U.S. patent application number 12/180730 was filed with the patent office on 2009-03-19 for antitumor effect of mutant bik.
Invention is credited to Mien-Chie Hung, Yan Li, Yong Wen.
Application Number | 20090075885 12/180730 |
Document ID | / |
Family ID | 33159707 |
Filed Date | 2009-03-19 |
United States Patent
Application |
20090075885 |
Kind Code |
A1 |
Hung; Mien-Chie ; et
al. |
March 19, 2009 |
ANTITUMOR EFFECT OF MUTANT BIK
Abstract
The present invention regards mutant forms of Bik that comprise
anti-cell proliferation and/or pro-apoptotic activities. In
particular embodiments, the Bik polypeptides comprise a
substitution at Thr33 and Ser35 and, in some embodiments,
phosphorylation at these sites is inhibited. In more particular
embodiments, these forms are useful for cancer therapy,
particularly when administered in combination with liposomes. In
embodiments wherein a mutant Bik polynucleotide is administered for
cancer therapy, the polynucleotide may be regulated in a
tissue-specific manner.
Inventors: |
Hung; Mien-Chie; (Houston,
TX) ; Li; Yan; (Houston, TX) ; Wen; Yong;
(Burlingame, CA) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI, LLP
1301 MCKINNEY, SUITE 5100
HOUSTON
TX
77010-3095
US
|
Family ID: |
33159707 |
Appl. No.: |
12/180730 |
Filed: |
July 28, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10816698 |
Apr 2, 2004 |
|
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12180730 |
|
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60459901 |
Apr 2, 2003 |
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Current U.S.
Class: |
514/1.1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61P 35/02 20180101; C07K 14/4747 20130101; A61P 31/00 20180101;
A61K 38/1709 20130101 |
Class at
Publication: |
514/12 |
International
Class: |
A61K 38/00 20060101
A61K038/00; A61P 31/00 20060101 A61P031/00 |
Claims
1.-75. (canceled)
76. A method of inducing anti-tumor activity, anti-cell
proliferation activity, and/or pro-apoptotic activity in a subject,
comprising administering to the subject an effective amount of a
mutant Bik polypeptide having an altered amino acid sequence, said
polypeptide comprising: a) SEQ ID NO:7, SEQ ID NO:8, or SEQ ID
NO:9; or b) polypeptides having sequence that is between 91% and
99% identical to SEQ ID NO:7, SEQ ID NO:8, or SEQ ID NO:9, said
polypeptides having a mutation at Thr.sup.33, Ser.sup.35, or both
Thr.sup.33 and Ser.sup.35.
77. The method of claim 76, wherein the polypeptide further
comprises a protein transduction domain.
78. The method of claim 76, wherein the subject is a human.
79. The method of claim 78, wherein the human has a proliferative
cell disorder.
80. The method of claim 79, wherein the proliferative cell disorder
is cancer.
81. The method of claim 80, wherein the cancer is breast cancer,
prostate cancer, ovarian cancer, sarcoma, lung cancer, brain
cancer, pancreatic cancer, liver cancer, bladder cancer,
gastrointestinal cancer, leukemia, lymphoma, or myeloma.
82. The method of claim 80, wherein the cancer is estrogen receptor
positive, is EGF receptor overexpressing, is
Her2/neu-overexpressing, is not Her-2/neu-overexpressing, is Akt
overexpressing, is angrogen independent, or is androgen
dependent.
83. The method of claim 80, wherein the cancer is a solid tumors,
such as, for example, sarcoma, lung, brain, pancreatic, liver,
bladder, gastrointestinal cancers, or hematologic malignancies,
such as leukemia, lymphoma, and myeloma
84. The method of claim 79, wherein the proliferative cell disorder
is restenosis.
85. The method of claim 76, wherein the polypeptide is comprised in
pharmacologically acceptable excipient.
86. The method of claim 76, wherein the polypeptide is complexed
with a lipid.
87. The method of claim 76, further defined as a method of
preventing growth of a cell in an individual.
Description
[0001] This application claims priority to U.S. patent application
Ser. No. 10/816,698, filed Apr. 4, 2004, which claims priority to
U.S. Provisional Patent Application No. 60/459,901, filed Apr. 2,
2003, all of which are incorporated by reference herein in their
entirety.
FIELD OF THE INVENTION
[0002] The present invention is directed to the fields of cell
biology, molecular biology, cancer biology, and medicine. More
particularly, the present invention regards variant forms of Bik
comprising anti-cell proliferative and/or pro-apoptotic activities
that are useful for cancer therapy.
BACKGROUND OF THE INVENTION
[0003] Bik, also known as nbk, is one of the pro-apoptotic BH3-only
proteins, which have only one of the Bcl-2 homology regions, BH3
domains, and have recently been recognized as essential initiators
of apoptosis (Han et al., 1996; Boyd et al., 1995). Loss of
informative alleles on chromosome 22q where the Bik gene is located
may be related to the development of human breast and colorectal
cancers (Daniel et al., 1999). The 18-kDa Bik protein interacts
with E1B 19K and forms heterodimers with various anti-apoptotic
proteins, e.g. Bcl-2 and Bcl-XL, the association of which hinders
the function of the anti-apoptotic protein (Han et al., 1996).
Bik-mediated apoptosis requires BAX (Theodorakis et al., 2002) and
is independent of p53 (Han et al., 1996). Bik is also a downstream
apoptotic effector of p53 (Bartke et al., 2001) in response to a
physiological p53-mediated death stimulus provided by E1A. Elevated
Bcl-2 functioned downstream of p53 and Bik induction to inhibit the
E1A death pathway, with the ratio of anti-apoptotic Bcl-2 and
pro-apoptotic Bik determining cell death or survival in
E1A-expressing cells (Mathai et al., 2002). Moreover, Bik can
sensitize tumor cells to certain chemotherapeutic agents (Daniel et
al., 1999) and one of the chemotherapy drugs, doxorubicin, that
induces apoptosis is mediated by Bik gene (Panaretakis et al.,
2002). All of these suggest that Bik is a useful therapeutic gene
to target human cancer.
[0004] BH3-only proteins differ in their expression pattern and
mode of activation, and many BH3-only proteins activation needs
posttranslational modification (Puthalakath and Strasser, 2002).
Bik is one of the BH3-only proteins whose activity can be regulated
by phosphorylation (Verma et al., 2000). Verma et al. demonstrated
that Bik exists as a phosphoprotein and is phosphorylated at
residues threonine 33 and serine 35, which they determined is
required for the full apoptotic activity of Bik, possibly by a
casein kinase II-related enzyme. That is, Verma et al. mutated the
phosphorylation sites at the threonine and serine residues to
alanine residues, which reduced the apoptotic activity of Bik. They
concluded phosphorylation is required for the pro-apoptotic potency
of Bik by a presently unknown mechanism without significantly
affecting its affinity for Bcl-2.
BRIEF SUMMARY OF THE INVENTION
[0005] The present invention provides novel therapeutic Bik mutant
compositions and methods, particularly for cancer, and a skilled
artisan recognizes that any additional means in an arsenal to fight
cancer is beneficial to public health.
[0006] The present invention is directed to a system and method
related to mutants of Bik, and particularly to their anti-tumor
effects. Bik is a proapoptotic member of the Bcl-2 family of
proteins. In the present invention, the inventors demonstrate the
novel finding that non-wildtype forms of Bik, such as mutant forms,
exerted strong antitumor activity in both in vivo and in vitro
systems. In contrast to the teachings of Verma et al. (2000), the
exemplary mutant Bik polypeptides described herein similarly
abolish phosphorylation sites in Bik yet still comprise potent
anti-tumor, anti-cell proliferation, and/or pro-apoptotic activity
and, in some embodiments, are more potent than wild-type Bik. That
is, the Examples presented herein indicate that the transfection
with a polynucleotide encoding a mutant bik polypeptide induces
apoptosis in various human cancers. This provides compositions,
such as therapeutics, and methods of using same for the present
invention, such as the mutant bik polynucleotide in gene therapy
for cancer, such as ovarian, breast, pancreatic, and prostate
cancer.
[0007] Thus, the present invention generally relates to methods for
inhibiting proliferation in a cancer cell and/or tumor cell, the
method comprising contacting the cell with a mutant Bik polypeptide
in an amount effective to inhibit proliferation. The mutant Bik
polypeptide referred to herein is a mutant form having anti-cell
proliferative, pro-apoptotic, and/or anti-tumor activity.
Inhibition of proliferation may be indicated by, for example, an
induction of apoptosis of a cell, such as, for example, in cell
culture, inhibition of growth of a cancer cell line, reduction in
size of a tumor, and/or an increase in survivability, in exemplary
embodiments. More preferably, in some embodiments the cell in which
proliferation is to be inhibited is a cell in a living organism,
for example a human. The inhibition of such transformation has
great utility in the prevention and/or treatment of such
transformation-driven events as cancer, tumorigenesis, and/or
metastasis.
[0008] A mutant Bik polypeptide may be contacted with or introduced
to a cell through any of a variety of manners known to those of
skill. The mutant Bik polypeptide may be introduced through direct
introduction of a mutant Bik polypeptide to a cell. In this case,
the mutant Bik polypeptide may be obtained through any method known
in the art, although it is expected that in vitro production of the
mutant Bik polypeptide in a cell, for example in a cell culture
system, may be a preferred manner of obtaining mutant Bik.
[0009] Mutant Bik may also be introduced to a cell via the
introduction of a polynucleotide that encodes the mutant Bik
polypeptide to the cell. For example, RNA or DNA encoding Bik may
be introduced to the cell by any manner known in the art. In
certain preferred embodiments, the mutant Bik is introduced into
the cell through the introduction of a DNA segment that encodes
mutant Bik. In some such embodiments, it is envisioned that the DNA
segment further comprises the mutant Bik gene (or mutant Bik
polynucleotide) operatively linked to its associated control
sequences. For example, the bik gene may be operatively linked to a
suitable promoter and a suitable terminator sequence. The
construction of such gene/control sequence DNA constructs is
well-known within the art. In particular embodiments, the promoter
is selected from the group comprising of CMV, telomerase, TCF-4, or
VEGF. However, in particular embodiments, the constructs comprise
promoters that are tissue-specific, such as, for example,
tissue-specific for cancers including the exemplary breast,
prostate, or pancreatic cancers.
[0010] In certain embodiments for introduction, the DNA segment may
be located on a vector, for example, a plasmid vector or a viral
vector. The virus vector may be, for example, selected from the
group comprising retrovirus, adenovirus, herpesvirus, vaccina
virus, and adeno-associated virus. Such a DNA segment may be used
in a variety of methods related to the invention. The vector may be
used to deliver a mutant bik gene to a cell in one of the
gene-therapy embodiments of the invention. Also, such vectors can
be used to transform cultured cells, and such cultured cells could
be used, inter alia, for the expression of mutant Bik in vitro.
[0011] The present invention is useful for all types of cancer,
since mutant Bik, as shown herein in exemplary embodiments, kills
cancer cells regardless of their survival tactics adopted by many
cancer cells, such as growth factor receptor and AKT pathways. In a
particular embodiment, mutant Bik is effective on solid tumors,
such as, for example, sarcoma, lung, brain, prostate, breast,
ovarian, pancreatic, liver, bladder, gastrointestinal cancers, and
hematologic malignancies, such as leukemia, lymphoma, and myeloma.
In exemplary embodiments, the present invention is useful for
cancers that are estrogen receptor positive, EGF receptor
overexpressing, Her2/neu-overexpressing,
Her-2/neu-nonoverexpressing, Akt overexpressing, androgen
dependent, or angrogen independent. That is, mutant Bik is
effective on cancer cells regardless of their status of oncogene
overexpression, such as Her-2/neu, EGFR, AKT, or whether their
growth is hormone dependent (such as, for example, MCF-7) or not
(such as, for example, PC3).
[0012] For example, contained herein are specific data showing
effectiveness of mutant Bik against cell lines tested from at least
six exemplary types of cancers or angiogenic cells, including: 1)
breast cancer, such as with MCF-7 (estrogen receptor positive),
MDA-MB-468 (EGF receptor overexpressing), and MDA-MB-231; 2)
endothelial cell, such as with Human Umbilical Vascular Endothelial
Cells (HUVEC) (which shows the anti-angiogenesis effect of mutant
Bik; 3) Head and Neck Cancer, such as with TU138 and TU167; 4)
Melanoma, such as with B16F10; 5) Ovarian cancer, such as with
SKOV-ip1 (Her-2/neu overexpression), SKOV (no Her-2/neu
overexpression), and 2774 (Akt overexpression); and 6) Prostate
Cancer, such as with PC3 (androgen independent growth).
[0013] In particular embodiments, mutant Bik is introduced into a
cell that is a human cell. In many embodiments the cell is a tumor
cell. In some presently preferred embodiments the tumor cell is a
breast tumor cell, a prostrate tumor cell, an ovarian tumor cell,
or a pancreatic tumor cell. However, mutant Bik may be introduced
into other cells including, but not limited to, a bladder cancer
cell, a testicular cancer cell, a colon cancer cell, a skin cancer
cell, a lung cancer cell, a stomach cancer cell, an esophageal
cancer cell, a brain cancer cell, a leukemia cancer cell, a liver
cancer cell, an endometrial cancer cell, or a head and neck cancer
cell. In some embodiments, the mutant Bik composition is introduced
by injection. In particular embodiments, the mutant Bik composition
is comprised in a liposome.
[0014] In some embodiments of the present invention, the inventors'
discovery that Bik mutants are able to inhibit proliferation will
be used in combination with other anti-transformation/anti-cancer
therapies. These other therapies may be known at the time of this
application, or may become apparent after the date of this
application. Bik mutants may be used in combination with other
therapeutic polypeptides, polynucleotides encoding other
therapeutic polypeptides, chemotherapeutic agents, surgical
methods, or radiation, for example. For example, mutant Bik may be
used in conjunction with other known polypeptides, such as
TNF.alpha. or p53. Other polypeptide inducers of apoptosis that may
be used in combination with Bik include, but are not limited to,
p53, Bax, Bak, Bcl-x, Bad, Bim, Bok, Bid, Harakiri, Ad E1B, Bad and
ICE-CED3 proteases.
[0015] Mutant Bik may be used in conjunction with any suitable
chemotherapeutic agent. In one representative embodiment, the
chemotherapeutic agent is taxol. Mutant Bik also may be used in
conjunction with radiotherapy. The type of ionizing radiation
constituting the radiotherapy may be selected from the group
comprising x-rays, .gamma.-rays, and microwaves. In certain
embodiments, the ionizing radiation may be delivered by external
beam irradiation or by administration of a radionuclide. Mutant Bik
also may be used with other gene-therapy regimes. In particular
embodiments the mutant Bik is introduced into a tumor. The tumor
may be in an animal, in particular, a human.
[0016] The Bik mutant gene products and polynucleotides of the
present invention may also be introduced using any suitable method.
A "suitable method" of introduction is one that places a mutant bik
gene product in a position to reduce the proliferation of a tumor
cell. For example, injection, oral, and inhalation methods may be
employed, with the skilled artisan being able to determine an
appropriate method of introduction for a given circumstance. In the
embodiments where injection will be used, this injection may be
intravenous, intraperitoneal, intramuscular, subcutaneous,
intratumoral, intrapleural, or of any other appropriate form.
[0017] In certain other aspects of the present invention there are
provided therapeutic kits comprising in a suitable container a
pharmaceutical formulation of a mutant Bik gene product or a
polynucleotide encoding a mutant Bik gene product. Such a kit may
further comprise a pharmaceutical formulation of a therapeutic
polypeptide, polynucleotide encoding a therapeutic polypeptide,
and/or chemotherapeutic agent.
[0018] The term "mutant Bik" as used herein refers to a Bik
polynucleotide or polypeptide from any organism that comprises
such, so long as the mutant Bik comprises anti-tumor activity,
anti-cell proliferation activity, and/or pro-apoptotic activity,
wherein the mutant Bik comprises at least one altered amino acid
compared to native Bik, or the corresponding polynucleotide
encoding same. The alteration may comprise a modified amino acid,
such as one comprising an additional acetyl group, for example, or
the alteration may comprise at least one substituted amino acid on
at least one particular amino acid position. In particular
embodiments, the mutant Bik will comprise at least one alteration
for at least one amino acid, yet retain anti-cell proliferation
activity, anti-tumor activity, pro-apoptotic activity, and/or a
combination thereof that is useful for the purposes described
herein. In some cases, the mutant Bik will exhibit better anti-cell
proliferation activity, anti-tumor activity, pro-apoptotic
activity, and/or a combination thereof than native Bik. In other
circumstances, the mutant Bik may have similar anti-cell
proliferation activity, anti-tumor activity, pro-apoptotic
activity, and/or a combination thereof to native Bik. In other
cases, the mutant Bik may have reduced anti-cell proliferation
activity, anti-tumor activity, pro-apoptotic activity, and/or a
combination thereof relative to native Bik. Of course, most mutants
that comprise one or more relevant activities that are
substantially less than the same one or more relevant activities in
native Bik or other mutant Biks will likely not be useful in all
embodiments of the invention. However, those of skill in the art
will, in view of the teachings of the specification and the
knowledge of skill in the art, be able to select such mutants that
will have utility in certain specific embodiments of the
invention.
[0019] The anti-tumor activity, anti-cell proliferation activity,
and/or pro-apoptotic activity may be useful for an organism other
than the one from which the mutant Bik is derived. For example, the
human Bik is utilized herein in exemplary embodiments, although a
murine Bik may be used alternatively or in addition for human
treatment. The mutant Bik from any organism may be altered at any
amino acid. Furthermore, the human Bik may be mutated at a
particular residue(s) and found useful for therapy, and the mutant
murine Bik with its analogous residue(s) substitution may also be
effective. In a particular embodiment, the murine Bik polypeptide
comprises at least one altered phosphorylation site, and based on
the teachings provided herein related to human Bik, one of skill
would know how to generate analogous changes in murine Bik. In a
further particular embodiment, serine 27, threonine 29, serine 31,
and/or a combination thereof are substituted in mutant murine
Bik.
[0020] Of course, Bik may be mutated for any number of reasons, and
one of skill in the art is aware that there may be desirable
mutations generated in the Bik polypeptide or a nucleic acid
encoding same that are for purposes other than removing
phosphorylation sites and/or for effecting or retaining anti-tumor
activity, anti-cell proliferation activity, and/or pro-apoptotic
activity. For example, mutations may be made to render the Bik
polynucleotide and/or polypeptide more amenable for a therapeutic
purpose. For example, modifications may be made that reduce
antigenicity of the polypeptide, that remove regions of the
polypeptide, that enhance nuclear localization of the polypeptide,
that increase the half-life of the polypeptide, and so forth.
[0021] At least one assay for determining effectiveness of a
particular mutant in comparison to wild type is described herein at
least in the Examples, and others in the art may be utilized. In
particular embodiments, mutants that comprise at least one activity
that is substantially less than native Bik are not desirable and
are not in the scope of the present invention.
[0022] Specifically, the present invention is directed to methods
and compositions regarding particular mutant forms of Bik that are
associated with control of cell growth, survival or proliferation.
In specific embodiments, the control of cell growth is useful in
the treatment of cancer or restenosis. Specifically, the present
invention teaches a skilled artisan that mutant Bik polypeptides
that have one or more amino acid substitutions that result in the
polypeptide either not being phosphorylated or being phosphorylated
at a lower level are useful for anti-tumor applications. In
specific embodiments, the inventors have shown that mutant Bik can
reduce or abolish phosphorylation and lead to suppression of growth
of transformed cells treated with such mutant Bik. In particular
embodiments, substitution with Asp or Glu, for example, results in
failure of the polypeptide to be phosphorylated at those sites and
leads to suppression of growth of transformed cells upon treatment
with such Bik mutants. Of course, the invention is not limited to
embodiments in which the amino acid substitution is either Asp or
Glu. Rather, any amino acid substitution that prevents
phosphorylation of Bik by any amino acid is contemplated within the
scope of the invention. In a specific embodiment, the amino acid
for substitution at Thr33 and/or Ser35 is not alanine. In another
specific embodiment, the at least one amino acid substitution has
at least one acidic property, such as, for example, the amino acids
aspartate or glutamate. Therefore, an object of the present
invention is directed to at least one modification in Bik
polypeptide that results in failure of the polypeptide to be
phosphorylated, and preferably for that resultant Bik mutant to
comprise anti-cell proliferation capability, anti-tumor capability,
pro-apoptotic capability, or a combination thereof.
[0023] One of skill in the art recognizes that mutations, either to
similar amino acids or not, may be made elsewhere in Bik, and that
some of these mutants will have the same activities as the
exemplary embodiments provided herein. For example, threonine,
serine, or other appropriate amino acids anywhere within Bik can be
substituted, such as with the exemplary aspartate or glutamate. A
skilled artisan is aware of publicly available databases that
provide Bik sequences for making alterations different from native
Bik, such as the National Center for Biotechnology Information's
GenBank database or commercially available databases such as from
Celera Genomics, Inc. (Rockville, Md.). Exemplary polypeptide Bik
sequences include (as identified by their GenBank Accession
numbers): SEQ ID NO:3 (AAC50413; NP.sub.--001188; AAF01156;
AAC79124; CAA62013; and S58214); and SEQ ID NO:4 (AAC40079 and
NP.sub.--031572). Exemplary polynucleotide Bik sequences include
(as identified by their GenBank Accession numbers): SEQ ID NO:5
(AY245248) and SEQ ID NO:6 (NM.sub.--001197).
[0024] Thus, the present invention provides guidance regarding
different mutations in Bik, and, therefore, the present invention
is directed to a novel improvement to the overall arts of cell
growth control, including inhibition of cell proliferation and/or
facilitation of cell death. In a specific embodiment, the
inhibition of a cell proliferation comprises a delay in its rate of
proliferation, a delay in its total cell numbers of proliferation,
or both.
[0025] A skilled artisan recognizes that any site in the Bik
polypeptide may be modified to generate such compositions as
described, and furthermore that multiple sites may be modified. A
skilled artisan is cognizant that a limited number of sites for
modification exist in the approximately 160 amino acid Bik
polypeptide (depending on the organism). In addition, a skilled
artisan recognizes that there are only twenty standard amino acids
from which to modify to, and guidance is provided herein directed
to methods to generate those modifications. Furthermore, a skilled
artisan in the teachings of the present invention knows how to test
for anti-tumor, anti-cell growth, and/or pro apoptotic effects,
based on the teachings provided herein and other methods in the
art, and therefore assaying a particular modification would not
subject one skilled in the art to undue experimentation.
[0026] Thus, based on the guidance provided herein, the present
invention is directed to mutant polypeptides of Bik that result in
inhibition of proliferation of a cell or enhancement of cell
survival. In specific embodiments, the present invention is
directed to mutants including, for example, Bik T33D, S35D, and
double mutant T33DS35D.
[0027] In accordance with the objects of the present invention,
there is as a composition of matter a mutant Bik polypeptide. For
example, the composition comprises an amino acid substitution. In a
specific embodiment, the substitution prevents phosphorylation of
the Bik polypeptide under conditions that would result in
phosphorylation of an unsubstituted Bik polypeptide. In other
specific embodiments, the substitution is a Thr33 to Asp33
substitution or a Ser35 to Asp35 substitution, or both. In other
specific embodiments, the compositions are further defined as
compositions in a pharmacologically acceptable excipient in which
the Bik polypeptide is dispersed. In additional specific
embodiments, the compositions are confined in a suitable container
in a kit.
[0028] In an additional object of the present invention, there is a
method of preventing growth of a cell in an individual comprising
the step of administering to the individual a mutant Bik
polypeptide. In another specific embodiment, the administration of
the polypeptide is by a liposome. In an additional specific
embodiment, the polypeptide further comprises a protein
transduction domain, such as HIV Tat or penetratin, for
example.
[0029] In another object of the present invention, there is a
method of preventing growth of a cell in an individual comprising
the step of administering to the individual a nucleic acid encoding
a mutant Bik polypeptide. In another specific embodiment, the
administration of the nucleic acid is by a vector selected from the
group consisting of a plasmid, a retroviral vector, an adenoviral
vector, an adeno-associated viral vector, a liposome, and a
combination thereof.
[0030] In an additional object of the present invention, there is a
method of using a mutant Bik polypeptide composition wherein the
Bik polypeptide composition is dispersed in a pharmacologically
acceptable excipient, and wherein the composition is administered
to an animal having a proliferative cell disorder.
[0031] In another object of the present invention, there is a
method of treating a proliferative cell disorder in an individual
comprising the step of administering to the individual a mutant Bik
polypeptide. In another specific embodiment, the proliferative cell
disorder is cancer. In a further specific embodiment, the
proliferative cell disorder is restenosis. In a further specific
embodiment, the cancer is breast cancer, prostate cancer, ovarian
cancer, or pancreatic cancer.
[0032] In an additional object of the present invention, there is a
method of treating a cell comprising contacting the cell with a
mutant Bik polypeptide. In a specific embodiment, the cell is a
human cell. In another specific embodiment, the cell is comprised
in an animal. In a further specific embodiment, the animal is a
human. In a further specific embodiment, the human has a
proliferative cell disorder. In an additional specific embodiment,
the proliferative cell disorder is cancer. In a further specific
embodiment, the cancer is breast cancer, ovarian cancer, or
prostate cancer. In another specific embodiment, the proliferative
cell disorder is restenosis.
[0033] In a further object of the invention, a polynucleotide
encoding a mutant Bik polypeptide is regulated by a tissue-specific
promoter, such as one that targets cancerous tissue. Although any
promoter that targets cancerous tissue preferentially over
non-cancerous tissue, in a specific embodiment the cancer-specific
promoter is a breast cancer specific promoter, a prostate
cancer-specific promoter, a or pancreatic-specific promoter, for
example.
[0034] In a particular embodiment, a breast cancer-specific
promoter comprises a breast cancer-specific sequence and, in
further embodiments, an enhancer sequence that augments expression,
such as the expression level, of the tissue-specific sequence. In a
particular embodiment, a CMV promoter enhancer sequence is linked
with a breast cancer-specific segment from the exemplary
topoisomerase II.alpha. (topII.quadrature.) promoter or the
exemplary transferrin receptor promoter. They are useful for gene
targeting to target and treat primary and metastatic breast cancers
with less toxicity to normal tissues.
[0035] In another embodiment, the expression of a polynucleotide
encoding a mutant Bik polypeptide is regulated by a
pancreatic-cancer specific promoter. In a particular embodiment, a
novel pancreatic cancer specific promoter is utilized, such as one
referred to herein as CTP, which is comprised of at least the
minimal Cholecystokinin A receptor (CCKAR, -726 to +1 nucleotides),
a two-step transcriptional system sequence and the post
translational regulatory element of the woodchuck hepatitis virus
(WPRE). This engineered construct has a strong promoter activity
and demonstrates specificity to pancreatic cancer cells in vitro
and in vivo for the expression of mutant Bik.
[0036] In another specific embodiment of the present invention, a
prostate cancer-specific promoter regulates expression of a
polynucleotide that encodes a mutant Bik polypeptide. In a
particular embodiment, the invention utilizes a novel prostate
cancer specific promoter, such as one referred to herein as ATTP,
comprised of at least the minimal human telomerase reverse
transcriptase promoter (hTert), the post translational regulatory
element of the woodchuck hepatitis virus (WPRE), and an ARR2
control sequence, which is responsive to androgen stimulation. This
engineered construct has a strong promoter activity and
demonstrates specificity to both androgen-dependent and
androgen-independent prostate cancer cells in vitro. This promoter
can be used to specifically drive gene expression of mutant Bik in
prostate cancer in vivo.
[0037] The foregoing has outlined rather broadly the features and
technical advantages of the present invention in order that the
detailed description of the invention that follows may be better
understood. Additional features and advantages of the invention
will be described hereinafter which form the subject of the claims
of the invention. It should be appreciated by those skilled in the
art that the conception and specific embodiment disclosed may be
readily utilized as a basis for modifying or designing other
structures for carrying out the same purposes of the present
invention. It should also be realized by those skilled in the art
that such equivalent constructions do not depart from the spirit
and scope of the invention as set forth in the appended claims. The
novel features which are believed to be characteristic of the
invention, both as to its organization and method of operation,
together with further objects and advantages will be better
understood from the following description when considered in
connection with the accompanying figures. It is to be expressly
understood, however, that each of the figures is provided for the
purpose of illustration and description only and is not intended as
a definition of the limits of the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] For a more complete understanding of the present invention,
reference is now made to the following descriptions taken in
conjunction with the accompanying drawings.
[0039] FIGS. 1A through 1F illustrate that the expression of Bik
mutants exhibited stronger growth inhibitory effect in different
human cancer cell lines.
[0040] FIG. 2 shows that the Bik mutants exhibited stronger
cell-killing effect in different human cancer cell lines.
[0041] FIG. 3 illustrates that the Bik mutants exhibit strong
cell-killing in additional human cancer cell lines.
[0042] FIGS. 4A through 4B illustrate that the Bik mutants
exhibited stronger tumor suppression effect in ex vivo assay.
[0043] FIGS. 5A through 5B demonstrate that the mutant Bik gene
delivered by SN significantly inhibited growth of human tumors in
mice.
[0044] FIGS. 6A and 6B show that an exemplary Bik mutant (BikDD)
polynucleotide demonstrated a significant suppression of tumor
growth and an increase in survival in a breast cancer orthotopic
model. In FIG. 6A, tumor volume was measured and recorded weekly.
FIG. 6B shows that BikDD increased the survival rate of mice
bearing MDA-MB-231 orthotopic tumors.
[0045] FIGS. 7A and 7B show that the Bik mutant (BikDD) gene
demonstrated a significant suppression of tumor growth and an
increase in survival in a breast cancer orthotopic model.
[0046] FIG. 7A shows tumor volume that was measured and recorded
weekly. FIG. 2B shows that BikDD increased the survival rate of
mice bearing MDA-MB-231 orthotopic tumors.
[0047] FIG. 8 shows that treatment with the Bik mutant (BikDD) gene
increases the survival of mice in a ovarian cancer orthotopic
model.
[0048] FIG. 9 demonstrates that treatment with the Bik mutant
(BikDD) gene increases the survival of mice in a pancreatic cancer
orthotopic model.
[0049] FIG. 10 shows in vitro killing assay of CT90BikDD and
CMV-BikDD in different cell lines. The Y-axis value indicates the
percentage of vital cells after treatment.
[0050] FIG. 11 shows in vivo anti-tumor effect of CT90-BikDD gene
therapy. Breast cancer cell line MDA-MB-231 was inoculated
2.5.times.106 per mouse and mice were treated once per week by
liposome-complexed CT0-BikDD, CMV-BikDD, empty vector pGL3, and
dextrose buffer D5W as no-treatment control. Tumor size (Y-axis
value) was measured twice per week during treatment and showed in
the figure. The X-axis indicates the treatment dates.
[0051] FIGS. 12A-12E show therapeutic effects of CT90-BikDD breast
cancer targeting gene therapy in an exemplary orthotopic mouse
model. FIG. 12A demonstrates that liposome-complexed CT90-BikDD
targets breast cancer cells in orthotopic mouse model. In FIGS. 12B
and 12D, tumor size record during gene therapy treatment is
demonstrated wherein the mice were treated once a week (QW, FIG.
12B) or twice a week (BIW, FIG. 12D).
