaaaaaacgccgtcaacgccgccgtggtttccgtacgaaacgcagcgcc G S G
R K K R R Q R R R G F R T K R S A
ctggtccgtcgcctgtggcgcagccgtgctccgggtggtgaagatgaagaagaaggtgct L V R
R L W R S R A P G G E D E E E G A
ggcggcggtggcggtggcggtgaactgcgtggcgagggtgcaaccgatagccgtgcacat G G G
G G G G E L R G E G A T D S R A H
ggtgcaggcggtggcggtccgggtcgtgctggttgctgtctgggtaaagctgtgcgcggc G A G
G G G P G R A G C C L G K A V R G
gcgaaaggtcatcatcatccgcatccgccggcagcaggtgcaggtgcagctggcggtgcg A K G
H H H P H P P A A G A G A A G G A
gaagccgatctgaaagccctgacccatagcgtcctgaaaaaactgaaagaacgtcagctg E A D
L K A L T H S V L K K L K E R Q L
gagctgctgctgcaagcagtagaaagccgtggcggtacccgtacggcttgtctgctgctg E L L
L Q A V E S R G G T R T A C L L L
ccgggtcgtctggattgccgtctgggtccgggtgcaccggctggtgcgcagccggcacaa P G R
L D C R L G P G A P A G A Q P A Q
ccgccgagcagctacagcctgccgctgctgctgtgtaaagtgtttcgttggccggacctg P P S
S Y S L P L L L C K V F R W P D L
cgccatagcagcgaagttaaacgcctgtgctgttgcgagagctatggcaaaattaacccg R H S
S E V K R L C C C E S Y G K I N P
gaactggtttgttgcaatccgcatcatctgagccgtctgtgtgaactggagagcccgccg E L V
C C N P H H L S R L C E L E S P P
ccgccgtatagccgttacccgctggatttcctgaaaccgactgcagattgcccggacgca P P Y
S R Y P L D F L K P T A D C P D A
gtcccgagcagcgctgagaccggcggcaccaactatctggcaccgggcggtctgagcgat V P S
S A E T G G T N Y L A P G G L S D
agccagctgctgctggaaccgggcgaccgtagccattggtgtgtggttgcctattgggaa S Q L
L L E P G D R S H W C V V A Y W E
gagaaaacgcgtgtcggtcgcctgtactgcgtacaggaaccgagcctggatatcttttat E K T
R V G R L Y C V Q E P S L D I F Y
gacctgccgcagggcaatggtttctgtctgggccaactgaacagcgataataaaagccag D L P
Q G N G F C L G Q L N S D N K S Q
ctggtgcaaaaagttcgcagcaaaattggctgcggtatccagctgacccgtgaagttgac L V Q
K V R S K I G C G I Q L T R E V D
ggtgtctgggtatataaccgcagcagctacccgatttttatcaaaagcgccaccctggat G V W
V Y N R S S Y P I F I K S A T L D
aatccggacagccgtacgctgctggtccataaagtatttccgggcttcagcatcaaagcg N P D
S R T L L V H K V F P G F S I K A
ttcgattacgagaaagcctacagcctgcagcgcccgaacgaccatgaattcatgcagcaa F D Y
E K A Y S L Q R P N D H E F M Q Q
ccgtggacgggttttactgtgcagatcagcttcgttaaaggctggggtcaatgctacacc P W T
G F T V Q I S F V K G W G Q C Y T
cgtcagtttatcagcagctgtccgtgctggctggaagtgattttcaatagccgcaagggc R Q F
I S S C P C W L E V I F N S R K G
gagctcaatagcaagcttgaaggtaagcctatccctaaccctctcctcggtctcgatagc E L N
S K L E G K P I P N P L L G L D S acgtgagtcgac
T--(SEQ ID NO: 31)
[0309] The optimized nucleotide and amino acid sequences will also
be used to make a variety of N-terminal and C-terminal Tat-Smad7
fragments. Representative examples are provided below.
[0310] The Tat-N-Smad7-V5 most optimized nucleotide and amino acid
sequences are provided. The protein sequence includes the V5
epitope, which is indicated by bold capital letters.
TABLE-US-00016
ggatccggccgtaaaaaacgccgtcaacgccgccgtggtttccgtacgaaacgcagcgcc G S G
R K K R R Q R R R G F R T K R S A
ctggtccgtcgcctgtggcgcagccgtgctccgggtggtgaagatgaagaagaaggtgct L V R
R L W R S R A P G G E D E E E G A
ggcggcggtggcggtggcggtgaactgcgtggcgagggtgcaaccgatagccgtgcacat G G G
G G G G E L R G E G A T D S R A H
ggtgcaggcggtggcggtccgggtcgtgctggttgctgtctgggtaaagctgtgcgcggc G A G
G G G P G R A G C C L G K A V R G
gcgaaaggtcatcatcatccgcatccgccggcagcaggtgcaggtgcagctggcggtgcg A K G
H H H P H P P A A G A G A A G G A
gaagccgatctgaaagccctgacccatagcgtcctgaaaaaactgaaagaacgtcagctg E A D
L K A L T H S V L K K L K E R Q L
gagctgctgctgcaagcagtagaaagccgtggcggtacccgtacggcttgtctgctgctg E L L
L Q A V E S R G G T R T A C L L L
ccgggtcgtctggattgccgtctgggtccgggtgcaccggctggtgcgcagccggcacaa P G R
L D C R L G P G A P A G A Q P A Q
ccgccgagcagctacagcctgccgctgctgctgtgtaaagtgtttcgttggccggacctg P P S
S Y S L P L L L C K V F R W P D L
cgccatagcagcgaagttaaacgcctgtgctgttgcgagagctatggcaaaattaacccg R H S
S E V K R L C C C E S Y G K I N P
gaactggtttgttgcaatccgcatcatctgagccgtctgtgtgaactggagagcccgccg E L V
C C N P H H L S R L C E L E S P P
ccgccgtatagccgttacccgatggatttcctgaaaccgactgcagattgcccggacgca P P Y
S R Y P M D F L K P T A D C P D A
gtcccgagcagcgctgagaccggcggcaccaactatctggcaccgggcggtctgagcgat V P S
S A E T G G T N Y L A P G G L S D
agccagctgctgctggaaccgggcgaccgtggtaagcctatccctaaccctctcctcggt S Q L
L L E P G D R G K P I P N P L L G ctcgattctacgtgagtcgac (SEQ ID NO:
32) L D S T - (SEQ ID NO: 27)
[0311] Tat-C-Smad7-V5 most optimized nucleotide and amino acid
sequences are provided. The protein sequence includes the V5
epitope (indicated by bold capital letters), and the pET101-Topo
backbone (indicated by underlined italics).
TABLE-US-00017
ggatccggccgtaaaaaacgccgtcaacgccgccgttcacattggtgtgtggttgcctat G S G
R K K R R Q R R R S H W C V V A Y
tgggaagagaaaacgcgtgtcggtcgcctgtactgcgtacaggaaccgagcctggatatc W E E
K T R V G R L Y C V Q E P S L D I
ttttatgacctgccgcagggcaatggtttctgtctgggccaactgaacagcgataataaa F Y D
L P Q G N G F C L G Q L N S D N K
agccagctggtgcaaaaagttcgcagcaaaattggctgcggtatccagctgacccgtgaa S Q L
V Q K V R S K I G C G I Q L T R E
gttgacggtgtctgggtatataaccgcagcagctacccgatttttatcaaaagcgccacc V D G
V W V Y N R S S Y P I F I K S A T
ctggataatccggacagccgtacgctgctggtccataaagtatttccgggcttcagcatc L D N
P D S R T L L V H K V F P G F S I
aaagcgttcgattacgagaaagcctacagcctgcagcgcccgaacgaccatgaattcatg K A F
D Y E K A Y S L Q R P N D H E F M
cagcaaccgtggacgggttttactgtgcagatcagcttcgttaaaggctggggtcaatgc Q Q P
W T G F T V Q I S F V K G W G Q C
tacacccgtcagtttatcagcagctgtccgtgctggctggaagtgattttcaatagccgc Y T R
Q F I S S C P C W L E V I F N S R
aagggcgagctcaatagcaagcttgaaggtaagcctatccctaaccctctcctcggtctc K G E
L N S K L E G K P I P N P L L G L gatagcacgtgagtcgac (SEQ ID NO:
34) D S T - (SEQ ID NO: 25)
[0312] The comparisons before and after the above optimizations are
provided below. C-terminal optimization (top strand) (alignment
discloses SEQ ID NOs: 34 and 24, respectively, in order of
appearance):
TABLE-US-00018 Alignment: Local DNA homologies. Parameters: Both
strands. Method: FastScan-Max Score Mol 1 Tat-C-Smad7-ser-his
optimized (1-618) Mol 2 Tat-C-termal Smad7-V5 (1-618) Number of
sequences to align: 2 Settings: Similarity significance value
cutoff: >= 60% Homology Flock: Percent Matches 93 Score 541
Length 618 ##STR00003##
N-terminal optimization (top strand) (alignment discloses SEQ ID
NOs: 32 and 26, respectively, in order of appearance):
TABLE-US-00019 Alignment: Local DNA homologies. Parameters: Both
strands. Method: FastScan-Max Score Mol 1 Tat-N-Smad7-V5-Ser-His
optimized (1-861) Mol 2 Tat-N-Smad7-V5 (1-861) Number of sequences
to align: 2 Settings: Similarity significance value cutoff: >=
60% Homology Block: Percent Matches 95 Score 781 Length 861
##STR00004##
[0313] In addition, other codon-optimized nucleic acids will also
be assessed for their ability to produce Smad7 protein in one or
more expression systems. Provided below is another example of such
a sequence.