[0052] FIG. 13 illustrates gene therapy of Bik-DD in MDA-MB-468
xenograft mice.
DETAILED DESCRIPTION OF THE INVENTION
[0053] As used herein the specification, "a" or "an" may mean one
or more. As used herein in the claim(s), when used in conjunction
with the word "comprising", the words "a" or "an" may mean one or
more than one. As used herein "another" may mean at least a second
or more.
[0054] The present invention regards mutant Bik polypeptides and
the nucleic acids that encode them, as well as methods regarding
the use of mutant Bik. Thus, in exemplary embodiments, the present
inventors changed the residues threonine 33 and serine 35 to
aspartate amino acids as potentially constitutive active forms, and
these Bik mutants were demonstrated as therapeutic agents in cancer
gene therapy. Although the inventors previously demonstrated that
systemically administrated nonviral gene delivery system (SN)-bik
significantly inhibited the growth and metastasis of human breast
cancer cells implanted in nude mice and prolonged the life span of
the treated animals (Zou et al., 2002), as described herein Bik
mutants could also enhance anti-tumor function of Bik gene product
in in vitro and in vivo models, compared to wild type Bik.
[0055] The present invention regards variant forms of the
pro-apoptotic bik polynucleotide as a tumor suppressor gene to
treat human ovarian cancer, pancreatic cancer, breast cancer,
prostate cancer, and other cancers. In some embodiments it is
delivered by, for example, either a viral or non-viral delivery
system into an appropriate recipient animal to suppress tumor
growth and development. In one exemplary embodiment of the present
invention, the delivered Bik mutants act through an apoptosis
mechanism to suppress tumor growth and development. Thus, herein
the inventors demonstrate that the bik variants exerted strong
anti-tumor activity and behaved like a classic tumor
suppressor.
[0056] Bik was initially identified as a BH3-domain-only protein
that interacts with E1B 19K and Bcl-2. Systemically administrated
Bik significantly inhibited the tumor growth and metastasis in
human breast cancer nude mice model. Recently, it has been reported
that post-translational phosphorylation can regulate the
pro-apoptotic potency of Bik. Here, the present inventors
demonstrated that Bik mutants were more potent than wild type (wt)
Bik to inhibit cell proliferation and enhance apoptosis induction
in various human cancer cells. They also demonstrated that the Bik
mutants suppressed the tumorigenicity and tumor taking rate in mice
ex vivo model. Finally, the inventors demonstrated the Bik
mutant-SN liposome inhibited the tumor growth and prolonged the
life span in mice in vivo model. Thus, the present invention
provides mutant Bik gene product, and polynucleotides that encode
same, are more potent than wt Bik to induce cell death.
[0057] Exemplary mutants of Bik, Bik-T33D (threonine 33 to
aspartate), Bik-S35D (serine 35 to aspartate) and Bik-T33DS35D
(threonine 33 and serine 35 to aspartate), were generated. These
mutants, in preferred embodiments of the present invention,
selectively inhibit cancer cell growth and also preferably are more
potent anti-cancer agents than the wild-type Bik. In other
embodiments the mutant Biks inhibit other cell proliferation and is
useful for the treatment of restenosis or to inhibit angiogenesis.
One skilled in the art following the teachings of this
specification can generate other exemplary mutants of Bik
polypeptide.
[0058] A skilled artisan recognizes that a multitude of Bik nucleic
acid and polypeptide sequences may be used in the present
invention. A skilled artisan is aware of publicly available
databases that provide these sequences, such as the National Center
for Biotechnology Information's GenBank database or commercially
available databases such as from Celera Genomics, Inc. (Rockville,
Md.). Exemplary polypeptide sequences include (as identified by
their GenBank Accession numbers): SEQ ID NO:3 (AAC50413;
NP.sub.--001188; AAF01156; AAC79124; CAA62013; and S58214) and SEQ
ID NO:4 (AAC40079). Exemplary polynucleotide sequences include (as
identified by their GenBank Accession numbers): SEQ ID NO:5
(AY245248) and SEQ ID NO:6 (NM.sub.--001197).
[0059] In specific embodiments, the Bik polypeptide comprises at
least one of the following domains: BH3 domain (for example,
ALRLACIGDEMD; SEQ ID NO:10); at least one E1B 19K-interacting
domain (for example, LRLACIGDEMDV; SEQ ID NO:11); at least one
Bcl-2-interacting domain (for example, ALALRLACIGDEMDVSLR; SEQ ID
NO:12); and/or at least one heterodimerization domain (for example,
LALRLACIGDEMDVSLRA; SEQ ID NO:13), such as with various
anti-apoptotic proteins, e.g. Bcl-2 and Bcl-XL; and/or a
transmembrane domain (for example, EQVLLALLLLLALLLPLLSGGLHLLLK; SEQ
ID NO:14), and at least some of the domains may overlap. In
particular embodiments, the Bik polypeptide comprises no
substantially functional phosphorylation sites at particular amino
acids, although in alternative embodiments the Bik may comprise one
or more functional phosphorylation sites. As used herein the term
"no substantially functional phosphorylation sites" refers to the
majority of Bik molecules lacking phosphorylation ability at one or
more particular amino acids capable of being phosphorylated. In
specific embodiments, the phosphorylation sites in question include
Thr33 and Ser35. A skilled artisan recognizes how to assay for
phosphorylation capability, such as providing antibodies specific
for the phosphorylated species or for the non-phosphorylated
species to the Bik form in question. A skilled artisan may also use
two-dimensional gel electrophoresis or mass spectrum, for example,
to identify the phosphorylation of these residues, in some
embodiments.
[0060] A skilled artisan recognizes that the mutants of Bik, such
as the exemplary BikT33D (threonine 33 to aspartate) (SEQ ID NO:7);
BikS35D (serine 35 to aspartate) (SEQ ID NO:8); and Bik T33DS35D
(both threonine 33 and serine 35 to aspartate) (SEQ ID NO:9), may
be generated by a variety of means. In a specific embodiment, a
nucleic acid sequence as set forth in, for example, SEQ ID NO:5 or
SEQ ID NO:6, is mutated at least at the codon that encodes a
particular amino acid desired to be altered, such as the threonine
at residue 33 to encode an aspartic acid, and so forth. Table 1
presents codons for all standard amino acids, and a skilled artisan
would be well aware how to manipulate a starting nucleic acid to
generate a desired mutation using standard site-directed
mutagenesis techniques, for example.
[0061] In an embodiment of the present invention, the Bik wild type
gene product is phosphorylated under native conditions. In
particular, the inventors have shown herein that mutant Bik, such
as one or more comprising a mutation that encodes the T33 residue
and/or the S35 residue, in spite of an inability to be
phosphorylated, renders the mutant Bik gene product also useful for
inhibition of cell growth and/or useful for pro-apoptotic activity.
A skilled artisan recognizes that the T33 and/or S35 residues may
be altered to prohibit phosphorylation. For example, the T33 amino
acid residue may be changed by altering the nucleic acid codon that
encodes it, such as by site-directed mutagenesis. Alternatively,
the T33 and/or S35 amino acid(s) may be blocked with at least one
compound that prevents phosphorylation, for example with blocking
agents such as carbodiamide and/or by acetylation of the residue
with acetylchloride in trifluoroacetic acid.
[0062] A skilled artisan recognizes that the substitution at Thr33
and/or Ser35 may prevent phosphorylation of the Bik polypeptide
under conditions that would result in phosphorylation of an
unsubstituted Bik polypeptide, and furthermore would know methods
standard in the art to determine these conditions.
[0063] In one aspect of the invention, mutant Bik polypeptide
having at least one defective phosphorylation site is administered
as a polynucleotide that is regulated in a tissue-specific manner.
In particular, mutant Bik may be targeted for expression in breast
cancer, pancreatic cancer, or prostate cancer, for example. In
certain aspects of the invention, a breast cancer-specific promoter
controls expression of mutant Bik. Although any breast
cancer-specific control sequence is contemplated by the present
inventors, in a particular embodiment mutant Bik expression is
controlled by a composite (chimeric) promoter. For example, breast
cancer specific promoters comprised of a CMV promoter enhancer
sequence linked with breast cancer specific segments in either
topoisomerase II.alpha. promoter (named as CT90) or transferring
receptor promoter (named as CTR116) may be utilized. Both of these
chimeric promoters drive gene expression selectively in breast
cancer cells and possess activity levels comparable to the CMV
promoter. The mutant Bik constructs employing the CT90 or CTR116
chimeric promoters are used in gene transfer to target and treat
primary and metastatic breast cancers with less toxicity to normal
tissues, preferably by selectively killing breast cancer cells
and/or significantly reducing breast tumor growth and/or growth
rate.
[0064] In other aspects of the invention, a prostate
cancer-specific or pancreatic cancer-specific promoter controls
expression of mutant Bik. Although any respective prostate
cancer-specific or pancreatic cancer-specific promoter is
contemplated by the present inventors, in a particular embodiment a
composite prostate cancer-specific or pancreatic cancer-specific
promoter, respectively, is utilized. For example, the prostate
cancer-specific promoter may comprise an ARR2 control sequence,
whereas the pancreatic cancer-specific promoter may comprise a
CCKAR control sequence.
[0065] Any promoter or control sequence utilized to regulate
expression of a polynucleotide encoding a mutant Bik polypeptide
may utilize specific regulatory sequences that enhance expression
and/or post-transcriptional processes, for example. Particular but
exemplary sequences include enhancers, two-step transcriptional
amplification system, elements that regulate RNA polyadenylation,
half-life, and so forth, such as the WPRE, and others in the
art.
[0066] In other embodiments of the present invention, there are
methods of preventing growth of a cell in an individual comprising
administering to the individual a mutant Bik polypeptide. In
specific embodiments, the polypeptide is administered in a liposome
and/or the polypeptide further comprises a protein transduction
domain (Schwarze et al., 1999), such as HIV Tat or penetratin. In
alternative embodiments, mutant Bik is administered as a
polynucleotide, wherein the polynucleotide comprises the alteration
that effects modification at the amino acid level, such as is
generated by site-directed mutagenesis, for example. The modified
Bik polynucleotide is administered in a vector such as a plasmid,
retroviral vector, adenoviral vector, adeno-associated viral
vector, liposome, or a combination thereof.
[0067] There are also embodiments of the present invention wherein
there are methods of treating a cell comprising contacting the cell
with a mutant Bik polypeptide. In specific embodiments, the cell is
a human cell, the cell is comprised in an animal, and/or the animal
is human.
[0068] It is contemplated herein that the compositions of the
present invention preferably have an activity similar from a native
Bik polypeptide in the cell, and which may be approximately the
same or more potent against a cancer cell than native Bik. That is,
the scope of the present invention, in some embodiments, is
directed to a change in the native Bik polypeptide for use in a
manner similar to the wildtype Bik polypeptide. In an alternative
embodiment, the mutant Bik forms (e.g. different from the wild type
sequence) comprise an activity different from the native Bik
polypeptide.
I. Definitions and Techniques Affecting Bik Gene Products and
Genes
[0069] A. Bik Gene Products and Genes
[0070] As used herein, the terms "mutant Bik gene product" and
"mutant Bik" refer to proteins having amino acid sequences that are
not identical to the native Bik but that are biologically active in
that they are capable of performing similar activities to native
Bik. For example, they are preferably capable of pro-apoptotic
activity, anti-cell proliferative activity, anti-tumor activity
and/or cross-reactive antibody activity with anti-Bik antibody
raised against Bik. The term "Bik gene product" includes analogs of
Bik molecules that exhibit at least some biological activity in
common with native Bik. Furthermore, those skilled in the art of
mutagenesis will appreciate that other analogs, as yet undisclosed
or undiscovered, may be used to construct Bik analogs.
[0071] The term "mutant form of Bik" refers to any DNA sequence
that is substantially identical to a DNA sequence encoding a Bik
gene product as defined above. The term also refers to RNA or
antisense sequences compatible with such DNA sequences. A "Bik
gene" may also comprise any combination of associated control
sequences.
[0072] The term "substantially identical", when used to define
either a Bik amino acid sequence or Bik nucleic acid sequence,
means that a particular subject sequence, for example, a mutant
sequence, varies from the sequence of natural Bik by, for example,
one or more substitutions, deletions, additions, or a combination
thereof, the net effect of which is to retain at least some
biological activity of the Bik protein. Alternatively, DNA analog
sequences are "substantially identical" to specific DNA sequences
disclosed herein if: (a) the DNA analog sequence is derived from
coding regions of the natural Bik gene; or (b) the DNA analog
sequence is capable of hybridization of DNA sequences of (a) under
moderately stringent conditions and which encode biologically
active Bik; or (c) DNA sequences that are degenerative as a result
of the genetic code to the DNA analog sequences defined in (a) or
(b). Substantially identical analog proteins will be greater than
about 80% similar to the corresponding sequence of the native
protein. Sequences having lesser degrees of similarity but
comparable biological activity are considered to be equivalents. In
determining nucleic acid sequences, all subject nucleic acid
sequences capable of encoding substantially similar amino acid
sequences are considered to be substantially similar to a reference
nucleic acid sequence, regardless of differences in codon
sequence.
[0073] B. Percent Similarity
[0074] Percent similarity may be determined, for example, by
comparing sequence information using the GAP computer program,
available from the University of Wisconsin Geneticist Computer
Group. The GAP program utilizes the alignment method of Needleman
et al., 1970, as revised by Smith et al., 1981. Briefly, the GAP
program defines similarity as the number of aligned symbols (i.e.
nucleotides or amino acids) which are similar, divided by the total
number of symbols in the shorter of the two sequences. The
preferred default parameters for the GAP program include (1) a
unitary comparison matrix (containing a value of 1 for identities
and 0 for non-identities) of nucleotides and the weighted
comparison matrix of Gribskov et al., 1986, as described by
Schwartz et al., 1979; (2) a penalty of 3.0 for each gap and an
additional 0.01 penalty for each symbol and each gap; and (3) no
penalty for end gaps.
II. Nucleic Acid Sequences
[0075] In certain embodiments, the invention concerns the use of
mutant Bik nucleic acids, genes and gene products, such as the
mutant Bik that includes a sequence that is different from that of
the known Bik gene, or the corresponding protein. The term "a
sequence essentially as Bik" means that the sequence substantially
corresponds to a portion of the Bik gene and has relatively few
bases or amino acids (whether DNA or protein) that are not
identical to those of Bik (or a biologically functional equivalent
thereof, when referring to proteins). The term "biologically
functional equivalent" is well understood in the art and is further
defined in detail herein. Accordingly, sequences that have between
about 70% and about 80%; or more preferably, between about 81% and
about 90%; or even more preferably, between about 91% and about
99%; of amino acids that are identical or functionally equivalent
to the amino acids of Bik will be sequences that are "essentially
the same".
[0076] Mutant bik nucleic acids that have functionally equivalent
codons are covered by the invention. The term "functionally
equivalent codon" is used herein to refer to codons that encode the
same amino acid, such as the six codons for arginine or serine, and
also refers to codons that encode biologically equivalent amino
acids (Table 1).
TABLE-US-00001 TABLE 1 FUNCTIONALLY EQUIVALENT CODONS Amino Acids
Codons Alanine Ala A GCA GCC GCG GCU Cysteine Cys C UGC UGU
Aspartic Acid Asp D GAC GAU Glutamic Acid Glu E GAA GAG
Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine
His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAA AAG
Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG
Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCU Glutamine Gln Q
CAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU
UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA
GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr Y UAC UAU
[0077] It will also be understood that amino acid and nucleic acid
sequences may include additional residues, such as additional N- or
C-terminal amino acids or 5' or 3' sequences, and yet still be
essentially as set forth in one of the sequences disclosed herein,
so long as the sequence meets the criteria set forth above,
including the maintenance of biological protein activity where
protein expression is concerned. The addition of terminal sequences
particularly applies to nucleic acid sequences which may, for
example, include various non-coding sequences flanking either of
the 5' or 3' portions of the coding region or may include various
internal sequences, i.e., introns, which are known to occur within
genes.
[0078] The present invention also encompasses the use of DNA
segments that are complementary, or essentially complementary, to
the sequences set forth in the specification. Nucleic acid
sequences that are "complementary" are those that are capable of
base-pairing according to the standard Watson-Crick complementarity
rules. As used herein, the term "complementary sequences" means
nucleic acid sequences that are substantially complementary, as may
be assessed by the same nucleotide comparison set forth above, or
as defined as being capable of hybridizing to the nucleic acid
segment in question under relatively stringent conditions such as
those described herein.
[0079] C. Biologically Functional Equivalents
[0080] As mentioned above, modification and changes may be made in
the structure of Bik and still obtain a molecule having like or
otherwise desirable characteristics. For example, certain amino
acids may be substituted for other amino acids in a protein
structure without appreciable loss of interactive binding capacity
with structures such as, for example, E1B 19K or Bcl-2. Since, in
many embodiments, it is the interactive capacity and nature of a
protein that defines that protein's biological functional activity,
certain amino acid sequence substitutions can be made in a protein
sequence (or, of course, its underlying DNA coding sequence) and
nevertheless obtain a protein with like or even countervailing
properties (e.g., antagonistic vs. agonistic). It is thus
contemplated by the inventors that various changes may be made in
the sequence of the mutant Bik proteins or peptides (or underlying
DNA) without appreciable loss of their desired biological utility
or activity.
[0081] It is also well understood by the skilled artisan that,
inherent in the definition of a biologically functional equivalent
protein or peptide, is the concept that there is a limit to the
number of changes that may be made within a defined portion of the
molecule and still result in a molecule with an acceptable level of
equivalent biological activity. Biologically functional equivalent
peptides are thus defined herein as those peptides in which
certain, not most or all, of the amino acids may be substituted. Of
course, a plurality of distinct proteins/peptides with different
substitutions may easily be made and used in accordance with the
invention.
[0082] It is also well understood that where certain residues are
shown to be particularly important to the biological or structural
properties of a protein or peptide, e.g., residues in active sites,
such residues may not generally be exchanged.
[0083] Amino acid substitutions, such as those that might be
employed in modifying Bik, are generally based on the relative
similarity of the amino acid side-chain substituents, for example,
their hydrophobicity, hydrophilicity, charge, size, and the like.
An analysis of the size, shape and type of the amino acid
side-chain substituents reveals that arginine, lysine and histidine
are all positively charged residues; that alanine, glycine and
serine are all a similar size; and that phenylalanine, tryptophan
and tyrosine all have a generally similar shape. Therefore, based
upon these considerations, arginine, lysine and histidine; alanine,
glycine and serine; and phenylalanine, tryptophan and tyrosine; are
defined herein as biologically functional equivalents.
[0084] In making such changes, the hydropathic index of amino acids
may be considered. Each amino acid has been assigned a hydropathic
index on the basis of their hydrophobicity and charge
characteristics, these are: isoleucine (+4.5); valine (+4.2);
leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5);
methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine
(-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline
(-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5);
aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine
(-4.5).
[0085] The importance of the hydropathic amino acid index in
conferring interactive biological function on a protein is
generally understood in the art (Kyte and Doolittle, 1982,
incorporated herein by reference). It is known that certain amino
acids may be substituted for other amino acids having a similar
hydropathic index or score and still retain a similar biological
activity. In making changes based upon the hydropathic index, the
substitution of amino acids whose hydropathic indices are within
.+-.2 is preferred, those that are within .+-.1 are particularly
preferred, and those within .+-.0.5 are even more particularly
preferred.
[0086] It is also understood in the art that the substitution of
like amino acids can be made effectively on the basis of
hydrophilicity. U.S. Pat. No. 4,554,101, incorporated herein by
reference, states that the greatest local average hydrophilicity of
a protein, as governed by the hydrophilicity of its adjacent amino
acids, correlates with its immunogenicity and antigenicity, i.e.
with a biological property of the protein. It is understood that an
amino acid can be substituted for another having a similar
hydrophilicity value and still obtain a biologically equivalent
protein.
[0087] As detailed in U.S. Pat. No. 4,554,101, the following
hydrophilicity values have been assigned to amino acid residues:
arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-0.1); glutamate
(+3.0.+-0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2);
glycine (0); threonine (-0.4); proline (-0.5.+-0.1); alanine
(-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3);
valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3);
phenylalanine (-2.5); tryptophan (-3.4).
[0088] In making changes based upon similar hydrophilicity values,
the substitution of amino acids whose hydrophilicity values are
within .+-.2 is preferred, those that are within .+-.1 are
particularly preferred, and those within .+-.0.5 are even more
particularly preferred.
[0089] While discussion has focused on functionally equivalent
polypeptides arising from amino acid changes, it will be
appreciated that these changes may be effected by alteration of the
encoding DNA; taking into consideration also that the genetic code
is degenerate and that two or more codons may code for the same
amino acid.
III. Nucleic Acid-Based Expression Systems
[0090] The present invention utilizes, in some embodiments, systems
for expressing mutant Bik-comprising polynucleotides. Particular
exemplary aspects for these polynucleotides are described
herein.
[0091] A. Vectors
[0092] 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 in Maniatis et al., 1988 and Ausubel et al., 1994,
both incorporated herein by reference.
[0093] 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. In addition to control sequences that govern
transcription and translation, vectors and expression vectors may
contain nucleic acid sequences that serve other functions as well
and are described infra.
[0094] 1. Promoters and Enhancers
[0095] A "promoter" is a control sequence that is a region of a
nucleic acid sequence at which initiation and rate of transcription
are controlled. In a specific embodiment, a control sequence, such
as a promoter, regulates the tissue specificity within which the
nucleic acid sequence is expressed. A promoter, or control
sequence, may comprise genetic elements at which regulatory
proteins and molecules may bind, such as RNA polymerase and other
transcription factors. The phrases "operatively positioned,"
"operatively linked," "under control," and "under transcriptional
control" mean that a promoter or other control sequence is in a
correct functional location and/or orientation in relation to a
nucleic acid sequence to control transcriptional initiation and/or
expression of that sequence. A promoter may or may not be used in
conjunction with an "enhancer," which refers to a cis-acting
regulatory sequence involved in the transcriptional activation of a
nucleic acid sequence.
[0096] A promoter may be one naturally associated with a gene or
sequence, as may be obtained by isolating the 5' non-coding
sequences located upstream of the coding segment and/or exon. Such
a promoter can be referred to as "endogenous." Similarly, an
enhancer may be one naturally associated with a nucleic acid
sequence, located either downstream or upstream of that sequence.
Alternatively, certain advantages will be gained by positioning the
coding nucleic acid segment under the control of a recombinant or
heterologous promoter, which refers to a promoter that is not
normally associated with a nucleic acid sequence in its natural
environment. A recombinant or heterologous enhancer refers also to
an enhancer not normally associated with a nucleic acid sequence in
its natural environment. Such promoters or enhancers may include
promoters or enhancers of other genes, and promoters or enhancers
isolated from any other prokaryotic, viral, or eukaryotic cell, and
promoters or enhancers not "naturally occurring," i.e., containing
different elements of different transcriptional regulatory regions,
and/or mutations that alter expression. In addition to producing
nucleic acid sequences of promoters and enhancers synthetically,
sequences may be produced using recombinant cloning and/or nucleic
acid amplification technology, including PCR.TM., in connection
with the compositions disclosed herein (see U.S. Pat. No.
4,683,202; U.S. Pat. No. 5,928,906, each incorporated herein by
reference). Furthermore, it is contemplated the control sequences
that direct transcription and/or expression of sequences within
non-nuclear organelles such as mitochondria, chloroplasts, and the
like, can be employed as well.
[0097] Naturally, it will be important to employ a promoter and/or
enhancer that effectively directs the expression of the DNA segment
in the cell type, organelle, and organism chosen for expression.
Those of skill in the art of molecular biology generally know the
use of promoters, enhancers, and cell type combinations for protein
expression, for example, see Sambrook et al. (1989), incorporated
herein by reference. The promoters employed may be constitutive,
tissue-specific, inducible, and/or useful under the appropriate
conditions to direct high level expression of the introduced DNA
segment, such as is advantageous in the large-scale production of
recombinant proteins and/or peptides. The promoter may be
heterologous or endogenous.
[0098] Table 2 lists several elements/promoters that may be
employed, in the context of the present invention, to regulate the
expression of a gene. This list is not intended to be exhaustive of
all the possible elements involved in the promotion of expression
but, merely, to be exemplary thereof. Table 3 provides examples of
inducible elements, which are regions of a nucleic acid sequence
that can be activated in response to a specific stimulus.
TABLE-US-00002 TABLE 2 Promoter and/or Enhancer Promoter/Enhancer
References Immunoglobulin Heavy Chain Banerji et al., 1983; Gilles
et al., 1983; Grosschedl et al., 1985; Atchinson et al., 1986,
1987; Imler et al., 1987; Weinberger et al., 1984; Kiledjian et
al., 1988; Porton et al.; 1990 Immunoglobulin Light Chain Queen et
al., 1983; Picard et al., 1984 T-Cell Receptor Luria et al., 1987;
Winoto et al., 1989; Redondo et al.; 1990 HLA DQ a and/or DQ .beta.
Sullivan et al., 1987 .beta.-Interferon Goodbourn et al., 1986;
Fujita et al., 1987; Goodbourn et al., 1988 Interleukin-2 Greene et
al., 1989 Interleukin-2 Receptor Greene et al., 1989; Lin et al.,
1990 MHC Class II 5 Koch et al., 1989 MHC Class II HLA-DRa Sherman
et al., 1989 .beta.-Actin Kawamoto et al., 1988; Ng et al.; 1989
Muscle Creatine Kinase (MCK) Jaynes et al., 1988; Horlick et al.,
1989; Johnson et al., 1989 Prealbumin (Transthyretin) Costa et al.,
1988 Elastase I Omitz et al., 1987 Metallothionein (MTII) Karin et
al., 1987; Culotta et al., 1989 Collagenase Pinkert et al., 1987;
Angel et al., 1987 Albumin Pinkert et al., 1987; Tronche et al.,
1989, 1990 .alpha.-Fetoprotein Godbout et al., 1988; Campere et
al., 1989 t-Globin Bodine et al., 1987; Perez-Stable et al., 1990
.beta.-Globin Trudel et al., 1987 c-fos Cohen et al., 1987 c-HA-ras
Triesman, 1986; Deschamps et al., 1985 Insulin Edlund et al., 1985
Neural Cell Adhesion Molecule Hirsh et al., 1990 (NCAM)
.alpha.1-Antitrypain Latimer et al., 1990 H2B (TH2B) Histone Hwang
et al., 1990 Mouse and/or Type I Collagen Ripe et al., 1989
Glucose-Regulated Proteins Chang et al., 1989 (GRP94 and GRP78) Rat
Growth Hormone Larsen et al., 1986 Human Serum Amyloid A Edbrooke
et al., 1989 (SAA) Troponin I (TN I) Yutzey et al., 1989
Platelet-Derived Growth Factor Pech et al., 1989 (PDGF) Duchenne
Muscular Dystrophy Klamut et al., 1990 SV40 Banerji et al., 1981;
Moreau et al., 1981; Sleigh et al., 1985; Firak et al., 1986; Herr
et al., 1986; Imbra et al., 1986; Kadesch et al., 1986; Wang et
al., 1986; Ondek et al., 1987; Kuhl et al., 1987; Schaffner et al.,
1988 Polyoma Swartzendruber et al., 1975; Vasseur et al., 1980;
Katinka et al., 1980, 1981; Tyndell et al., 1981; Dandolo et al.,
1983; de Villiers et al., 1984; Hen et al., 1986; Satake et al.,
1988; Campbell and Villarreal, 1988 Retroviruses Kriegler et al.,
1982, 1983; Levinson et al., 1982; Kriegler et al., 1983, 1984a, b,
1988; Bosze et al., 1986; Miksicek et al., 1986; Celander et al.,
1987; Thiesen et al., 1988; Celander et al., 1988; Chol et al.,
1988; Reisman et al., 1989 Papilloma Virus Campo et al., 1983;
Lusky et al., 1983; Spandidos and Wilkie, 1983; Spalholz et al.,
1985; Lusky et al., 1986; Cripe et al., 1987; Gloss et al., 1987;
Hirochika et al., 1987; Stephens et al., 1987; Glue et al., 1988
Hepatitis B Virus Bulla et al., 1986; Jameel et al., 1986; Shaul et
al., 1987; Spandau et al., 1988; Vannice et al., 1988 Human
Immunodeficiency Muesing et al., 1987; Hauber et al., 1988;
Jakobovits Virus et al., 1988; Feng et al., 1988; Takebe et al.,
1988; Rosen et al., 1988; Berkhout et al., 1989; Laspia et al.,
1989; Sharp et al., 1989; Braddock et al., 1989 Cytomegalovirus
(CMV) Weber et al., 1984; Boshart et al., 1985; Foecking et al.,
1986 Gibbon Ape Leukemia Virus Holbrook et al., 1987; Quinn et al.,
1989
TABLE-US-00003 TABLE 3 Inducible Elements Element Inducer
References MT II Phorbol Ester (TFA) Palmiter et al., 1982;
Haslinger Heavy metals et al., 1985; Searle et al., 1985; Stuart et
al., 1985; Imagawa et al., 1987, Karin et al., 1987; Angel et al.,
1987b; McNeall et al., 1989 MMTV (mouse mammary Glucocorticoids
Huang et al., 1981; Lee et al., tumor virus) 1981; Majors et al.,
1983; Chandler et al., 1983; Lee et al., 1984; Ponta et al., 1985;
Sakai et al., 1988 .beta.-Interferon poly(rI) .times. Tavernier et
al., 1983 poly(rc) Adenovirus 5 E2 ElA Imperiale et al., 1984
Collagenase Phorbol Ester (TPA) Angel et al., 1987a Stromelysin
Phorbol Ester (TPA) Angel et al., 1987b SV40 Phorbol Ester (TPA)
Angel et al., 1987b Murine MX Gene Interferon, Newcastle Hug et
al., 1988 Disease Virus GRP78 Gene A23187 Resendez et al., 1988
.alpha.-2-Macroglobulin IL-6 Kunz et al., 1989 Vimentin Serum
Rittling et al., 1989 MHC Class I Gene H- Interferon Blanar et al.,
1989 2.kappa.b HSP70 ElA, SV40 Large T Taylor et al., 1989, 1990a,
Antigen 1990b Proliferin Phorbol Ester-TPA Mordacq et al., 1989
Tumor Necrosis Factor PMA Hensel et al., 1989 Thyroid Stimulating
Thyroid Hormone Chatterjee et al., 1989 Hormone .alpha. Gene
[0099] The identity of tissue-specific promoters or elements, as
well as assays to characterize their activity, is well known to
those of skill in the art. Examples of such regions include the
human LIMK2 gene (Nomoto et al. 1999), the somatostatin receptor 2
gene (Kraus et al., 1998), murine epididymal retinoic acid-binding
gene (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998),
mouse alpha2 (XI) collagen (Tsumaki, et al., 1998), DIA dopamine
receptor gene (Lee, et al., 1997), insulin-like growth factor II
(Wu et al., 1997), or human platelet endothelial cell adhesion
molecule-1 (Almendro et al., 1996), for example.