TABLE-US-00020 Tat-Smad.sup.7M7216L-V5 optimized by Optimizer
program:
ggatcoggtcgtaaaaaacgtcgtcagcgtcgtcgtggtttccgtaccaaacgttctgcg G S G
R K K R R Q R R R G F R T K R S A
ctggttcgtcgtctgtggcgttctcgtgcgccgggtggtgaagacgaagaagaaggtgcg L V R
R L W R S R A P G G E D E E E G A
ggtggtggtggtggtggtggtgaactgcgtggtgaaggtgcgaccgactctcgtgcgcac G G G
G G G G E L R G E G A T D S R A H
ggtgcgggtggtggtggtccgggtcgtgcgggttgctgcctgggtaaagcggttcgtggt G A G
G G G P G R A G C C L G K A V R G
gcgaaaggtcaccaccacccgcacccgccggcggcgggtgcgggtgcggcgggtggtgcg A K G
H H H P H P P A A G A G A A G G A
gaagcggacctgaaagcgctgacccactctgttctgaaaaaactgaaagaacgtcagctg E A D
L K A L T H S V L K K L K E R Q L
gaactgctgctgcaggcggttgaatctcgtggtggtacccgtaccgcgtgcctgctgctg E L L
L Q A V E S R G G T R T A C L L L
ccgggtcgtctggactgccgtctgggtccgggtgcgccggcgggtgcgcagccggcgcag P G R
L D C R L G P G A P A G A Q P A Q
ccgccgtcttcttactctctgccgctgctgctgtgcaaagttaccgttggccggacctg P P S S
Y S L P L L L C K V F R W P D L
cgtcactcttctgaagttaaacgtctgtgctgctgcgaatcttacggtaaaatcaacccg R H S
S E V K R L C C C E S Y G K I N P
gaactggtttgctgcaacccgcaccacctgtctcgtctgtgcgaactggaatctccgccg E L V
C C N P H H L S R L C E L E S P P
ccgccgtactctcgttacccgctggacttcctgaaaccgaccgcggactgcccggacgcg P P Y
S R Y P L D F L K P T A D C P D A
gttccgtcttctgcggaaaccggtggtaccaactacctggcgccgggtggtctgtctgac V P S
S A E T G G T N Y L A P G G L S D
tctcagctgctgctggaaccgggtgaccgttctcactggtgcgttgttgcgtactgggaa S Q L
L L E P G D R S H W C V V A Y W E
gaaaaaacccgtgttggtcgtctgtactgcgttcaggaaccgtctctggacatcttctac E K T
R V G R L Y C V Q E P S L D I F Y
gacctgccgcagggtaacggtttctgcctgggtcagctgaactctgacaacaaatctcag D L P
Q G N G F C L G Q L N S D N K S Q
ctggttcagaaagttcgttctaaaatcggttgcggtatccagctgacccgtgaagttgac L V Q
K V R S K I G C G I Q L T R E V D
ggtgtttgggtttacaaccgttcttcttacccgatcttcatcaaatctgcgaccctggac G V W
V Y N R S S Y P I F I K S A T L D
aacccggactctcgtaccctgctggttcacaaagttttcccgggtttctctatcaaagcg N P D
S R T L L V H K V F P G F S I K A
ttcgactacgaaaaagcgtactctctgcagcgtccgaacgaccacgaattcatgcagcag F D Y
E K A Y S L Q R P N D H E F M Q Q
ccgtggaccggtttcaccgttcagatctctttcgttaaaggttggggtcagtgctacacc P W T
G F T V Q I S F V K G W G Q C Y T
cgtcagttcatctcttcttgcccgtgctggctggaagttatcttcaactctcgtggtaaa R Q F
I S S C P C W L E V I F N S R G K
ccgatcccgaacccgctgctgggtctggactctacctgagtcgac (SEQ ID NO: 36) P I P
N P L L G L D S T - - (SEQ ID NO: 37)
[0314] Nucleotide Sequence:
TABLE-US-00021 1-6: BamHI; 7-36: Tat; 37-1314: codon-optimized
human Smad7; 1315-1356: V5; 137-1359: stop; 1360-1365 SalI ATG is
removed to be used with GST; 682 ATG to CTG (M216 to L) (SEQ ID NO:
36) 1 ggatccggtc gtaaaaaacg tcgtcagcgt cgtcgtggtt tccgtaccaa
acgttctgcg 61 ctggttcgtc gtctgtggcg ttctcgtgcg ccgggtggtg
aagacgaaga agaaggtgcg 121 ggtggtggtg gtggtggtgg tgaactgcgt
ggtgaaggtg cgaccgactc tcgtgcgcac 181 ggtgcgggtg gtggtggtcc
gggtcgtgcg ggttgctgcc tgggtaaagc ggttcgtggt 241 gcgaaaggtc
accaccaccc gcacccgccg gcggcgggtg cgggtgcggc gggtggtgcg 301
gaagcggacc tgaaagcgct gacccactct gttctgaaaa aactgaaaga acgtcagctg
361 gaactgctgc tgcaggcggt tgaatctcgt ggtggtaccc gtaccgcgtg
cctgctgctg 421 ccgggtcgtc tggactgccg tctgggtccg ggtgcgccgg
cgggtgcgca gccggcgcag 481 ccgccgtctt cttactctct gccgctgctg
ctgtgcaaag ttttccgftg gccggacctg 541 cgtcactctt ctgaagttaa
acgtctgtgc tgctgcgaat cttacggtaa aatcaacccg 601 gaactggttt
gctgcaaccc gcaccacctg tctcgtctgt gcgaactgga atctccgccg 661
ccgccgtact ctcgttaccc gctggacttc ctgaaaccga ccgcggactg cccggacgcg
721 gttccgtctt ctgcggaaac cggtggtacc aactacctgg cgccgggtgg
tctgtctgac 781 tctcagctgc tgctggaacc gggtgaccgt tctcactggt
gcgttgttgc gtactgggaa 841 gaaaaaaccc gtgttggtcg tctgtactgc
gttcaggaac cgtctctgga catcttctac 901 gacctgccgc agggtaacgg
tttctgcctg ggtcagctga actctgacaa caaatctcag 961 ctggttcaga
aagttcgttc taaaatcggt tgcggtatcc agctgacccg tgaagttgac 1021
ggtgtttggg tttacaaccg ttcttcttac ccgatcttca tcaaatctgc gaccctggac
1081 aacccggact ctcgtaccct gctggttcac aaagttttcc cgggtttctc
tatcaaagcg 1141 ttcgactacg aaaaagcgta ctctctgcag cgtccgaacg
accacgaatt catgcagcag 1201 ccgtggaccg gtttcaccgt tcagatctct
ttcgttaaag gttggggtca gtgctacacc 1261 cgtcagttca tctcttcttg
cccgtgctgg ctggaagtta tcttcaactc tcgtggtaaa 1321 ccgatcccga
acccgctgct gggtctggac tctacctgag tcgac
[0315] Sequence comparison with Tat-Smad7.sup.M7216L-V5 described
in Example 4 (alignment discloses SEQ ID NOs: 36 and 30,
respectively, in order of appearance):
TABLE-US-00022 Alignment: Global DNA alignment against reference
molecule Parameters: Scoring matrix: Linear (Mismatch 2, OpenGap 4,
ExtGap 1) Reference molecule: Tat-Smad7-216L-V5-optimizer, Region
1-1365 Number of sequences to align: 2 Settings: Similarity
significance value cutoff: >= 60% Summary of Percent Matches:
Reference: Tat-Smad7-216L-V5-optimizer 1-1365 (1365 bps) --
Sequence 2: TatSmad7 Ser-His optimized-682 mutant 1-1392 (1392 bps)
79% ##STR00005##
[0316] The foregoing discussion of the present technology has been
presented for purposes of illustration and description. The
foregoing is not intended to limit the present technology to the
form or forms disclosed herein. Although the description of the
present technology has included description of one or more
embodiments and certain variations and modifications, other
variations and modifications are within the scope of the present
technology, e.g., as may be within the skill and knowledge of those
in the art, after understanding the present disclosure. It is
intended to obtain rights which include alternative embodiments to
the extent permitted, including alternate, interchangeable and/or
equivalent structures, functions, ranges or steps to those claimed,
whether or not such alternate, interchangeable and/or equivalent
structures, functions, ranges or steps are disclosed herein, and
without intending to publicly dedicate any patentable subject
matter.
Sequence CWU 1
1
87130DNAHuman immunodeficiency virusCDS(1)..(30) 1ggc cgt aaa aaa
cgc cgt caa cgc cgc cgt 30Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1
5 10 210PRTHuman immunodeficiency virus 2Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg 1 5 10 333DNAHuman immunodeficiency virus
3tatggccgta aaaaacgccg tcaacgccgc cgt 33411PRTHuman
immunodeficiency virus 4Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg
1 5 10 521DNAHuman immunodeficiency virus 5ggccgtaaaa aacgccgtca a
2167PRTHuman immunodeficiency virus 6Gly Arg Lys Lys Arg Arg Gln 1
5 730DNAHuman immunodeficiency virus 7ggccgtaaaa aacgccgtca
acgccgccgt 30830DNAHuman immunodeficiency virus 8ggccgtaaaa
aacgccgtca acgccgccgt 3091497DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 9ttcccctcta gaaataattt
tgtttaactt taagaaggaa ttcaggagcc cttcacc 57atg cgt aaa aaa cgc cgt
caa cgc cgc cgt ggt ttc cgt acg aaa cgc 105Met Arg Lys Lys Arg Arg
Gln Arg Arg Arg Gly Phe Arg Thr Lys Arg 1 5 10 15 tcg gcc ctg gtc
cgt cgc ctg tgg cgc tcc cgt gct ccg ggt ggt gaa 153Ser Ala Leu Val
Arg Arg Leu Trp Arg Ser Arg Ala Pro Gly Gly Glu 20 25 30 gat gaa
gaa gaa ggt gct ggc ggc ggt ggc ggt ggc ggt gaa ctg cgt 201Asp Glu
Glu Glu Gly Ala Gly Gly Gly Gly Gly Gly Gly Glu Leu Arg 35 40 45
ggc gag ggt gca acc gat agt cgt gca cac ggt gca ggc ggt ggc ggt
249Gly Glu Gly Ala Thr Asp Ser Arg Ala His Gly Ala Gly Gly Gly Gly
50 55 60 ccg ggt cgt gct ggt tgc tgt ctg ggt aaa gct gtg cgc ggc
gcg aaa 297Pro Gly Arg Ala Gly Cys Cys Leu Gly Lys Ala Val Arg Gly
Ala Lys 65 70 75 80 ggt cat cac cat ccg cac ccg ccg gca gca ggt gca
ggt gca gct ggc 345Gly His His His Pro His Pro Pro Ala Ala Gly Ala
Gly Ala Ala Gly 85 90 95 ggt gcg gaa gcc gat ctg aaa gcc ctg acc
cat agt gtc ctg aaa aaa 393Gly Ala Glu Ala Asp Leu Lys Ala Leu Thr
His Ser Val Leu Lys Lys 100 105 110 ctg aaa gaa cgt cag ctg gag ctg
ctg ctg caa gca gta gaa tcc cgt 441Leu Lys Glu Arg Gln Leu Glu Leu
Leu Leu Gln Ala Val Glu Ser Arg 115 120 125 ggc ggt acc cgt acg gct
tgt ctg ctg ctg ccg ggt cgt ctg gat tgc 489Gly Gly Thr Arg Thr Ala
Cys Leu Leu Leu Pro Gly Arg Leu Asp Cys 130 135 140 cgt ctg ggt ccg
ggt gca ccg gct ggt gcg cag ccg gca caa ccg ccg 537Arg Leu Gly Pro
Gly Ala Pro Ala Gly Ala Gln Pro Ala Gln Pro Pro 145 150 155 160 agc
tct tac agc ctg ccg ctg ctg ctg tgt aaa gtg ttt cgt tgg ccg 585Ser
Ser Tyr Ser Leu Pro Leu Leu Leu Cys Lys Val Phe Arg Trp Pro 165 170
175 gac ctg cgc cac agt tcc gaa gtt aaa cgc ctg tgc tgt tgc gag agc
633Asp Leu Arg His Ser Ser Glu Val Lys Arg Leu Cys Cys Cys Glu Ser
180 185 190 tat ggc aaa att aac ccg gaa ctg gtt tgt tgc aat ccg cac
cat ctg 681Tyr Gly Lys Ile Asn Pro Glu Leu Val Cys Cys Asn Pro His
His Leu 195 200 205 tct cgt ctg tgt gaa ctg gag agc ccg ccg ccg ccg
tat tct cgt tac 729Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro Pro Pro
Tyr Ser Arg Tyr 210 215 220 ccg atg gat ttc ctg aaa ccg act gca gat
tgc ccg gac gca gtc ccg 777Pro Met Asp Phe Leu Lys Pro Thr Ala Asp
Cys Pro Asp Ala Val Pro 225 230 235 240 tca tcg gct gag acc ggc ggc
acc aac tat ctg gca ccg ggc ggt ctg 825Ser Ser Ala Glu Thr Gly Gly
Thr Asn Tyr Leu Ala Pro Gly Gly Leu 245 250 255 agt gat tcc cag ctg
ctg ctg gaa ccg ggc gac cgt tca cat tgg tgt 873Ser Asp Ser Gln Leu
Leu Leu Glu Pro Gly Asp Arg Ser His Trp Cys 260 265 270 gtg gtt gcc
tat tgg gaa gag aaa acg cgt gtc ggt cgc ctg tac tgc 921Val Val Ala
Tyr Trp Glu Glu Lys Thr Arg Val Gly Arg Leu Tyr Cys 275 280 285 gta
cag gaa ccg tcg ctg gat atc ttt tat gac ctg ccg cag ggc aat 969Val
Gln Glu Pro Ser Leu Asp Ile Phe Tyr Asp Leu Pro Gln Gly Asn 290 295
300 ggt ttc tgt ctg ggc caa ctg aac tca gat aat aaa tcg cag ctg gtg
1017Gly Phe Cys Leu Gly Gln Leu Asn Ser Asp Asn Lys Ser Gln Leu Val
305 310 315 320 caa aaa gtt cgc tca aaa att ggc tgc ggt atc cag ctg
acc cgt gaa 1065Gln Lys Val Arg Ser Lys Ile Gly Cys Gly Ile Gln Leu
Thr Arg Glu 325 330 335 gtt gac ggt gtc tgg gta tat aac cgc agc tct
tac ccg att ttt atc 1113Val Asp Gly Val Trp Val Tyr Asn Arg Ser Ser
Tyr Pro Ile Phe Ile 340 345 350 aaa agt gcc acc ctg gat aat ccg gac
tcc cgt acg ctg ctg gtc cac 1161Lys Ser Ala Thr Leu Asp Asn Pro Asp
Ser Arg Thr Leu Leu Val His 355 360 365 aaa gta ttt ccg ggc ttc tca
atc aaa gcg ttc gat tac gag aaa gcc 1209Lys Val Phe Pro Gly Phe Ser
Ile Lys Ala Phe Asp Tyr Glu Lys Ala 370 375 380 tac tcg ctg cag cgc
ccg aac gac cat gaa ttc atg cag caa ccg tgg 1257Tyr Ser Leu Gln Arg
Pro Asn Asp His Glu Phe Met Gln Gln Pro Trp 385 390 395 400 acg ggt
ttt act gtg cag atc tct ttc gtt aaa ggc tgg ggt caa tgc 1305Thr Gly
Phe Thr Val Gln Ile Ser Phe Val Lys Gly Trp Gly Gln Cys 405 410 415
tac acc cgt cag ttt atc tcg tcc tgt ccg tgc tgg ctg gaa gtg att
1353Tyr Thr Arg Gln Phe Ile Ser Ser Cys Pro Cys Trp Leu Glu Val Ile
420 425 430 ttc aat agc cgc aag ggc gag ctc aat tcg aag ctt gaa ggt
aag cct 1401Phe Asn Ser Arg Lys Gly Glu Leu Asn Ser Lys Leu Glu Gly
Lys Pro 435 440 445 atc cct aac cct ctc ctc ggt ctc gat tct acg cgt
acc ggt cat cat 1449Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Arg
Thr Gly His His 450 455 460 cac cat cac cat tgagtttgat ccggctgcta
acaaagcccg aaagga 1497His His His His 465 10468PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
10Met Arg Lys Lys Arg Arg Gln Arg Arg Arg Gly Phe Arg Thr Lys Arg 1
5 10 15 Ser Ala Leu Val Arg Arg Leu Trp Arg Ser Arg Ala Pro Gly Gly
Glu 20 25 30 Asp Glu Glu Glu Gly Ala Gly Gly Gly Gly Gly Gly Gly
Glu Leu Arg 35 40 45 Gly Glu Gly Ala Thr Asp Ser Arg Ala His Gly
Ala Gly Gly Gly Gly 50 55 60 Pro Gly Arg Ala Gly Cys Cys Leu Gly
Lys Ala Val Arg Gly Ala Lys 65 70 75 80 Gly His His His Pro His Pro
Pro Ala Ala Gly Ala Gly Ala Ala Gly 85 90 95 Gly Ala Glu Ala Asp
Leu Lys Ala Leu Thr His Ser Val Leu Lys Lys 100 105 110 Leu Lys Glu
Arg Gln Leu Glu Leu Leu Leu Gln Ala Val Glu Ser Arg 115 120 125 Gly
Gly Thr Arg Thr Ala Cys Leu Leu Leu Pro Gly Arg Leu Asp Cys 130 135
140 Arg Leu Gly Pro Gly Ala Pro Ala Gly Ala Gln Pro Ala Gln Pro Pro
145 150 155 160 Ser Ser Tyr Ser Leu Pro Leu Leu Leu Cys Lys Val Phe
Arg Trp Pro 165 170 175 Asp Leu Arg His Ser Ser Glu Val Lys Arg Leu
Cys Cys Cys Glu Ser 180 185 190 Tyr Gly Lys Ile Asn Pro Glu Leu Val
Cys Cys Asn Pro His His Leu 195 200 205 Ser Arg Leu Cys Glu Leu Glu
Ser Pro Pro Pro Pro Tyr Ser Arg Tyr 210 215 220 Pro Met Asp Phe Leu
Lys Pro Thr Ala Asp Cys Pro Asp Ala Val Pro 225 230 235 240 Ser Ser
Ala Glu Thr Gly Gly Thr Asn Tyr Leu Ala Pro Gly Gly Leu 245 250 255
Ser Asp Ser Gln Leu Leu Leu Glu Pro Gly Asp Arg Ser His Trp Cys 260
265 270 Val Val Ala Tyr Trp Glu Glu Lys Thr Arg Val Gly Arg Leu Tyr
Cys 275 280 285 Val Gln Glu Pro Ser Leu Asp Ile Phe Tyr Asp Leu Pro
Gln Gly Asn 290 295 300 Gly Phe Cys Leu Gly Gln Leu Asn Ser Asp Asn
Lys Ser Gln Leu Val 305 310 315 320 Gln Lys Val Arg Ser Lys Ile Gly
Cys Gly Ile Gln Leu Thr Arg Glu 325 330 335 Val Asp Gly Val Trp Val
Tyr Asn Arg Ser Ser Tyr Pro Ile Phe Ile 340 345 350 Lys Ser Ala Thr
Leu Asp Asn Pro Asp Ser Arg Thr Leu Leu Val His 355 360 365 Lys Val
Phe Pro Gly Phe Ser Ile Lys Ala Phe Asp Tyr Glu Lys Ala 370 375 380
Tyr Ser Leu Gln Arg Pro Asn Asp His Glu Phe Met Gln Gln Pro Trp 385
390 395 400 Thr Gly Phe Thr Val Gln Ile Ser Phe Val Lys Gly Trp Gly
Gln Cys 405 410 415 Tyr Thr Arg Gln Phe Ile Ser Ser Cys Pro Cys Trp
Leu Glu Val Ile 420 425 430 Phe Asn Ser Arg Lys Gly Glu Leu Asn Ser
Lys Leu Glu Gly Lys Pro 435 440 445 Ile Pro Asn Pro Leu Leu Gly Leu
Asp Ser Thr Arg Thr Gly His His 450 455 460 His His His His 465
11461PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 11Gly Ser Gly Arg Lys Lys Arg Arg Gln Arg Arg
Arg Gly Phe Arg Thr 1 5 10 15 Lys Arg Ser Ala Leu Val Arg Arg Leu
Trp Arg Ser Arg Ala Pro Gly 20 25 30 Gly Glu Asp Glu Glu Glu Gly
Ala Gly Gly Gly Gly Gly Gly Gly Glu 35 40 45 Leu Arg Gly Glu Gly
Ala Thr Asp Ser Arg Ala His Gly Ala Gly Gly 50 55 60 Gly Gly Pro
Gly Arg Ala Gly Cys Cys Leu Gly Lys Ala Val Arg Gly 65 70 75 80 Ala
Lys Gly His His His Pro His Pro Pro Ala Ala Gly Ala Gly Ala 85 90
95 Ala Gly Gly Ala Glu Ala Asp Leu Lys Ala Leu Thr His Ser Val Leu
100 105 110 Lys Lys Leu Lys Glu Arg Gln Leu Glu Leu Leu Leu Gln Ala
Val Glu 115 120 125 Ser Arg Gly Gly Thr Arg Thr Ala Cys Leu Leu Leu
Pro Gly Arg Leu 130 135 140 Asp Cys Arg Leu Gly Pro Gly Ala Pro Ala
Gly Ala Gln Pro Ala Gln 145 150 155 160 Pro Pro Ser Ser Tyr Ser Leu
Pro Leu Leu Leu Cys Lys Val Phe Arg 165 170 175 Trp Pro Asp Leu Arg
His Ser Ser Glu Val Lys Arg Leu Cys Cys Cys 180 185 190 Glu Ser Tyr
Gly Lys Ile Asn Pro Glu Leu Val Cys Cys Asn Pro His 195 200 205 His
Leu Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro Pro Pro Tyr Ser 210 215
220 Arg Tyr Pro Met Asp Phe Leu Lys Pro Thr Ala Asp Cys Pro Asp Ala
225 230 235 240 Val Pro Ser Ser Ala Glu Thr Gly Gly Thr Asn Tyr Leu
Ala Pro Gly 245 250 255 Gly Leu Ser Asp Ser Gln Leu Leu Leu Glu Pro
Gly Asp Arg Ser His 260 265 270 Trp Cys Val Val Ala Tyr Trp Glu Glu
Lys Thr Arg Val Gly Arg Leu 275 280 285 Tyr Cys Val Gln Glu Pro Ser
Leu Asp Ile Phe Tyr Asp Leu Pro Gln 290 295 300 Gly Asn Gly Phe Cys
Leu Gly Gln Leu Asn Ser Asp Asn Lys Ser Gln 305 310 315 320 Leu Val
Gln Lys Val Arg Ser Lys Ile Gly Cys Gly Ile Gln Leu Thr 325 330 335
Arg Glu Val Asp Gly Val Trp Val Tyr Asn Arg Ser Ser Tyr Pro Ile 340
345 350 Phe Ile Lys Ser Ala Thr Leu Asp Asn Pro Asp Ser Arg Thr Leu
Leu 355 360 365 Val His Lys Val Phe Pro Gly Phe Ser Ile Lys Ala Phe
Asp Tyr Glu 370 375 380 Lys Ala Tyr Ser Leu Gln Arg Pro Asn Asp His
Glu Phe Met Gln Gln 385 390 395 400 Pro Trp Thr Gly Phe Thr Val Gln
Ile Ser Phe Val Lys Gly Trp Gly 405 410 415 Gln Cys Tyr Thr Arg Gln
Phe Ile Ser Ser Cys Pro Cys Trp Leu Glu 420 425 430 Val Ile Phe Asn
Ser Arg Lys Gly Glu Leu Asn Ser Lys Leu Glu Gly 435 440 445 Lys Pro
Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 450 455 460
12426PRTHomo sapiens 12Met Phe Arg Thr Lys Arg Ser Ala Leu Val Arg
Arg Leu Trp Arg Ser 1 5 10 15 Arg Ala Pro Gly Gly Glu Asp Glu Glu
Glu Gly Ala Gly Gly Gly Gly 20 25 30 Gly Gly Gly Glu Leu Arg Gly
Glu Gly Ala Thr Asp Ser Arg Ala His 35 40 45 Gly Ala Gly Gly Gly
Gly Pro Gly Arg Ala Gly Cys Cys Leu Gly Lys 50 55 60 Ala Val Arg
Gly Ala Lys Gly His His His Pro His Pro Pro Ala Ala 65 70 75 80 Gly
Ala Gly Ala Ala Gly Gly Ala Glu Ala Asp Leu Lys Ala Leu Thr 85 90
95 His Ser Val Leu Lys Lys Leu Lys Glu Arg Gln Leu Glu Leu Leu Leu
100 105 110 Gln Ala Val Glu Ser Arg Gly Gly Thr Arg Thr Ala Cys Leu
Leu Leu 115 120 125 Pro Gly Arg Leu Asp Cys Arg Leu Gly Pro Gly Ala
Pro Ala Gly Ala 130 135 140 Gln Pro Ala Gln Pro Pro Ser Ser Tyr Ser
Leu Pro Leu Leu Leu Cys 145 150 155 160 Lys Val Phe Arg Trp Pro Asp
Leu Arg His Ser Ser Glu Val Lys Arg 165 170 175 Leu Cys Cys Cys Glu
Ser Tyr Gly Lys Ile Asn Pro Glu Leu Val Cys 180 185 190 Cys Asn Pro
His His Leu Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro 195 200 205 Pro
Pro Tyr Ser Arg Tyr Pro Met Asp Phe Leu Lys Pro Thr Ala Asp 210 215
220 Cys Pro Asp Ala Val Pro Ser Ser Ala Glu Thr Gly Gly Thr Asn Tyr
225 230 235 240 Leu Ala Pro Gly Gly Leu Ser Asp Ser Gln Leu Leu Leu
Glu Pro Gly 245 250 255 Asp Arg Ser His Trp Cys Val Val Ala Tyr Trp
Glu Glu Lys Thr Arg 260 265 270 Val Gly Arg Leu Tyr Cys Val Gln Glu
Pro Ser Leu Asp Ile Phe Tyr 275 280 285 Asp Leu Pro Gln Gly Asn Gly
Phe Cys Leu Gly Gln Leu Asn Ser Asp 290 295 300 Asn Lys Ser Gln Leu
Val Gln Lys Val Arg Ser Lys Ile Gly Cys Gly 305 310 315 320 Ile Gln
Leu Thr Arg Glu Val Asp Gly Val Trp Val Tyr Asn Arg Ser 325 330 335
Ser Tyr Pro Ile Phe Ile Lys Ser Ala Thr Leu Asp Asn Pro Asp Ser 340
345 350 Arg Thr Leu Leu Val His Lys Val Phe Pro Gly Phe Ser Ile Lys
Ala 355 360 365
Phe Asp Tyr Glu Lys Ala Tyr Ser Leu Gln Arg Pro Asn Asp His Glu 370
375 380 Phe Met Gln Gln Pro Trp Thr Gly Phe Thr Val Gln Ile Ser Phe