[0100] Tissue-specific promoters utilized to control expression
targeting and/or levels of mutant Bik may be endogenous wild-type
promoters, mutant promoters, or synthetic promoters, so long as the
expression of mutant Bik is preferentially retained in one or more
tissues of interest compared to tissues that are not the desired
target. Synthetic promoters may be further defined as composite
promoters, referred to herein as a promoter comprising at least two
separate regions originating from different endogenous and/or
synthetic promoters yet operably linked to control expression of a
mutant Bik. In a particular embodiment, the tissue specificity
refers to specificity for cancerous tissue, as opposed to
non-cancerous tissue. The term "cancerous tissue" as used herein
refers to a tissue comprising at least one cancer cell.
[0101] a. Breast Cancer Tissue-Specific Promoters
[0102] Most of the promoters currently used in cancer gene therapy
possess strong but unselective activity (e.g. CMV and .beta.-actin
promoters) in both normal and tumor cells. Thus, in some aspects of
the present invention, a breast tissue-specific promoter is
utilized in the invention, such as to control expression of a
mutant form of Bik, including the exemplary BikT33D, BikS35D, and
Bik T33DS35D mutants. In a particular aspect, the breast
tissue-specific promoter is a breast cancer tissue-specific
promoter. As such, the desired promoter for this embodiment targets
expression specifically to breast cancer tissue.
[0103] Any breast cancer tissue-specific promoter may be employed
in the invention so long as it preferentially directs expression of
mutant Bik in breast cancer tissue. Examples of breast cancer
tissue-specific promoters that may direct expression of mutant Bik
in breast cancer tissue include at least hALA, GLG, HK-II, and HER2
promoters (Anderson et al., 2000; Katabi et al., 1999; Lu et al.,
2002; Maeda et al., 2001).
[0104] In one particular embodiment of the present invention,
composite promoters utilizing either topoisomerase II.alpha.
(topoII.alpha.) and transferrin receptor (TfR) breast
cancer-specific control sequences are employed. The topoisomerase
II.alpha. (topoII.alpha.) and transferrin receptor (TfR) levels are
elevated in breast cancer, as determined using SAGE analysis and
cDNA microarray, for example. The present inventors identified a 90
base pair segment (SEQ ID NO:26) and a 116 base pair segment (SEQ
ID NO:27) in the 5'-end of topoII.alpha. and TfR promoter,
respectively, as a minimally required breast-cancer specific
control sequence. In particular embodiments, the promoter activity
is enhanced by operatively linking these two short promoters with
an enhancer sequence, such as the cytomegalovirus (CMV) promoter
enhancer sequence (SEQ ID NO:25); these chimeric promoters are
referred to herein as CT90 and CTR116, respectively. The full CT90
promoter is comprised in SEQ ID NO:37, and the full CTR116 promoter
is comprised in SEQ ID NO:38. These promoters are described herein
but are further characterized in detail in U.S. Provisional Patent
Application No. 60/559,111, entitled "Cancer-Specific Promoters" by
Mien-Chie Hung, Yan Li, Yong Wen, Chi-Ping Day, Kun-Ming Rau,
Xiaoming Xie, Zheng Li, filed simultaneously herewith and
incorporated by reference herein in its entirety. To demonstrate
its use in cancer gene therapy, the present inventors generated a
DNA construct using CT90 to drive mutant Bik expression. When
transfected into cell lines, this construct selectively killed
breast cancer cells. Moreover, the present inventors demonstrated
that this construct had an anti-tumor effect on breast tumor
xenograft in mouse by intravenous injection with an exemplary
non-viral delivery system. This indicates that CT90 and CTR116 can
drive the expression of a therapeutic gene such as mutant Bik
selectively in breast cancer cells.
[0105] Thus, the current invention encompasses breast
cancer-specific promoters for control of expression of mutant Bik
to target breast cancer cells for treatment that is less toxic or
non-toxic to normal tissues.
[0106] b. Pancreatic Cancer Tissue-Specific Promoters
[0107] Pancreatic-specific promoters can be used for targeted
expression of mutant Bik, including the exemplary BikT33D, BikS35D,
and Bik T33DS35D mutants. Any pancreatic cancer tissue-specific
promoter may be employed in the invention so long as it
preferentially directs expression of mutant Bik in pancreatic
cancer tissue. Examples of pancreatic cancer tissue-specific
promoters that may direct expression of mutant Bik in pancreatic
cancer tissue include the insulin promoter, such as the rat insulin
promoter (Wang et al., 2004); midkine and cyclooxygenase-2
promoters (Wesseling et al., 2001); and carcinoembryonic antigen
(CEA) promoter (Takeuchi et al., 2000), for example.
[0108] The present inventors developed a pancreatic cancer-specific
promoter that is described herein but provided in further detail in
U.S. Provisional Patent Application 60/559,111, entitled
"Cancer-Specific Promoters" by Mien-Chie Hung, Yan Li, Yong Wen,
Chi-Ping Day, Kun-Ming Rau, Xiaoming Xie, Zheng Li, filed
simultaneously herewith, which is incorporated by reference herein
in its entirety. The promoter comprises Cholecystoskinin A receptor
(CCKAR) promoter sequence, particularly CCKAR promoter ranging from
nt -726 to +1 (SEQ ID NO:28) operatively linked to an enhancer,
such as CMV enhancer. The CCKAR-CMV composite is then engineered
with a particular two-step transcriptional amplification (TSTA)
system (Iyer et al., 2001; Zhang et al., 2002; Sato et al., 2003;
and references cited therein), such as the exemplary GAL4-VP16 or
GAL4-VP2 fusion protein, to augment the transcriptional activity
and, it is also operatively linked to the post-transcriptional
regulatory element of the woodchuck hepatitis virus (WPRE) (SEQ ID
NO:29) to modify RNA polyadenylation signal, RNA export, and/or RNA
translation. A skilled artisan recognizes that the term "two-step
transcriptional amplification (TSTA) system" may also be referred
to as "two-step transcriptional activation (TSTA) system" or
"recombinant transcriptional activation approach" (Nettelbeck et
al., 2000). In a particular aspect, the CCAKAR-TSTA-WPRE (CTP)
promoter is utilized, and an example of such a composite promoter
is comprised in SEQ ID NO:34. Thus, the molecularly engineered CTP
promoter is employed for effective treatment modalities for
pancreatic cancer gene therapy with mutant Bik.
[0109] c. Prostate Cancer Tissue-Specific Promoters
[0110] Prostate cancer tissue-specific promoters can be used to
control expression of polynucleotides that encode mutant Bik.
Prostate-specific promoters, like PSA, probasin and hK2, for
example, have been recently developed. The activities of these
promoters are androgen-dependent. For numerous disease stages,
patients are androgen-dependent (ADPC), allowing the use of
androgen-responsive vectors to direct expression of therapeutic
genes to prostatic tissue. Although robust prostate-specific
promoters responsive to androgen receptor have been developed by
the present inventors (Xie et al., Cancer Res 2001) and other
groups (Zhang et al., Mol Endocrinol 2000), these
androgen-dependent promoters may be less active after castration or
androgen ablation therapy, which are the main modalities for
progressive prostate cancer treatment. These patients treated with
compositions comprising these promoters may fail this kind of
therapy and die of recurrent androgen-independent prostate cancer
(AIPC).
[0111] The inventors have developed prostate cancer-specific
promoters that may be expected to have benefit for both ADPC and
androgen-independent prostate cancer (AIPC) to treat metastatic and
recurrent hormonal refractory prostate cancer, particularly to
regulate expression of mutant Bik. This promoter is described
herein and characterized in further detail in U.S. Provisional
Patent Application No. 60/559,111, entitled "Cancer-Specific
Promoters" by Mien-Chie Hung, Yan Li, Yong Wen, Chi-Ping Day,
Kun-Ming Rau, Xiaoming Xie, Zheng Li, filed simultaneously herewith
and incorporated by reference herein in its entirety. The promoter,
referred to herein as ATTP, comprises at least the minimal promoter
fragment (hTERTp) of the human telomerase reverse transcriptase
(hTERT) (SEQ ID NO:32) operably linked to a two-step
transcriptional amplification (TSTA) system, such as the exemplary
GAL4-VP16 or GAL4-VP2 (two examples of GAL4-VP2 are SEQ ID NO:30 or
SEQ ID NO:33) fusion protein-encoding sequences, and it is also
operatively linked to the post-transcriptional regulatory element
of the woodchuck hepatitis virus (WPRE) to modify RNA
polyadenylation signal, RNA export, and/or RNA translation. These
regulatory sequences are effective in both ADPC and AIPC cell
lines. Given that in most cases of recurrent prostate cancers the
AR gene is amplified and/or AR is overexpressed, this particular
promoter greatly improves the effective index for the embodiment
wherein the activity of this system is stimulated by androgen. In
preferred embodiments the tissue-specificity region comprises at
least, and for example, the ARR2 regulatory element (SEQ ID NO:31)
from ARR2 gene. In a particular aspect of the invention, the
TSTA-hTERT-ARR2 and WPRE elements are utilized as the prostate
cancer-specific regulatory elements, which in specific embodiments
are comprised in SEQ ID NO:35. Thus, the present inventors have
developed a novel prostate cancer-specific regulatory system that
will target mutant Bik to not only ADPC but also AIPC.
[0112] 2. Initiation Signals and Internal Ribosome Binding
Sites
[0113] A specific initiation signal also may be required for
efficient translation of coding sequences. These signals include
the ATG initiation codon or adjacent sequences. Exogenous
translational control signals, including the ATG initiation codon,
may need to be provided. One of ordinary skill in the art would
readily be capable of determining this and providing the necessary
signals. It is well known that the initiation codon must be
"in-frame" with the reading frame of the desired coding sequence to
ensure translation of the entire insert. The exogenous
translational control signals and initiation codons can be either
natural or synthetic. The efficiency of expression may be enhanced
by the inclusion of appropriate transcription enhancer
elements.
[0114] In certain embodiments of the invention, the use of internal
ribosome entry sites (IRES) elements are used to create multigene,
or polycistronic, messages. IRES elements are able to bypass the
ribosome scanning model of 5' methylated Cap dependent translation
and begin translation at internal sites (Pelletier and Sonenberg,
1988). IRES elements from two members of the picornavirus family
(polio and encephalomyocarditis) have been described (Pelletier and
Sonenberg, 1988), as well an IRES from a mammalian message (Macejak
and Sarnow, 1991). IRES elements can be linked to heterologous open
reading frames. Multiple open reading frames can be transcribed
together, each separated by an IRES, creating polycistronic
messages. By virtue of the IRES element, each open reading frame is
accessible to ribosomes for efficient translation. Multiple genes
can be efficiently expressed using a single promoter/enhancer to
transcribe a single message (see U.S. Pat. Nos. 5,925,565 and
5,935,819, herein incorporated by reference).
[0115] 3. Multiple Cloning Sites
[0116] Vectors can include a multiple cloning site (MCS), which is
a nucleic acid region that contains multiple restriction enzyme
sites, any of which can be used in conjunction with standard
recombinant technology to digest the vector. (See Carbonelli et
al., 1999, Levenson et al., 1998, and Cocea, 1997, incorporated
herein by reference.) "Restriction enzyme digestion" refers to
catalytic cleavage of a nucleic acid molecule with an enzyme that
functions only at specific locations in a nucleic acid molecule.
Many of these restriction enzymes are commercially available. Use
of such enzymes is widely understood by those of skill in the art.
Frequently, a vector is linearized or fragmented using a
restriction enzyme that cuts within the MCS to enable exogenous
sequences to be ligated to the vector. "Ligation" refers to the
process of forming phosphodiester bonds between two nucleic acid
fragments, which may or may not be contiguous with each other.
Techniques involving restriction enzymes and ligation reactions are
well known to those of skill in the art of recombinant
technology.
[0117] 4. Splicing Sites
[0118] Most transcribed eukaryotic RNA molecules will undergo RNA
splicing to remove introns from the primary transcripts. Vectors
containing genomic eukaryotic sequences may require donor and/or
acceptor splicing sites to ensure proper processing of the
transcript for protein expression. (See Chandler et al., 1997,
herein incorporated by reference.)
[0119] 5. Polyadenylation Signals
[0120] In expression, one will typically include a polyadenylation
signal to effect proper polyadenylation of the transcript. The
nature of the polyadenylation signal is not believed to be crucial
to the successful practice of the invention, and/or any such
sequence may be employed. Preferred embodiments include the SV40
polyadenylation signal and/or the bovine growth hormone
polyadenylation signal, convenient and/or known to function well in
various target cells. Also contemplated as an element of the
expression cassette is a transcriptional termination site. These
elements can serve to enhance message levels and/or to minimize
read through from the cassette into other sequences.
[0121] 6. Origins of Replication
[0122] In order to propagate a vector in a host cell, it may
contain one or more origins of replication sites (often termed
"ori"), which is a specific nucleic acid sequence at which
replication is initiated. Alternatively an autonomously replicating
sequence (ARS) can be employed if the host cell is yeast.
[0123] 7. Selectable and Screenable Markers
[0124] In certain embodiments of the invention, the cells contain
nucleic acid construct of the present invention, a cell may be
identified in vitro or in vivo by including a marker in the
expression vector. Such markers would confer an identifiable change
to the cell permitting easy identification of cells containing the
expression vector. Generally, a selectable marker is one that
confers a property that allows for selection. A positive selectable
marker is one in which the presence of the marker allows for its
selection, while a negative selectable marker is one in which its
presence prevents its selection. An example of a positive
selectable marker is a drug resistance marker.
[0125] Usually the inclusion of a drug selection marker aids in the
cloning and identification of transformants, for example, genes
that confer resistance to neomycin, puromycin, hygromycin, DHFR,
GPT, zeocin and histidinol are useful selectable markers. In
addition to markers conferring a phenotype that allows for the
discrimination of transformants based on the implementation of
conditions, other types of markers including screenable markers
such as GFP, whose basis is colorimetric analysis, are also
contemplated. Alternatively, screenable enzymes such as herpes
simplex virus thymidine kinase (tk) or chloramphenicol
acetyltransferase (CAT) may be utilized. One of skill in the art
would also know how to employ immunologic markers, possibly in
conjunction with FACS analysis. The marker used is not believed to
be important, so long as it is capable of being expressed
simultaneously with the nucleic acid encoding a gene product.
Further examples of selectable and screenable markers are well
known to one of skill in the art.
[0126] B. Host Cells
[0127] As used herein, the terms "cell," "cell line," and "cell
culture" may be used interchangeably. All of these term also
include their progeny, which is 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
organisms 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.
[0128] Host cells may be derived from prokaryotes or eukaryotes,
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
(www.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.
[0129] 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.
[0130] 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.
[0131] C. Expression Systems
[0132] 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
invention to produce nucleic acid sequences, or their cognate
polypeptides, proteins and peptides. Many such systems are
commercially and widely available.
[0133] 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, 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..
[0134] 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.
IV. Nucleic Acid Delivery
[0135] The general approach to the aspects of the present invention
concerning compositions and/or therapeutics is to provide a cell
with a gene construct encoding a specific and/or desired mutant Bik
protein, polypeptide, or peptide, thereby permitting the desired
activity of the protein, polypeptide, or peptide to take effect.
While it is conceivable that the gene construct and/or protein may
be delivered directly, a preferred embodiment involves providing a
nucleic acid encoding a specific and desired protein, polypeptide,
or peptide to the cell. Following this provision, the proteinaceous
composition is synthesized by the transcriptional and translational
machinery of the cell, as well as any that may be provided by the
expression construct. In providing antisense, ribozymes and other
inhibitors, the preferred mode is also to provide a nucleic acid
encoding the construct to the cell.
[0136] In certain embodiments of the invention, the nucleic acid
encoding the gene may be stably integrated into the genome of the
cell. In yet further embodiments, the nucleic acid may be stably
maintained in the cell as a separate, episomal segment of DNA. Such
nucleic acid segments and "episomes" encode sequences sufficient to
permit maintenance and replication independent of and in
synchronization with the host cell cycle. How the expression
construct is delivered to a cell and/or where in the cell the
nucleic acid remains is dependent on the type of expression
construct employed.
[0137] A. DNA Delivery Using Viral Vectors
[0138] The ability of certain viruses to infect cells and enter
cells via receptor-mediated endocytosis, and to integrate into host
cell genome and/or express viral genes stably and/or efficiently
have made them attractive candidates for the transfer of foreign
genes into mammalian cells. Preferred gene therapy vectors of the
present invention will generally be viral vectors.
[0139] Although some viruses that can accept foreign genetic
material are limited in the number of nucleotides they can
accommodate and/or in the range of cells they infect, these viruses
have been demonstrated to successfully effect gene expression.
However, adenoviruses do not integrate their genetic material into
the host genome and/or therefore do not require host replication
for gene expression, making them ideally suited for rapid,
efficient, heterologous gene expression. Techniques for preparing
replication-defective infective viruses are well known in the
art.
[0140] Of course, in using viral delivery systems, one will desire
to purify the virion sufficiently to render it essentially free of
undesirable contaminants, such as defective interfering viral
particles and endotoxins and other pyrogens such that it will not
cause any untoward reactions in the cell, animal and/or individual
receiving the vector construct. A preferred means of purifying the
vector involves the use of buoyant density gradients, such as
cesium chloride gradient centrifugation.
[0141] 1. Adenoviral Vectors
[0142] A particular method for delivery of the expression
constructs involves the use of an adenovirus expression vector.
Although adenovirus vectors are known to have a low capacity for
integration into genomic DNA, this feature is counterbalanced by
the high efficiency of gene transfer afforded by these vectors.
"Adenovirus expression vector" is meant to include those constructs
containing adenovirus sequences sufficient to (a) support packaging
of the construct and/or (b) to ultimately express a tissue and/or
cell-specific construct that has been cloned therein.
[0143] The expression vector comprises a genetically engineered
form of adenovirus. Knowledge of the genetic organization and
adenovirus, a 36 kb, linear, double-stranded DNA virus, allows
substitution of large pieces of adenoviral DNA with foreign
sequences up to 7 kb (Grunhaus and Horwitz, 1992). In contrast to
retrovirus, the adenoviral infection of host cells does not result
in chromosomal integration because adenoviral DNA can replicate in
an episomal manner without potential genotoxicity. Also,
adenoviruses are structurally stable, and/or no genome
rearrangement has been detected after extensive amplification.
[0144] Adenovirus is particularly suitable for use as a gene
transfer vector because of its mid-sized genome, ease of
manipulation, high titer, wide target-cell range and/or high
infectivity. Both ends of the viral genome contain 100-200 base
pair inverted repeats (ITRs), which are cis elements necessary for
viral DNA replication and/or packaging. The early (E) and/or late
(L) regions of the genome contain different transcription units
that are divided by the onset of viral DNA replication. The E1
region (E1A and/or E1B) encodes proteins responsible for the
regulation of transcription of the viral genome and/or a few
cellular genes. The expression of the E2 region (E2A and/or E2B)
results in the synthesis of the proteins for viral DNA replication.
These proteins are involved in DNA replication, late gene
expression and/or host cell shut-off (Renan, 1990). The products of
the late genes, including the majority of the viral capsid
proteins, are expressed only after significant processing of a
single primary transcript issued by the major late promoter (MLP).
The MLP (located at 16.8 m.u.) is particularly efficient during the
late phase of infection, and/or all the mRNA's issued from this
promoter possess a 5'-tripartite leader (TPL) sequence which makes
them preferred mRNA's for translation.
[0145] In a current system, recombinant adenovirus is generated
from homologous recombination between shuttle vector and provirus
vector. Due to the possible recombination between two proviral
vectors, wild-type adenovirus may be generated from this process.
Therefore, it is critical to isolate a single clone of virus from
an individual plaque and/or examine its genomic structure.
[0146] Generation and/or propagation of the current adenovirus
vectors, which are replication deficient, depend on a unique helper
cell line, designated 293, which was transformed from human
embryonic kidney cells by Ad5 DNA fragments and constitutively
expresses E1 proteins (E1A and/or E1B; Graham et al., 1977). Since
the E3 region is dispensable from the adenovirus genome (Jones and
Shenk, 1978), the current adenovirus vectors, with the help of 293
cells, carry foreign DNA in either the E1, the D3 and both regions
(Graham and Prevec, 1991). Recently, adenoviral vectors comprising
deletions in the E4 region have been described (U.S. Pat. No.
5,670,488, incorporated herein by reference).
[0147] In nature, adenovirus can package approximately 105% of the
wild-type genome (Ghosh-Choudhury et al., 1987), providing capacity
for about 2 extra kb of DNA. Combined with the approximately 5.5 kb
of DNA that is replaceable in the E1 and/or E3 regions, the maximum
capacity of the current adenovirus vector is under 7.5 kb, and/or
about 15% of the total length of the vector. More than 80% of the
adenovirus viral genome remains in the vector backbone.
[0148] Helper cell lines may be derived from human cells such as
human embryonic kidney cells, muscle cells, hematopoietic cells and
other human embryonic mesenchymal and epithelial cells.
Alternatively, the helper cells may be derived from the cells of
other mammalian species that are permissive for human adenovirus.
Such cells include, e.g., Vero cells and other monkey embryonic
mesenchymal and/or epithelial cells. As stated above, the preferred
helper cell line is 293.
[0149] Recently, Racher et al. (1995) disclosed improved methods
for culturing 293 cells and/or propagating adenovirus. In one
format, natural cell aggregates are grown by inoculating individual
cells into 1 liter siliconized spinner flasks (Techne, Cambridge,
UK) containing 100-200 ml of medium. Following stirring at 40 rpm,
the cell viability is estimated with trypan blue. In another
format, Fibra-Cel microcarriers (Bibby Sterlin, Stone, UK) (5 g/l)
is employed as follows. A cell inoculum, resuspended in 5 ml of
medium, is added to the carrier (50 ml) in a 250 ml Erlenmeyer
flask and/or left stationary, with occasional agitation, for 1 to 4
h. The medium is then replaced with 50 ml of fresh medium and/or
shaking initiated. For virus production, cells are allowed to grow
to about 80% confluence, after which time the medium is replaced
(to 25% of the final volume) and/or adenovirus added at an MOI of
0.05. Cultures are left stationary overnight, following which the
volume is increased to 100% and/or shaking commenced for another 72
h.
[0150] Other than the requirement that the adenovirus vector be
replication defective, and at least conditionally defective, the
nature of the adenovirus vector is not believed to be crucial to
the successful practice of the invention. The adenovirus may be of
any of the 42 different known serotypes and subgroups A-F.
Adenovirus type 5 of subgroup C is the preferred starting material
in order to obtain the conditional replication-defective adenovirus
vector for use in the present invention. This is because Adenovirus
type 5 is a human adenovirus about which a great deal of
biochemical and genetic information is known, and it has
historically been used for most constructions employing adenovirus
as a vector.
[0151] As stated above, the typical vector according to the present
invention is replication defective and will not have an adenovirus
E1 region. Thus, it will be most convenient to introduce the
transforming construct at the position from which the E1-coding
sequences have been removed. However, the position of insertion of
the construct within the adenovirus sequences is not critical to
the invention. The polynucleotide encoding the gene of interest may
also be inserted in lieu of the deleted E3 region in E3 replacement
vectors as described by Karlsson et al. (1986) and in the E4 region
where a helper cell line and helper virus complements the E4
defect.
[0152] Adenovirus growth and/or manipulation is known to those of
skill in the art, and/or exhibits broad host range in vitro and in
vivo. This group of viruses can be obtained in high titers, e.g.,
109 to 1011 plaque-forming units per ml, and they are highly
infective. The life cycle of adenovirus does not require
integration into the host cell genome. The foreign genes delivered
by adenovirus vectors are episomal and, therefore, have low
genotoxicity to host cells. No side effects have been reported in
studies of vaccination with wild-type adenovirus (Couch et al.,
1963; Top et al., 1971), demonstrating their safety and/or
therapeutic potential as in vivo gene transfer vectors.
[0153] Adenovirus vectors have been used in eukaryotic gene
expression (Levrero et al., 1991; Gomez-Foix et al., 1992) and
vaccine development (Grunhaus and Horwitz, 1992; Graham and Prevec,
1992). Recently, animal studies suggested that recombinant
adenovirus could be used for gene therapy (Stratford-Perricaudet
and Perricaudet, 1991a; Stratford-Perricaudet et al., 1991b; Rich
et al., 1993). Studies in administering recombinant adenovirus to
different tissues include trachea instillation (Rosenfeld et al.,
1991; Rosenfeld et al., 1992), muscle injection (Ragot et al.,
1993), peripheral intravenous injections (Herz and Gerard, 1993)
and/or stereotactic inoculation into the brain (Le Gal La Salle et
al., 1993). Recombinant adenovirus and adeno-associated virus (see
below) can both infect and transduce non-dividing human primary
cells.
[0154] 2. AAV Vectors
[0155] Adeno-associated virus (AAV) is an attractive vector system
for use in the cell transduction of the present invention as it has
a high frequency of integration and it can infect nondividing
cells, thus making it useful for delivery of genes into mammalian
cells, for example, in tissue culture (Muzyczka, 1992) and in vivo.
AAV has a broad host range for infectivity (Tratschin et al., 1984;
Laughlin et al., 1986; Lebkowski et al., 1988; McLaughlin et al.,
1988). Details concerning the generation and use of rAAV vectors
are described in U.S. Pat. No. 5,139,941 and/or U.S. Pat. No.
4,797,368, each incorporated herein by reference.
[0156] Studies demonstrating the use of AAV in gene delivery
include LaFace et al. (1988); Zhou et al. (1993); Flotte et al.
(1993); and Walsh et al. (1994). Recombinant AAV vectors have been
used successfully for in vitro and/or in vivo transduction of
marker genes (Kaplitt et al., 1994; Lebkowski et al., 1988;
Samulski et al., 1989; Yoder et al., 1994; Zhou et al., 1994;
Hermonat and Muzyczka, 1984; Tratschin et al., 1985; McLaughlin et
al., 1988) and genes involved in human diseases (Flotte et al.,
1992; Luo et al., 1994; Ohi et al., 1990; Walsh et al., 1994; Wei
et al., 1994). Recently, an AAV vector has been approved for phase
I human trials for the treatment of cystic fibrosis.
[0157] AAV is a dependent parvovirus in that it requires
coinfection with another virus (either adenovirus and a member of
the herpes virus family) to undergo a productive infection in
cultured cells (Muzyczka, 1992). In the absence of coinfection with
helper virus, the wild type AAV genome integrates through its ends
into human chromosome 19 where it resides in a latent state as a
provirus (Kotin et al., 1990; Samulski et al., 1991). rAAV,
however, is not restricted to chromosome 19 for integration unless
the AAV Rep protein is also expressed (Shelling and Smith, 1994).
When a cell carrying an AAV provirus is superinfected with a helper
virus, the AAV genome is "rescued" from the chromosome and from a
recombinant plasmid, and/or a normal productive infection is
established (Samulski et al., 1989; McLaughlin et al., 1988; Kotin
et al., 1990; Muzyczka, 1992).
[0158] Typically, recombinant AAV (rAAV) virus is made by
cotransfecting a plasmid containing the gene of interest flanked by
the two AAV terminal repeats (McLaughlin et al., 1988; Samulski et
al., 1989; each incorporated herein by reference) and/or an
expression plasmid containing the wild type AAV coding sequences
without the terminal repeats, for example pIM45 (McCarty et al.,
1991; incorporated herein by reference). The cells are also
infected and transfected with adenovirus and plasmids carrying the
adenovirus genes required for AAV helper function. rAAV virus
stocks made in such fashion are contaminated with adenovirus which
must be physically separated from the rAAV particles (for example,
by cesium chloride density centrifugation). Alternatively,
adenovirus vectors containing the AAV coding regions and cell lines
containing the AAV coding regions and some and all of the
adenovirus helper genes could be used (Yang et al., 1994; Clark et
al., 1995). Cell lines carrying the rAAV DNA as an integrated
provirus can also be used (Flotte et al., 1995).
[0159] 3. Retroviral Vectors
[0160] Retroviruses have promise as gene delivery vectors due to
their ability to integrate their genes into the host genome,
transferring a large amount of foreign genetic material, infecting
a broad spectrum of species and cell types and of being packaged in
special cell-lines (Miller, 1992).
[0161] The retroviruses are a group of single-stranded RNA viruses
characterized by an ability to convert their RNA to double-stranded
DNA in infected cells by a process of reverse-transcription
(Coffin, 1990). The resulting DNA then stably integrates into
cellular chromosomes as a provirus and/or directs synthesis of
viral proteins. The integration results in the retention of the
viral gene sequences in the recipient cell and/or its descendants.
The retroviral genome contains three genes, gag, pol, and/or env
that code for capsid proteins, polymerase enzyme, and envelope
components, respectively. A sequence found upstream from the gag
gene contains a signal for packaging of the genome into virions.
Two long terminal repeat (LTR) sequences are present at the 5' and
3' ends of the viral genome. These contain strong promoter and
enhancer sequences and are also required for integration in the
host cell genome (Coffin, 1990).
[0162] In order to construct a retroviral vector, a nucleic acid
encoding a gene of interest is inserted into the viral genome in
the place of certain viral sequences to produce a virus that is
replication-defective. In order to produce virions, a packaging
cell line containing the gag, pol, and env genes but without the
LTR and packaging components is constructed (Mann et al., 1983).
When a recombinant plasmid containing a cDNA, together with the
retroviral LTR and packaging sequences is introduced into this cell
line (by calcium phosphate precipitation for example), the
packaging sequence allows the RNA transcript of the recombinant
plasmid to be packaged into viral particles, which are then
secreted into the culture media (Nicolas and Rubenstein, 1988;
Temin, 1986; Mann et al., 1983). The media containing the
recombinant retroviruses is then collected, optionally
concentrated, and used for gene transfer. Retroviral vectors are
able to infect a broad variety of cell types. However, integration
and/or stable expression require the division of host cells
(Paskind et al., 1975).
[0163] Concern with the use of defective retrovirus vectors is the
potential appearance of wild-type replication-competent virus in
the packaging cells. This can result from recombination events in
which the intact sequence from the recombinant virus inserts
upstream from the gag, pol, env sequence integrated in the host
cell genome. However, new packaging cell lines are now available
that should greatly decrease the likelihood of recombination
(Markowitz et al., 1988; Hersdorffer et al., 1990).
[0164] Gene delivery using second generation retroviral vectors has
been reported. Kasahara et al. (1994) prepared an engineered
variant of the Moloney murine leukemia virus, that normally infects
only mouse cells, and modified an envelope protein so that the
virus specifically bound to, and infected, human cells bearing the
erythropoietin (EPO) receptor. This was achieved by inserting a
portion of the EPO sequence into an envelope protein to create a
chimeric protein with a new binding specificity.
[0165] 4. Other Viral Vectors
[0166] Other viral vectors may be employed as expression constructs
in the present invention. Vectors derived from viruses such as
vaccinia virus (Ridgeway, 1988; Baichwal and Sugden, 1986; Coupar
et al., 1988), sindbis virus, cytomegalovirus and/or herpes simplex
virus may be employed. They offer several attractive features for
various mammalian cells (Friedmann, 1989; Ridgeway, 1988; Baichwal
and Sugden, 1986; Coupar et al., 1988; Horwich et al., 1990).
[0167] With the recent recognition of defective hepatitis B
viruses, new insight was gained into the structure-function
relationship of different viral sequences. In vitro studies showed
that the virus could retain the ability for helper-dependent
packaging and reverse transcription despite the deletion of up to
80% of its genome (Horwich et al., 1990). This suggested that large
portions of the genome could be replaced with foreign genetic
material. Chang et al. recently introduced the chloramphenicol
acetyltransferase (CAT) gene into duck hepatitis B virus genome in
the place of the polymerase, surface, and/or pre-surface coding
sequences. It was cotransfected with wild-type virus into an avian
hepatoma cell line. Culture media containing high titers of the
recombinant virus were used to infect primary duckling hepatocytes.
Stable CAT gene expression was detected for at least 24 days after
transfection (Chang et al., 1991).
[0168] In certain further embodiments, the gene therapy vector will
be HSV. A factor that makes HSV an attractive vector is the size
and organization of the genome. Because HSV is large, incorporation
of multiple genes and expression cassettes is less problematic than
in other smaller viral systems. In addition, the availability of
different viral control sequences with varying performance
(temporal, strength, etc.) makes it possible to control expression
to a greater extent than in other systems. It also is an advantage
that the virus has relatively few spliced messages, further easing
genetic manipulations. HSV also is relatively easy to manipulate
and/or can be grown to high titers. Thus, delivery is less of a
problem, both in terms of volumes needed to attain sufficient MOI
and in a lessened need for repeat dosings.
[0169] 5. Modified Viruses
[0170] In still further embodiments of the present invention, the
nucleic acids to be delivered are housed within an infective virus
that has been engineered to express a specific binding ligand. The
virus particle will thus bind specifically to the cognate receptors
of the target cell and deliver the contents to the cell. A novel
approach designed to allow specific targeting of retrovirus vectors
was recently developed based on the chemical modification of a
retrovirus by the chemical addition of lactose residues to the
viral envelope. This modification can permit the specific infection
of hepatocytes via sialoglycoprotein receptors.
[0171] Another approach to targeting of recombinant retroviruses
was designed in which biotinylated antibodies against a retroviral
envelope protein and/or against a specific cell receptor were used.
The antibodies were coupled via the biotin components by using
streptavidin (Roux et al., 1989). Using antibodies against major
histocompatibility complex class I and class II antigens, they
demonstrated the infection of a variety of human cells that bore
those surface antigens with an ecotropic virus in vitro (Roux et
al., 1989).
[0172] B. Other Methods of DNA Delivery
[0173] In various embodiments of the invention, DNA is delivered to
a cell as an expression construct. In order to effect expression of
a gene construct, the expression construct must be delivered into a
cell. As described herein, the preferred mechanism for delivery is
via viral infection, where the expression construct is encapsidated
in an infectious viral particle. However, several non-viral methods
for the transfer of expression constructs into cells also are
contemplated by the present invention. In one embodiment of the
present invention, the expression construct may consist only of
naked recombinant DNA and/or plasmids. Transfer of the construct
may be performed by any of the methods mentioned which physically
and/or chemically permeabilize the cell membrane. Some of these
techniques may be successfully adapted for in vivo and/or ex vivo
use, as discussed below.
[0174] C. Liposome-Mediated Transfection
[0175] In a further embodiment of the invention, the expression
construct may be entrapped in a liposome. Liposomes are vesicular
structures characterized by a phospholipid bilayer membrane and/or
an inner aqueous medium. Multilamellar liposomes have multiple
lipid layers separated by aqueous medium. They form spontaneously
when phospholipids are suspended in an excess of aqueous solution.
The lipid components undergo self-rearrangement before the
formation of closed structures and/or entrap water and/or dissolved
solutes between the lipid bilayers (Ghosh and Bachhawat, 1991).
Also contemplated is an expression construct complexed with
Lipofectamine (Gibco BRL).
[0176] Liposome-mediated nucleic acid delivery and expression of
foreign DNA in vitro has been very successful (Nicolau and Sene,
1982; Fraley et al., 1979; Nicolau et al., 1987). Wong et al.
(1980) demonstrated the feasibility of liposome-mediated delivery
and/or expression of foreign DNA in cultured chick embryo, HeLa and
hepatoma cells.
[0177] In certain embodiments of the invention, the liposome may be
complexed with a hemagglutinating virus (HVJ). This has been shown
to facilitate fusion with the cell membrane and/or promote cell
entry of liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the liposome may be complexed and/or employed in
conjunction with nuclear non-histone chromosomal proteins (HMG-1)
(Kato et al., 1991). In yet further embodiments, the liposome may
be complexed and/or employed in conjunction with both HVJ and
HMG-1. In other embodiments, the delivery vehicle may comprise a
ligand and a liposome. Where a bacterial promoter is employed in
the DNA construct, it also will be desirable to include within the
liposome an appropriate bacterial polymerase.
[0178] The inventors contemplate that neu-suppressing gene products
can be introduced into cells using liposome-mediated gene transfer.
It is proposed that such constructs can be coupled with liposomes
and directly introduced via a catheter, as described by Nabel et
al. (1990). By employing these methods, the neu-suppressing gene
products can be expressed efficiently at a specific site in vivo,
not just the liver and spleen cells which are accessible via
intravenous injection. Therefore, this invention also encompasses
compositions of DNA constructs encoding a neu-suppressing gene
product formulated as a DNA/liposome complex and methods of using
such constructs.
[0179] As described in U.S. Pat. No. 5,641,484, liposomes are
particularly well suited for the treatment of HER2/neu-mediated
cancer
[0180] a. Preparation of Liposomes
[0181] Catatonic liposomes that are efficient transfection reagents
for Bik for animal cells can be prepared using the method of Gao et
al. (1991). Gao et al. describes a novel catatonic cholesterol
derivative that can be synthesized in a single step. Liposomes made
of this lipid are reportedly more efficient in transfection and
less toxic to treated cells than those made with the reagent
Lipofectin. These lipids are a mixture of DC-Chol
("3.quadrature.(N--(N'N'-dimethylaminoethane)-carbamoyl
cholesterol") and DOPE ("dioleoylphosphatidylethanolamine"). The
steps in producing these liposomes are as follows.
[0182] DC-Chol is synthesized by a simple reaction from cholesteryl
chloroformate and N,N-Dimethylethylenediamine. A solution of
cholesteryl chloroformate (2.25 g, 5 mmol in 5 ml dry chloroform)
is added dropwise to a solution of excess
N,N-Dimethylethylenediamine (2 ml, 18.2 mmol in 3 ml dry
chloroform) at 0.degree. C. Following removal of the solvent by
evaporation, the residue is purified by recrystallization in
absolute ethanol at 4.degree. C. and dried in vacuo. The yield is a
white powder of DC-Chol.
[0183] Cationic liposomes are prepared by mixing 1.2
.quadrature.mol of DC-Chol and 8.0 .quadrature.mol of DOPE in
chloroform. This mixture is then dried, vacuum desiccated, and
resuspended in 1 ml sterol 20 mM Hepes buffer (pH 7.8) in a tube.
After 24 hours of hydration at 4.degree. C., the dispersion is
sonicated for 5-10 minutes in a sonicator form liposomes with an
average diameter of 150-200 nm.
[0184] To prepare a liposome/DNA complex, the inventors use the
following steps. The DNA to be transfected is placed in DMEM/F12
medium in a ratio of 15 .mu.g DNA to 50 .mu.l DMEM/F12. DMEM/F12 is
then used to dilute the DC-Chol/DOPE liposome mixture to a ratio of
50 .mu.l DMEZM/F12 to 100 .mu.l liposome. The DNA dilution and the
liposome dilution are then gently mixed, and incubated at
37.degree. C. for 10 minutes. Following incubation, the
DNA/liposome complex is ready for injection.
[0185] Liposomal transfection can be via liposomes composed of, for
example, phosphatidylcholine (PC), phosphatidylserine (PS),
cholesterol (Chol),
N-[1-(2,3-dioleyloxy)propyl]-N,N-trimethylammonium chloride
(DOTMA), dioleoylphosphatidylethanolamine (DOPE), and/or
3.beta.[N--(N'N'-dimethylaminoethane)-carbarmoyl cholesterol
(DC-Chol), as well as other lipids known to those of skill in the
art. Those of skill in the art will recognize that there are a
variety of liposomal transfection techniques that will be useful in
the present invention. Among these techniques are those described
in Nicolau et al., 1987, Nabel et al., 1990, and Gao et al., 1991.
In a specific embodiment, the liposomes comprise DC-Chol. More
particularly, the inventors the liposomes comprise DC-Chol and DOPE
that have been prepared following the teaching of Gao et al. (1991)
in the manner described in the Preferred Embodiments Section. The
inventors also anticipate utility for liposomes comprised of DOTMA,
such as those that are available commercially under the trademark
Lipofectin.TM., from Vical, Inc., in San Diego, Calif.
[0186] Liposomes may be introduced into contact with cells to be
transfected by a variety of methods. In cell culture, the
liposome-DNA complex can simply be dispersed in the cell culture
solution. For application in vivo, liposome-DNA complex are
typically injected. Intravenous injection allow liposome-mediated
transfer of DNA complex, for example, the liver and the spleen. In
order to allow transfection of DNA into cells that are not
accessible through intravenous injection, it is possible to
directly inject the liposome-DNA complexes into a specific location
in an animal's body. For example, Nabel et al. teach injection via
a catheter into the arterial wall. In another example, the
inventors have used intraperitoneal injection to allow for gene
transfer into mice.
[0187] The present invention also contemplates compositions
comprising a liposomal complex. This liposomal complex will
comprise a lipid component and a DNA segment encoding a nucleic
acid encoding a mutant form of Bik. The nucleic acid encoding the
mutant form of Bik employed in the liposomal complex can be, for
example, one that encodes Bik-T145A or Bik-T145D.
[0188] The lipid employed to make the liposomal complex can be any
of the above-discussed lipids. In particular, DOTMA, DOPE, and/or
DC-Chol may form all or part of the liposomal complex. The
inventors have had particular success with complexes comprising
DC-Chol. In a preferred embodiment, the lipid will comprise DC-Chol
and DOPE. While any ratio of DC-Chol to DOPE is anticipated to have
utility, it is anticipated that those comprising a ratio of
DC-Chol:DOPE between 1:20 and 20:1 will be particularly
advantageous. The inventors have found that liposomes prepared from
a ratio of DC-Chol:DOPE of about 1:10 to about 1:5 have been
useful.
[0189] In a specific embodiment, one employs the smallest region
needed to enhance retention of Bik in the nucleus of a cell so that
one is not introducing unnecessary DNA into cells which receive a
Bik gene construct. Techniques well known to those of skill in the
art, such as the use of restriction enzymes, will allow for the
generation of small regions of Bik. The ability of these regions to
inhibit neu can easily be determined by the assays reported in the
Examples.
[0190] In certain embodiments of the invention, the liposome may be
complexed with a hemagglutinatin virus (HVJ). This has been shown
to facilitate fusion with the cell membrane and promote cell entry
of liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the liposome may be complexed or employed in
conjunction with nuclear non-histone chromosomal proteins (HMG-1)
(Kato et al., 1991). In yet further embodiments, the liposome may
be complexed or employed in conjunction with both HVJ and HMG-1. In
that such expression constructs have been successfully employed in
transfer and expression of nucleic acid in vitro and in vivo, then
they are applicable for the present invention. Where a bacterial
promoter is employed in the DNA construct, it also will be
desirable to include within the liposome an appropriate bacterial
polymerase.
[0191] b. In Vivo Treatment of Cancer Via Liposomes with Bik
Mutants
[0192] Based on the teachings provided herein, a skilled artisan
recognizes that any cell may be treated with at least one Bik
mutant, and in particular embodiments, any cancer cell may be
treated with such. For example, in some embodiments the nature of
the treated cell is irrespective of being HER2/neu-positive or
HER2/neu-negative. However, in one specific embodiment it is
HER2/neu-positive.
[0193] U.S. Pat. No. 5,641,484, incorporated in its entirety by
reference herein, teaches that liposome-mediated direct gene
transfer techniques can be employed to obtain suppression of
HER2/neu-overexpressing human cancer cells in living host. The
protocol for described therein was as follows. Female nude mice
(5-6 weeks old) were given intraperitoneal injections of SK-OV-3
cells (2.times.106/100 .mu.l). SK-OV-3 cells are human ovarian
cancer cells that have been shown to grow within the peritoneal
cavity of nude mice. After five days, the mice were given
intraperitoneal injections of various compounds. Some mice were
injected with the therapeutic DNA alone, some were injected with
liposome/therapeutic DNA complex prepared in the manner described
above, and some were injected with liposome/mutant therapeutic DNA
complex. 200 .mu.l of a given compound was injected into a given
mouse. After the initial injections, injections were repeated every
seven days throughout the life of the mouse.
[0194] The results described therein indicate that
liposome-mediated gene transfer can inhibit HER2/neu-overexpressing
human ovarian cancer cell growth. Therefore, it is predictable that
liposome-mediated mutant Bik gene therapy may serve as a powerful
therapeutic agent for HER-2 neu-overexpressing human ovarian
cancers by direct targeting of mutant Bik at the HER-2
neu-oncogene.
[0195] c. Liposomal Transfection with Mutant Bik to Treat
Humans
[0196] Based on the results of the in vivo animal studies described
in U.S. Pat. No. 5,641,484, those of skill in the art will
understand and predict the enormous potential for human treatment
of HER2/neu-mediated cancers with Bik T33D, S35D, and/or T33DS35D
DNA complexed to liposomes. Clinical studies to demonstrate these
affects are contemplated. Those of skill in the art will recognize
that the best treatment regimens for using Bik T33D, S35D, and/or
T33DS35D to suppress HER2/neu-mediated cancers can be
straightforwardly determined. This is not a question of
experimentation, but rather one of optimization, which is routinely
conducted in the medical arts. In vivo studies in nude mice provide
a starting point from which to begin to optimize the dosage and
delivery regimes. The frequency of injection is initially once a
week, as was done in the mice studies described in U.S. Pat. No.
5,641,484. However, this frequency might be optimally adjusted from
one day to every two weeks to monthly, depending upon the results
obtained from the initial clinical trials and the needs of a
particular patient. Human dosage amounts can initially be
determined by extrapolating from the amount of Bik T33D, S35D,
and/or T33DS35D used in mice, approximately 15 .mu.g of plasmid DNA
per 50 g body weight. Based on this, a 50 kg woman would require
treatment with 15 mg of DNA per dose. Of course, this dosage amount
may be adjusted upward or downward, as is routinely done in such
treatment protocols, depending on the results of the initial
clinical trials and the needs of a particular patient. These
clinical trials are anticipated to show utility of Bik T33D, S35D,
and/or T33DS35D and other neu-suppressing gene products for the
treatment of HER2/neu-overexpressing cancers in humans. Dosage and
frequency regimes will initially be based on the data obtained from
in vivo animal studies, as is done frequently in the art.
[0197] D. Electroporation
[0198] In certain embodiments of the present invention, the
expression construct is introduced into the cell via
electroporation. Electroporation involves the exposure of a
suspension of cells and/or DNA to a high-voltage electric
discharge.
[0199] Transfection of eukaryotic cells using electroporation has
been quite successful. Mouse pre-B lymphocytes have been
transfected with humankappa-immunoglobulin genes (Potter et al.,
1984), and/or rat hepatocytes have been transfected with the
chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986) in
this manner.
[0200] E. Calcium Phosphate and/or DEAE-Dextran
[0201] In other embodiments of the present invention, the
expression construct is introduced to the cells using calcium
phosphate precipitation. HumanKB cells have been transfected with
adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this
technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1,
BHK, NIH3T3 and/or HeLa cells were transfected with a neomycin
marker gene (Chen and Okayama, 1987), and/or rat hepatocytes were
transfected with a variety of marker genes (Rippe et al.,
1990).
[0202] In another embodiment, the expression construct is delivered
into the cell using DEAE-dextran followed by polyethylene glycol.
In this manner, reporter plasmids were introduced into mouse
myeloma and/or erythroleukemia cells (Gopal, 1985).
[0203] F. Particle Bombardment
[0204] Another embodiment of the invention for transferring a naked
DNA expression construct into cells may involve particle
bombardment. This method depends on the ability to accelerate
DNA-coated microprojectiles to a high velocity allowing them to
pierce cell membranes and/or enter cells without killing them
(Klein et al., 1987). Several devices for accelerating small
particles have been developed. One such device relies on a high
voltage discharge to generate an electrical current, which in turn
provides the motive force (Yang et al., 1990). The microprojectiles
used have consisted of biologically inert substances such as
tungsten and/or gold beads.
[0205] G. Direct Microinjection and/or Sonication Loading
[0206] Further embodiments of the present invention include the
introduction of the expression construct by direct microinjection
and/or sonication loading. Direct microinjection has been used to
introduce nucleic acid constructs into Xenopus oocytes (Harland and
Weintraub, 1985), and/or LTK-fibroblasts have been transfected with
the thymidine kinase gene by sonication loading (Fechheimer et al.,
1987).
[0207] H. Adenoviral Assisted Transfection
[0208] In certain embodiments of the present invention, the
expression construct is introduced into the cell using adenovirus
assisted transfection. Increased transfection efficiencies have
been reported in cell systems using adenovirus coupled systems
(Kelleher and Vos, 1994; Cotten et al., 1992; Curiel, 1994).
V. Combination Treatments
[0209] In order to increase the effectiveness of a mutant form of
Bik, or expression construct coding therefore, it may be desirable
to combine these compositions with other agents effective in the
treatment of hyperproliferative disease, such as anti-cancer
agents. An "anti-cancer" agent is capable of negatively affecting
cancer in a subject, for example, by killing cancer cells, inducing
apoptosis in cancer cells, reducing the growth rate of cancer
cells, reducing the incidence or number of metastases, reducing
tumor size, inhibiting tumor growth, reducing the blood supply to a
tumor or cancer cells, promoting an immune response against cancer
cells or a tumor, preventing or inhibiting the progression of
cancer, or increasing the lifespan of a subject with cancer. More
generally, these other compositions would be provided in a combined
amount effective to kill or inhibit proliferation of the cell. This
process may involve contacting the cells with the expression
construct and the agent(s) or multiple factor(s) at the same time.
This may be achieved by contacting the cell with a single
composition or pharmacological formulation that includes both
agents, or by contacting the cell with two distinct compositions or
formulations, at the same time, wherein one composition includes
the expression construct and the other includes the second
agent(s).
[0210] Tumor cell resistance to chemotherapy and radiotherapy
agents represents a major problem in clinical oncology. One goal of
current cancer research is to find ways to improve the efficacy of
chemo- and radiotherapy by combining it with gene therapy. For
example, the herpes simplex-thymidine kinase (HS-tK) gene, when
delivered to brain tumors by a retroviral vector system,
successfully induced susceptibility to the antiviral agent
ganciclovir (Culver et al., 1992). In the context of the present
invention, it is contemplated that Bik gene therapy could be used
similarly in conjunction with chemotherapeutic, radiotherapeutic,
or immunotherapeutic intervention, in addition to other
pro-apoptotic or cell cycle regulating agents.
[0211] Alternatively, the gene therapy may precede or follow the
other agent treatment by intervals ranging from minutes to weeks.
In embodiments where the other agent and expression construct are
applied separately to the cell, one would generally ensure that a
significant period of time did not expire between the time of each
delivery, such that the agent and expression construct would still
be able to exert an advantageously combined effect on the cell. In
such instances, it is contemplated that one may contact the cell
with both modalities within about 12-24 h of each other and, more
preferably, within about 6-12 h of each other. In some situations,
it may be desirable to extend the time period for treatment
significantly, however, where several d (2, 3, 4, 5, 6 or 7) to
several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective
administrations.
[0212] Various combinations may be employed, gene therapy is "A"
and the secondary agent, such as radio- or chemotherapy, is
"B":
TABLE-US-00004 A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/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
A/A/A/B B/A/A/A A/B/A/A A/A/B/A
[0213] Administration of the therapeutic expression constructs of
the present invention to a patient will follow general protocols
for the administration of chemotherapeutics, taking into account
the toxicity, if any, of the vector. It is expected that the
treatment cycles would be repeated as necessary. It also is
contemplated that various standard therapies, as well as surgical
intervention, may be applied in combination with the described
hyperproliferative cell therapy.
[0214] A. Chemotherapy
[0215] A skilled artisan recognizes that in addition to the Bik
mutant forms described herein for the purpose of inhibiting cell
growth, other chemotherapeutic agents are useful in the treatment
of neoplastic disease. Examples of such chemotherapeutic agents are
described in the following Table 4.
[0216] Cancer therapies include a variety of combination therapies
with both chemical and radiation based treatments. Exemplary
embodiments include, for example, cisplatin (CDDP), carboplatin,
procarbazine, mechlorethamine, cyclophosphamide, camptothecin,
ifosfamide, melphalan, chlorambucil, busulfan, nitrosurea,
dactinomycin, daunorubicin, doxorubicin, bleomycin, plicomycin,
mitomycin, etoposide (VP16), tamoxifen, raloxifene, estrogen
receptor binding agents, taxol, gemcitabien, navelbine,
farnesyl-protein tansferase inhibitors, transplatinum,
5-fluorouracil, vincristin, vinblastin and methotrexate, or any
analog or derivative variant of the foregoing.
TABLE-US-00005 TABLE 4 Chemotherapeutic Agents Useful In Neoplastic
Disease NONPROPRIETARY TYPE OF NAMES CLASS AGENT (OTHER NAMES)
DISEASE Alkylating Nitrogen Mechlorethamine Hodgkin's disease,
Agents Mustards (HN2) non-Hodgkin's lymphomas Cyclophosphamide
Acute and chronic Ifosfamide lymphocytic leukemias, Hodgkin's
disease, non-Hodgkin's lymphomas, multiple myeloma, neuroblastoma,
breast, ovary, lung, Wilms' tumor, cervix, testis, soft-tissue
sarcomas Melphalan Multiple myeloma, (l-sarcolysin) breast, ovary
Chlorambucil Chronic lymphocytic leukemia, primary
macroglobulinemia, Hodgkin's disease, non-Hodgkin's lymphomas
Ethylenimenes Hexamethylmelamine Ovary and Thiotepa Bladder,
breast, ovary Methylmelamines Alkyl Busulfan Chronic granulocytic
Sulfonates leukemia Nitrosoureas Carmustine Hodgkin's disease,
(BCNU) non-Hodgkin's lymphomas, primary brain tumors, multiple
myeloma, malignant melanoma Lomustine (CCNU) Hodgkin's disease,
non-Hodgkin's lymphomas, primary brain tumors, small-cell lung
Semustine Primary brain tumors, (methyl-CCNU) stomach, colon
Streptozocin Malignant pancreatic (streptozotocin) insulinoma,
malignant carcinoid Triazines Dacarbazine (DTIC; Malignant
melanoma, dimethyltriazenoimi Hodgkin's disease, dazolecarboxamide)
soft-tissue sarcomas Antimetabolites Folic Acid Methotrexate Acute
lymphocytic Analogs (amethopterin) leukemia, choriocarcinoma,
mycosis fungoides, breast, head and neck, lung, osteogenic sarcoma
Pyrimidine Fluouracil Breast, colon, stomach, Analogs
(5-fluorouracil; pancreas, ovary, head 5-FU) and neck, urinary
Floxuridine bladder, premalignant (fluorode-oxyuridine; skin
lesions (topical) FUdR) Cytarabine (cytosine Acute granulocytic and
arabinoside) acute lymphocytic leukemias Purine Analogs
Mercaptopurine Acute lymphocytic, acute and Related
(6-mercaptopurine; granulocytic and chronic Inhibitors 6-MP)
granulocytic leukemias Thioguanine Acute granulocytic,
(6-thioguanine; TG) acute lymphocytic and chronic granulocytic
leukemias Pentostatin Hairy cell leukemia, (2-deoxycoformycin)
mycosis fungoides, chronic lymphocytic leukemia Vinca Vinblastine
(VLB) Hodgkin's disease, Alkaloids non-Hodgkin's lymphomas, breast,
testis Vincristine Acute lymphocytic leukemia, neuroblastoma,
Wilms' tumor, rhabdomyosarcoma, Hodgkin's disease, non-Hodgkin's
lymphomas, small-cell lung Epipodophyllotoxins Etoposide Testis,
small-cell lung Tertiposide and other lung, breast, Hodgkin's
disease, non-Hodgkin's lymphomas, acute granulocytic leukemia,
Kaposi's sarcoma Natural Antibiotics Dactinomycin Choriocarcinoma,
Products (actinomycin D) Wilms' tumor, rhabdomyosarcoma, testis,
Kaposi's sarcoma Daunorubicin Acute granulocytic and (daunomycin;
acute lymphocytic rubidomycin) leukemias Doxorubicin Soft-tissue,
osteogenic and other sarcomas; Hodgkin's disease, non-Hodgkin's
lymphomas, acute leukemias, breast, genitourinary, thyroid, lung,
stomach, neuroblastoma Bleomycin Testis, head and neck, skin,
esophagus, lung and genitourinary tract; Hodgkin's disease,
non-Hodgkin's lymphomas Plicamycin Testis, malignant (mithramycin)
hypercalcemia Mitomycin Stomach, cervix, colon, (mitomycin C)
breast, pancreas, bladder, head and neck Enzymes 1-Asparaginase
Acute lymphocytic leukemia Biological Interferon alfa Hairy cell
leukemia., Response Kaposi's sarcoma, Modifiers melanoma,
carcinoid, renal cell, ovary, bladder, non-Hodgkin's lymphomas,
mycosis fungoides, multiple myeloma, chronic granulocytic leukemia
Miscellaneous Platinum Cisplatin (cis-DDP) Testis, ovary, bladder,
Agents Coordination Carboplatin head and neck, lung, Complexes
thyroid, cervix, endometrium, neuroblastoma, osteogenic sarcoma
Anthracenedione Mitoxantrone Acute granulocytic leukemia, breast
Substituted Hydroxyurea Chronic granulocytic Urea leukemia,
polycythemia vera, essental thrombocytosis, malignant melanoma
Methyl Procarbazine Hodgkin's disease Hydrazine (N-methylhydrazine,
Derivative MIH) Adrenocortical Mitotane Adrenal cortex (o,p'-DDD)
Suppressant Aminoglutethimide Breast Hormones Adrenocorticosteroids
Prednisone (several Acute and chronic and other equivalent
lymphocytic leukemias, Antagonists preparations non-Hodgkin's
available) lymphomas, Hodgkin's disease, breast Progestins
Hydroxyprogesterone Endometrium, breast caproate
Medroxyprogesterone acetate Megestrol acetate Estrogens
Diethylstilbestrol Breast, prostate Ethinyl estradiol (other
preparations available) Antiestrogen Tamoxifen Breast Androgens
Testosterone Breast propionate Fluoxymesterone (other preparations
available) Antiandrogen Flutamide Prostate Gonadotropin- Leuprolide
Prostate releasing hormone analog
[0217] B. Radiotherapy
[0218] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .quadrature.-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 on DNA, on the
precursors of DNA, on the replication and repair of DNA, and on 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 wk), 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.
[0219] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
construct and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing or stasis, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0220] C. Immunotherapy
[0221] Immunotherapeutics, generally, rely on the use of immune
effector cells and molecules to target and destroy cancer cells.
The immune effector may be, for example, an antibody specific for
some marker on the surface of a tumor cell. The antibody alone may
serve as an effector of therapy or it may recruit other cells to
actually effect cell killing. The antibody also may be conjugated
to a drug or toxin (chemotherapeutic, radionuclide, ricin A chain,
cholera toxin, pertussis toxin, etc.) and serve merely as a
targeting agent. Alternatively, the effector may be a lymphocyte
carrying a surface molecule that interacts, either directly or
indirectly, with a tumor cell target. Various effector cells
include cytotoxic T cells and NK cells.
[0222] Immunotherapy, thus, could be used as part of a combined
therapy, in conjunction with Ad-Bik gene therapy. The general
approach for combined therapy is discussed below. Generally, the
tumor cell must bear some marker that is amenable to targeting,
i.e., is not present on the majority of other cells. Many tumor
markers exist and any of these may be suitable for targeting in the
context of the present invention. Common tumor markers include
carcinoembryonic antigen, prostate specific antigen, urinary tumor
associated antigen, fetal antigen, tyrosinase (p97), gp68, TAG-72,
HMFG, Sialyl Lewis Antigen, MucA, MucB, PLAP, estrogen receptor,
laminin receptor, erb B and p155.
[0223] D. Genes
[0224] In yet another embodiment, the secondary treatment is a
secondary gene therapy in which a second therapeutic polynucleotide
is administered before, after, or at the same time a first
therapeutic polynucleotide encoding all of part of a mutant form of
Bik. Delivery of a vector encoding either a full length or
truncated mutant form of Bik in conjunction with a second vector
encoding one of the following gene products will have a combined
anti-hyperproliferative effect on target tissues. Alternatively, a
single vector encoding both genes may be used. A variety of
proteins are encompassed within the invention, some of which are
described below.
[0225] 1. Inducers of Cellular Proliferation
[0226] The proteins that induce cellular proliferation further fall
into various categories dependent on function. The commonality of
all of these proteins is their ability to regulate cellular
proliferation. For example, a form of PDGF, the sis oncogene, is a
secreted growth factor. Oncogenes rarely arise from genes encoding
growth factors, and at the present, sis is the only known
naturally-occurring oncogenic growth factor. In one embodiment of
the present invention, it is contemplated that anti-sense mRNA
directed to a particular inducer of cellular proliferation is used
to prevent expression of the inducer of cellular proliferation.
[0227] The proteins FMS, ErbA, ErbB and neu are growth factor
receptors. Mutations to these receptors result in loss of
regulatable function. For example, a point mutation affecting the
transmembrane domain of the Neu receptor protein results in the neu
oncogene. The erbA oncogene is derived from the intracellular
receptor for thyroid hormone. The modified oncogenic ErbA receptor
is believed to compete with the endogenous thyroid hormone
receptor, causing uncontrolled growth.
[0228] The largest class of oncogenes includes the signal
transducing proteins (e.g., Src, Abl and Ras). The protein Src is a
cytoplasmic protein-tyrosine kinase, and its transformation from
proto-oncogene to oncogene in some cases, results via mutations at
tyrosine residue 527. In contrast, transformation of GTPase protein
ras from proto-oncogene to oncogene, in one example, results from a
valine to glycine mutation at amino acid 12 in the sequence,
reducing ras GTPase activity.
[0229] The proteins Jun, Fos and Myc are proteins that directly
exert their effects on nuclear functions as transcription
factors.
[0230] 2. Inhibitors of Cellular Proliferation
[0231] The tumor suppressor oncogenes function to inhibit excessive
cellular proliferation. The inactivation of these genes destroys
their inhibitory activity, resulting in unregulated proliferation.
The tumor suppressors p53, p16 and C-CAM are described below.
[0232] High levels of mutant p53 have been found in many cells
transformed by chemical carcinogenesis, ultraviolet radiation, and
several viruses. The p53 gene is a frequent target of mutational
inactivation in a wide variety of human tumors and is already
documented to be the most frequently mutated gene in common human
cancers. It is mutated in over 50% of human NSCLC (Hollstein et
al., 1991) and in a wide spectrum of other tumors.
[0233] The p53 gene encodes a 393-amino acid phosphoprotein that
can form complexes with host proteins such as large-T antigen and
E1B. The protein is found in normal tissues and cells, but at
concentrations which are minute by comparison with transformed
cells or tumor tissue.