Val 385 390 395 400 Lys Gly Trp Gly Gln Cys Tyr Thr Arg Gln Phe Ile
Ser Ser Cys Pro 405 410 415 Cys Trp Leu Glu Val Ile Phe Asn Ser Arg
420 425 1320DNAArtificial SequenceDescription of Artificial
Sequence Synthetic primer 13tggaattcct ggtctggttt
201420DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 14gccaagctgc tcttccagta 201521DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
15tctcaggggg ccaaaggtgt t 211622DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 16tcccagcacc tgaatcacat gg
221710DNAUnknownDescription of Unknown Smad binding element
oligonucleotide 17tgtctgtgct 101810DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 18tgatagagct 101926DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
19atcctcgagt atcctccagg tctggg 262028DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
20gccaagctta gcgtccagcg ttaacctg 28211392DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
21ggatccggcc gtaaaaaacg ccgtcaacgc cgccgtggtt tccgtacgaa acgctcggcc
60ctggtccgtc gcctgtggcg ctcccgtgct ccgggtggtg aagatgaaga agaaggtgct
120ggcggcggtg gcggtggcgg tgaactgcgt ggcgagggtg caaccgatag
tcgtgcacac 180ggtgcaggcg gtggcggtcc gggtcgtgct ggttgctgtc
tgggtaaagc tgtgcgcggc 240gcgaaaggtc atcaccatcc gcacccgccg
gcagcaggtg caggtgcagc tggcggtgcg 300gaagccgatc tgaaagccct
gacccatagt gtcctgaaaa aactgaaaga acgtcagctg 360gagctgctgc
tgcaagcagt agaatcccgt ggcggtaccc gtacggcttg tctgctgctg
420ccgggtcgtc tggattgccg tctgggtccg ggtgcaccgg ctggtgcgca
gccggcacaa 480ccgccgagct cttacagcct gccgctgctg ctgtgtaaag
tgtttcgttg gccggacctg 540cgccacagtt ccgaagttaa acgcctgtgc
tgttgcgaga gctatggcaa aattaacccg 600gaactggttt gttgcaatcc
gcaccatctg tctcgtctgt gtgaactgga gagcccgccg 660ccgccgtatt
ctcgttaccc gatggatttc ctgaaaccga ctgcagattg cccggacgca
720gtcccgtcat cggctgagac cggcggcacc aactatctgg caccgggcgg
tctgagtgat 780tcccagctgc tgctggaacc gggcgaccgt tcacattggt
gtgtggttgc ctattgggaa 840gagaaaacgc gtgtcggtcg cctgtactgc
gtacaggaac cgtcgctgga tatcttttat 900gacctgccgc agggcaatgg
tttctgtctg ggccaactga actcagataa taaatcgcag 960ctggtgcaaa
aagttcgctc aaaaattggc tgcggtatcc agctgacccg tgaagttgac
1020ggtgtctggg tatataaccg cagctcttac ccgattttta tcaaaagtgc
caccctggat 1080ggtgtctggg tatataaccg cagctcttac ccgattttta
tcaaaagtgc caccctggat 1140aatccggact cccgtacgct gctggtccac
aaagtatttc cgggcttctc aatcaaagcg 1200ccgtggacgg gttttactgt
gcagatctct ttcgttaaag gctggggtca atgctacacc 1260cgtcagttta
tctcgtcctg tccgtgctgg ctggaagtga ttttcaatag ccgcaagggc
1320gagctcaatt cgaagcttga aggtaagcct atccctaacc ctctcctcgg
tctcgattct 1380acgtgagtcg ac 1392221281DNAHomo sapiens 22atgttcagga
ccaaacgatc tgcgctcgtc cggcgtctct ggaggagccg tgcgcccggc 60ggcgaggacg
aggaggaggg cgcaggggga ggtggaggag gaggcgagct gcggggagaa
120ggggcgacgg acagccgagc gcatggggcc ggtggcggcg gcccgggcag
ggctggatgc 180tgcctgggca aggcggtgcg aggtgccaaa ggtcaccacc
atccccaccc gccagccgcg 240ggcgccggcg cggccggggg cgccgaggcg
gatctgaagg cgctcacgca ctcggtgctc 300aagaaactga aggagcggca
gctggagctg ctgctccagg ccgtggagtc ccgcggcggg 360acgcgcaccg
cgtgcctcct gctgcccggc cgcctggact gcaggctggg cccgggggcg
420cccgccggcg cgcagcctgc gcagccgccc tcgtcctact cgctccccct
cctgctgtgc 480aaagtgttca ggtggccgga tctcaggcat tcctcggaag
tcaagaggct gtgttgctgt 540gaatcttacg ggaagatcaa ccccgagctg
gtgtgctgca acccccatca ccttagccga 600ctctgcgaac tagagtctcc
cccccctcct tactccagat acccgatgga ttttctcaaa 660ccaactgcag
actgtccaga tgctgtgcct tcctccgctg aaacaggggg aacgaattat
720ctggcccctg gggggctttc agattcccaa cttcttctgg agcctgggga
tcggtcacac 780tggtgcgtgg tggcatactg ggaggagaag acgagagtgg
ggaggctcta ctgtgtccag 840gagccctctc tggatatctt ctatgatcta
cctcagggga atggcttttg cctcggacag 900ctcaattcgg acaacaagag
tcagctggtg cagaaggtgc ggagcaaaat cggctgcggc 960atccagctga
cgcgggaggt ggatggtgtg tgggtgtaca accgcagcag ttaccccatc
1020ttcatcaagt ccgccacact ggacaacccg gactccagga cgctgttggt
acacaaggtg 1080ttccccggtt tctccatcaa ggctttcgac tacgagaagg
cgtacagcct gcagcggccc 1140aatgaccacg agtttatgca gcagccgtgg
acgggcttta ccgtgcagat cagctttgtg 1200aagggctggg gtcagtgcta
cacccgccag ttcatcagca gctgcccgtg ctggctagag 1260gtcatcttca
acagccggta g 1281231392DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 23ggatccggcc
gtaaaaaacg ccgtcaacgc cgccgtggtt tccgtacgaa acgctcggcc 60ctggtccgtc
gcctgtggcg ctcccgtgct ccgggtggtg aagatgaaga agaaggtgct
120ggcggcggtg gcggtggcgg tgaactgcgt ggcgagggtg caaccgatag
tcgtgcacac 180ggtgcaggcg gtggcggtcc gggtcgtgct ggttgctgtc
tgggtaaagc tgtgcgcggc 240gcgaaaggtc atcaccatcc gcacccgccg
gcagcaggtg caggtgcagc tggcggtgcg 300gaagccgatc tgaaagccct
gacccatagt gtcctgaaaa aactgaaaga acgtcagctg 360gagctgctgc
tgcaagcagt agaatcccgt ggcggtaccc gtacggcttg tctgctgctg
420ccgggtcgtc tggattgccg tctgggtccg ggtgcaccgg ctggtgcgca
gccggcacaa 480ccgccgagct cttacagcct gccgctgctg ctgtgtaaag
tgtttcgttg gccggacctg 540cgccacagtt ccgaagttaa acgcctgtgc
tgttgcgaga gctatggcaa aattaacccg 600gaactggttt gttgcaatcc
gcaccatctg tctcgtctgt gtgaactgga gagcccgccg 660ccgccgtatt
ctcgttaccc gatggatttc ctgaaaccga ctgcagattg cccggacgca
720gtcccgtcat cggctgagac cggcggcacc aactatctgg caccgggcgg
tctgagtgat 780tcccagctgc tgctggaacc gggcgaccgt tcacattggt
gtgtggttgc ctattgggaa 840gagaaaacgc gtgtcggtcg cctgtactgc
gtacaggaac cgtcgctgga tatcttttat 900gacctgccgc agggcaatgg
tttctgtctg ggccaactga actcagataa taaatcgcag 960ctggtgcaaa
aagttcgctc aaaaattggc tgcggtatcc agctgacccg tgaagttgac
1020ggtgtctggg tatataaccg cagctcttac ccgattttta tcaaaagtgc
caccctggat 1080aatccggact cccgtacgct gctggtccac aaagtatttc
cgggcttctc aatcaaagcg 1140ttcgattacg agaaagccta ctcgctgcag
cgcccgaacg accatgaatt catgcagcaa 1200ccgtggacgg gttttactgt
gcagatctct ttcgttaaag gctggggtca atgctacacc 1260cgtcagttta
tctcgtcctg tccgtgctgg ctggaagtga ttttcaatag ccgcaagggc
1320gagctcaatt cgaagcttga aggtaagcct atccctaacc ctctcctcgg
tctcgattct 1380acgtgagtcg ac 139224618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
24gga tcc ggc cgt aaa aaa cgc cgt caa cgc cgc cgt tca cat tgg tgt
48Gly Ser Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser His Trp Cys 1
5 10 15 gtg gtt gcc tat tgg gaa gag aaa acg cgt gtc ggt cgc ctg tac
tgc 96Val Val Ala Tyr Trp Glu Glu Lys Thr Arg Val Gly Arg Leu Tyr
Cys 20 25 30 gta cag gaa ccg tcg ctg gat atc ttt tat gac ctg ccg
cag ggc aat 144Val Gln Glu Pro Ser Leu Asp Ile Phe Tyr Asp Leu Pro
Gln Gly Asn 35 40 45 ggt ttc tgt ctg ggc caa ctg aac tca gat aat
aaa tcg cag ctg gtg 192Gly Phe Cys Leu Gly Gln Leu Asn Ser Asp Asn
Lys Ser Gln Leu Val 50 55 60 caa aaa gtt cgc tca aaa att ggc tgc
ggt atc cag ctg acc cgt gaa 240Gln Lys Val Arg Ser Lys Ile Gly Cys
Gly Ile Gln Leu Thr Arg Glu 65 70 75 80 gtt gac ggt gtc tgg gta tat
aac cgc agc tct tac ccg att ttt atc 288Val Asp Gly Val Trp Val Tyr
Asn Arg Ser Ser Tyr Pro Ile Phe Ile 85 90 95 aaa agt gcc acc ctg
gat aat ccg gac tcc cgt acg ctg ctg gtc cac 336Lys Ser Ala Thr Leu
Asp Asn Pro Asp Ser Arg Thr Leu Leu Val His 100 105 110 aaa gta ttt
ccg ggc ttc tca atc aaa gcg ttc gat tac gag aaa gcc 384Lys Val Phe
Pro Gly Phe Ser Ile Lys Ala Phe Asp Tyr Glu Lys Ala 115 120 125 tac
tcg ctg cag cgc ccg aac gac cat gaa ttc atg cag caa ccg tgg 432Tyr
Ser Leu Gln Arg Pro Asn Asp His Glu Phe Met Gln Gln Pro Trp 130 135
140 acg ggt ttt act gtg cag atc tct ttc gtt aaa ggc tgg ggt caa tgc
480Thr Gly Phe Thr Val Gln Ile Ser Phe Val Lys Gly Trp Gly Gln Cys
145 150 155 160 tac acc cgt cag ttt atc tcg tcc tgt ccg tgc tgg ctg
gaa gtg att 528Tyr Thr Arg Gln Phe Ile Ser Ser Cys Pro Cys Trp Leu
Glu Val Ile 165 170 175 ttc aat agc cgc aag ggc gag ctc aat tcg aag
ctt gaa ggt aag cct 576Phe Asn Ser Arg Lys Gly Glu Leu Asn Ser Lys
Leu Glu Gly Lys Pro 180 185 190 atc cct aac cct ctc ctc ggt ctc gat
tct acg tgagtcgac 618Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr
195 200 25203PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 25Gly Ser Gly Arg Lys Lys Arg Arg
Gln Arg Arg Arg Ser His Trp Cys 1 5 10 15 Val Val Ala Tyr Trp Glu
Glu Lys Thr Arg Val Gly Arg Leu Tyr Cys 20 25 30 Val Gln Glu Pro
Ser Leu Asp Ile Phe Tyr Asp Leu Pro Gln Gly Asn 35 40 45 Gly Phe
Cys Leu Gly Gln Leu Asn Ser Asp Asn Lys Ser Gln Leu Val 50 55 60
Gln Lys Val Arg Ser Lys Ile Gly Cys Gly Ile Gln Leu Thr Arg Glu 65
70 75 80 Val Asp Gly Val Trp Val Tyr Asn Arg Ser Ser Tyr Pro Ile
Phe Ile 85 90 95 Lys Ser Ala Thr Leu Asp Asn Pro Asp Ser Arg Thr
Leu Leu Val His 100 105 110 Lys Val Phe Pro Gly Phe Ser Ile Lys Ala
Phe Asp Tyr Glu Lys Ala 115 120 125 Tyr Ser Leu Gln Arg Pro Asn Asp
His Glu Phe Met Gln Gln Pro Trp 130 135 140 Thr Gly Phe Thr Val Gln
Ile Ser Phe Val Lys Gly Trp Gly Gln Cys 145 150 155 160 Tyr Thr Arg
Gln Phe Ile Ser Ser Cys Pro Cys Trp Leu Glu Val Ile 165 170 175 Phe
Asn Ser Arg Lys Gly Glu Leu Asn Ser Lys Leu Glu Gly Lys Pro 180 185
190 Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 195 200
26861DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 26gga tcc ggc cgt aaa aaa cgc cgt caa cgc