[0234] Wild-type p53 is recognized as an important growth regulator
in many cell types. Missense mutations are common for the p53 gene
and are essential for the transforming ability of the oncogene. A
single genetic change prompted by point mutations can create
carcinogenic p53. Unlike other oncogenes, however, p53 point
mutations are known to occur in at least 30 distinct codons, often
creating dominant alleles that produce shifts in cell phenotype
without a reduction to homozygosity. Additionally, many of these
dominant negative alleles appear to be tolerated in the organism
and passed on in the germ line. Various mutant alleles appear to
range from minimally dysfunctional to strongly penetrant, dominant
negative alleles (Weinberg, 1991).
[0235] Another inhibitor of cellular proliferation is p16. The
major transitions of the eukaryotic cell cycle are triggered by
cyclin-dependent kinases, or CDK's. One CDK, cyclin-dependent
kinase 4 (CDK4), regulates progression through the G1. The activity
of this enzyme may be to phosphorylate Rb at late G1. The activity
of CDK4 is controlled by an activating subunit, D-type cyclin, and
by an inhibitory subunit, the p16INK4 has been biochemically
characterized as a protein that specifically binds to and inhibits
CDK4, and thus may regulate Rb phosphorylation (Serrano et al.,
1993; Serrano et al., 1995). Since the p16INK4 protein is a CDK4
inhibitor (Serrano, 1993), deletion of this gene may increase the
activity of CDK4, resulting in hyperphosphorylation of the Rb
protein. p16 also is known to regulate the function of CDK6.
[0236] p16INK4 belongs to a newly described class of CDK-inhibitory
proteins that also includes p16B, p19, p21Waf1/Cip1, and p27KIP1.
The p16INK4 gene maps to 9Bik, a chromosome region frequently
deleted in many tumor types. Homozygous deletions and mutations of
the p16INK4 gene are frequent in human tumor cell lines. This
evidence suggests that the p16INK4 gene is a tumor suppressor gene.
This interpretation has been challenged, however, by the
observation that the frequency of the p16INK4 gene alterations is
much lower in primary uncultured tumors than in cultured cell lines
(Caldas et al., 1994; Cheng et al., 1994; Hussussian et al., 1994;
Kamb et al., 1994; Kamb et al., 1994; Mori et al., 1994; Okamoto et
al., 1994; Nobori et al., 1995; Orlow et al., 1994; Arap et al.,
1995). Restoration of wild-type p16INK4 function by transfection
with a plasmid expression vector reduced colony formation by some
human cancer cell lines (Okamoto, 1994; Arap, 1995).
[0237] Other genes that may be employed according to the present
invention include Rb, APC, DCC, NF-1, NF-2, WT-1, MEN-I, MEN-II,
zac1, p73, VHL, MMAC1/PTEN, DBCCR-1, FCC, rsk-3, p27, p27/p16
fusions, Bik/p27 fusions, anti-thrombotic genes (e.g., COX-1,
TFPI), PGS, Dp, E2F, ras, myc, neu, raf, erb, fms, trk, ret, gsp,
hst, abl, E1A, p300, genes involved in angiogenesis (e.g., VEGF,
FGF, thrombospondin, BAI-1, GDAIF, or their receptors) and MCC.
[0238] 3. Regulators of Programmed Cell Death
[0239] Apoptosis, or programmed cell death, is an essential process
for normal embryonic development, maintaining homeostasis in adult
tissues, and suppressing carcinogenesis (Kerr et al., 1972). The
Bcl-2 family of proteins and ICE-like proteases have been
demonstrated to be important regulators and effectors of apoptosis
in other systems. The Bcl-2 protein, discovered in association with
follicular lymphoma, plays a prominent role in controlling
apoptosis and enhancing cell survival in response to diverse
apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar, 1985;
Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce,
1986). The evolutionarily conserved Bcl-2 protein now is recognized
to be a member of a family of related proteins, which can be
categorized as death agonists or death antagonists.
[0240] Subsequent to its discovery, it was shown that Bcl-2 acts to
suppress cell death triggered by a variety of stimuli. Also, it now
is apparent that there is a family of Bcl-2 cell death regulatory
proteins which share in common structural and sequence homologies.
These different family members have been shown to either possess
similar functions to Bcl-2 (e.g., BclXL, BclW, BclS, Mcl-1, A1,
Bfl-1) or counteract Bcl-2 function and promote cell death (e.g.,
Bax, Bak, Bik, Bim, Bid, Bad, Harakiri).
[0241] E. Surgery
[0242] Approximately 60% of persons with cancer will undergo
surgery of some type, which includes preventative, diagnostic or
staging, curative and palliative surgery. Curative surgery is a
cancer treatment that may be used in conjunction with other
therapies, such as the treatment of the present invention,
chemotherapy, radiotherapy, hormonal therapy, gene therapy,
immunotherapy and/or alternative therapies.
[0243] Curative surgery includes resection in which all or part of
cancerous tissue is physically removed, excised, and/or destroyed.
Tumor resection refers to physical removal of at least part of a
tumor. In addition to tumor resection, treatment by surgery
includes laser surgery, cryosurgery, electrosurgery, and
miscopically controlled surgery (Mohs' surgery). It is further
contemplated that the present invention may be used in conjunction
with removal of superficial cancers, precancers, or incidental
amounts of normal tissue.
[0244] Upon excision of part of all of cancerous cells, tissue, or
tumor, a cavity may be formed in the body. Treatment may be
accomplished by perfusion, direct injection or local application of
the area with an additional anti-cancer therapy. Such treatment may
be repeated, for example, every 1, 2, 3, 4, 5, 6, or 7 days, or
every 1, 2, 3, 4, and 5 weeks or every 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, or 12 months. These treatments may be of varying dosages as
well.
[0245] F. Other Agents
[0246] It is contemplated that other agents may be used in
combination with the present invention to improve the therapeutic
efficacy of treatment. These additional agents include
immunomodulatory agents, agents that affect the upregulation of
cell surface receptors and GAP junctions, cytostatic and
differentiation agents, inhibitors of cell adhesion, or agents that
increase the sensitivity of the hyperproliferative cells to
apoptotic inducers. Immunomodulatory agents include tumor necrosis
factor; interferon alpha, beta, and gamma; IL-2 and other
cytokines; F42K and other cytokine analogs; or MIP-1, MIP-1beta,
MCP-1, RANTES, and other chemokines. It is further contemplated
that the upregulation of cell surface receptors or their ligands
such as Fas/Fas ligand, DR4 or DR5/TRAIL would potentiate the
apoptotic inducing abilities of the present invention by
establishment of an autocrine or paracrine effect on
hyperproliferative cells. Increases intercellular signaling by
elevating the number of GAP junctions would increase the
anti-hyperproliferative effects on the neighboring
hyperproliferative cell population. In other embodiments,
cytostatic or differentiation agents can be used in combination
with the present invention to improve the anti-hyerproliferative
efficacy of the treatments. Inhibitors of cell adhesion are
contemplated to improve the efficacy of the present invention.
Examples of cell adhesion inhibitors are focal adhesion kinase
(FAKs) inhibitors and Lovastatin. It is further contemplated that
other agents that increase the sensitivity of a hyperproliferative
cell to apoptosis, such as the antibody c225, could be used in
combination with the present invention to improve the treatment
efficacy.
[0247] Hormonal therapy may also be used in conjunction with the
present invention or in combination with any other cancer therapy
previously described. The use of hormones may be employed in the
treatment of certain cancers such as breast, prostate, ovarian, or
cervical cancer to lower the level or block the effects of certain
hormones such as testosterone or estrogen. This treatment is often
used in combination with at least one other cancer therapy as a
treatment option or to reduce the risk of metastases.
VI. Pharmaceutical Preparations
[0248] Pharmaceutical compositions of the present invention
comprise an effective amount of one or more forms of mutant Bik or
additional agent dissolved or dispersed in a pharmaceutically
acceptable carrier or excipient. The phrases "pharmaceutical or
pharmacologically acceptable" refers to molecular entities and
compositions that do not produce an adverse, allergic or other
untoward reaction when administered to an animal, such as, for
example, a human, as appropriate. The preparation of an
pharmaceutical composition that contains at least one Bik mutant
form or additional active ingredient will be known to those of
skill in the art in light of the present disclosure, as exemplified
by Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing
Company, 1990, incorporated herein by reference. Moreover, for
animal (e.g., human) administration, it will be understood that
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biological
Standards.
[0249] As used herein, "pharmaceutically acceptable carrier"
includes any and all solvents, dispersion media, coatings,
surfactants, antioxidants, preservatives (e.g., antibacterial
agents, antifungal agents), isotonic agents, absorption delaying
agents, salts, preservatives, drugs, drug stabilizers, binders,
excipients, disintegration agents, lubricants, sweetening agents,
flavoring agents, dyes, such like materials and combinations
thereof, as would be known to one of ordinary skill in the art
(see, for example, Remington's Pharmaceutical Sciences, 18th Ed.
Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by
reference). Except insofar as any conventional carrier is
incompatible with the active ingredient, its use in the therapeutic
or pharmaceutical compositions is contemplated. In a specific
embodiment, the mutant Bik composition is administered in a
liposome.
[0250] The Bik mutant form may comprise different types of carriers
depending on whether it is to be administered in solid, liquid or
aerosol form, and whether it need to be sterile for such routes of
administration as injection. The present invention can be
administered intravenously, intradermally, intraarterially,
intraperitoneally, intralesionally, intracranially,
intraarticularly, intraprostaticaly, intrapleurally,
intratracheally, intranasally, intravitreally, intravaginally,
rectally, topically, intratumorally, intramuscularly,
intraperitoneally, subcutaneously, intravesicularlly, mucosally,
intrapericardially, orally, topically, locally, using aerosol,
injection, infusion, continuous infusion, localized perfusion
bathing target cells directly, via a catheter, via a lavage, in
cremes, in lipid compositions (e.g., liposomes), or by other method
or any combination of the forgoing as would be known to one of
ordinary skill in the art (see, for example, Remington's
Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990,
incorporated herein by reference).
[0251] The actual dosage amount of a composition of the present
invention administered to an animal patient can be determined by
physical and physiological factors such as body weight, severity of
condition, the type of disease being treated, previous or
concurrent therapeutic interventions, idiopathy of the patient and
on the route of administration. The practitioner responsible for
administration will, in any event, determine the concentration of
active ingredient(s) in a composition and appropriate dose(s) for
the individual subject.
[0252] In certain embodiments, pharmaceutical compositions may
comprise, for example, at least about 0.1% of an active compound.
In other embodiments, the an active compound may comprise between
about 2% to about 75% of the weight of the unit, or between about
25% to about 60%, for example, and any range derivable therein. In
other non-limiting examples, a dose may also comprise from about 1
microgram/kg/body weight, about 5 microgram/kg/body weight, about
10 microgram/kg/body weight, about 50 microgram/kg/body weight,
about 100 microgram/kg/body weight, about 200 microgram/kg/body
weight, about 350 microgram/kg/body weight, about 500
microgram/kg/body weight, about 1 milligram/kg/body weight, about 5
milligram/kg/body weight, about 10 milligram/kg/body weight, about
50 milligram/kg/body weight, about 100 milligram/kg/body weight,
about 200 milligram/kg/body weight, about 350 milligram/kg/body
weight, about 500 milligram/kg/body weight, to about 1000
mg/kg/body weight or more per administration, and any range
derivable therein. In non-limiting examples of a derivable range
from the numbers listed herein, a range of about 5 mg/kg/body
weight to about 100 mg/kg/body weight, about 5 microgram/kg/body
weight to about 500 milligram/kg/body weight, etc., can be
administered, based on the numbers described above.
[0253] In any case, the composition may comprise various
antioxidants to retard oxidation of one or more component.
Additionally, the prevention of the action of microorganisms can be
brought about by preservatives such as various antibacterial and
antifungal agents, including but not limited to parabens (e.g.,
methylparabens, propylparabens), chlorobutanol, phenol, sorbic
acid, thimerosal or combinations thereof.
[0254] The Bik mutant form may be formulated into a composition in
a free base, neutral or salt form. Pharmaceutically acceptable
salts, include the acid addition salts, e.g., those formed with the
free amino groups of a proteinaceous composition, or which are
formed with inorganic acids such as for example, hydrochloric or
phosphoric acids, or such organic acids as acetic, oxalic, tartaric
or mandelic acid. 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; or such organic
bases as isopropylamine, trimethylamine, histidine or procaine.
[0255] In embodiments where the composition is in a liquid form, a
carrier can be a solvent or dispersion medium comprising but not
limited to, water, ethanol, polyol (e.g., glycerol, propylene
glycol, liquid polyethylene glycol, etc), lipids (e.g.,
triglycerides, vegetable oils, liposomes) and combinations thereof.
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 by dispersion in carriers such as, for example liquid
polyol or lipids; by the use of surfactants such as, for example
hydroxypropylcellulose; or combinations thereof such methods. In
many cases, it will be preferable to include isotonic agents, such
as, for example, sugars, sodium chloride or combinations
thereof.
[0256] In other embodiments, one may use eye drops, nasal solutions
or sprays, aerosols, mouthwashes, or inhalants in the present
invention. Such compositions are generally designed to be
compatible with the target tissue type. In a non-limiting example,
nasal solutions are usually aqueous solutions designed to be
administered to the nasal passages in drops or sprays. Nasal
solutions are prepared so that they are similar in many respects to
nasal secretions, so that normal ciliary action is maintained.
Thus, in preferred embodiments the aqueous nasal solutions usually
are isotonic or slightly buffered to maintain a pH of about 5.5 to
about 6.5. In addition, antimicrobial preservatives, similar to
those used in ophthalmic preparations, drugs, or appropriate drug
stabilizers, if required, may be included in the formulation. For
example, various commercial nasal preparations are known and
include drugs such as antibiotics or antihistamines.
[0257] In certain embodiments the Bik mutant form is prepared for
administration by such routes as oral ingestion. In these
embodiments, the solid composition may comprise, for example,
solutions, suspensions, emulsions, tablets, pills, capsules (e.g.,
hard or soft shelled gelatin capsules), sustained release
formulations, buccal compositions, troches, elixirs, suspensions,
syrups, wafers, or combinations thereof. Oral compositions may be
incorporated directly with the food of the diet. Preferred carriers
for oral administration comprise inert diluents, assimilable edible
carriers or combinations thereof. In other aspects of the
invention, the oral composition may be prepared as a syrup or
elixir. A syrup or elixir, and may comprise, for example, at least
one active agent, a sweetening agent, a preservative, a flavoring
agent, a dye, a preservative, or combinations thereof.
[0258] In certain preferred embodiments an oral composition may
comprise one or more binders, excipients, disintegration agents,
lubricants, flavoring agents, and combinations thereof. In certain
embodiments, a composition may comprise one or more of the
following: a binder, such as, for example, gum tragacanth, acacia,
cornstarch, gelatin or combinations thereof; an excipient, such as,
for example, dicalcium phosphate, mannitol, lactose, starch,
magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate or combinations thereof; a disintegrating agent, such as,
for example, corn starch, potato starch, alginic acid or
combinations thereof; a lubricant, such as, for example, magnesium
stearate; a sweetening agent, such as, for example, sucrose,
lactose, saccharin or combinations thereof; a flavoring agent, such
as, for example peppermint, oil of wintergreen, cherry flavoring,
orange flavoring, etc.; or combinations thereof the foregoing. When
the dosage unit form is a capsule, it may contain, in addition to
materials of the above type, carriers such as a liquid carrier.
Various other materials may be present as coatings or to otherwise
modify the physical form of the dosage unit. For instance, tablets,
pills, or capsules may be coated with shellac, sugar or both.
[0259] Additional formulations which are suitable for other modes
of administration include suppositories. Suppositories are solid
dosage forms of various weights and shapes, usually medicated, for
insertion into the rectum, vagina or urethra. After insertion,
suppositories soften, melt or dissolve in the cavity fluids. In
general, for suppositories, traditional carriers may include, for
example, polyalkylene glycols, triglycerides or combinations
thereof. In certain embodiments, suppositories may be formed from
mixtures containing, for example, the active ingredient in the
range of about 0.5% to about 10%, and preferably about 1% to about
2%.
[0260] 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 that contains the basic
dispersion medium and/or the other ingredients. In the case of
sterile powders for the preparation of sterile injectable
solutions, suspensions or emulsion, the preferred methods of
preparation are vacuum-drying or freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered liquid medium
thereof. The liquid medium should be suitably buffered if necessary
and the liquid diluent first rendered isotonic prior to injection
with sufficient saline or glucose. The preparation of highly
concentrated compositions for direct injection is also
contemplated, where the use of DMSO as solvent is envisioned to
result in extremely rapid penetration, delivering high
concentrations of the active agents to a small area.
[0261] The composition must be stable under the conditions of
manufacture and storage, and preserved against the contaminating
action of microorganisms, such as bacteria and fungi. It will be
appreciated that endotoxin contamination should be kept minimally
at a safe level, for example, less that 0.5 ng/mg protein.
[0262] In particular embodiments, prolonged absorption of an
injectable composition can be brought about by the use in the
compositions of agents delaying absorption, such as, for example,
aluminum monostearate, gelatin or combinations thereof.
VII. Site-Directed Mutagenesis
[0263] Structure-guided site-specific mutagenesis represents a
powerful tool for the dissection and engineering of protein-ligand
interactions (Wells, 1996, Braisted et al., 1996). The technique
provides for the preparation and testing of sequence variants by
introducing one or more nucleotide sequence changes into a selected
DNA.
[0264] Site-specific mutagenesis uses specific oligonucleotide
sequences which encode the DNA sequence of the desired mutation, as
well as a sufficient number of adjacent, unmodified nucleotides. In
this way, a primer sequence is provided with sufficient size and
complexity to form a stable duplex on both sides of the deletion
junction being traversed. A primer of about 17 to 25 nucleotides in
length is preferred, with about 5 to 10 residues on both sides of
the junction of the sequence being altered.
[0265] The technique typically employs a bacteriophage vector that
exists in both a single-stranded and double-stranded form. Vectors
useful in site-directed mutagenesis include vectors such as the M13
phage. These phage vectors are commercially available and their use
is generally well known to those skilled in the art.
Double-stranded plasmids are also routinely employed in
site-directed mutagenesis, which eliminates the step of
transferring the gene of interest from a phage to a plasmid.
[0266] In general, one first obtains a single-stranded vector, or
melts two strands of a double-stranded vector, which includes
within its sequence a DNA sequence encoding the desired protein or
genetic element. An oligonucleotide primer bearing the desired
mutated sequence, synthetically prepared, is then annealed with the
single-stranded DNA preparation, taking into account the degree of
mismatch when selecting hybridization conditions. The hybridized
product is subjected to DNA polymerizing enzymes such as E. coli
polymerase I (Klenow fragment) in order to complete the synthesis
of the mutation-bearing strand. Thus, a heteroduplex is formed,
wherein one strand encodes the original non-mutated sequence, and
the second strand bears the desired mutation. This heteroduplex
vector is then used to transform appropriate host cells, such as E.
coli cells, and clones are selected that include recombinant
vectors bearing the mutated sequence arrangement.
[0267] Comprehensive information on the functional significance and
information content of a given residue of protein can best be
obtained by saturation mutagenesis in which all 19 amino acid
substitutions are examined. The shortcoming of this approach is
that the logistics of multiresidue saturation mutagenesis are
daunting (Warren et al., 1996, Brown et al., 1996; Zeng et al.,
1996; Burton and Barbas, 1994; Yelton et al., 1995; Jackson et al.,
1995; Short et al., 1995; Wong et al., 1996; Hilton et al., 1996).
Hundreds, and possibly even thousands, of site specific mutants
must be studied. However, improved techniques make production and
rapid screening of mutants much more straightforward. See also,
U.S. Pat. Nos. 5,798,208 and 5,830,650, for a description of
"walk-through" mutagenesis.
[0268] Other methods of site-directed mutagenesis are disclosed in
U.S. Pat. Nos. 5,220,007; 5,284,760; 5,354,670; 5,366,878;
5,389,514; 5,635,377; and 5,789,166.
EXAMPLES
[0269] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventors to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Exemplary Materials and Methods
Cell Lines
[0270] Human breast cancer cell lines MCF-7, MDA-MB-231,
MDA-MB-435, ovarian cancer cell line SKOV-ip1 and the human
prostate carcinoma cell line PC-3 were purchased from the American
Type Culture Collection (Rockville, Md.) and cultured according to
the vendor's instructions.
Plasmids Construction and Site-Directed Mutagenesis
[0271] Bik, Luc, and GFP-expressing plasmids were constructed by
inserting the cDNAs of Bik, Luc, and GFP, respectively, into the
pcDNA3 vector containing a cytomegalovirus promoter. Site-directed
mutagenesis was performed according to the manufacturer's protocol
(Clontech Inc.). Bik residues threonine 33 and serine 35 aspartate
by using the following primers: for T33D,
5'-GGCATGACTGACGATGAAGAGGACCTG-3' (SEQ ID NO:1), and for S35D,
5'-GTTCTTGGCATGGATGACTCTGAACAGG-3' (SEQ ID NO:2). The sequences of
three Bik mutant constructs were verified by automated
sequencing.
Formulation
[0272] The non-viral gene delivery system, termed SN, was
essentially a cationic liposome, which is formulated as described
previously (Zou et al., 2002).
Transfection
[0273] Cells were cultured for 24 h in six-well plates with 1
ml/well of DMEM/F12 medium with 10% FBS (Life Technologies, Inc.,
Gaithersburg, Md.) until 60-70% confluence was reached. The
liposomal DNA (SN-DNA) was directly added into the culture plates
at a ratio of 2 .mu.g of DNA/106 cells. Twenty-four h later, the
transfection efficiency was determined by counting the GFP-positive
cells under a fluorescence microscope and expressing the result as
a percentage of total cells. Six random fields with >200
cells/field were counted for each sample. All experiments were
repeated three times independently.
Western Blot Analysis
[0274] Protein lysate was prepared with RIPA-B cell lysis buffer
containing 20 mM Na2PO4 (pH 7.4), 150 mM NaCl, 1% Triton X-100, 100
mM NaF, 2 mM Na3VO4, 5 mM phenylmethylsulfonyl fluoride, 1%
aprotinin, and 10 pg/ml leupeptin. Goat anti-Bik polyclonal
antibody was perchanced from Santa Cruz Biotechnology (Santa Cruz,
Calif.). Donkey anti-goat IgG peroxidase (Jackson) was used as
secondary antibody. Western blots were developed by enhanced
chemiluminescence (ECL; Amersham).
Luciferase Assays
[0275] To determine the Bik dose effect on cell proliferation,
different cancer cells were co-transfected with 50 ng of CMV-luc
and an increasing amount (0, 0.5, or 2 .mu.g) of CMV-Bik. The total
amount of DNA transfected at each dose was kept constant (2.05
.mu.g) by adding an appropriate amount of pcDNA3 vector.
Forty-eight hrs after transfection, cells were harvested, and luc
activity was measured using the luc assay system (Promega)
according to the protocol supplied by the manufacturer. The
relative activities were calculated by setting the luc activities
obtained from transfections without CMV-Bik (0 .mu.g) at 100%. The
data represent mean.+-.SD of three independent experiments.
Apoptosis Assay
[0276] For in vitro studies, standard fluorescence-activated cell
sorter analysis was used to determine the apoptosis of the cells.
Briefly, the cells were transfected with SN-bik or other agents. 12
or 24 hrs after transfection, the apoptotic cells were assessed by
flow cytometric detection of sub-G1 DNA content after being stained
with propidium iodide. Fields with >2000 cells in each were
randomly selected, and the apoptotic versus nonapoptotic cells were
counted.
Ex Vivo Tumor Inhibition
[0277] Human breast cancer cell line MCF-7 and prostate cancer cell
lines PC-3 cells were transfected by SN-bik or SN-luc. Twenty-four
h after transfection, the cells were carefully trypsinized,
harvested, and inoculated into the MFPs of nude mice (2.times.106
cells/tumor). The volume of the resulting tumor was measured
weekly.
Anti-Tumor Activity Tests
[0278] To study tumor growth inhibition, female nude mice were
inoculated with 2.times.106 of breast cancer cells/tumor into the
MFPs. Two weeks later, when most tumors exceeded 4.times.4 mm, the
tumor-bearing mice were randomly divided into three groups with 5
mice in each group. The mice in all treatment groups received i.v.
injections of SN-bik three times a week for 3 weeks, at a dose of
15 .mu.g of DNA/mouse. The mice in control groups were injected
with the same dose of SN-luc or the same volume of PBS. The tumor
volume was measured weekly. To assess animal survival and the
increase in life span, the same tumor models and the same
therapeutic treatments were used. The experiment was terminated on
day 200 after tumor inoculation.
Statistical Analysis
[0279] All statistical tests used in this study are two-sided
log-rank statistical tests.
Example 2
Construction of Exemplary Bik Mutants
[0280] The Bik single mutant T33D, S35D and double mutant T33DS35D,
in which the residues 33 (threonine) and 35 (serine) were changed
to aspartate acid residues respectively or together, were
constructed by site-directed mutagenesis (Clontech; La Jolla,
Calif.). After confirming the mutant Bik constructs by DNA
sequencing, Western blot with a Bik antibody demonstrated
production of the different Bik mutants expressed in HBK293 cell
after transient transfection. A formulation of cationic lipid (SN)
was used to deliver the pro-apoptotic mutant Bik gene into
different human cancer cells in vitro and in vivo (Zou et al.,
2002).
Example 3
In Vitro Testing of Exemplary Bik Mutants
[0281] To test whether the Bik mutants inhibited human cancer cell
line growth in vitro better than wide type Bik, transient
transfection assay was performed to evaluate the cell growth
inhibition effect of mutant Bik, in which a fixed amount (50 ng) of
CMV-luc was co-transfected with an increasing amount (0, 0.5, and 2
.mu.g) of CMV-Bik wide type and mutants in different human cancer
cells, including human breast cancer cell lines MDA-MB-231,
MDA-MB-468, MCF-7, prostate cancer cell line PC-3 and ovary cancer
cell line SKOV-ip1. Because the apparent luc activity is indicative
of living cells, the relative luciferase activity could be used as
the index of cell growth and proliferation (FIG. 1).
[0282] In FIG. 1A, western blot analysis of Bik and mutants protein
expression after transient transfection in 293T cells is shown.
Actin was used as an equal loading control. In FIGS. 1B through 1F,
human breast cancer cell lines MDA-MB-468 (FIG. 1B), MCF-7 (FIG.
1C), MDA-MB-231 (FIG. 1D), ovary cancer cell line skov-ip1 (FIG.
1E) and prostate cancer cell line PC-3 (FIG. 1F) were cotransfected
with 50 ng of CMV-luc and an increasing amount (0, 0.5, or 2 .mu.g)
of CMV-Bik wide type or mutants. The relative activities were
calculated by setting the luciferase activities obtained from
transfections without CMV-Bik (0 .mu.g) at 100%. The data represent
means of three independent experiments; bars, SD; stars (*) mean
significant difference, compared with wide type (P<0.05).
[0283] While the wild type and mutant Bik expression caused overall
growth inhibition in a dose-dependent manner, the Bik mutants,
especially the T33DS35D mutant, exhibited stronger growth
inhibitory effect on different cancer cells, and the expression
level was proportional to the growth-inhibitory activity. Thus,
mutant Bik is very potent against numerous cancer cell types both
in vitro and in vivo. Its effect is independent of the expression
level of Her-2/neu oncogene.
[0284] Using similar methods, and as illustrated in FIG. 2, Bik
mutant T33DS35D also exhibits potent anticancer effect on head and
neck cancer cells (TU138 and TU167), melanoma (B16F10), ovarian
(2774 and SKOV. SKOV is not a Her-2/neu overexpressing cell, as
opposed to SKOV-ip1), and endothelial cells (Human umbilical
vascular endothelial cells, HUVEC). Thus, in addition to SKOV-ip1,
which is a Her-2/neu overexpressing cancer cell, the bik mutants
also demonstrate potent inhibitory effects on cells without
Her-2/neu overexpression.
Example 4
FACS Analysis of Activity of Bik Mutants
[0285] The pro-apoptotic gene Bik is known to elicit apoptosis in a
variety of malignant cells. To examine whether the mutant Bik will
cause improved cell-killing effect or apoptosis on different cancer
cells, standard fluorescence-activated cell sorter analysis (FACS)
was used to assay apoptosis of the cancer cells. Briefly, the cells
were transfected with SN-Bik or other agents.
[0286] Specifically, human breast cancer cell lines MCF-7 and
prostate cancer cell line PC-3 were cotransfected with 100 ng of
CMV-GFP and 2 .mu.g of CMV-Bik wide type or mutants. pcDNA3
transfected cells were used as control. Twelve or twenty-four hours
after transfection, the apoptotic cells were harvested and assessed
by flow cytometric detection of sub-G1 DNA content after being
stained with propidium iodide. The data represent means of three
independent experiments; bars, SD; stars (*) mean significant
difference, compared with wide type (P<0.05).
[0287] Twelve or twenty-four hours after transfection, the
apoptotic cells were assessed by flow cytometric detection of
sub-G1 DNA content after being stained with propidium iodide. In
the MCF-7 cell, the different Bik mutants-induced 40% to 80% more
apoptosis than the wild type Bik (FIG. 3). The onset of apoptosis
was also early than the wild type, as early as 8 h after the
transfection. In the PC-3 cell lines similar phenomena was also
observed. The results indicate that mutant Bik can induce
significant cancer cell apoptosis, stronger and earlier than the wt
Bik.
Example 5
Ex Vivo Testing of Exemplary Bik Mutants
[0288] One of the most critical biological properties for a tumor
suppressor gene is its ability to reduced tumorigenicity in vivo.
To test the possibly better anti-tumor activity of different Bik
mutants, an ex vivo tumorigenicity assay was performed in a nude
mice cancer model. Human breast cancer cell lines MCF-7 and
prostate cancer cell line PC-3 were transfected with CMV-Bik wild
type or mutants delivered by SN liposome in culture plates. pcDNA3
transfected cells were used as control. Twenty-four hours later,
the treated cells were carefully harvested and inoculated into the
mammary fat pads (mfp) (for MCF-7) or subcutaneous connective
tissue (for PC-3) of nude mice. Four million cells were inoculated
for MCF-7 and one million cells for PC-3. Empty vector
pcDNA3-transfected cells were used as a control. The inoculated
tumor size was measured weekly.
[0289] Specifically, human breast cancer cell lines MCF-7
(4.times.10.sup.6 cells, each mouse implanted 0.72 mg 17
.beta.-estradiol pellet subcutaneously 2 weeks before inoculation)
and prostate cancer cell line PC-3 (1.times.10.sup.6 cells) were
transfected with CMV-Bik wide type or mutants delivered by SN
liposome in culture plates. pcDNA3 transfected cells were used as
control. Twenty-four hours later, the treated cells were harvested
and inoculated in the mammary fat pads (mfp) (for MCF-7) or
subcutaneous connective tissue (for PC-3) of nude mice, with 8 mice
in each group. Tumor sizes were measured weekly, as showed in FIG.
4A. After 7 weeks, the mice were scarified and the tumor weight
were measured as showed in FIG. 4B. In the each group, there was
tumor-formatting rate. bars, SD; stars (*) denote mutants having
significant difference, compared with wide type (P<0.05); stars
(**) denote wild type Bik having significant difference, compared
with pcDNA3 control (P<0.05).