cgc cgt ggt ttc cgt acg 48Gly Ser Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Gly Phe Arg Thr 1 5 10 15 aaa cgc tcg gcc ctg gtc cgt cgc
ctg tgg cgc tcc cgt gct ccg ggt 96Lys Arg Ser Ala Leu Val Arg Arg
Leu Trp Arg Ser Arg Ala Pro Gly 20 25 30 ggt gaa gat gaa gaa gaa
ggt gct ggc ggc ggt ggc ggt ggc ggt gaa 144Gly Glu Asp Glu Glu Glu
Gly Ala Gly Gly Gly Gly Gly Gly Gly Glu 35 40 45 ctg cgt ggc gag
ggt gca acc gat agt cgt gca cac ggt gca ggc ggt 192Leu Arg Gly Glu
Gly Ala Thr Asp Ser Arg Ala His Gly Ala Gly Gly 50 55 60 ggc ggt
ccg ggt cgt gct ggt tgc tgt ctg ggt aaa gct gtg cgc ggc 240Gly Gly
Pro Gly Arg Ala Gly Cys Cys Leu Gly Lys Ala Val Arg Gly 65 70 75 80
gcg aaa ggt cat cac cat ccg cac ccg ccg gca gca ggt gca ggt gca
288Ala Lys Gly His His His Pro His Pro Pro Ala Ala Gly Ala Gly Ala
85 90 95 gct ggc ggt gcg gaa gcc gat ctg aaa gcc ctg acc cat agt
gtc ctg 336Ala Gly Gly Ala Glu Ala Asp Leu Lys Ala Leu Thr His Ser
Val Leu 100 105 110 aaa aaa ctg aaa gaa cgt cag ctg gag ctg ctg ctg
caa gca gta gaa 384Lys Lys Leu Lys Glu Arg Gln Leu Glu Leu Leu Leu
Gln Ala Val Glu 115 120 125 tcc cgt ggc ggt acc cgt acg gct tgt ctg
ctg ctg ccg ggt cgt ctg 432Ser Arg Gly Gly Thr Arg Thr Ala Cys Leu
Leu Leu Pro Gly Arg Leu 130 135 140 gat tgc cgt ctg ggt ccg ggt gca
ccg gct ggt gcg cag ccg gca caa 480Asp Cys Arg Leu Gly Pro Gly Ala
Pro Ala Gly Ala Gln Pro Ala Gln 145 150 155 160 ccg ccg agc tct tac
agc ctg ccg ctg ctg ctg tgt aaa gtg ttt cgt 528Pro Pro Ser Ser Tyr
Ser Leu Pro Leu Leu Leu Cys Lys Val Phe Arg 165 170 175 tgg ccg gac
ctg cgc cac agt tcc gaa gtt aaa cgc ctg tgc tgt tgc 576Trp Pro Asp
Leu Arg His Ser Ser Glu Val Lys Arg Leu Cys Cys Cys 180 185 190 gag
agc tat ggc aaa att aac ccg gaa ctg gtt tgt tgc aat ccg cac 624Glu
Ser Tyr Gly Lys Ile Asn Pro Glu Leu Val Cys Cys Asn Pro His 195 200
205 cat ctg tct cgt ctg tgt gaa ctg gag agc ccg ccg ccg ccg tat tct
672His Leu Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro Pro Pro Tyr Ser
210 215 220 cgt tac ccg atg gat ttc ctg aaa ccg act gca gat tgc ccg
gac gca 720Arg Tyr Pro Met Asp Phe Leu Lys Pro Thr Ala Asp Cys Pro
Asp Ala 225 230 235 240 gtc ccg tca tcg gct gag acc ggc ggc acc aac
tat ctg gca ccg ggc 768Val Pro Ser Ser Ala Glu Thr Gly Gly Thr Asn
Tyr Leu Ala Pro Gly 245 250 255 ggt ctg agt gat tcc cag ctg ctg ctg
gaa ccg ggc gac cgt ggt aag 816Gly Leu Ser Asp Ser Gln Leu Leu Leu
Glu Pro Gly Asp Arg Gly Lys 260 265 270 cct atc cct aac cct ctc ctc
ggt ctc gat tct acg tgagtcgac 861Pro Ile Pro Asn Pro Leu Leu Gly
Leu Asp Ser Thr 275 280 27284PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 27Gly Ser Gly Arg Lys Lys
Arg Arg Gln Arg Arg Arg Gly Phe Arg Thr 1 5 10 15 Lys Arg Ser Ala
Leu Val Arg Arg Leu Trp Arg Ser Arg Ala Pro Gly 20 25 30 Gly Glu
Asp Glu Glu Glu Gly Ala Gly Gly Gly Gly Gly Gly Gly Glu 35 40 45
Leu Arg Gly Glu Gly Ala Thr Asp Ser Arg Ala His Gly Ala Gly Gly 50
55 60 Gly Gly Pro Gly Arg Ala Gly Cys Cys Leu Gly Lys Ala Val Arg
Gly 65 70 75 80 Ala Lys Gly His His His Pro His Pro Pro Ala Ala Gly
Ala Gly Ala 85 90 95 Ala Gly Gly Ala Glu Ala Asp Leu Lys Ala Leu
Thr His Ser Val Leu 100 105 110 Lys Lys Leu Lys Glu Arg Gln Leu Glu
Leu Leu Leu Gln Ala Val Glu 115 120 125 Ser Arg Gly Gly Thr Arg Thr
Ala Cys Leu Leu Leu Pro Gly Arg Leu 130 135 140 Asp Cys Arg Leu Gly
Pro Gly Ala Pro Ala Gly Ala Gln Pro Ala Gln 145 150 155 160 Pro Pro
Ser Ser Tyr Ser Leu Pro Leu Leu Leu Cys Lys Val Phe Arg 165 170 175
Trp Pro Asp Leu Arg His Ser Ser Glu Val Lys Arg Leu Cys Cys Cys 180
185 190 Glu Ser Tyr Gly Lys Ile Asn Pro Glu Leu Val Cys Cys Asn Pro
His 195 200 205 His Leu Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro Pro
Pro Tyr Ser 210 215 220 Arg Tyr Pro Met Asp Phe Leu Lys Pro Thr Ala
Asp Cys Pro Asp Ala 225 230 235 240 Val Pro Ser Ser Ala Glu Thr Gly
Gly Thr Asn Tyr Leu Ala Pro Gly 245 250 255 Gly Leu Ser Asp Ser Gln
Leu Leu Leu Glu Pro Gly Asp Arg Gly Lys 260 265 270 Pro Ile Pro Asn
Pro Leu Leu Gly Leu Asp Ser Thr 275 280 28 1442DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
28ggatccggcc gtaaaaaacg
ccgtcaacgc cgccgtggtt tccgtacgaa acgctcggcc 60ctggtccgtc gcctgtggcg
ctcccgtgct ccgggtggtg aagatgaaga agaaggtgct 120ggcggcggtg
gcggtggcgg tgaactgcgt aagatgaaga agaaggtgct ggcggcggtg
180gcggtggcgg tgaactgcgt ggcgagggtg caaccgatag tcgtgcacac
ggtgcaggcg 240gtggcggtcc gggtcgtgct ggttgctgtc tgggtaaagc
tgtgcgcggc gcgaaaggtc 300atcaccatcc gcacccgccg gcagcaggtg
caggtgcagc tggcggtgcg gaagccgatc 360tgaaagccct gacccatagt
gtcctgaaaa aactgaaaga acgtcagctg gagctgctgc 420tgcaagcagt
agaatcccgt ggcggtaccc gtacggcttg tctgctgctg ccgggtcgtc
480tggattgccg tctgggtccg ggtgcaccgg ctggtgcgca gccggcacaa
ccgccgagct 540cttacagcct gccgctgctg ctgtgtaaag tgtttcgttg
gccggacctg cgccacagtt 600ccgaagttaa acgcctgtgc tgttgcgaga
gctatggcaa aattaacccg gaactggttt 660gttgcaatcc gcaccatctg
tctcgtctgt gtgaactgga gagcccgccg ccgccgtatt 720ctcgttaccc
gatggatttc ctgaaaccga ctgcagattg cccggacgca gtcccgtcat
780cggctgagac cggcggcacc aactatctgg caccgggcgg tctgagtgat
tcccagctgc 840tgctggaacc gggcgaccgt tcacattggt gtgtggttgc
ctattgggaa gagaaaacgc 900gtgtcggtcg cctgtactgc gtacaggaac
cgtcgctgga tatcttttat gacctgccgc 960agggcaatgg tttctgtctg
ggccaactga actcagataa taaatcgcag ctggtgcaaa 1020aagttcgctc
aaaaattggc tgcggtatcc agctgacccg tgaagttgac ggtgtctggg
1080tatataaccg cagctcttac ccgattttta tcaaaagtgc caccctggat
aatccggact 1140cccgtacgct gctggtccac aaagtatttc cgggcttctc
aatcaaagcg ttcgattacg 1200agaaagccta ctcgctgcag cgcccgaacg
accatgaatt catgcagcaa ccgtggacgg 1260gttttactgt gcagatctct
ttcgttaaag gctggggtca atgctacacc cgtcagttta 1320tctcgtcctg
tccgtgctgg ctggaagtga ttttcaatag ccgcaagggc gagctcaatt
1380cgaagcttga aggtaagcct atccctaacc ctctcctcgg tctcgattct
acgtgagtcg 1440ac 14422966DNAArtificial SequenceDescription of
Artificial Sequence Synthetic oligonucleotide 29agcagctttc
agtcagtagc agccagcagc cagagcagca gccagtagca gcagcagcag 60agcagc
66301392DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 30gga tcc ggc cgt aaa aaa cgc cgt caa cgc
cgc cgt ggt ttc cgt acg 48Gly Ser Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Gly Phe Arg Thr 1 5 10 15 aaa cgc agc gcc ctg gtc cgt cgc
ctg tgg cgc agc cgt gct ccg ggt 96Lys Arg Ser Ala Leu Val Arg Arg
Leu Trp Arg Ser Arg Ala Pro Gly 20 25 30 ggt gaa gat gaa gaa gaa
ggt gct ggc ggc ggt ggc ggt ggc ggt gaa 144Gly Glu Asp Glu Glu Glu
Gly Ala Gly Gly Gly Gly Gly Gly Gly Glu 35 40 45 ctg cgt ggc gag
ggt gca acc gat agc cgt gca cat ggt gca ggc ggt 192Leu Arg Gly Glu
Gly Ala Thr Asp Ser Arg Ala His Gly Ala Gly Gly 50 55 60 ggc ggt
ccg ggt cgt gct ggt tgc tgt ctg ggt aaa gct gtg cgc ggc 240Gly Gly
Pro Gly Arg Ala Gly Cys Cys Leu Gly Lys Ala Val Arg Gly 65 70 75 80
gcg aaa ggt cat cat cat ccg cat ccg ccg gca gca ggt gca ggt gca
288Ala Lys Gly His His His Pro His Pro Pro Ala Ala Gly Ala Gly Ala
85 90 95 gct ggc ggt gcg gaa gcc gat ctg aaa gcc ctg acc cat agc
gtc ctg 336Ala Gly Gly Ala Glu Ala Asp Leu Lys Ala Leu Thr His Ser
Val Leu 100 105 110 aaa aaa ctg aaa gaa cgt cag ctg gag ctg ctg ctg
caa gca gta gaa 384Lys Lys Leu Lys Glu Arg Gln Leu Glu Leu Leu Leu
Gln Ala Val Glu 115 120 125 agc cgt ggc ggt acc cgt acg gct tgt ctg
ctg ctg ccg ggt cgt ctg 432Ser Arg Gly Gly Thr Arg Thr Ala Cys Leu
Leu Leu Pro Gly Arg Leu 130 135 140 gat tgc cgt ctg ggt ccg ggt gca
ccg gct ggt gcg cag ccg gca caa 480Asp Cys Arg Leu Gly Pro Gly Ala
Pro Ala Gly Ala Gln Pro Ala Gln 145 150 155 160 ccg ccg agc agc tac
agc ctg ccg ctg ctg ctg tgt aaa gtg ttt cgt 528Pro Pro Ser Ser Tyr
Ser Leu Pro Leu Leu Leu Cys Lys Val Phe Arg 165 170 175 tgg ccg gac
ctg cgc cat agc agc gaa gtt aaa cgc ctg tgc tgt tgc 576Trp Pro Asp
Leu Arg His Ser Ser Glu Val Lys Arg Leu Cys Cys Cys 180 185 190 gag
agc tat ggc aaa att aac ccg gaa ctg gtt tgt tgc aat ccg cat 624Glu
Ser Tyr Gly Lys Ile Asn Pro Glu Leu Val Cys Cys Asn Pro His 195 200
205 cat ctg agc cgt ctg tgt gaa ctg gag agc ccg ccg ccg ccg tat agc
672His Leu Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro Pro Pro Tyr Ser
210 215 220 cgt tac ccg ctg gat ttc ctg aaa ccg act gca gat tgc ccg
gac gca 720Arg Tyr Pro Leu Asp Phe Leu Lys Pro Thr Ala Asp Cys Pro
Asp Ala 225 230 235 240 gtc ccg agc agc gct gag acc ggc ggc acc aac
tat ctg gca ccg ggc 768Val Pro Ser Ser Ala Glu Thr Gly Gly Thr Asn
Tyr Leu Ala Pro Gly 245 250 255 ggt ctg agc gat agc cag ctg ctg ctg
gaa ccg ggc gac cgt agc cat 816Gly Leu Ser Asp Ser Gln Leu Leu Leu
Glu Pro Gly Asp Arg Ser His 260 265 270 tgg tgt gtg gtt gcc tat tgg
gaa gag aaa acg cgt gtc ggt cgc ctg 864Trp Cys Val Val Ala Tyr Trp
Glu Glu Lys Thr Arg Val Gly Arg Leu 275 280 285 tac tgc gta cag gaa
ccg agc ctg gat atc ttt tat gac ctg ccg cag 912Tyr Cys Val Gln Glu
Pro Ser Leu Asp Ile Phe Tyr Asp Leu Pro Gln 290 295 300 ggc aat ggt