[0290] This "ex vivo test" bypassed the gene delivery problems in
vivo and showed that under the optimal gene delivery condition,
tumor cells with mutant Bik had less tumor growth ability than wide
type Bik in vivo (FIG. 4A). SN-Bik delayed tumor growth in mice by
at least 3 weeks compared with the pcDNA3 control. The tumor volume
ratios of wt Bik versus 3 Bik mutants treatment groups during weeks
7 ranged from 2.1 to 3.7 for MCF-7 cell and from 2.2 to 4.1 for
PC-3 cell, suggesting a strong tumor suppression activity by mutant
Bik treatment in vivo. The data in FIG. 4 represent the
mean.+-.standard deviation of tumors size of 8 mice in each group.
Furthermore, the average tumor size (measured by weight) of the wt
Bik was about 2 to 4 folds than that of different Bik mutant groups
(FIG. 4B). In MCF-7 group, mutant Bik also had less tumor
taking-rate.
Example 6
In Vivo Testing of Exemplary Bik Mutants
[0291] The above studies showed that three mutant Bik forms could
induce apoptosis and inhibit tumor cell growth better than wild
type Bik in vitro and ex vivo, and the anti-tumor activity of
SN-delivered Bik mutants was further compared with wt Bik in
orthotopic breast cancer model and subcutaneous prostate cancer
model.
[0292] Because the double mutant T33DS35D Bik is the strongest
mutant of the three Bik mutants in apoptosis assay in vitro and
tumor growth ex vivo assay, the Bik T33DS35D (Bik DD) was used as a
representative of the three Bik mutants to compare with the wt Bik
in the following in vivo study. Mice with established tumors were
then treated with SN-wt Bik, SN-Bik T33DS35D or SN-pcDNA3. SN-Bik
T33DS35D injection significantly inhibited tumor growth in mice
compared with the SN-Bik and SN-pcDNA-treated mice.
[0293] Specifically, in FIG. 5A, orthotopic human breast cancer
MCF-7 cell (mammary fat pads, 4.times.106 cells/mouse, each mouse
implanted 0.72 mg 17 .beta.-estradiol pellet subcutaneously 2 weeks
before inoculation) and ectopic human prostate cancer PC3 cell
(subcutaneous, 1.times.106 cells/mouse) models in mice were used.
One week later, the mice bearing tumors were randomly divided into
three groups with 5 mice in each. One group received multiple
injections of SN-DNA, 15 .mu.g DNA/mouse, three times a week for a
total of 12 treatments, orthotopic breast cancer by intravenous
injection and ectopic prostate cancer by intra-tumor injection, the
other group received same doses of SN-pcDNA3. The tumor volume was
measured weekly. In FIG. 5B, the mutant Bik gene delivered by SN
significantly prolonged the life of mice with orthotopic or ectopic
human cancer. The data shown in the FIG. represent the
mean.+-.standard deviations from 5 individual mice. Bars, SD.)
[0294] By weeks 3 to 5, the mean tumor volume of SN-Bik-treated
mice was higher than that of SN-Bik T33S35D-treated mice in two
models. The most significant tumor suppression effect could be
observed by week 8 and 9, with an approximate 2-3-fold difference
in tumor volumes between the wide type and mutant treatment groups
(P<0.05; FIG. 5A). Treatment by SN-Bik T33S35D significantly
increased the survival rate of the treated mice compared with the
control groups treated with SN-wt Bik (P<0.05; FIG. 5B). The
median survival time was 175 days for Bik T33S35D treatment vs. 112
days for Bik in MCF-7 cells, 140 days vs. 105 days in PC3 cells.
The results indicate that SN-Bik T33S35D inhibited about 50% of the
tumor growth in these human cancer models and significantly
increased the survival rates.
[0295] A systemic gene therapy approach for breast cancer was
developed, consisting of a nonviral gene delivery system (SN) and a
proapoptotic gene, bik. The SN-Bik gene complex induced significant
apoptosis in four breast cancer cell lines in vitro as well as in
orthotopic tumor tissues in nude mice (Zou et al., 2002).
Systemically administrated SN-Bik significantly inhibited the
growth and metastasis of human breast cancer cells implanted in
nude mice and prolonged the life span of the treated animals. The
Bik gene is a potent inducer for apoptosis, independent of p53.
Like Bad (Wang et al., 1999) and Bid (Desagher et al., 2001), Bik
is regulated by phosphorylation (residues threonine 33 and serine
35) Unlike Bad, phosphorylation increases the pro-apoptotic potency
of Bik. The mechanism is presently unknown, possibly by a casein
kinase II-related enzyme (Verma et al., 2000). The phosphatase PP2A
might negatively regulate its function (Klumpp and Krieglstein,
2002). The post-translational phosphorylation of Bik, in specific
embodiments, results in conformational changes to cause release
from an inactive complex and increased affinity or accessibility to
antiapoptotic Bcl-2 homologues.
[0296] The results showed that transfection of Bik mutants (T33D,
S35D and T33DS35D) was much more potent than wild type (wt) Bik to
inhibit cell proliferation and enhance apoptosis induction of
various human cancer cells and to inhibit tumor growth in mice in
ex vivo and in vivo models. Thus, this shows that mutant Bik gene
is more potent than wt Bik to induce cell death, and SN-Bik is
useful for a therapeutic agent of cancer.
[0297] Obviously, methods disclosed herein have proven useful for
specific Bik mutants in the context of the invention. Following the
teachings provided herein, one of skill in the art can prepare and
test any number of mutants for anti-cell proliferative activity,
antitumor activity, pro-apoptotic activity, or a combination
thereof.
Example 7
Testing of Exemplary Bik Mutants as Therapeutic Agents
[0298] Bik mutants as they relate to anti-tumor activity are tested
in an animal study, such as cell lines, cell culture, and/or models
in addition to or other than those described in the preceding
Examples. In general embodiments of the present invention, mutants
are delivered by a vector, such as a liposome, adenoviral vector,
or combination thereof, into nude mice models for their anti-tumor
activity. Once the anti-tumor activity is demonstrated, potential
toxicity is further examined using immunocompetent mice, followed
by clinical trials.
[0299] In a specific embodiment, the preferential growth inhibitory
activity of mutant Bik is tested in animal. Briefly, cancer cell
lines are administered into mammary fat-pad of nude mice to
generate a breast xenografted model. Although, as described herein,
any cancer cell is within the scope of the present invention
irrespective of its genotype or expression levels, (such as, for
example, whether it is HER-2/neu-positive or HER-2/neu-negative),
in a specific embodiment HER-2/neu overexpressing breast cancer
cell lines (such as, for example, SKBR3 and/or MDA-MB361) are
utilized, such as for testing. After the tumors reach a particular
size, the Bik mutant and/or wild-type Bik control is administered
into the mouse, such as, for example, intravenously injected in an
admixture with an acceptable carrier, such as liposomes. The tumor
sizes and survival curve from these treatments are compared and
statistically analyzed. In a preferred embodiment, the mutant Bik
is substantially the same as or better in its inhibition of the
growth of tumor compared to that of wild-type Bik.
Example 8
Preparation of Additional Bik Mutants
[0300] Based on the data in previous Examples and the teachings
elsewhere in the specification, in addition to the knowledge in the
art, a skilled artisan would be motivated and capable of generating
additional Bik mutants and, furthermore, able to determine the
usefulness in the context of the invention using methodology
disclosed herein.
Example 9
Testing of Additional Bik Mutants
[0301] Once Bik mutants other than the exemplary mutants disclosed
herein are generated, testing using a cell culture in a relevant
cell line(s) is performed, such as described herein. Furthermore,
testing of the Bik mutants using FACS analysis is performed, such
as described herein. Also, testing of the additional Bik mutants
using ex vivo systems or in vivo systems as described herein may be
employed, in specific embodiments.
Example 10
Anti-Cancer Effect of Mutant Bik in Breast, Ovarian and Pancreatic
Cancer Models
[0302] An exemplary Bik mutant (BikDD) polynucleotide demonstrated
a significant suppression of tumor growth and an increase in
survival in a breast cancer orthotopic model. Human breast cancer
cells MDA-MB-231 (2.times.10.sup.6 cells) were inoculated into the
mammary fat pads (MFP) of nude mice. After 1 week, the mice bearing
tumors were randomly divided into five groups with 5 or 8 mice in
each group. The mice in all treatment groups received weekly i.v.
injections of the either vector (pUK21) or BikDD (pUK/BikDD) (15
.mu.g DNA/mouse) delivered using the NIH-liposome for ten weeks.
The mice in the control group received injections of 5% Dextrose
(D5W). In FIG. 6A, tumor volume was measured and recorded weekly.
FIG. 6B shows that BikDD increased the survival rate of mice
bearing MDA-MB-231 orthotopic tumors. The data shown in the FIGS.
6A and 6B represent the mean and standard deviations from 5 or 8
individual mice.
[0303] Also, the Bik mutant (BikDD) gene demonstrated a significant
suppression of tumor growth and an increase in survival in a breast
cancer orthotopic model. Human breast cancer cells MDA-MB-468
(3.times.10.sup.6 cells), were inoculated into the mammary fat pads
(MFP) of nude mice. After 1 week, the mice bearing tumors were
randomly divided into five groups with 9 or 10 mice in each group.
The mice in all treatment groups received weekly i.v. injections of
the either vector (pUK21) or BikDD (pUK/BikDD) (45 .mu.g DNA/mouse)
delivered using the N1H-liposome for ten weeks. The mice in the
control group received injections of 5% Dextrose (D5W). In FIG. 7A,
tumor volume was measured and recorded weekly. In FIG. 2B, BikDD
increased the survival rate of mice bearing MDA-MB-231 orthotopic
tumors. The data shown in the FIGS. 7A and 7B represent the mean
and standard deviations from 9 or 10 individual mice.
[0304] In FIG. 8, treatment with the Bik mutant (BikDD) gene
increases the survival of mice in a ovarian cancer orthotopic
model. Human ovarian cancer cells, 2774 (2.times.10.sup.6 cells),
were inoculated intraperitononeally (i.p.) into nude mice. The mice
were randomly split into 3 groups of ten mice each. The mice in all
treatment groups received weekly i.p. injections of the either
vector (pUK21) or BikDD (pUK/BikDD) (15 .mu.g DNA/mouse) delivered
using the NIH-liposome for twelve weeks. The mice in the control
group received injections of 5% Dextrose (D5W). The data shown in
the figures represent the mean and standard deviations from 10
individual mice.
[0305] In FIG. 9, treatment with the Bik mutant (BikDD) gene
increases the survival of mice in a pancreatic cancer orthotopic
model. Pancreatic cancer cells, Pan02 (5.times.10.sup.4 cells),
were inoculated into nude mice. The mice were randomly split into 3
groups of ten mice each. The mice in all treatment groups received
weekly i.p. injections of the either vector (pUK21) or BikDD
(pUK/BikDD) (15 .mu.g DNA/mouse) delivered using the NIH-liposome
for eleven weeks. The mice in the control group received injections
of PBS.
Example 11
Cancer Tissue-Specific Expression of Mutant Bik
[0306] Current cancer therapies, such as chemotherapy (CT) and
radiotherapy, have low selectivity for tumor cells and side effects
for normal tissues. To minimize the side effects, these therapies
are generally given in an intermittent manner, allowing normal
cells to recover between treatment cycles. However, during the
recovery period, some surviving cancer cells become more resistant
to the treatment because of gene mutation. Consequently, cancer
recurrence or progression may occur. Tumor-targeting gene therapy
can minimize treatment side effects and the risk of developing
resistance by acting on the tumor-specific signaling pathways. In
the present invention, tissue-specific promoters are used for
targeting gene therapy of mutant Bik, such as for breast cancer,
pancreatic cancer, and prostate cancer.
Breast Cancer-Specific Expression of Mutant Bik
[0307] Breast cancer-specific expression of mutant Bik employs two
exemplary promoters that are described herein and presented in
further detail in U.S. Provisional Patent Application 60/559,111,
entitled "Cancer-Specific Promoters" by Mien-Chie Hung, Yan Li,
Yong Wen, Chi-Ping Day, Kun-Ming Rau, Xiaoming Xie, Zheng Li, filed
simultaneously herewith and incorporated by reference herein in its
entirety.
Topoisomerase II.alpha. Breast Cancer-Specific Expression
[0308] A therapeutic construct was generated that comprises
topoisomerase II.alpha.control sequence, such as the CT90 region
(SEQ ID NO:26), that was operatively linked to CMV enhancer (SEQ ID
NO:25), and the composite construct comprising both sequences (SEQ
ID NO:37) was operatively linked to a polynucleotide encoding
mutant Bik to regulate its expression. The construct is detailed
herein but described further in U.S. Provisional Patent Application
60/559,111, entitled "Cancer-Specific Promoters" by Mien-Chie Hung,
Yan Li, Yong Wen, Chi-Ping Day, Kun-Ming Rau, Xiaoming Xie, Zheng
Li, filed simultaneously herewith and incorporated by reference
herein in its entirety. A particular mutant Bik construct
comprising sequence encoding the BikDD mutant is hereinafter
referred to as CT90-BikDD. This construct was co-transfected with a
luciferase reporter vector into breast cancer cell lines MDA-MB-231
and 468, and the normal breast epithelium cell line 184A1, and then
the cell-killing effect was determined by a luciferase vitality
assay. The CMV promoter-driven BikDD vector (CMV-BikDD) and empty
vector were used as positive and negative controls, respectively.
While CMV-BikDD killed all three cell lines to a nearly equal
extent, CT90-BikDD killed breast cancer cells preferentially (FIG.
10), indicating that the killing effect of CT90-BikDD is selective
for breast cancer cells. Therefore, CT90 is useful in breast
cancer-targeting gene therapy.
[0309] Next, the anti-tumor effect of this breast cancer-targeting
gene therapy was characterized in vivo. One week after inoculating
breast cancer MDA-MB-231 cells into mammary fat pads, the nude mice
were treated once per week with liposome-complexed CT90-BikDD
(therapeutic group), CMV-BikDD (positive control), and CMV-PGL3
(mock treatment), or dextrose buffer D5W as a no-treatment control.
Each mouse was intravenously injected with 15 .quadrature.g of
liposome-complexed DNA construct, once per week, and tumor size was
measured regularly. The CT90-BikDD group showed a superior tumor
suppressive effect compared to CMV-BikDD or CMV-PGL3 (FIG. 11).
[0310] Therapeutic effects of CT90-BikDD breast cancer construct
were also demonstrated in an orthotopic mouse model wherein
liposome-complexed CT90-BikDD targets breast cancer cells (FIG.
12A). Liposome-complexed CT90-BikDD or CMV-BikDD constructs were
administered into mice carrying MDA-MB-468 breast cancer xenograft.
The mice were sacrificed 72 hours after injection, and tumor and
major organs were removed and fixed. In situ hybridization was
performed on the tissue sections to detect BikDD mRNA expression.
The results of tumor and heart were shown. The arrows indicate
positive cells. In FIGS. 12B and 12D, tumor size record during gene
therapy treatment is demonstrated. Mice carrying MDA-MB-231 breast
cancer xenograft received treatment of 15-.mu.g liposome complexed
CT90-BikDD, CMV-BikDD, empty vector pGL3, or 5% dextrose in water
through i.v. injection. Each treatment group had ten mice. The mice
were treated once a week (QW, FIG. 12B) or twice a week (BIW, FIG.
12D) for eight weeks, and the tumor size was measured and recorded
twice a week. The p values from T-test for CT90 vs. pGL3
(P(CT90:pGL3)) and CMV vs. pGL3 (P(CMV:pGL3)) were shown. FIGS. 12C
and 12E show survival record of mice in QW (FIG. 12C) or BIW (FIG.
12E) group. The treatment was stopped in the eighth week, and the
mice were kept alive until morbid status defined by institute
regulation. The survival number each week was recorded.
[0311] Gene therapy of Bik-DD in MDA-MB-468 xenograft mice is
illustrated in FIG. 13. Mice carrying MDA-MB-468 breast cancer
xenograft received treatment of 15-.mu.g liposome-complexed
CT90-BikDD, CMV-BikDD, empty vector pUK21, or 5% dextrose in water
through i.v. injection, for example. Each treatment group had ten
mice. The mice were treated once a week for eight weeks, and the
tumor size was measured and recorded twice a week.
Transferrin Receptor Breast Cancer-Specific Expression
[0312] The present inventors also demonstrate in U.S. Provisional
Patent Application 60/559,111, entitled "Cancer-Specific Promoters"
by Mien-Chie Hung, Yan Li, Yong Wen, Chi-Ping Day, Kun-Ming Rau,
Xiaoming Xie, Zheng Li, filed simultaneously herewith and
incorporated by reference herein, that at least part of the
transferrin receptor (TR) promoter, such as that comprising SEQ ID
NO:27 (CTR116), possesses breast cancer specificity, and in
combination with a CMV promoter enhancer (SEQ ID NO:25), for
example, it can regulate expression of mutant Bik for effective
breast cancer-specific expression. The full CTR116 control sequence
(SEQ ID NO:38) comprises SEQ ID NO:25 operatively linked to SEQ ID
NO:27.
[0313] In further embodiments of the present invention, the
respective CT90 and CTR116 elements are also narrowed further to
identify even smaller segments within that retain breast
cancer-specific expression activity. For example, deletion
constructs may be made of these respective regions, and their
tissue specificity is tested to identify the smaller segments that
maintain the ability to direct expression in breast cancer
tissue.
Pancreatic Cancer-Specific Expression of Mutant Bik
[0314] The present inventors may utilize pancreatic cancer-specific
promoter sequences to control expression of a polynucleotide
encoding a mutant Bik polypeptide. One particular but exemplary
pancreatic cancer-specific promoter is described herein and is
presented in further detail in U.S. Provisional Patent Application
60/559,111, entitled "Cancer-Specific Promoters" by Mien-Chie Hung,
Yan Li, Yong Wen, Chi-Ping Day, Kun-Ming Rau, Xiaoming Xie, Zheng
Li, filed simultaneously herewith and incorporated by reference
herein in its entirety.
[0315] Generally, an operably linked minimal CCKAR promoter and
TSTA construct (wherein the TSTA sequence may be Gal4VP2, for
example) operably linked to WPRE regulates expression of mutant Bik
polypeptide. The two-step transcriptional amplification
(activation) (TSTA) sequence preferably augments the
transcriptional activity of cellular promoters (Iyer, Wu et al.
2001; Zhang, Adams et al. 2002) such as CCKAR. In this system, the
first step involves the tissue-specific expression of the fusion
protein of GAL4-VP16 or GAL4-VP2, for example. In the second step,
GAL4-VP16 or GAL4-VP2 in turn, drives target gene expression under
the control of GAL4 response elements in a minimal promoter. Also,
the TSTA sequence may comprise the G5E4T sequence (SEQ ID NO:36),
as an example, which comprises five 17-bp GAL4 binding sites
positioned 23 bases from the TAT box of the E4 gene of adenovirus
(Carey et al., 1990). Post-transcriptional regulation of mutant Bik
may also be employed in pancreatic cancer utilizing a regulatory
element such as WPRE. Thus, a composite construct for pancreatic
cancer-specific expression is comprised in SEQ ID NO:34, which
includes at least CCKAR (SEQ ID NO:28), GAL4-VP2 (SEQ ID NO:30 or
SEQ ID NO:33), G5E4T (SEQ ID NO:36), and WPRE (SEQ ID NO:29).
[0316] In particular embodiments of the present invention,
constructs are similarly generated comprising these or similar
pancreatic-specific promoters operatively linked to a
polynucleotide encoding a mutant Bik, followed by introduction into
a mammal in need of pancreatic cancer therapy treatment based on
analogous methods described herein. Parameters are easily optimized
by those of skill in the art, such as delivery mode, concentration
of composition, and so forth.
[0317] In further embodiments of the present invention, the
pancreatic cancer-specific element(s) is narrowed further to
identify one or more smaller segments within that retain pancreatic
cancer-specific expression activity. For example, deletion
constructs may be made of these respective regions, and their
tissue specificity is characterized to identify the smaller
segments that maintain the ability to direct expression in
pancreatic cancer tissue.
Prostate Cancer-Specific Expression of Mutant Bik
[0318] A prostate cancer-specific promoter sequence is employed to
control expression of a polynucleotide encoding a mutant Bik
polypeptide. One particular but exemplary pancreatic
cancer-specific promoter is described herein and is presented in
further detail in U.S. Provisional Patent Application 60/559,111,
entitled "Cancer-Specific Promoters" by Mien-Chie Hung, Yan Li,
Yong Wen, Chi-Ping Day, Kun-Ming Rau, Xiaoming Xie, Zheng Li, filed
simultaneously herewith and incorporated by reference herein in its
entirety. In specific embodiments, a prostate cancer-specific
promoter that regulates expression of mutant Bik in both
androgen-dependent and androgen-independent manners is
utilized.
[0319] Generally, the hTERT promoter is operatively linked to a
two-step transcriptional amplification (activation) (TSTA) sequence
that preferably augments the transcriptional activity of cellular
promoters (Iyer, Wu et al. 2001; Zhang, Adams et al. 2002).
GAL4-VP16 fusion protein or GAL4-VP2 fusion protein may be
utilized. In this system, the first step involves the
tissue-specific expression of the GAL4-VP16 (or GAL4-VP2, for
example) fusion protein. In the second step, GAL4-VP16, in turn,
drives target gene expression under the control of GAL4 response
elements in a minimal promoter. The use of TSTA preferably leads to
amplified levels of the transgene expression. The promoter further
comprises ARR2 prostate-cancer specific element (SEQ ID NO:31). The
post-transcriptional regulatory element of the woodchuck hepatitis
virus (WPRE), which involves modification of RNA polyadenylation,
RNA export, and/or RNA translation (Donello, Loeb et al., 1998), is
operatively linked to pARR2-hTERT-TSTA to produce
pARR2.hTERTp-TSTA-mutant Bik-WPRE. In a specific aspect of the
invention, the promoter is effective in both androgen-dependent and
androgen-independent prostate cancers. Thus, a composite construct
for prostate cancer-specific expression is comprised in SEQ ID
NO:35, which includes at least ARR2 (SEQ ID NO:31), hTERT (SEQ ID
NO:32), GAL4-VP2 (SEQ ID NO:30 or SEQ ID NO:33), G5E4T (SEQ ID
NO:36), and WPRE (SEQ ID NO:29).
[0320] In further embodiments of the present invention, the
respective prostate cancer-specific element(s) is narrowed further
to identify one or more even smaller segments within that retain
prostate cancer-specific expression activity. For example, deletion
constructs may be made of these respective regions, and their
tissue specificity is tested to identify the one or more smaller
segments that maintain the ability to direct expression in prostate
cancer tissue.
Example 12
Clinical Trials
[0321] This example is concerned with the development of human
treatment protocols using the Bik mutant protein, peptide, or
polypeptide or a nucleic acid encoding the Bik mutant protein,
peptide, or polypeptides, alone or in combination with other
anti-cancer drugs. The Bik mutant protein, peptide, or polypeptide
or a nucleic acid encoding the Bik mutant protein, peptide, or
polypeptides, and anti-cancer drug treatment will be of use in the
clinical treatment of various cancers involving, for example, Akt
activation in which transformed or cancerous cells play a role.
Such treatment will be particularly useful tools in anti-tumor
therapy, for example, in treating patients with ovarian, breast,
prostate, pancreatic, brain, colon, and lung cancers that are
resistant to conventional chemotherapeutic regimens.
[0322] The various elements of conducting a clinical trial,
including patient treatment and monitoring, will be known to those
of skill in the art in light of the present disclosure. The
following information is being presented as a general guideline for
use in establishing the Bik mutant protein, peptide, or polypeptide
or a nucleic acid encoding the Bik mutant protein, peptide, or
polypeptides, in clinical trials.
[0323] Patients with advanced, metastatic breast, epithelial
ovarian carcinoma, pancreatic, colon, or other cancers chosen for
clinical study will typically be at high risk for developing the
cancer, will have been treated previously for the cancer which is
presently in remission, or will have failed to respond to at least
one course of conventional therapy. In an exemplary clinical
protocol, patients may undergo placement of a Tenckhoff catheter,
or other suitable device, in the pleural or peritoneal cavity and
undergo serial sampling of pleural/peritoneal effusion. Typically,
one will wish to determine the absence of known loculation of the
pleural or peritoneal cavity, creatinine levels that are below 2
mg/dl, and bilirubin levels that are below 2 mg/dl. The patient
should exhibit a normal coagulation profile.
[0324] In regard to the Bik mutant protein, peptide, or polypeptide
or a nucleic acid encoding the Bik mutant protein, peptide, or
polypeptides, and other anti-cancer drug administration, a
Tenckhoff catheter, or alternative device may be placed in the
pleural cavity or in the peritoneal cavity, unless such a device is
already in place from prior surgery. A sample of pleural or
peritoneal fluid can be obtained, so that baseline cellularity,
cytology, LDH, and appropriate markers in the fluid (CEA, CA15-3,
CA 125, PSA, p38 (phosphorylated and un-phosphorylated forms), Akt
(phosphorylated and un-phosphorylated forms) and in the cells (Bik
mutant proteins, peptides or polypeptides or nucleic acids encoding
the same) may be assessed and recorded.
[0325] In the same procedure, the Bik mutant protein, peptide, or
polypeptide or a nucleic acid encoding the Bik mutant protein,
peptide, or polypeptides, may be administered alone or in
combination with the other anti-cancer drug. The administration may
be in the pleural/peritoneal cavity, directly into the tumor, or in
a systemic manner. The starting dose may be 0.5 mg/kg body weight.
Three patients may be treated at each dose level in the absence of
grade>3 toxicity. Dose escalation may be done by 100% increments
(0.5 mg, 1 mg, 2 mg, 4 mg) until drug related grade 2 toxicity is
detected. Thereafter dose escalation may proceed by 25% increments.
The administered dose may be fractionated equally into two
infusions, separated by six hours if the combined endotoxin levels
determined for the lot of the Bik protein, peptide, or polypeptide
or a nucleic acid encoding the Bik mutant protein, peptide, or
polypeptides, and the lot of anti-cancer drug exceed 5 EU/kg for
any given patient.
[0326] The Bik mutant protein, peptide, or polypeptide or a nucleic
acid encoding the Bik mutant protein, peptide, or polypeptides,
and/or the other anti-cancer drug combination, may be administered
over a short infusion time or at a steady rate of infusion over a 7
to 21 day period. The Bik mutant protein, peptide, or polypeptide
or a nucleic acid encoding the Bik mutant protein, peptide, or
polypeptides, infusion may be administered alone or in combination
with the anti-cancer drug and/or emodin like tyrosine kinase
inhibitor. The infusion given at any dose level will be dependent
upon the toxicity achieved after each. Hence, if Grade II toxicity
was reached after any single infusion, or at a particular period of
time for a steady rate infusion, further doses should be withheld
or the steady rate infusion stopped unless toxicity improved.
Increasing doses of the Bik mutant protein, peptide, or polypeptide
or a nucleic acid encoding the mutant protein, peptide, or
polypeptides, in combination with an anti-cancer drug will be
administered to groups of patients until approximately 60% of
patients show unacceptable Grade III or IV toxicity in any
category. Doses that are 2/3 of this value could be defined as the
safe dose.
[0327] Physical examination, tumor measurements, and laboratory
tests should, of course, be performed before treatment and at
intervals of about 3-4 weeks later. Laboratory studies should
include CBC, differential and platelet count, urinalysis,
SMA-12-100 (liver and renal function tests), coagulation profile,
and any other appropriate chemistry studies to determine the extent
of disease, or determine the cause of existing symptoms. Also
appropriate biological markers in serum should be monitored e.g.
CEA, CA 15-3, p38 (phosphorylated and non-phopshorylated forms) and
Akt (phosphorylated and non-phosphorylated forms), p185, etc.
[0328] To monitor disease course and evaluate the anti-tumor
responses, it is contemplated that the patients should be examined
for appropriate tumor markers every 4 weeks, if initially abnormal,
with twice weekly CBC, differential and platelet count for the 4
weeks; then, if no myelosuppression has been observed, weekly. If
any patient has prolonged myelosuppression, a bone marrow
examination is advised to rule out the possibility of tumor
invasion of the marrow as the cause of pancytopenia. Coagulation
profile shall be obtained every 4 weeks. An SMA-12-100 shall be
performed weekly. Pleural/peritoneal effusion may be sampled 72
hours after the first dose, weekly thereafter for the first two
courses, then every 4 weeks until progression or off study.
Cellularity, cytology, LDH, and appropriate markers in the fluid
(CEA, CA15-3, CA 125, ki67 and Tunel assay to measure apoptosis,
Akt) and in the cells (Akt) may be assessed. When measurable
disease is present, tumor measurements are to be recorded every 4
weeks. Appropriate radiological studies should be repeated every 8
weeks to evaluate tumor response. Spirometry and DLCO may be
repeated 4 and 8 weeks after initiation of therapy and at the time
study participation ends. An urinalysis may be performed every 4
weeks.
[0329] Clinical responses may be defined by acceptable measure. For
example, a complete response may be defined by the disappearance of
all measurable disease for at least a month. Whereas a partial
response may be defined by a 50% or greater reduction of the sum of
the products of perpendicular diameters of all evaluable tumor
nodules or at least 1 month with no tumor sites showing
enlargement. Similarly, a mixed response may be defined by a
reduction of the product of perpendicular diameters of all
measurable lesions by 50% or greater with progression in one or
more sites.
REFERENCES
[0330] All patents and publications mentioned in the specification
are indicative of the level of those skilled in the art to which
the invention pertains. All patents and publications are herein
incorporated by reference in their entirety to the same extent as
if each individual publication was specifically and individually
indicated to be incorporated by reference.
Patents
[0331] U.S. Pat. No. 4,683,202 [0332] U.S. Pat. No. 4,797,368
[0333] U.S. Pat. No. 4,879,236 [0334] U.S. Pat. No. 5,139,941
[0335] U.S. Pat. No. 5,220,007 [0336] U.S. Pat. No. 5,284,760
[0337] U.S. Pat. No. 5,354,670 [0338] U.S. Pat. No. 5,366,878
[0339] U.S. Pat. No. 5,389,514 [0340] U.S. Pat. No. 5,635,377
[0341] U.S. Pat. No. 5,641,484 [0342] U.S. Pat. No. 5,670,488
[0343] U.S. Pat. No. 5,789,166 [0344] U.S. Pat. No. 5,798,208
[0345] U.S. Pat. No. 5,830,650 [0346] U.S. Pat. No. 5,871,986
[0347] U.S. Pat. No. 5,925,565 [0348] U.S. Pat. No. 5,928,906
[0349] U.S. Pat. No. 5,935,819
Publications
[0349] [0350] Anderson, L. M., Krotz, S., Weitzman, S. A., and
Thimmapaya, B. (2000). Breast cancer-specific expression of the
Candida albicans cytosine deaminase gene using a transcriptional
targeting approach. Cancer Gene Ther 7, 845-852. [0351] Bartke, T.,
Siegmund, D., Peters, N., Reichwein, M., Henkler, F., Scheurich,
P., and Wajant, H. p53 upregulates cFLIP, inhibits transcription of
NF-kappaB-regulated genes and induces caspase-8-independent cell
death in DLD-1 cells, Oncogene. 20: 571-80., 2001. [0352] Boyd, J.
M., Gallo, G. J., Elangovan, B., Houghton, A. B., Malstrom, S.,
Avery, B. J., Ebb, R. G., Subramanian, T., Chittenden, T., Lutz, R.
J., and et al. Bik, a novel death-inducing protein shares a
distinct sequence motif with Bcl-2 family proteins and interacts
with viral and cellular survival-promoting proteins, Oncogene. 11:
1921-8., 1995. [0353] Carey, M., Leatherwood, J., and Ptashne, M. A
potent GAL4 derivative activates transcription at a distance in
vitro, Science 247:710-712, 1990. [0354] Daniel, P. T., Pun, K. T.,
Ritschel, S., Sturm, I., Holler, J., Dorken, B., and Brown, R.
Expression of the death gene Bik/Nbk promotes sensitivity to
drug-induced apoptosis in corticosteroid-resistant T-cell lymphoma
and prevents tumor growth in severe combined immunodeficient mice,
Blood. 94: 1100-7., 1999. [0355] Desagher, S., Osen-Sand, A.,
Montessuit, S., Magnenat, E., Vilbois, F., Hochmann, A., Journot,
L., Antonsson, B., and Martinou, J. C. Phosphorylation of bid by
casein kinases I and II regulates its cleavage by caspase 8, Mol.
Cell. 8: 601-11, 2001. [0356] Emami, K. H. and Carey, M. (1992) A
synergistic increase in potency of a multimerized VP16
transcriptional activation domain. EMBO J. 11:5005-5012. [0357]
Han, J., Sabbatini, P., and White, E. Induction of apoptosis by
human Nbk/Bik, a BH3-containing protein that interacts with E1B
19K, Mol Cell Biol. 16: 5857-64., 1996. [0358] Iyer, M., Wu, L.,
Carey, M., Wang, Y., Smallwood, A., and Gambhir, S. S. (2001).
Two-step transcriptional amplification as a method for imaging
reporter gene expression using weak promoters. Proc. Natl. Acad.
Sci. 98:14595-14600. [0359] Katabi, M. M., Chan, H. L., Karp, S.
E., and Batist, G. (1999). Hexokinase type II: a novel
tumor-specific promoter for gene-targeted therapy differentially
expressed and regulated in human cancer cells. Hum Gene Ther 10,
155-164. [0360] Klumpp, S, and Krieglstein, J. Serine/threonine
protein phosphatases in apoptosis, Curr Opin Pharmacol. 2: 458-62.,
2002. [0361] Lu, H., Zhang, Y., Roberts, D. D., Osborne, C. K., and
Templeton, N. S. (2002). Enhanced gene expression in breast cancer
cells in vitro and tumors in vivo. Mol Ther 6, 783-792. [0362]
Maeda, T., J, O. W., Matsubara, H., Asano, T., Ochiai, T.,
Sakiyama, S., and Tagawa, M. (2001). A minimum c-erbB-2
promoter-mediated expression of herpes simplex virus thymidine
kinase gene confers selective cytotoxicity of human breast cancer
cells to ganciclovir. Cancer Gene Ther 8, 890-896. [0363] Mathai,
J. P., Germain, M., Marcellus, R. C., and Shore, G. C. Induction
and endoplasmic reticulum location of BIK/NBK in response to
apoptotic signaling by E1A and p53, Oncogene. 21: 2534-44., 2002.
[0364] Nettelbeck, D. M., Jerome, V. and Muller, R. (2000) Gene
therapy: designer promoters for tumour targeting. Trends Genet.
16:174-181. [0365] Panaretakis, T., Pokrovskaja, K., Shoshan, M.
C., and Grander, D. Activation of Bak, Bax, and BH3-only Proteins
in the Apoptotic Response to Doxorubicin, J Biol. Chem. 277:
44317-26., 2002. [0366] Puthalakath, H. and Strasser, A. Keeping
killers on a tight leash: transcriptional and post-translational
control of the pro-apoptotic activity of BH3-only proteins, Cell
Death Differ. 9: 505-12, 2002. [0367] Qiao, J., Doubrovin, M.,
Sauter, B. V., Huang, Y., Guo, Z. S., Balatoni, J., Akhurst, T.,
Blasberg, R. G., Tjuvajev, J. G., Chen, S. H., and Woo, S. L.
(2002). Tumor-specific transcriptional targeting of suicide gene
therapy. Gene Ther 9, 168-175. [0368] Sadowski I, Ma J, Triezenberg
S, Ptashne M. GAL4-VP16 is an unusually potent transcriptional
activator. Nature. 1988 Oct. 6; 335(6190):563-4. [0369] Sato, M.,
Johnson, M., Zhang, L., Zhang, B., Le, K., Gambhir, S. S., Carey,
M., Wu, L. (2003) Optimization of adenoviral vectors to direct
highly amplified prostate-specific expression for imaging and gene
therapy. Mol. Ther. 8(5):726-737. [0370] Takeuchi M, Shichinohe T,
Senmaru N, Miyamoto M, Fujita H, Takimoto M, Kondo S, Katoh H,
Kuzumaki N. The dominant negative H-ras mutant, N116Y, suppresses
growth of metastatic human pancreatic cancer cells in the liver of
nude mice. Gene Ther. 2000 March; 7(6):518-26. [0371] Theodorakis,
P., Lomonosova, E., and Chinnadurai, G. Critical requirement of BAX
for manifestation of apoptosis induced by multiple stimuli in human
epithelial cancer cells, Cancer Res. 62: 3373-6., 2002. [0372]
Verma, S., Zhao, L., and Chinnadurai, G. Phosphorylation of the
Pro-Apoptotic Protein BIK: Mapping of Phosphorylation sites and
Effect on Apoptosis, J Biol. Chem. 17: 17, 2000. [0373] Wang, H.
G., Pathan, N., Ethell, I. M., Krajewski, S., Yamaguchi, Y.,
Shibasaki, F., McKeon, F., Bobo, T., Franke, T. F., and Reed, J. C.
Ca2+-induced apoptosis through calcineurin dephosphorylation of
BAD, Science. 284: 339-43., 1999. [0374] Wang X P, Yazawa K, Yang
J, Kohn D, Fisher W E, Brunicardi F C. Specific gene expression and
therapy for pancreatic cancer using the cytosine deaminase gene
directed by the rat insulin promoter. J Gastrointest Surg. 2004
January; 8(1):98-108. [0375] Wesseling J G, Yamamoto M, Adachi Y,
Bosma P J, van Wijland M, Blackwell J L, Li H, Reynolds P N,
Dmitriev I, Vickers S M, Huibregtse K, Curiel D T. Midkine and
cyclooxygenase-2 promoters are promising for adenoviral vector gene
delivery of pancreatic carcinoma. Cancer Gene Ther. 2001 December;
8(12):990-6. [0376] Zhang, L., Adams, J. Y., Billick, E., Ilagan,
R., Iyer, M., Le, K., Smallwood, A., Gambhir, S. S., Carey, M., Wu,
L. (2002) Molecular engineering of a two-step transcription
amplification (TSTA) system for transgene delivery in prostate
cancer. Mol. Ther. 5(3): 223-232. [0377] Zou, Y., Peng, H., Zhou,
B., Wen, Y., Wang, S. C., Tsai, E. M., and Hung, M. C. Systemic
tumor suppression by the proapoptotic gene bik, Cancer Res. 62:
8-12., 2002.
[0378] Although the present invention and its advantages have been
described in detail, it should be understood that various changes,
substitutions and alterations can be made herein without departing
from the spirit and scope of the invention as defined by the
appended claims. Moreover, the scope of the present application is
not intended to be limited to the particular embodiments of the
process, machine, manufacture, composition of matter, means,
methods and steps described in the specification. As one of
ordinary skill in the art will readily appreciate from the
disclosure of the present invention, processes, machines,
manufacture, compositions of matter, means, methods, or steps,
presently existing or later to be developed that perform
substantially the same function or achieve substantially the same
result as the corresponding embodiments described herein may be
utilized according to the present invention. Accordingly, the
appended claims are intended to include within their scope such
processes, machines, manufacture, compositions of matter, means,
methods, or steps.
Sequence CWU 1
1
38127DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 1ggcatgactg acgatgaaga ggacctg 27228DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
2gttcttggca tggatgactc tgaacagg 283160PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 3Met
Ser Glu Val Arg Pro Leu Ser Arg Asp Ile Leu Met Glu Thr Leu 1 5 10
15Leu Tyr Glu Gln Leu Leu Glu Pro Pro Thr Met Glu Val Leu Gly Met
20 25 30Thr Asp Ser Glu Glu Asp Leu Asp Pro Met Glu Asp Phe Asp Ser
Leu 35 40 45Glu Cys Met Glu Gly Ser Asp Ala Leu Ala Leu Arg Leu Ala
Cys Ile 50 55 60Gly Asp Glu Met Asp Val Ser Leu Arg Ala Pro Arg Leu
Ala Gln Leu 65 70 75 80Ser Glu Val Ala Met His Ser Leu Gly Leu Ala
Phe Ile Tyr Asp Gln 85 90 95Thr Glu Asp Ile Arg Asp Val Leu Arg Ser
Phe Met Asp Gly Phe Thr 100 105 110Thr Leu Lys Glu Asn Ile Met Arg
Phe Trp Arg Ser Pro Asn Pro Gly 115 120 125Ser Trp Val Ser Cys Glu
Gln Val Leu Leu Ala Leu Leu Leu Leu Leu 130 135 140Ala Leu Leu Leu
Pro Leu Leu Ser Gly Gly Leu His Leu Leu Leu Lys145 150 155
1604150PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 4Met Ser Glu Ala Arg Leu Met Ala Arg Asp Val Ile
Lys Thr Val Pro 1 5 10 15His Asp Gln Val Pro Gln Pro Pro Val Ala
Ser Glu Thr Pro Ser Met 20 25 30Lys Glu Pro Val Arg Asp Val Asp Leu
Met Glu Cys Val Glu Gly Arg 35 40 45Asn Gln Val Ala Leu Arg Leu Ala
Cys Ile Gly Asp Glu Met Asp Leu 50 55 60Cys Leu Arg Ser Pro Arg Leu
Val Gln Leu Pro Gly Ile Ala Ile His 65 70 75 80Arg Leu Ala Val Thr
Tyr Ser Arg Thr Gly Val Arg Gly Ile Phe Arg 85 90 95Ser Leu Ile Arg
Ser Leu Thr Asn Leu Arg Glu Asn Ile Trp Ser Trp 100 105 110Arg Val
Leu Thr Pro Gly Ala Trp Val Ser Pro Asp Gln Asp Pro Gly 115 120
125Gln Leu Phe Pro Met Val Leu Leu Val Phe Leu Leu Leu Gly Gly Ala
130 135 140Trp Tyr Leu Gln Leu Gln145 150521560DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
5tgatcagcat attgtcttgg gattttgcaa agtaaataag tactgtattt gcacccactc
60tgcccttgaa tcatccagtg tccccaaacg gtccttcttt ctccatcttt tctgctaatg
120tttactgaac atctcctaca tacctggcac tgtgcctcca gccttaagac
ccctctgcaa 180gcatctcaca tatatcaggt ctttcggacc catgagaccc
cgtgtcattg ctttacctcc 240ctgagtctca attttttcat ctgcaaaatg
cattcagagg gaagccccca ttatgaaggg 300ttccctactc ctgactgtaa
ggcactctgt ggctgttagc cacagtcagg gcgagctgct 360ttgtaggggt
tgatgtagtt tggaggccct agaagaaaag actacagggc cgggagtggt
420ggctcacgcc tgtaatccca gcactttggg agcccgaggc cggtggatca
cctggtgtca 480ggagttcaag accagcctgg ccaatatggc aaaaccccgt
ctctactaaa aatacaaaaa 540ctagccgggc atggtggcac acgcctgtaa
tccagctact aggggggctg aggcgggagg 600attgcttcaa cctgggagac
agacgttgca gtgagccaag attgtgccac tgcactccag 660cctgggcaac
agggctagac tctgtctcaa aaaaacaaaa acaaaaacaa acaaaaaaac
720cccacaaaag caaagaaggt ccagagcccg gaaggcggga ggggctctga
agactttatc 780acactttacc tcctctgttc ctcccaacag cccagctctt
ggtaggtgct gttgtccatt 840tcacagatga gaacattgaa ggggaagtaa
ttaccctaga ggtgggaaga acagagtgag 900ggtacccaac aggtagcaaa
cgacagagct gggggagggg aacccctgtc cctgcccctc 960ccctaatctc
tgaggggatc aagacagaag taaacaagct ttgccgtgcc caggacaatt
1020gttactttgt tattccagga gcgctctgcc ttctcccacc cccaatatac
cccagggctg 1080gagttaggtc ctacccatcc ccgcgtagca ggctgcccac
ccgcccaccc cgcctggaag 1140ctttctgatt tctctgttcg ccccgccagg
cgctgtgggg tccgtctcac caggtctgca 1200cgtgagcccc ctgcccccaa
tccctcccag tcccgcccgc ctctcgcgga cccggagccc 1260cgacgggagg
gggaaggcag tgggtgtgtc atgcagctgg aaggctgcgg acgggcggca
1320gtggaggggc agccccctgg cttcgggtat ggatcactgg atgctgctgc
taaccaaatg 1380tgaacctcgg ttttcatatt tgtgaaatag gcttaaaaac
acctaaatcc caaagctgcc 1440agcctaagga gcacacgtct ttgaacgctg
gcttcacgct gtcatttaag tcatttcgtc 1500ctcttggagc ctcggtttcc
acgtgggtaa aatgatcgtg aaaaaaagca ccgaggagta 1560ctttgagctc
gaacggaggc catccgtgta aagggccaga ttctgtcaat ggatcgatcc
1620ccccgatatt gatggaaacc cctgagtgca cgcccgtgct gggcgcaggg
gaaacagcga 1680cgcacgggac aaaacaagct tgcagaacag cagggggcag
agaggctgta aacaagccaa 1740cgggctgcac ttgtagcggt tctgttgcca
atgccattca gaccccagtc cgggattccg 1800cgctcggggt gcgagaggcc
gctcccgggg aggggcggga cccgggcggg gcgggagggg 1860cggggcgccc
gggcctatta ggtcccgcgc cggcagccgg gccgcagaca cgaagcctcc
1920cgggtggctt acagacgctg ccagcatcgc cgccgccagg tgagtgcccc
cgaccctgct 1980gccgcccgct gctccgccca gctcgagccc ggcggttagc
gcccaggccg cggcccgggt 2040gccgagcggc tggaagtgtg gggagccgtg
cccaggtttt accgctccag caagtcgctg 2100gcggggcgac gtctcgggac
tccggctccg aaacgtaccc tctctgtccg gggctccagg 2160tccccgacgg
gcgatcgtgc caggcgcggc ccctgcgggc ctcagtctcc gcgcctgtgc
2220aatggggtgg tccctctgcg tctcgccgcg acggctggac gccccatcgc
cgccgcacag 2280tcctccagtg cggcttccgg gcacccggct ccgaactctg
gggtcgtgcg gacccggcca 2340gaccgtagcg cggcaacgcc agcccacggc
cgcggccgca cagccccggt gcccttaaaa 2400gagggaagat ggcgtcgtgc
ccggtgccgc cgggaccggc tcccggaggc gctgcgcacc 2460tgaggaaggg
ccgaggaaag gcttcgtaac gggacgccca gaaagtccgg aacaggaacg
2520tgcacacgga gcggcgcgca gccgcccgcc ctcgcttgcc cacgcgccct
gcacaggtgc 2580gccccagact gggcggggac tagagcccgg ctggggcatg
cgaccttgtt tggtaaattg 2640gaggtgcgcc cctgcgggtg acccgcgagg
gggccccccg cctgcggggc gcggtcacga 2700caaggcttgt ggggttcgga
gctggtcgtc gtctgggctc tggcctccta aatgcgatcg 2760ctttcaagcc
cttcattgtc aattttgcta gaatgagcca ctcgtggggt aggacgtggt
2820tgttgatgtt actgttgtag ctcttactat tgtaacattt ttgttgctgt
tttaacgcag 2880tgtttactgt ggtctgggag cccgagcttc ccagggagca
ttggtgtcag cctcatgtcc 2940ccgcattagg aggagcgcag tctggtgggc
actagcgccg ggaagttctt aacatctgtc 3000tgcccctggg agcaggaggc
ctcatcttcc aggtggagaa acagagattt cagtcccagg 3060aggcacagaa
atgccagggt tgtctagcta gaagccaagg gccctttatg gaacccaagc
3120caaaacctct ttccactctc cagccctgtg gcctcggaag ggccactcca
tctctctgaa 3180cctcagtttg ttctgttcac tctgcaaaat gtatccctga
ggtttctggc caggtgaaag 3240cccctctcaa gttagtagcc tccctggagt
taagactcac cccgatttct acttaatttg 3300ggcacgcctt tggaataagt
tctcaggaat ggcacagttt gggtgttttt tgtttctttt 3360tgtttttttt
tgaaacagag cttcgctctt gttgcccagg ctggagtgca atggcgccgt
3420ctcggctaac tgcaacctcg gcctcctggg ttcaaacggt tctcctgcct
cagcctccta 3480gtagctggga ttacaggcgc ctaccacgag gcccagctaa
ttttttgtat ttttagtaga 3540gatggggttt caccatgttg gccaagctag
tctcgaactc ctgacctcag gcgatccacc 3600cgcctcagcc tcccaaagtg
ctgggattac aggtgtgacc caccccgccc ggcttgagtg 3660tttaaatact
cctgcctcag cctcccaagg tgctgggatt acagacgtga gcccctatgc
3720ctggcccttt tttttttttt aattgtctta aggatgttaa gactaagatt
ctcacacatt 3780tgactcgtgc ctgtaatcct agcactttgg gaggctgagg
caggaggatt gcttgagctc 3840aggagactag actgggctag accagactgg
gtaacatggt aaaaccctgt ctctaccaaa 3900aatacaaaaa attaaccggg
catggtggca tgtgcctctg gtcctagcta cgcaggaggc 3960tgagttcaga
ggcctacctg agcttggagg ggttgaggct gcagtgaacc ctgatcatgc
4020cactgcactc tagcctggga gacagagtga gaccctgtct caaaaaaagg
agcattaggc 4080tgggcatggt ggctcacatt tgtaatccta gcactttggg
aggccgaggg agatggatca 4140cctgaggtca ggagttgtga gacgagcctg
gccaacatgg caaaaccagt ctcttttaaa 4200aatgcaaaaa ttagccggtc
gtggtggcgt gtgtctgtaa tcccagctac ttgggaggct 4260gaggcaggag
aactgcttga atctgggagg cagaggttgc actgagcgga gatcacacca
4320tggcactcca gcctgggcca catagggaga ctctctgtca aaagaaaatt
taaaaaagca 4380tcaaatggta gatccccagt gtctgttaca gcttgggtac
tctatggctg gatagagagg 4440ctgcatacat gttagttatt gtaagtttgt
ttgttttttt gagacggagt ttcgctcttg 4500ttgcccaggc tggagtgcaa
tggctgggaa tacaggcgtg agccacctca cccggctcaa 4560gattcttaca
catttgactt gcctgtaatc ccagcacttt gggaggctaa ggcgggagaa
4620ttgcttgaac ccaggaggcg gagattgcag tgagcagaga tggtgccact
gcactcagcc 4680tgggctaacg tgagactcca tctcaaaaaa aaaaaaatta
cgtcttgtgt cacagagctg 4740cagataaacc agagactact cataaccgtg
tacttttttt ttcccccatt tctgatcccg 4800tctcaacgga aggatgtaat
tgaggaaggg cttgattggc cagtgagtct tgaagctgac 4860accactgctg
gtggtatttt cccctctccc tggaaagcat cctgttttta gtgaacctat
4920taaatgtgta gacaattaat ggctttttgc ttcccctgct gcagacttag
cggatcccat 4980gagattttga ctaccgtctg tgctcagaac agtttgagcc
gtatggagga agtctccgca 5040ccagtcttac tgttggtggt caccaggaag
ccagcagtgt gtctgaactg gacacatgtg 5100gccacttcct agcctccctt
tgtcctgcca ctggttggtt ggggctgggc cctgggagaa 5160acatagaatg
tgagcaaatc agtccgtggc ccctaagttc cttgttggcc ccgaggcagg
5220caggagaggg gcgggcacac agggcagctg attttcctag tgtcaatatt
aggatgtgac 5280aatacaaaac accactgggc ctcgccaggc actgcatggt
aaacctgttt ttctgggggt 5340gggacagaac ttggcaccca gttagccaaa
ctgaaaagcc atgcagcaaa gagagaaaag 5400ctcacttgtt tatttgagtg
agggtccctg tcattttgga tatttaagat caagtattct 5460tgaccgcccc
ctccccgaga tggagtcttg ctctgttgcc caggctggag tgcagtggtg
5520tgatctcagc tcactgcaac ctctgcctcc cgggttcagg tgattctcct
gcctcagcct 5580cccgagtagc tgagattaca ggcgcgtgcc accacaccag
gctaattttt gtatttttaa 5640tagagatggg gtttcaccac gttggccagg
ttggtcttga actcctgacc ttgtgatccg 5700cccgcctcag cctcccaaag
tgctgggatt acaggtgcga gccaccgtgc ctggccaaga 5760gatgacattt
attgtgtgtg acagtatctt cgagattaga ggctcccttt catgtgaggg
5820caaagagagc tacctgattc caggaggagc tgaaattccc tgtgcagaca
atggcgcatc 5880atgaagataa atccgagctc ggaccgtgag gtggtcagca
cgggtgtgcg cctcgcagag 5940caacaggctc acagaatgag agctgcctct
tagtgggcac ttactgcata ccatgcatgc 6000agcatttcac ctccttctca
gggctacatc tgcccatggg gctttccctc ctgcccttta 6060acagaaggtg
atactgtcca gtccagggtc tcccagccag gaacagcctg ccatcgtgag
6120cccagcacca gagtaacccc ttccctcatt ctcaggattt tgcagtgaac
tctcaggctg 6180tctgcatcct ctcaccggct ttcactgggt ccctgctctg
ttctggggcc atgcaggttc 6240ccgactggct ggggcagaca cacaggtaaa
catttaacga gaccttgatg ggctgagtgc 6300ccaatccagg tctgtttacc
caaggtgctg ggacaacaca gatgaggagc atttaattct 6360gtcctgggga
gggtggggag gaagtgactt gtgcctgagc cccaaggaag cagtgagtcc
6420cccgtgcctg cctgggggat gttggtaaca gagggaggat gtaaaggagg
agctggggtg 6480tgaaagggct gggtgcaagt tcatagggga gacccaaaca
ctgaagtgga gcccaggggc 6540cgtacctgcc tctcaagcct cagggtcggg
gccagggtga gcagtcttca tattcttcag 6600cctcagcatt tcagaacttg
ttttattttt tttttgagat ggagccttac tcctgtcgcg 6660ggggctggag
tgcagtggcg cgatctcggc tcactgcaac ctccacctcc tgggttcaag
6720tgattctcct tcctcagcct ccctagtagc tgggattaca ggcgtgcgcc
accacgcctg 6780gctagttttt gtatttttag tagagatgag gtttcgccat
gttggccagg atggttgtga 6840actcctgacc tcagttgatc cacctgcctc
aacctcccaa agtgctagga ttataggtgt 6900gagccaccgc gtctggccta
ttttttattt ttgattgagt cttgctctgt tgcccaggtt 6960agagtgcggt
ggcacgatct tggctcactg caacctctgc ctctcaggtt taagtgattc
7020tcctgcctta gtctcccgag tagcttggac tacaggtgcc cgccaccacg
cccggctaac 7080tttggtattt ttagtagaga cactgtttca ccatgttgac
cggggggtct tgaactcctg 7140accttagttg atccatctgc cttgacctcc
caaagtgctg ggattacagg agggagccac 7200cgcgccccgg cccagaactt
gttttaaata tgaacttttg aaacttaaca actgtaggcc 7260caggtggtgg
cttggcattc tctgcttcct tcatggtgat aaaaaggcac aggcttcccc
7320tttttggggt catttcaaaa tcagtcaaga gaattattag tctgttagac
ttcctctacg 7380gttaggatta tttttatagg tgttcgaaca ggaaaggaca
tagaataaaa tctcctcccc 7440taacttattg atacagggtc tcactctgtc
gcccaggctg gagtgcagtg gcgcagtcac 7500agctcactgc agcctcaacc
tcctgggctc aagtgatcct ttcgccttgt gctcttaaag 7560tgctgggagc
ctccaaaagg ctaagctgtg acgttgggta ggttacatgt attaaatgca
7620tttttttttt tttgagacag ttttgttctt gtcacccagg ctggagtgta
atggcataat 7680ctcggctcac tacaacctct gcctcccagg ttcaagtgat
tctcctgcct cagcctcccg 7740agtagctggg attacaggcg cccaccccca
cgcccggcta atttttgtat ttttagtaga 7800gacagagttt caccgtgtta
gccagaatgg tttcgatctc ctgacctcgt gatccgcccg 7860cctcggcctc
ccaaagtgct gggattacag gcgtaagcca ccgtgcccgg cctggtgaaa
7920ccccgtcttt actaaaaata caaaattagc caggtgtggt ggcgtgcacc
tgtagtccca 7980ggtacttggg aagttgaggt ggaaagatca cctgagccca
gggaggtgga gactacagtg 8040agccatgttc atgccactgc actccagcct
gggtgacaga gagaccctgt ctcaaaatag 8100taatactcca tattgggcct
ctcacagggg aatcttgggg ggagctgcag ctcagggtga 8160ctcccatctt
gtcactagcc aggtgacccc ttcattctgg agccttagct ctgaaagccg
8220caggtggggg tgccgtttca gatgcccctt ttccatttca aaggctctga
ttctagatct 8280tgaagccgga tgcggcactg gcacttggct tcagtttcca
ctgtgacgga cggaggtctc 8340ccaggcccag cccaggcagc caagcccatc
ctggaatcag aacacgctga gcacattttg 8400tagggtggca cctttttatc
caagttacta gctacacatc agtgtttaaa gagaaaaaag 8460tgagctgtct
tttttttttc ttgaaacttg aggaaacaag gtacatacta cggatttttt
8520tttttctttt tctttttttt ttttgagaca gtctcactct gtcgcccaga
ccggagtgtg 8580gtggcatgat ctcggcttac tgaaagctct gcctccaggg
ttcaagccat tctcctgcct 8640cagcctcccg agtagctggg actacaggcg
cccgccacca cgcccagcta attttttttg 8700tatttttagt agagacaggg
tttcactgtg ttagccagga tgctctccat ctcctgacct 8760tgtgatctgc
acgcctcaac ctcccaaagt gctgggatta caggcgtgag ccaccgcgcc
8820cggcccatat tttttttttt tgagatggag tttttcgctc ttgtcgccca
ggttggagtg 8880taatggtgtg atctcggctc actgcaaact cctcctccca
ggttcaagcg attctcctgc 8940ctcagccacc cgagtagctg gattacaggc
gcgcaccacc acacctggct aatttttttt 9000gaaacggggt agagacggag
ttttattacc atgttagtca ggctggtctt gaactcctga 9060cctcatgatc
tgcccacctt ggcctcccaa agtgctggga ttacaggtgt aagccaccgc
9120ggctggccca tttcatatat ttttaaaatt ttttattgtt aatttatttt
tagagagggg 9180gtctcgttgt attgcccagg ctggtcttga actgggctcg
tgcgacgtgc ccgcctcggc 9240ctcccgaagg gctgtgatga cagcacagcg
gtgcctgtga tctatggctt caatttttca 9300aggctgccag gtccatgcag
gggtgtgtgt gggatgcatc cctagaaatc aaaggtcatg 9360tgcgtgtgtg
actggtggat gctgatggtc acactaccct ccagaaaggc agcaacatgt
9420tcagtccacc gtggcgtcct gagctgtgag tggcctctgg tctcctctct
tctaaggctg 9480ttgattgtgt taaccagctt ttatggagcc cctgctaggt
ggtggaaaat gggcagtagt 9540gtatgttttc atcctctcag cagccctgga
ggcccggctg tttctgcaag gctcagaggt 9600gagctggttg ttctgcctca
cacagcaggg gccgagcagg gggtggaact gaggtccagc 9660ctggctgcag
gctcagggcg tttcctgctg ggttacactg gagttgtgtg cgctccaagg
9720gctttgtgcc accccaccct catggtccag ggccaggctg tgactgggag
ggtggcacgt 9780ggaagactgg accagggttt tctagctggg cccactcagc
tggccctccc tgtgacccca 9840gcgtggcccc tgaccctctc ggtttgcctg
cctgtgaaac aggaagtgtc agatcccgcc 9900tgcttggctt tgacaggact
gggcgaggtc atgagagtct gagggtccct accaggagtt 9960cgacctcatc
ctcaggtcca gccccatcca cctctgcccc caaccccgct ctgacactcc
10020tgcagcctcg gccctcgaac acctgctgag ggctgagcag aacacatctc
tacttgcttg 10080tgcttctggg caacctggga ggggcttggg aggtagatgg
cgtttcccca tcttccagat 10140gagccatagg cgagctgtcg agtgcagagc
tgttcaccaa atgtaggctg ctctgtccag 10200aacctgccct ccctcccact
cggagccctg caggtgccct caaggccgtg ggtgtggtac 10260tcttttctgc
acatactaga tttgcatcct aacctcttag ggtaaaatag ggcttctgac
10320tggcgcctgg tgtttccact ggtgagtgtg taggaggcta agtgcgggct
gtagcctggg 10380tggggacaca tggctaggtg tgtcttcctt caccaaagaa
gggtttcaac tactcatttg 10440caagtttgct ggaaaaaaaa agataataaa
aaccagtcca gtttcctgta tctgcagctg 10500cagtgacctc acagtgggtc
ctctgggaat aggttgcctt catgtcttca gtcaacaaac 10560atatgtctgc
tctatgccag gccttgggct gacaaccaga aactgataag agcagcagcc
10620cctctcttag cggtttactc tgtgccggat catttcatcc ccacaacagt
cctgtgcaat 10680aggtgttact agtatctcca ttctaatttt ttttttttct
gatatggagt cttgccctgt 10740agcccagcct ggagtgcagt ggtcagctgg
agccagaggg gagcagagac aggggctgca 10800ggacccccag gaggccccca
agcagactct gagggtccaa gaaggtgatg cccgaatagg 10860ccagcctcat
acccatcctc tcagtacttt gggaggccca ggcgggagtt gaagccagga
10920gttccgagac aagcctgggc aacaaagcaa gaccctatct ctacagcaat
tctataaaaa 10980tgagccaagt gcagtggtac atacctgtcg agttaccctg
gaggctgagg tgggatgatt 11040acctgagccc aggagtttga ggctgtagtg
agcatgatca ggccactgca ctccagcctg 11100ggcaaacggg tgagaccctg