ttc tgt ctg ggc caa ctg aac agc gat aat aaa agc cag 960Gly Asn Gly
Phe Cys Leu Gly Gln Leu Asn Ser Asp Asn Lys Ser Gln 305 310 315 320
ctg gtg caa aaa gtt cgc agc aaa att ggc tgc ggt atc cag ctg acc
1008Leu Val Gln Lys Val Arg Ser Lys Ile Gly Cys Gly Ile Gln Leu Thr
325 330 335 cgt gaa gtt gac ggt gtc tgg gta tat aac cgc agc agc tac
ccg att 1056Arg Glu Val Asp Gly Val Trp Val Tyr Asn Arg Ser Ser Tyr
Pro Ile 340 345 350 ttt atc aaa agc gcc acc ctg gat aat ccg gac agc
cgt acg ctg ctg 1104Phe Ile Lys Ser Ala Thr Leu Asp Asn Pro Asp Ser
Arg Thr Leu Leu 355 360 365 gtc cat aaa gta ttt ccg ggc ttc agc atc
aaa gcg ttc gat tac gag 1152Val His Lys Val Phe Pro Gly Phe Ser Ile
Lys Ala Phe Asp Tyr Glu 370 375 380 aaa gcc tac agc ctg cag cgc ccg
aac gac cat gaa ttc atg cag caa 1200Lys Ala Tyr Ser Leu Gln Arg Pro
Asn Asp His Glu Phe Met Gln Gln 385 390 395 400 ccg tgg acg ggt ttt
act gtg cag atc agc ttc gtt aaa ggc tgg ggt 1248Pro Trp Thr Gly Phe
Thr Val Gln Ile Ser Phe Val Lys Gly Trp Gly 405 410 415 caa tgc tac
acc cgt cag ttt atc agc agc tgt ccg tgc tgg ctg gaa 1296Gln Cys Tyr
Thr Arg Gln Phe Ile Ser Ser Cys Pro Cys Trp Leu Glu 420 425 430 gtg
att ttc aat agc cgc aag ggc gag ctc aat agc aag ctt gaa ggt 1344Val
Ile Phe Asn Ser Arg Lys Gly Glu Leu Asn Ser Lys Leu Glu Gly 435 440
445 aag cct atc cct aac cct ctc ctc ggt ctc gat agc acg tgagtcgac
1392Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 450 455 460
31461PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 31Gly Ser Gly Arg Lys Lys Arg Arg Gln Arg Arg
Arg Gly Phe Arg Thr 1 5 10 15 Lys Arg Ser Ala Leu Val Arg Arg Leu
Trp Arg Ser Arg Ala Pro Gly 20 25 30 Gly Glu Asp Glu Glu Glu Gly
Ala Gly Gly Gly Gly Gly Gly Gly Glu 35 40 45 Leu Arg Gly Glu Gly
Ala Thr Asp Ser Arg Ala His Gly Ala Gly Gly 50 55 60 Gly Gly Pro
Gly Arg Ala Gly Cys Cys Leu Gly Lys Ala Val Arg Gly 65 70 75 80 Ala
Lys Gly His His His Pro His Pro Pro Ala Ala Gly Ala Gly Ala 85 90
95 Ala Gly Gly Ala Glu Ala Asp Leu Lys Ala Leu Thr His Ser Val Leu
100 105 110 Lys Lys Leu Lys Glu Arg Gln Leu Glu Leu Leu Leu Gln Ala
Val Glu 115 120 125 Ser Arg Gly Gly Thr Arg Thr Ala Cys Leu Leu Leu
Pro Gly Arg Leu 130 135 140 Asp Cys Arg Leu Gly Pro Gly Ala Pro Ala
Gly Ala Gln Pro Ala Gln 145 150 155 160 Pro Pro Ser Ser Tyr Ser Leu
Pro Leu Leu Leu Cys Lys Val Phe Arg 165 170 175 Trp Pro Asp Leu Arg
His Ser Ser Glu Val Lys Arg Leu Cys Cys Cys 180 185 190 Glu Ser Tyr
Gly Lys Ile Asn Pro Glu Leu Val Cys Cys Asn Pro His 195 200 205 His
Leu Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro Pro Pro Tyr Ser 210 215
220 Arg Tyr Pro Leu Asp Phe Leu Lys Pro Thr Ala Asp Cys Pro Asp Ala
225 230 235 240 Val Pro Ser Ser Ala Glu Thr Gly Gly Thr Asn Tyr Leu
Ala Pro Gly 245 250 255 Gly Leu Ser Asp Ser Gln Leu Leu Leu Glu Pro
Gly Asp Arg Ser His 260 265 270 Trp Cys Val Val Ala Tyr Trp Glu Glu
Lys Thr Arg Val Gly Arg Leu 275 280 285 Tyr Cys Val Gln Glu Pro Ser
Leu Asp Ile Phe Tyr Asp Leu Pro Gln 290 295 300 Gly Asn Gly Phe Cys
Leu Gly Gln Leu Asn Ser Asp Asn Lys Ser Gln 305 310 315 320 Leu Val
Gln Lys Val Arg Ser Lys Ile Gly Cys Gly Ile Gln Leu Thr 325 330 335
Arg Glu Val Asp Gly Val Trp Val Tyr Asn Arg Ser Ser Tyr Pro Ile 340
345 350 Phe Ile Lys Ser Ala Thr Leu Asp Asn Pro Asp Ser Arg Thr Leu
Leu 355 360 365 Val His Lys Val Phe Pro Gly Phe Ser Ile Lys Ala Phe
Asp Tyr Glu 370 375 380 Lys Ala Tyr Ser Leu Gln Arg Pro Asn Asp His
Glu Phe Met Gln Gln 385 390 395 400 Pro Trp Thr Gly Phe Thr Val Gln
Ile Ser Phe Val Lys Gly Trp Gly 405 410 415 Gln Cys Tyr Thr Arg Gln
Phe Ile Ser Ser Cys Pro Cys Trp Leu Glu 420 425 430 Val Ile Phe Asn
Ser Arg Lys Gly Glu Leu Asn Ser Lys Leu Glu Gly 435 440 445 Lys Pro
Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr 450 455 460
32861DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 32gga tcc ggc cgt aaa aaa cgc cgt caa cgc
cgc cgt ggt ttc cgt acg 48Gly Ser Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Gly Phe Arg Thr 1 5 10 15 aaa cgc agc gcc ctg gtc cgt cgc
ctg tgg cgc agc cgt gct ccg ggt 96Lys Arg Ser Ala Leu Val Arg Arg
Leu Trp Arg Ser Arg Ala Pro Gly 20 25 30 ggt gaa gat gaa gaa gaa
ggt gct ggc ggc ggt ggc ggt ggc ggt gaa 144Gly Glu Asp Glu Glu Glu
Gly Ala Gly Gly Gly Gly Gly Gly Gly Glu 35 40 45 ctg cgt ggc gag
ggt gca acc gat agc cgt gca cat ggt gca ggc ggt 192Leu Arg Gly Glu
Gly Ala Thr Asp Ser Arg Ala His Gly Ala Gly Gly 50 55 60 ggc ggt
ccg ggt cgt gct ggt tgc tgt ctg ggt aaa gct gtg cgc ggc 240Gly Gly
Pro Gly Arg Ala Gly Cys Cys Leu Gly Lys Ala Val Arg Gly 65 70 75 80
gcg aaa ggt cat cat cat ccg cat ccg ccg gca gca ggt gca ggt gca
288Ala Lys Gly His His His Pro His Pro Pro Ala Ala Gly Ala Gly Ala
85 90 95 gct ggc ggt gcg gaa gcc gat ctg aaa gcc ctg acc cat agc
gtc ctg 336Ala Gly Gly Ala Glu Ala Asp Leu Lys Ala Leu Thr His Ser
Val Leu 100 105 110 aaa aaa ctg aaa gaa cgt cag ctg gag ctg ctg ctg
caa gca gta gaa 384Lys Lys Leu Lys Glu Arg Gln Leu Glu Leu Leu Leu
Gln Ala Val Glu 115 120 125 agc cgt ggc ggt acc cgt acg gct tgt ctg
ctg ctg ccg ggt cgt ctg 432Ser Arg Gly Gly Thr Arg Thr Ala Cys Leu
Leu Leu Pro Gly Arg Leu 130 135 140 gat tgc cgt ctg ggt ccg ggt gca
ccg gct ggt gcg cag ccg gca caa 480Asp Cys Arg Leu Gly Pro Gly Ala
Pro Ala Gly Ala Gln Pro Ala Gln 145 150 155 160 ccg ccg agc agc tac
agc ctg ccg ctg ctg ctg tgt aaa gtg ttt cgt 528Pro Pro Ser Ser Tyr
Ser Leu Pro Leu Leu Leu Cys Lys Val Phe Arg 165 170 175 tgg ccg gac
ctg cgc cat agc agc gaa gtt aaa cgc ctg tgc tgt tgc 576Trp Pro Asp
Leu Arg His Ser Ser Glu Val Lys Arg Leu Cys Cys Cys 180 185 190 gag
agc tat ggc aaa att aac ccg gaa ctg gtt tgt tgc aat ccg cat 624Glu
Ser Tyr Gly Lys Ile Asn Pro Glu Leu Val Cys Cys Asn Pro His 195 200
205 cat ctg agc cgt ctg tgt gaa ctg gag agc ccg ccg ccg ccg tat agc
672His Leu Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro Pro Pro Tyr Ser
210 215 220 cgt tac ccg atg gat ttc ctg aaa ccg act gca gat tgc ccg
gac gca 720Arg Tyr Pro Met Asp Phe Leu Lys Pro Thr Ala Asp Cys Pro
Asp Ala 225 230 235 240 gtc ccg agc agc gct gag acc ggc ggc acc aac
tat ctg gca ccg ggc 768Val Pro Ser Ser Ala Glu Thr Gly Gly Thr Asn
Tyr Leu Ala Pro Gly 245 250 255 ggt ctg agc gat agc cag ctg ctg ctg
gaa ccg ggc gac cgt ggt aag 816Gly Leu Ser Asp Ser Gln Leu Leu Leu
Glu Pro Gly Asp Arg Gly Lys 260 265 270 cct atc cct aac cct ctc ctc
ggt ctc gat tct acg tgagtcgac 861Pro Ile Pro Asn Pro Leu Leu Gly
Leu Asp Ser Thr 275 280 33558DNAArtificial SequenceDescription of
Artificial Sequence Synthetic polynucleotide 33ggatccggcc
gtaaaaaacg ccgtcaacgc cgccgttcac attggtgtgt ggttgcctat 60tgggaagaga
aaacgcgtgt cggtcgcctg tactgcgtac aggaaccgag cctggatatc
120ttttatgacc tgccgcaggg caatggtttc tgtctgggcc aactgaacag
cgataataaa 180agccagctgg tgcaaaaagt tcgcagcaaa attggctgcg
gtatccagct gacccgtgaa 240gttgacggtg tctgggtata taaccgcagc
agctacccga tttttatcaa aagcgccacc 300ctggataatc cggacagccg
tacgctgctg gtccataaag tatttccggg cttcagcatc 360aaagcgttcg
attacgagaa agcctacagc ctgcagcgcc cgaacgacca tgaattcatg
420cagcaaccgt ggacgggttt tactgtgcag atcagcttcg ttaaaggctg
gggtcaatgc 480aagggcgagc tcaatagcaa gcttgaaggt aagcctatcc
ctaaccctct cctcggtctc 540gatagcacgt gagtcgac 55834618DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
34gga tcc ggc cgt aaa aaa cgc cgt caa cgc cgc cgt tca cat tgg tgt
48Gly Ser Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Ser His Trp Cys 1
5 10 15 gtg gtt gcc tat tgg gaa gag aaa acg cgt gtc ggt cgc ctg tac
tgc 96Val Val Ala Tyr Trp Glu Glu Lys Thr Arg Val Gly Arg Leu Tyr
Cys 20 25 30 gta cag gaa ccg agc ctg gat atc ttt tat gac ctg ccg
cag ggc aat 144Val Gln Glu Pro Ser Leu Asp Ile Phe Tyr Asp Leu Pro
Gln Gly Asn 35 40 45 ggt ttc tgt ctg ggc caa ctg aac agc gat aat
aaa agc cag ctg gtg 192Gly Phe Cys Leu Gly Gln Leu Asn Ser Asp Asn
Lys Ser Gln Leu Val 50 55 60 caa aaa gtt cgc agc aaa att ggc tgc
ggt atc cag ctg acc cgt gaa 240Gln Lys Val Arg Ser Lys Ile Gly Cys
Gly Ile Gln Leu Thr Arg Glu 65 70 75 80
gtt gac ggt gtc tgg gta tat aac cgc agc agc tac ccg att ttt atc
288Val Asp Gly Val Trp Val Tyr Asn Arg Ser Ser Tyr Pro Ile Phe Ile
85 90 95 aaa agc gcc acc ctg gat aat ccg gac agc cgt acg ctg ctg
gtc cat 336Lys Ser Ala Thr Leu Asp Asn Pro Asp Ser Arg Thr Leu Leu
Val His 100 105 110 aaa gta ttt ccg ggc ttc agc atc aaa gcg ttc gat
tac gag aaa gcc 384Lys Val Phe Pro Gly Phe Ser Ile Lys Ala Phe Asp
Tyr Glu Lys Ala 115 120 125 tac agc ctg cag cgc ccg aac gac cat gaa
ttc atg cag caa ccg tgg 432Tyr Ser Leu Gln Arg Pro Asn Asp His Glu
Phe Met Gln Gln Pro Trp 130 135 140 acg ggt ttt act gtg cag atc agc
ttc gtt aaa ggc tgg ggt caa tgc 480Thr Gly Phe Thr Val Gln Ile Ser
Phe Val Lys Gly Trp Gly Gln Cys 145 150 155 160 tac acc cgt cag ttt
atc agc agc tgt ccg tgc tgg ctg gaa gtg att 528Tyr Thr Arg Gln Phe
Ile Ser Ser Cys Pro Cys Trp Leu Glu Val Ile 165 170 175 ttc aat agc
cgc aag ggc gag ctc aat agc aag ctt gaa ggt aag cct 576Phe Asn Ser
Arg Lys Gly Glu Leu Asn Ser Lys Leu Glu Gly Lys Pro 180 185 190 atc
cct aac cct ctc ctc ggt ctc gat agc acg tgagtcgac 618Ile Pro Asn
Pro Leu Leu Gly Leu Asp Ser Thr 195 200 3539DNAArtificial
SequenceDescription of Artificial Sequence Synthetic
oligonucleotide 35tcgtcatcgt catctttcct catcgtcttc gtctcgtct
39361365DNAArtificial SequenceDescription of Artificial Sequence
Synthetic polynucleotide 36gga tcc ggt cgt aaa aaa cgt cgt cag cgt
cgt cgt ggt ttc cgt acc 48Gly Ser Gly Arg Lys Lys Arg Arg Gln Arg
Arg Arg Gly Phe Arg Thr 1 5 10 15 aaa cgt tct gcg ctg gtt cgt cgt
ctg tgg cgt tct cgt gcg ccg ggt 96Lys Arg Ser Ala Leu Val Arg Arg
Leu Trp Arg Ser Arg Ala Pro Gly 20 25 30 ggt gaa gac gaa gaa gaa
ggt gcg ggt ggt ggt ggt ggt ggt ggt gaa 144Gly Glu Asp Glu Glu Glu
Gly Ala Gly Gly Gly Gly Gly Gly Gly Glu 35 40 45 ctg cgt ggt gaa
ggt gcg acc gac tct cgt gcg cac ggt gcg ggt ggt 192Leu Arg Gly Glu
Gly Ala Thr Asp Ser Arg Ala His Gly Ala Gly Gly 50 55 60 ggt ggt
ccg ggt cgt gcg ggt tgc tgc ctg ggt aaa gcg gtt cgt ggt 240Gly Gly
Pro Gly Arg Ala Gly Cys Cys Leu Gly Lys Ala Val Arg Gly 65 70 75 80
gcg aaa ggt cac cac cac ccg cac ccg ccg gcg gcg ggt gcg ggt gcg
288Ala Lys Gly His His His Pro His Pro Pro Ala Ala Gly Ala Gly Ala
85 90 95 gcg ggt ggt gcg gaa gcg gac ctg aaa gcg ctg acc cac tct
gtt ctg 336Ala Gly Gly Ala Glu Ala Asp Leu Lys Ala Leu Thr His Ser
Val Leu 100 105 110 aaa aaa ctg aaa gaa cgt cag ctg gaa ctg ctg ctg
cag gcg gtt gaa 384Lys Lys Leu Lys Glu Arg Gln Leu Glu Leu Leu Leu
Gln Ala Val Glu 115 120 125 tct cgt ggt ggt acc cgt acc gcg tgc ctg
ctg ctg ccg ggt cgt ctg 432Ser Arg Gly Gly Thr Arg Thr Ala Cys Leu
Leu Leu Pro Gly Arg Leu 130 135 140 gac tgc cgt ctg ggt ccg ggt gcg
ccg gcg ggt gcg cag ccg gcg cag 480Asp Cys Arg Leu Gly Pro Gly Ala
Pro Ala Gly Ala Gln Pro Ala Gln 145 150 155 160 ccg ccg tct tct tac
tct ctg ccg ctg ctg ctg tgc aaa gtt ttc cgt 528Pro Pro Ser Ser Tyr
Ser Leu Pro Leu Leu Leu Cys Lys Val Phe Arg 165 170 175 tgg ccg gac
ctg cgt cac tct tct gaa gtt aaa cgt ctg tgc tgc tgc 576Trp Pro Asp
Leu Arg His Ser Ser Glu Val Lys Arg Leu Cys Cys Cys 180 185 190 gaa
tct tac ggt aaa atc aac ccg gaa ctg gtt tgc tgc aac ccg cac 624Glu
Ser Tyr Gly Lys Ile Asn Pro Glu Leu Val Cys Cys Asn Pro His 195 200
205 cac ctg tct cgt ctg tgc gaa ctg gaa tct ccg ccg ccg ccg tac tct
672His Leu Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro Pro Pro Tyr Ser
210 215 220 cgt tac ccg ctg gac ttc ctg aaa ccg acc gcg gac tgc ccg
gac gcg 720Arg Tyr Pro Leu Asp Phe Leu Lys Pro Thr Ala Asp Cys Pro
Asp Ala 225 230 235 240 gtt ccg tct tct gcg gaa acc ggt ggt acc aac
tac ctg gcg ccg ggt 768Val Pro Ser Ser Ala Glu Thr Gly Gly Thr Asn
Tyr Leu Ala Pro Gly 245 250 255 ggt ctg tct gac tct cag ctg ctg ctg
gaa ccg ggt gac cgt tct cac 816Gly Leu Ser Asp Ser Gln Leu Leu Leu
Glu Pro Gly Asp Arg Ser His 260 265 270 tgg tgc gtt gtt gcg tac tgg
gaa gaa aaa acc cgt gtt ggt cgt ctg 864Trp Cys Val Val Ala Tyr Trp
Glu Glu Lys Thr Arg Val Gly Arg Leu 275 280 285 tac tgc gtt cag gaa
ccg tct ctg gac atc ttc tac gac ctg ccg cag 912Tyr Cys Val Gln Glu
Pro Ser Leu Asp Ile Phe Tyr Asp Leu Pro Gln 290 295 300 ggt aac ggt
ttc tgc ctg ggt cag ctg aac tct gac aac aaa tct cag 960Gly Asn Gly
Phe Cys Leu Gly Gln Leu Asn Ser Asp Asn Lys Ser Gln 305 310 315 320
ctg gtt cag aaa gtt cgt tct aaa atc ggt tgc ggt atc cag ctg acc
1008Leu Val Gln Lys Val Arg Ser Lys Ile Gly Cys Gly Ile Gln Leu Thr
325 330 335 cgt gaa gtt gac ggt gtt tgg gtt tac aac cgt tct tct tac
ccg atc 1056Arg Glu Val Asp Gly Val Trp Val Tyr Asn Arg Ser Ser Tyr
Pro Ile 340 345 350 ttc atc aaa tct gcg acc ctg gac aac ccg gac tct
cgt acc ctg ctg 1104Phe Ile Lys Ser Ala Thr Leu Asp Asn Pro Asp Ser
Arg Thr Leu Leu 355 360 365 gtt cac aaa gtt ttc ccg ggt ttc tct atc
aaa gcg ttc gac tac gaa 1152Val His Lys Val Phe Pro Gly Phe Ser Ile
Lys Ala Phe Asp Tyr Glu 370 375 380 aaa gcg tac tct ctg cag cgt ccg
aac gac cac gaa ttc atg cag cag 1200Lys Ala Tyr Ser Leu Gln Arg Pro
Asn Asp His Glu Phe Met Gln Gln 385 390 395 400 ccg tgg acc ggt ttc
acc gtt cag atc tct ttc gtt aaa ggt tgg ggt 1248Pro Trp Thr Gly Phe
Thr Val Gln Ile Ser Phe Val Lys Gly Trp Gly 405 410 415 cag tgc tac
acc cgt cag ttc atc tct tct tgc ccg tgc tgg ctg gaa 1296Gln Cys Tyr
Thr Arg Gln Phe Ile Ser Ser Cys Pro Cys Trp Leu Glu 420 425 430 gtt
atc ttc aac tct cgt ggt aaa ccg atc ccg aac ccg ctg ctg ggt 1344Val
Ile Phe Asn Ser Arg Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly 435 440
445 ctg gac tct acc tgagtcgac 1365Leu Asp Ser Thr 450
37452PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 37Gly Ser Gly Arg Lys Lys Arg Arg Gln Arg Arg
Arg Gly Phe Arg Thr 1 5 10 15 Lys Arg Ser Ala Leu Val Arg Arg Leu
Trp Arg Ser Arg Ala Pro Gly 20 25 30 Gly Glu Asp Glu Glu Glu Gly
Ala Gly Gly Gly Gly Gly Gly Gly Glu 35 40 45 Leu Arg Gly Glu Gly
Ala Thr Asp Ser Arg Ala His Gly Ala Gly Gly 50 55 60 Gly Gly Pro
Gly Arg Ala Gly Cys Cys Leu Gly Lys Ala Val Arg Gly 65 70 75 80 Ala
Lys Gly His His His Pro His Pro Pro Ala Ala Gly Ala Gly Ala 85 90
95 Ala Gly Gly Ala Glu Ala Asp Leu Lys Ala Leu Thr His Ser Val Leu
100 105 110 Lys Lys Leu Lys Glu Arg Gln Leu Glu Leu Leu Leu Gln Ala
Val Glu 115 120 125 Ser Arg Gly Gly Thr Arg Thr Ala Cys Leu Leu Leu
Pro Gly Arg Leu 130 135 140 Asp Cys Arg Leu Gly Pro Gly Ala Pro Ala
Gly Ala Gln Pro Ala Gln 145 150 155 160 Pro Pro Ser Ser Tyr Ser Leu
Pro Leu Leu Leu Cys Lys Val Phe Arg 165 170 175 Trp Pro Asp Leu Arg
His Ser Ser Glu Val Lys Arg Leu Cys Cys Cys 180 185 190 Glu Ser Tyr
Gly Lys Ile Asn Pro Glu Leu Val Cys Cys Asn Pro His 195 200 205 His
Leu Ser Arg Leu Cys Glu Leu Glu Ser Pro Pro Pro Pro Tyr Ser 210 215
220 Arg Tyr Pro Leu Asp Phe Leu Lys Pro Thr Ala Asp Cys Pro Asp Ala
225 230 235 240 Val Pro Ser Ser Ala Glu Thr Gly Gly Thr Asn Tyr Leu
Ala Pro Gly 245 250 255 Gly Leu Ser Asp Ser Gln Leu Leu Leu Glu Pro
Gly Asp Arg Ser His 260 265 270 Trp Cys Val Val Ala Tyr Trp Glu Glu
Lys Thr Arg Val Gly Arg Leu 275 280 285 Tyr Cys Val Gln Glu Pro Ser
Leu Asp Ile Phe Tyr Asp Leu Pro Gln 290 295 300 Gly Asn Gly Phe Cys
Leu Gly Gln Leu Asn Ser Asp Asn Lys Ser Gln 305 310 315 320 Leu Val
Gln Lys Val Arg Ser Lys Ile Gly Cys Gly Ile Gln Leu Thr 325 330 335
Arg Glu Val Asp Gly Val Trp Val Tyr Asn Arg Ser Ser Tyr Pro Ile 340
345 350 Phe Ile Lys Ser Ala Thr Leu Asp Asn Pro Asp Ser Arg Thr Leu
Leu 355 360 365 Val His Lys Val Phe Pro Gly Phe Ser Ile Lys Ala Phe
Asp Tyr Glu 370 375 380 Lys Ala Tyr Ser Leu Gln Arg Pro Asn Asp His
Glu Phe Met Gln Gln 385 390 395 400 Pro Trp Thr Gly Phe Thr Val Gln
Ile Ser Phe Val Lys Gly Trp Gly 405 410 415 Gln Cys Tyr Thr Arg Gln
Phe Ile Ser Ser Cys Pro Cys Trp Leu Glu 420 425 430 Val Ile Phe Asn
Ser Arg Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly 435 440 445 Leu Asp
Ser Thr 450 381365DNAArtificial SequenceDescription of Artificial
Sequence Synthetic polynucleotide 38ggatccggtc gtaaaaaacg
tcgtcagcgt cgtcgtggtt tccgtaccaa acgttctgcg 60ctggttcgtc gtctgtggcg
ttctcgtgcg ccgggtggtg aagacgaaga agaaggtgcg 120ggtggtggtg
gtggtggtgg tgaactgcgt ggtgaaggtg cgaccgactc tcgtgcgcac
180ggtgcgggtg gtggtggtcc gggtcgtgcg ggttgctgcc tgggtaaagc
ggttcgtggt 240gcgaaaggtc accaccaccc gcacccgccg gcggcgggtg
cgggtgcggc gggtggtgcg 300gaagcggacc tgaaagcgct gacccactct
gttctgaaaa aactgaaaga acgtcagctg 360gaactgctgc tgcaggcggt
tgaatctcgt ggtggtaccc gtaccgcgtg cctgctgctg 420ccgggtcgtc
tggactgccg tctgggtccg ggtgcgccgg cgggtgcgca gccggcgcag
480ccgccgtctt cttactctct gccgctgctg ctgtgcaaag ttttccgttg
gccggacctg 540cgtcactctt ctgaagttaa acgtctgtgc tgctgcgaat
cttacggtaa aatcaacccg 600gaactggttt gctgcaaccc gcaccacctg
tctcgtctgt gcgaactgga atctccgccg 660ccgccgtact ctcgttaccc
gctggacttc ctgaaaccga ccgcggactg cccggacgcg 720gttccgtctt
ctgcggaaac cggtggtacc aactacctgg cgccgggtgg tctgtctgac
780tctcagctgc tgctggaacc gggtgaccgt tctcactggt gcgttgttgc
gtactgggaa 840gaaaaaaccc gtgttggtcg tctgtactgc gttcaggaac
cgtctctgga catcttctac 900gacctgccgc agggtaacgg tttctgcctg
ggtcagctga actctgacaa caaatctcag 960ctggttcaga aagttcgttc
taaaatcggt tgcggtatcc agctgacccg tgaagttgac 1020ggtgtttggg
tttacaaccg ttcttcttac ccgatcttca tcaaatctgc gaccctggac
1080aacccggact ctcgtaccct gctggttcac aaagttttcc cgggtttctc
tatcaaagcg 1140ttcgactacg aaaaagcgta ctctctgcag cgtccgaacg
accacgaatt catgcagcag 1200ccgtggaccg gtttcaccgt tcagatctct
ttcgttaaag gttggggtca gtgctacacc 1260cgtcagttca tctcttcttg
cccgtgctgg ctggaagtta tcttcaactc tcgtggtaaa 1320ccgatcccga
acccgctgct gggtctggac tctacctgag tcgac 1365391392DNAArtificial
SequenceDescription of Artificial Sequence Synthetic polynucleotide
39ggatccggcc gtaaaaaacg ccgtcaacgc cgccgtggtt tccgtacgaa acgcagcgcc
60ctggtccgtc gcctgtggcg cagccgtgct ccgggtggtg aagatgaaga agaaggtgct
120ggcggcggtg gcggtggcgg tgaactgcgt ggcgagggtg caaccgatag
ccgtgcacat 180ggtgcaggcg gtggcggtcc gggtcgtgct ggttgctgtc
tgggtaaagc tgtgcgcggc 240gcgaaaggtc atcatcatcc gcatccgccg
gcagcaggtg caggtgcagc tggcggtgcg 300gaagccgatc tgaaagccct
gacccatagc gtcctgaaaa aactgaaaga acgtcagctg 360gagctgctgc
tgcaagcagt agaaagccgt ggcggtaccc gtacggcttg tctgctgctg
420ccgggtcgtc tggattgccg tctgggtccg ggtgcaccgg ctggtgcgca
gccggcacaa 480ccgccgagca gctacagcct gccgctgctg ctgtgtaaag
tgtttcgttg gccggacctg 540cgccatagca gcgaagttaa acgcctgtgc
tgttgcgaga gctatggcaa aattaacccg 600gaactggttt gttgcaatcc
gcatcatctg agccgtctgt gtgaactgga gagcccgccg 660ccgccgtata
gccgttaccc gctggatttc ctgaaaccga ctgcagattg cccggacgca
720gtcccgagca gcgctgagac cggcggcacc aactatctgg caccgggcgg
tctgagcgat 780agccagctgc tgctggaacc gggcgaccgt agccattggt
gtgtggttgc ctattgggaa 840gagaaaacgc gtgtcggtcg cctgtactgc
gtacaggaac cgagcctgga tatcttttat 900gacctgccgc agggcaatgg
tttctgtctg ggccaactga acagcgataa taaaagccag 960ctggtgcaaa
aagttcgcag caaaattggc tgcggtatcc agctgacccg tgaagttgac
1020ggtgtctggg tatataaccg cagcagctac ccgattttta tcaaaagcgc
caccctggat 1080aatccggaca gccgtacgct gctggtccat aaagtatttc
cgggcttcag catcaaagcg 1140ttcgattacg agaaagccta cagcctgcag
cgcccgaacg accatgaatt catgcagcaa 1200ccgtggacgg gttttactgt
gcagatcagc ttcgttaaag gctggggtca atgctacacc 1260cgtcagttta
tcagcagctg tccgtgctgg ctggaagtga ttttcaatag ccgcaagggc
1320gagctcaata gcaagcttga aggtaagcct atccctaacc ctctcctcgg
tctcgatagc 1380acgtgagtcg ac 1392406PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
40His His His His His His 1 5 4114PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 41Gly Lys Pro Ile Pro Asn
Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 428PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 42Lys
Tyr Lys Asp Asp Asp Asp Lys 1 5 439PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 43Tyr
Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 4423PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 44Gly
Ala Leu Phe Leu Gly Trp Leu Gly Ala Ala Gly Ser Thr Met Gly 1 5 10
15 Ala Lys Lys Lys Arg Lys Val 20 4516PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 45Arg
Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys 1 5 10
15 467PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 46Arg Arg Met Lys Trp Lys Lys 1 5
4716PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 47Arg Arg Trp Arg Arg Trp Trp Arg Arg Trp Trp Arg
Arg Trp Arg Arg 1 5 10 15 4818PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 48Arg Gly Gly Arg Leu Ser Tyr
Ser Arg Arg Arg Phe Ser Thr Ser Thr 1 5 10 15 Gly Arg
499PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 49Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5
5011PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 50Tyr Ala Arg Ala Ala Ala Arg Gln Ala Arg Ala 1 5
10 518PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 51Arg Arg Arg Arg Arg Arg Arg Arg 1 5
528PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 52Lys Lys Lys Lys Lys Lys Lys Lys 1 5
5327PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 53Gly Trp Thr Leu Asn Ser Ala Gly Tyr Leu Leu Gly
Lys Ile Asn Leu 1 5 10 15 Lys
Ala Leu Ala Ala Leu Ala Lys Xaa Ile Leu 20 25 5418PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 54Leu
Leu Ile Leu Leu Arg Arg Arg Ile Arg Lys Gln Ala Asn Ala His 1 5 10
15 Ser Lys 5516PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 55Ser Arg Arg His His Cys Arg Ser Lys
Ala Lys Arg Ser Arg His His 1 5 10 15 5611PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 56Asn
Arg Ala Arg Arg Asn Arg Arg Arg Val Arg 1 5 10 5715PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 57Arg
Gln Leu Arg Ile Ala Gly Arg Arg Leu Arg Gly Arg Ser Arg 1 5 10 15
5813PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 58Lys Leu Ile Lys Gly Arg Thr Pro Ile Lys Phe Gly
Lys 1 5 10 5910PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 59Arg Arg Ile Pro Asn Arg Arg Pro Arg
Arg 1 5 10 6018PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 60Lys Leu Ala Leu Lys Leu Ala Leu Lys
Ala Leu Lys Ala Ala Leu Lys 1 5 10 15 Leu Ala 6114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 61Lys
Leu Ala Lys Leu Ala Lys Lys Leu Ala Lys Leu Ala Lys 1 5 10
6227PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 62Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly
Ser Thr Asn Gly 1 5 10 15 Ala Trp Ser Gln Pro Lys Lys Lys Arg Lys
Val 20 25 6321PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 63Lys Glu Thr Trp Trp Glu Thr Trp Trp
Thr Glu Trp Ser Gln Pro Lys 1 5 10 15 Lys Lys Arg Lys Val 20
6420PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 64Leu Lys Lys Leu Leu Lys Lys Leu Leu Lys Lys Leu
Leu Lys Lys Leu 1 5 10 15 Leu Lys Lys Leu 20 6534PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
65Gln Ala Ala Thr Ala Thr Arg Gly Arg Ser Ala Ala Ser Arg Pro Thr 1
5 10 15 Glu Arg Pro Arg Ala Pro Ala Arg Ser Ala Ser Arg Pro Arg Arg
Pro 20 25 30 Val Glu 6623PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 66Met Gly Leu Gly Leu His Leu
Leu Val Leu Ala Ala Ala Leu Gln Gly 1 5 10 15 Ala Lys Ser Lys Arg
Lys Val 20 6726PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 67Ala Ala Val Ala Leu Leu Pro Ala Val
Leu Leu Ala Leu Leu Ala Pro 1 5 10 15 Ala Ala Ala Asn Tyr Lys Lys
Pro Lys Leu 20 25 6828PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 68Met Ala Asn Leu Gly Tyr Trp
Leu Leu Ala Leu Phe Val Thr Met Trp 1 5 10 15 Thr Asp Val Gly Leu
Cys Lys Lys Arg Pro Lys Pro 20 25 6924PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 69Leu
Gly Thr Tyr Thr Gln Asp Phe Asn Lys Phe His Thr Phe Pro Gln 1 5 10
15 Thr Ala Ile Gly Val Gly Ala Pro 20 7026PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 70Asp
Pro Lys Gly Asp Pro Lys Gly Val Thr Val Thr Val Thr Val Thr 1 5 10
15 Val Thr Gly Lys Gly Asp Pro Xaa Pro Asp 20 25 7114PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 71Pro
Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro Pro 1 5 10
7218PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 72Val Arg Leu Pro Pro Pro Val Arg Leu Pro Pro Pro
Val Arg Leu Pro 1 5 10 15 Pro Pro 7310PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 73Pro
Arg Pro Leu Pro Pro Pro Arg Pro Gly 1 5 10 7430PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
74Ser Val Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro 1
5 10 15 Pro Pro Phe Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro 20
25 30 7521PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 75Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val
Gly Arg Val His 1 5 10 15 Arg Leu Leu Arg Lys 20 7623PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 76Gly
Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe 1 5 10
15 Val Gly Glu Ile Met Asn Ser 20 7737PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
77Lys Trp Lys Leu Phe Lys Lys Ile Glu Lys Val Gly Gln Asn Ile Arg 1
5 10 15 Asp Gly Ile Ile Lys Ala Gly Pro Ala Val Ala Val Val Gly Gln
Ala 20 25 30 Thr Gln Ile Ala Lys 35 7828PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 78Ala
Leu Trp Met Thr Leu Leu Lys Lys Val Leu Lys Ala Ala Ala Lys 1 5 10
15 Ala Ala Leu Asn Ala Val Leu Val Gly Ala Asn Ala 20 25
7926PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 79Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly
Leu Pro Ala Leu 1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln
20 25 8014PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 80Ile Asn Leu Lys Ala Leu Ala Ala Leu Ala Lys Lys
Ile Leu 1 5 10 8133PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 81Gly Phe Phe Ala Leu Ile Pro Lys
Ile Ile Ser Ser Pro Leu Pro Lys 1 5 10 15 Thr Leu Leu Ser Ala Val
Gly Ser Ala Leu Gly Gly Ser Gly Gly Gln 20 25 30 Glu
8215PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 82Leu Ala Lys Trp Ala Leu Lys Gln Gly Phe Ala Lys
Leu Lys Ser 1 5 10 15 8327PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 83Ser Met Ala Gln Asp Ile Ile
Ser Thr Ile Gly Asp Leu Val Lys Trp 1 5 10 15 Ile Ile Gln Thr Val
Asn Xaa Phe Thr Lys Lys 20 25 8441PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 84Leu Leu Gly Asp Phe
Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu 1 5 10 15 Phe Lys Arg
Ile Val Gln Arg Ile Lys Gln Arg Ile Lys Asp Phe Leu 20 25 30 Ala
Asn Leu Val Pro Arg Thr Glu Ser 35 40 8518PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 85Pro
Ala Trp Arg Lys Ala Phe Arg Trp Ala Trp Arg Met Leu Lys Lys 1 5 10
15 Ala Ala 8618PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 86Lys Leu Lys Leu Lys Leu Lys Leu Lys
Leu Lys Leu Lys Leu Lys Leu 1 5 10 15 Lys Leu 8713PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 87Gly
Leu Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser 1 5 10
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