tctgtgaaaa aaataataat aaaatttaat ttttaaaaaa 11160ggaccaatag
gcgtaggcat gggccggtct gaggttggct ggtgagctct gcacatttga
11220agactttgga ggcatggcag gaccaggcac catccatgga aagcatactt
gagtgtggtt 11280agcgcgggaa ccaagattgg ggtggcaggg ccacaggggg
agggcctgtt agtggatgtg 11340gcacaacccc ccacttgttt ttgagacaag
tcttgctctg tcacccaggt tggagtgcaa 11400tggcgcggtc tcggctcact
gcatattctg cctcctggat tcaagtgatt ctcctgcctc 11460agcctcccaa
gtagctggga ttataggcga gcgccaccac acctggttaa tttttgtatt
11520tttagtggag atggcgtttc accatgttag ccaggctagt ctcaaactcc
taagctcagg 11580tgatccacct gcctccacct ctcaaagtgc tgggattaca
ggcgtgagcc gcactgcacc 11640aggccaggtc tagggagccg aacagagcag
tgacagggtc agggttgagg ctgctacctt 11700gagattaggt tggagagagc
tccactgaag gctggagacc agtaaggcat tcagagctgg 11760tgccaggtag
gagctgacag gactgggcct cccttgggct ggtggggtgg ccaggagaga
11820ccagagcagg aggtcgctgc tgcaaggacc ctcctgggca ggctccccac
cttccgctcc 11880ctggctccac acttttccca tgcagatctg ggccctaagg
ccagcctagg cccagttggt 11940gatgggatct ttgccattca tatcaagtca
atctgagggc ctgggcccag aggccagatg 12000gttaaaggtt cagattaggt
cccttctttg tggtagaggg taatgggtat ttaataaatt 12060aatccaaagt
gctgtaatcc cagtactttg ggaggctgag gcgggtggat cacttgagat
12120caggagttga gaccagcctg gccaacatgg taaaaccccg actctactaa
aaatacaaaa 12180attagccggg tgtggtggca tgtgcctgta atcccagcta
cctgggagtc tgaggcagga 12240gaatcacttg aacctgggag gcggaggttg
cacctgagct gagatcacac cactgcactc 12300cagcctgggc aatagagtga
gactgtctca aaaacaaaca aacaaacaaa caaaaaaaca 12360ctaccaccaa
caaaaacaca atagatgggg aaactgaggc tggtgcagga agggttgggc
12420tagggtatca acctaaggtg agatgtggtc ctgggtctac tcagatgggg
aggttggcag 12480ggtggggagc ttgaaagggt cttgcaggca gagcgtgggt
gactgactcc tcaggtaggc 12540ctgacctgca ctctgctgca gaccctcggc
agcgcagccc actctggtgg ctggggcctt 12600cttcctatct ggcgcctaag
gattctcgga atgcctgctg ggtaaggggg ctgtgggcac 12660agtgggtcaa
ggcgaggctc ggtgcagctc acagctcagc
tcacagtgag gtgaggttcc 12720actgttgctc tggctttgcg gggcgtggag
gtactggggt ctccaggagc acaaagcagg 12780agactgcctt ttcgagggga
agggaaaggg gggaaaagca aacaagggct ggagctgctt 12840tcctagggaa
atgccatttg catagacacc caaggcagct ggaagttctg gacacagttc
12900ttctaatgcc agcttaacac gcacactgct ggccgtctat acaatgtaaa
acatctcatc 12960tgtgaaacct tattttttct ttttgagtca gaatctagct
ctgcctccca ggttcaagct 13020gggattacag gcgtatgtca ccacgcctgg
ctaattttgt atttttatta gaggcggggt 13080tttaccatgt tggccaggct
ggtctcaaac tcctgacctc aggtgatccg cctgtctcag 13140cctcccaaag
tgctgggatt acaggcgtga gccaccgcac ccggcccatc catctccctt
13200gaatgctgaa agcaagtcgt gagcacacac ttgtgcctag gcctgtggga
gggcggccct 13260gtgggtcctg cctgccgtca gttttctctc tcctgggggt
cagggagact tggagctctg 13320tcccttcttg gcttagtcat gaacctctct
aagcctcagt gtcctgtgtg aacaaacagc 13380tcagtgagat ggtgggaggg
cggagtgtcc tgtcagcagg catggggaat taaaaattaa 13440gaacacccag
ggcattatgg taagaaaata atgagttgaa gcataaaggt aatggttagt
13500gccaacttcc tcccagcgtg ccaggccctg ttcaaagtac atgaaggagt
tccttatttc 13560gtcttcccaa tagcccaggg cggggctctc agaccatgaa
acccatttca cagatgaaga 13620aattgacaca ctgctgctaa ctgccagagc
cgggctttga actaggccat tagatttccg 13680tgccacagtc atcgtcatta
tcgtcatcta catctaaggc tgtttggtac cctgtagggt 13740aaatgtgggg
tacatgatgg tgaatcaggt cgcagaaccc ttctctgcca tcatgatgaa
13800acatgtactg gcatgggatt cgcaatgatc atagttagga aggcctttgg
tggcaaagaa 13860gaggaaactc agtgaatggt gatgaggaca catttgggtt
catttttctt acacacagtt 13920ggaggagaca gctcagtgac accctcaggc
ctggccgctc tctgcctccc cgctctgctc 13980ttcttggtca gccactgcag
ctccatcttt gaaggagctg ccctggctgt gtctactccc 14040tggcatccaa
cccctgcctc cagcctctga gccctcattg accagaactg ggttacatgg
14100ttgtcaccag ctgcaaggga tgctgggaag ctgagggaga ggagggccat
ggttggctta 14160gatgaatgag gatgcagggc caccccgaag aaaattgggg
ttctatcagc aaggagccag 14220gtggaatgga tatcggagag gtaaatagca
tctgccagta aagaaggctg taagacactg 14280tatggggtga tcccatttgt
gaagaaggat gtgtgatgcg cacaagtgtt taggaaaaac 14340actagaagaa
ttagcgatgg tctctctggg aatggggatt gtgatacgtt tttctcatgc
14400tgcctctctg cctttcttct caagagaaac agcagacacc aaagaacagg
tgtagccttc 14460tgttaagtgg tacagaggaa tcgccctgca gcctggcctc
tgtcccctgt gctgacagca 14520ggtgcctggg ttttctgctt tgacaggttc
ttgagggctg gtgtccgagg agaacttcta 14580atatttattt atactctgat
tttgtttcac aaagaagaaa aaggctgggc gtggtggctc 14640atgtctgtcg
tcccagcact ttgggaggcc gaggtggggt ggatcacttt gaggtcagga
14700gttcaagacc agcctggcca acatgatgaa acccccgtct ttactaaaaa
tacaaaataa 14760ttagccaggc atagctcatg cctgtagtcc cagctactcg
ggaggctgag gcaggagaat 14820tgcttgaacc caggaggcag aggttgcagt
gagccaagat tgtgccactg ccctccagcc 14880tccagcaaga ctcagtatcc
aaaaacaaag aaagaaagaa agggccctta tgaatcatct 14940tcaagttcct
cccttccaag aacagagatg ctaggcgact tgcccagagg cacacagcca
15000gcatgtagca ggattcatgc ctgtgggagc tggggttggg ggatattgac
ggctttagcg 15060gatcaaaaga gctggtgagt agggctatac aatctggggg
tcatcctgtg agagagcccc 15120cagactgctc agttcttagg ggtccagtca
tatgctgtct ttttgcccca gaggagaaat 15180gtctgaagta agacccctct
ccagagacat cttgatggag accctcctgt atgagcagct 15240cctggaaccc
ccgaccatgg aggttcttgg catgactgac tctgaagagg acctggaccc
15300tatggaggac ttcgattctt tggaatgcat ggagggcagg taggtcccca
tggcctgccc 15360taccccctgc ctgatagtga cttcaggggt gggctggatg
agcagacctt ctgtgagcgg 15420gggagcgcct gcagatgcct cccaggcagg
gcctccgaga ggaagatttc ccggatgtgg 15480gcatggaggc ttctgccctg
ggagcggctt cactttgctg ccccaccctc ccctgatacc 15540agctcacaga
cccctggaca gccagcatgt tcacagtctc aagatggacc tggggcccct
15600ggctgtcaaa atggcacagt gtttgggcct cagggagcta gacaggccct
tagcgacctg 15660ctgaggacat cagggcctct tgaaggaggt gactctgttg
ccagaaaggg gacccgatcc 15720agatcccaag agagggttct tagatctcaa
gcaagaaaga atttgggcca cacgcagtgg 15780ctcgcgcctg taatcccagc
acttagggag gctgaggcag gcggatcact tgaggtcagg 15840agctcgagac
cagcctggcc aacatggcaa aaccctgtct ctactaaaaa tacaaaaagt
15900agctgggcct ggtggcacat gtctgtagtc ccagctagtc gggaggctga
ggcaaggaga 15960atctcttgaa cccaggaggc agaggttgca gtgacccaag
gttgcaaaaa tgcactccag 16020cctgggcaac agagcgagac tccatctcaa
aaaaagaaaa cctactctat aggcagagca 16080gcggcatggg ctgctcaact
gaatatactt atagttattt cctgattaca tgctaaacaa 16140ggggtggatt
attcatgagt ttccgggtaa ggggtgggca gttcccggaa ccgagaattc
16200ctcctccttt tagacaatat agggcaactt cctgacgttg ccgtggcatt
tgtaaactgt 16260cgtggcactg gtgagtgact ccaacatgct aatatagttg
gtgcacaatg aatagtgagg 16320atgactggag gtcacttttg tgcccatctt
ggctttggtg ggctttggct ggcgtctttg 16380ccgaatcctg ttttatcagc
agggtctttg taacctgtat cttatgctga tgtcctatct 16440catcctgtga
cttagaattc ctagcctcct gggaatgcaa cccagtaggt ctcagccttt
16500tgttacccag cccctaatca agatggagtc gctctggttc aactgcctct
gacggctctc 16560attttagaaa accagaaccg ggccgggcgc cgtggctcat
gcctgtaatc ccagcacttt 16620gggaggctga ggtgggcgga tcacctgagg
ttgggagttc gagactagcc tggccaacat 16680ggtgaaaccc aacctctact
aaaaatacaa atgttaccca ggtatggggc cgggggtggg 16740gggatgggcg
ctggtagtcc cagctactca ggaggctgag gcaggaaaat cacttgaacc
16800cgggacgtgg aggttgcagt gagccctgag attgcaccac tgcactccag
cctgggtaac 16860agagtaagac tccgtttcac aaaaaaagag aaagagaaaa
gaaagaaaga aaaaagaaag 16920aaaaccagaa cctatggaag agcctgagga
ggccatgcag ttcctggtcc cggcttcaaa 16980gaatcactga ggcatgaaga
agggatcttc cttggactga caaggaccct ggaggcagca 17040ggatttgcct
ggggtttcag gggccaaggt ccagtaggac ctgacccttg cactctgcca
17100actgctccca gctatgtcca gcacacggca gcatacccag atgtccaggc
acacccggcc 17160atcccagcgg caggaaggac ctgcactttt tttttttttt
tttgagacag agtctctgtc 17220acccaggctg gagtgcagtg gcatgatctt
ggctcactgc aacctccacc tcccgggttc 17280aagtgatttt cctgcctcag
cctcctgagt agctgggagt gcaggcgtgc accagcatgc 17340ccggcttttt
tttttttttt gagacggagt ctcgctgtgt cgcccaggct ggagtacagt
17400ggcatgatct ctgctcacca caagctccac ctccctggtt tacgccattc
tcctgcctca 17460gcctcctgag tagctgggac tacaggcgcc tgccaccaca
cctggctaat tttttgtatt 17520ttcagtagag acagggtttc atcatgttag
ccaggatggt ctcgatctcc tgacctcgtg 17580atctgcctgc ctcggcctcc
caaaaagtgc tgggattaca ggcgtgagcc accacacccg 17640gccaggactg
gcactttcat tcttcccctg gtacagatga ggaacgtgaa ggcccaggag
17700cagctgacct cagtagtttc taccctgagc ctggaagaga atggtcaggc
ctgacctggg 17760ctgttgcttt ctgctgggga atcctgtctc tgatgtggca
cctcccacag gccagggacc 17820agctccatgt tccacaggat tcttttctgc
tcaggatttt ttttttcagc ctcaggattt 17880ttagtcatta aggattttag
aactggaggc acctgtccat taatggacaa ggttggagga 17940catgaagcct
ggggcagtat tttcctgagg tttcccaggg taacagaagg taggaccaca
18000gcccccaacc ctgattgcca gcccacacct gtacccctcc cacccgcatc
ccctccaagt 18060agggagtgta ggcccagggc attggcaggt caggtgggtg
gggccagggc ccagggccgg 18120cctcagctca gggcccctag gagtcccact
tccctgggag ttcccccggt gtagtcactg 18180ggatggaatg cacacaggtc
cctggtagag gcccagacca gcaagctggg agggtgggga 18240gtgtcttagt
ctttgtgtgg gagtgaatgc gggtggggtg taggatcaag gatgtggttt
18300agtggggggc ctactagggg cttcttagtc cccccattcg gagaggggtg
gtgccagcag 18360ggctggtttc aggattggga ccaggacaca ggtcttagag
ccccctcacc tcccacaaag 18420gaaggtaagc tgcaacccct gtgggaaggg
ggctcaaggg ggctcagcca tccgggggac 18480tcagcaccac cagacctgtc
cattcccctt ggtcagcctc agccatccgg gggctcagca 18540cccccagacc
tgcctgttcc ccttgtccac catttccacc aaagcccaca ggtgggcctg
18600ccccaggatt ctttttgaaa tagcttgcac ctctggggcc acccacggat
gggacctcat 18660gggtatctct gaatccaagt gggagccgga ggagggatac
agggaggccc actcactgcc 18720tgccccaaat ctgacaccat ctcttatcct
ctgggccact cccagctgca cacaatccgt 18780tggctgggtg ggtccaggtg
catgtggcct ccacggcaca gccacacccg actcctgtgt 18840gtgctccctg
cagtgacgca ttggccctgc ggctggcctg catcggggac gagatggacg
18900tgagcctcag ggccccgcgc ctggcccagc tctccgaggt ggccatgcac
aggtagccgg 18960cctatgccct atgcctctac acctggggag gggccctggg
cggtgggtgg aggccctgaa 19020cacagcacag ggctgggccc tgaggaagct
ctgtggggga tgtgccttga cactctgggg 19080ctatactgaa acccttggct
gcttcccgca cctctcctgg gagcccccag ctcctggcac 19140tcgccccctg
ccacctgtcc ctggcattcc tgggcaacaa agcagagccc agggcccttt
19200tctctctcgc cgtcctcatt gagcccagat ggtacatttc caccgtggtc
ttaagagggg 19260gtgctacgtg agctcggatc ctagatcctg ggggctgggc
ctctcagcga gtctccggtg 19320ggggaataga gtgggcagtg gcttgtgagg
gccctgtaga atgggggttt attttccacg 19380tggccagctc tggaagacag
ggccaaacac ccggtgggct ggcgggcggc tggctggcgg 19440aggaagcagg
aagttggaat gaatttgacc acaaacagtc cataccacgc ggcccctgca
19500ggtggaggcg cccacggaaa gggccccggg tggctgtggg gtgggagggc
acaggcccct 19560gctccccaca gctgtgatgg tgtcatctaa gtacaggctg
cccctacctg ctcctgcagt 19620aatggctttg tccccccatc ctctttgtct
atagcctggg tctggctttc atctacgacc 19680agactgagga catcagggat
gttcttagaa gtttcatgga cggtttcacc acacttaagg 19740agaacataat
gaggttctgg agatccccga accccgggtc ctgggtaaga gccttgagat
19800ccctgaccct gacttgcgct gcggccagtg ggggctgtca gagccgctcc
ttggggcgcc 19860acagtcccca ccactccgta tcatcatctg tgtcacctgt
gtccacatct gcctgatccc 19920atgggctttt gggtttgaga tgcctggttc
tgagtgcaca aaccagtgca tggtcctggg 19980tctccctctg gtccgagagc
cttcacctgg caggcaggac tcccgtctcc tggccagggc 20040aggggcctcc
ctgagcagcc ttcctggtag cctggtccca tggtgtccac tcggcaccgc
20100ccaccacaag ggcagctgac tgccctcacc tgtgcccacc gggtgtcttt
gcctgtgtcc 20160cgcagactgg caggcccagg ccacgctggc ctctctggcc
acgtcctcag ggccactttc 20220cccctctcct gaactccttc cttctctggt
cccctcgagc tccttcccag tccccaccct 20280cctgggcttc cccttggcac
tccgctgtca cccgtctggc cccattgctg gggcctgccc 20340cgagcctgac
tcctctgctt tgctcccaca ggtgtcctgc gaacaggtgc tgctggcgct
20400gctgctgctg ctggcgctgc tgctgccgct gctcagcggg ggcctgcacc
tgctgctcaa 20460gtgaggcccc ggcggctcag ggcggggctg gccccacccc
catgaccact gccctggagg 20520tggcggcctg ctgctgttat ctttttaact
gttttctcat gatgcctttt tatatttaaa 20580ccccgagata gtgctggaac
actgctgagg ttttatactc aggttttttg tttttttttt 20640attccagttt
tcgttttttc taaaagatga attcctatgg ctctgcaatt gtcaccggtt
20700aactgtggcc tgtgcccagg aagagccatt cactcctgcc cctgcccaca
cggcaggtag 20760cagggggagt gctggtcaca cccctgtgtg atatgtgatg
ccctcggcaa agaatctact 20820ggaatagatt ccgaggagca ggagtgctca
ataaaatgtt ggtttccagc agtctctggt 20880cctctctggg gggttggcag
caccagcacg ggcttcctct cgccccaggg aggcacactg 20940tgttggtggg
gagggcaggg cctgtgtgct cctaatcaga tccttccctg caaaagggga
21000ccgcaaatgc tgccttggtt tggcccgacg gtttgccttc tcccttgtcc
cggtctgtgg 21060cctaaaatcc actgttgggg gttatttcct ttgggctttc
agttcctctt taggggatgc 21120ctgcctctcc ctgcacagac tcctcaccac
agggatgcag ccgtggctcc gctcacaggg 21180agaggtgtgg tggggattgg
ggagcaggac aaggggcacc aggggcagga gggccaggac 21240cctgctttgt
acctttgtag gcttggtacc tgccctgggc cttggcctcg tgccatttgt
21300agaccccacc aggcctccct cccaggacat cggctctgtg tctgccctcc
accccaaatg 21360tcaaagctgg gctgggccgg ccaccattct ccagccccca
acccccctca tccccagagc 21420tgggagatgc agctgttcat acctcccagg
ctggctgggc agaccctgca tcctgctcac 21480tcctccctcc atcctggcct
ccaaaccaaa ggggacctcc aataggcttt cctgcctcct 21540tatgtcttct
ctccggtctg 215606963DNAArtificial SequenceDescription of Artificial
Sequence Synthetic Primer 6gacacgaagc ctcccgggtg gcttacagac
gctgccagca tcgccgccgc cagaggagaa 60atgtctgaag taagacccct ctccagagac
atcttgatgg agaccctcct gtatgagcag 120ctcctggaac ccccgaccat
ggaggttctt ggcatgactg actctgaaga ggacctggac 180cctatggagg
acttcgattc tttggaatgc atggagggca gtgacgcatt ggccctgcgg
240ctggcctgca tcggggacga gatggacgtg agcctcaggg ccccgcgcct
ggcccagctc 300tccgaggtgg ccatgcacag cctgggtctg gctttcatct
acgaccagac tgaggacatc 360agggatgttc ttagaagttt catggacggt
ttcaccacac ttaaggagaa cataatgagg 420ttctggagat ccccgaaccc
cgggtcctgg gtgtcctgcg aacaggtgct gctggcgctg 480ctgctgctgc
tggcgctgct gctgccgctg ctcagcgggg gcctgcacct gctgctcaag
540tgaggccccg gcggctcagg gcggggctgg ccccaccccc atgaccactg
ccctggaggt 600ggcggcctgc tgctgttatc tttttaactg ttttctcatg
atgccttttt atatttaaac 660cccgagatag tgctggaaca ctgctgaggt
tttatactca ggttttttgt ttttttttta 720ttccagtttt cgttttttct
aaaagatgaa ttcctatggc tctgcaattg tcaccggtta 780actgtggcct
gtgcccagga agagccattc actcctgccc ctgcccacac ggcaggtagc
840agggggagtg ctggtcacac ccctgtgtga tatgtgatgc cctcggcaaa
gaatctactg 900gaatagattc cgaggagcag gagtgctcaa taaaatgttg
gtttccagca aaaaaaaaaa 960aaa 9637160PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 7Met
Ser Glu Val Arg Pro Leu Ser Arg Asp Ile Leu Met Glu Thr Leu 1 5 10
15Leu Tyr Glu Gln Leu Leu Glu Pro Pro Thr Met Glu Val Leu Gly Met
20 25 30Asp Asp Ser Glu Glu Asp Leu Asp Pro Met Glu Asp Phe Asp Ser
Leu 35 40 45Glu Cys Met Glu Gly Ser Asp Ala Leu Ala Leu Arg Leu Ala
Cys Ile 50 55 60Gly Asp Glu Met Asp Val Ser Leu Arg Ala Pro Arg Leu
Ala Gln Leu 65 70 75 80Ser Glu Val Ala Met His Ser Leu Gly Leu Ala
Phe Ile Tyr Asp Gln 85 90 95Thr Glu Asp Ile Arg Asp Val Leu Arg Ser
Phe Met Asp Gly Phe Thr 100 105 110Thr Leu Lys Glu Asn Ile Met Arg
Phe Trp Arg Ser Pro Asn Pro Gly 115 120 125Ser Trp Val Ser Cys Glu
Gln Val Leu Leu Ala Leu Leu Leu Leu Leu 130 135 140Ala Leu Leu Leu
Pro Leu Leu Ser Gly Gly Leu His Leu Leu Leu Lys145 150 155
1608160PRTArtificial SequenceDescription of Artificial Sequence
Synthetic Peptide 8Met Ser Glu Val Arg Pro Leu Ser Arg Asp Ile Leu
Met Glu Thr Leu 1 5 10 15Leu Tyr Glu Gln Leu Leu Glu Pro Pro Thr
Met Glu Val Leu Gly Met 20 25 30Thr Asp Asp Glu Glu Asp Leu Asp Pro
Met Glu Asp Phe Asp Ser Leu 35 40 45Glu Cys Met Glu Gly Ser Asp Ala
Leu Ala Leu Arg Leu Ala Cys Ile 50 55 60Gly Asp Glu Met Asp Val Ser
Leu Arg Ala Pro Arg Leu Ala Gln Leu 65 70 75 80Ser Glu Val Ala Met
His Ser Leu Gly Leu Ala Phe Ile Tyr Asp Gln 85 90 95Thr Glu Asp Ile
Arg Asp Val Leu Arg Ser Phe Met Asp Gly Phe Thr 100 105 110Thr Leu
Lys Glu Asn Ile Met Arg Phe Trp Arg Ser Pro Asn Pro Gly 115 120
125Ser Trp Val Ser Cys Glu Gln Val Leu Leu Ala Leu Leu Leu Leu Leu
130 135 140Ala Leu Leu Leu Pro Leu Leu Ser Gly Gly Leu His Leu Leu
Leu Lys145 150 155 1609160PRTArtificial SequenceDescription of
Artificial Sequence Synthetic Peptide 9Met Ser Glu Val Arg Pro Leu
Ser Arg Asp Ile Leu Met Glu Thr Leu 1 5 10 15Leu Tyr Glu Gln Leu
Leu Glu Pro Pro Thr Met Glu Val Leu Gly Met 20 25 30Asp Asp Asp Glu
Glu Asp Leu Asp Pro Met Glu Asp Phe Asp Ser Leu 35 40 45Glu Cys Met
Glu Gly Ser Asp Ala Leu Ala Leu Arg Leu Ala Cys Ile 50 55 60Gly Asp
Glu Met Asp Val Ser Leu Arg Ala Pro Arg Leu Ala Gln Leu 65 70 75
80Ser Glu Val Ala Met His Ser Leu Gly Leu Ala Phe Ile Tyr Asp Gln
85 90 95Thr Glu Asp Ile Arg Asp Val Leu Arg Ser Phe Met Asp Gly Phe
Thr 100 105 110Thr Leu Lys Glu Asn Ile Met Arg Phe Trp Arg Ser Pro
Asn Pro Gly 115 120 125Ser Trp Val Ser Cys Glu Gln Val Leu Leu Ala
Leu Leu Leu Leu Leu 130 135 140Ala Leu Leu Leu Pro Leu Leu Ser Gly
Gly Leu His Leu Leu Leu Lys145 150 155 1601012PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 10Ala
Leu Arg Leu Ala Cys Ile Gly Asp Glu Met Asp 1 5 101112PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Primer 11Leu
Arg Leu Ala Cys Ile Gly Asp Glu Met Asp Val 1 5 101218PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Peptide 12Ala
Leu Ala Leu Arg Leu Ala Cys Ile Gly Asp Glu Met Asp Val Ser 1 5 10
15Leu Arg1318PRTArtificial SequenceDescription of Artificial
Sequence Synthetic Peptide 13Leu Ala Leu Arg Leu Ala Cys Ile Gly
Asp Glu Met Asp Val Ser Leu 1 5 10 15Arg Ala1427PRTArtificial
SequenceDescription of Artificial Sequence Synthetic Primer 14Glu
Gln Val Leu Leu Ala Leu Leu Leu Leu Leu Ala Leu Leu Leu Pro 1 5 10
15Leu Leu Ser Gly Gly Leu His Leu Leu Leu Lys 20
251532DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 15aggacccagg tacctatgtt caaaagtgcc tc
321628DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 16ccttgcctgc tgctttccac caagtgct
281731DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 17caggttggga aaatggtcag ccctcctgaa a
311830DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 18cgttctgagg cgggcaatca aatgacctat
301933DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 19cccgctagcc taattttatt ttatttttaa ttc
332036DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 20cccctcgagg tattttggaa aaatgtcctt atctag
362131DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 21cgcacgcgta ggcatcagct ctctacaatt c
312232DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 22agcctcgagc aggatctgag ataagaacca cg
322328DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 23acggcgctcg agtccatcag ttctcatc
282428DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 24ttacttaagc ttgtgtagga cgcctgtc
2825452DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 25tacgggccag atatacgcgt tgacattgat tattgactag
ttattaatag taatcaatta 60cggggtcatt agttcatagc ccatatatgg agttccgcgt
tacataactt acggtaaatg 120gcccgcctgg ctgaccgccc aacgaccccc
gcccattgac gtcaataatg acgtatgttc 180ccatagtaac gccaataggg
actttccatt gacgtcaatg ggtggagtat ttacggtaaa 240ctgcccactt
ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
300atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg
gactttccta 360cttggcagta catctacgta ttagtcatcg ctattaccat
ggtgatgcgg ttttggcagt 420acatcaatgg gcgtggatag cggtttgact ca
45226180DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 26agacggtgag agcgagtcag ggattggctg gtctgcttcg
ggcgggctaa aggaaggttc 60aagtggagct ctcctaaccg acgcgcgtct gtggagaagc
ggcttggtcg ggggtggtct 120cgtggggtcc tgcctgttta gtcgctttca
gggttcttga gccccttcac gaccgtcacc 18027231DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
27cggccgccag tgtgatggat atctgcagaa ttcgcccttg cgatctgtca gagcacctcg
60cgagcgtacg tgcctcagga agtgacgcac agcccccctg ggggccgggg gcggggccag
120gctataaacc gccggttagg ggccgccatc ccctcagagc gtcgggatat
cgggtgaagg 180gcgaattcca gcacactggc ggccgttact agtggatccg
agctcggtac c 231281757DNAArtificial SequenceDescription of
Artificial Sequence Synthetic Primer 28ccttgcctgc tgctttccac
caagtgctgg agagctggtg aattgctcac tcccggctca 60ttcctctaat gaccgaagcg
tctcgcagat gcaacctgcc gtggaggagc agggagggag 120tgatttccag
gtgtgggctt tttcagccat tcctaaaggc gacttgagtt cacctcactc
180actccagcat ttgtactcct gttgtggaaa aggcagtgag cacaagccaa
gcccgctcca 240ccttcacccc gccccacctc ccccggccct ttcctgggcc
agtcttaggg ccctgagtac 300agacagcctg gctacccgtt aaccattctc
agcgtgtggc tgctttttac acacatgtgt 360acatatgcac ggacacacac
acacacacag aggcttcccc agtactcctc tatataggaa 420cccgtcacca
tcccagacat atgcagaaga aagcccaaac cggctgtgtg agacaggaac
480aattaacacg gtaacagatc cgataatgca gaccatcagg cctaaagaac
acggagggac 540tgtgttctac ctccttatag aaaagcaatt agtgcctttt
tagctttgga accatgcccg 600gtggtgtgtg tgtggacaga actgctggct
ggttgttaag ttgctactaa acacagtgtt 660gtttctcgtg gtctctgccc
ttgttaacta ggattgaggc acttttgaac ataggtacct 720gggtcctaag
ggcgaattcc agcacactgg cggccgttac tagtggatcc gagctcggta
780ccaagctcca gctgggaata gagataggag gggacccagc tggatgcagt
gggcagtggg 840ggtcatagag tcaagagggt acagaataca atggggtcct
agtatcatgg tggaggtcag 900aaagagccct aaaagagagg gtcaaggtag
gaggttagtg aaggtccacc tccaccctct 960ccaggacagg gacatcaggc
cacaattaat ttctctgcag ttggtgagtg gtcatggtct 1020ctggagtccc
cagcatccag agtgtccctg gtctagtggt cccccctttc tgagccacag
1080ccactttctc catcaaatga ggccagtaat acccatccca tagtgatgct
gtgaggatga 1140gatgagcatc tgtaagtgct gaagataatc cctgacacat
cccaagcatt cagcagtgca 1200agcatacact tacacggcac tccccagagc
caggcatgtg ctggtgcctc atacacgtga 1260ccacatttga tcgtcacaat
gaccctgtga gggagactgt gcaacagagg actgaccttg 1320ctcaaagacc
tcaggcgttt cccctcagag cctgagaggt catctctttt tttttttttt
1380tttcctttct ttctttttct tttccatttc tttttctttg caagaggtca
tctctaatgc 1440tttggaatat cctgccagat tagagtccct ttgttcacct
gaaggtttgg gccacaccag 1500atagtctaac ggtgtgattt gtgctgaagg
ttttgagcca cactatatca gctagatttc 1560tagagcggcc ggccgcaata
aaatatcttt attttcatta catctgtgtg ttggtttttt 1620gtgtgaatcg
atagtactaa catacgctct ccatcaaaac aaaacgaaac aaaacaaact
1680agcaaaatag gctgtcccca gtgcaagtgc aggtgccaga acatttctct
atcgataggt 1740accgagctca tttaggt 175729646DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
29aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa ctatgttgct
60ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat tgcttcccgt
120atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta
tgaggagttg 180tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt
ttgctgacgc aacccccact 240ggttggggca ttgccaccac ctgtcagctc
ctttccggga ctttcgcttt ccccctccct 300attgccacgg cggaactcat
cgccgcctgc cttgcccgct gctggacagg ggctcggctg 360ttgggcactg
acaattccgt ggtgttgtcg gggaagctga cgtcctttcc atggctgctc
420gcctgtgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc
ttcggccctc 480aatccagcgg accttccttc ccgcggcctg ctgccggctc
tgcggcctct tccgcgtctt 540cgccttcgcc ctcagacgag tcggatctcc
ctttgggccg cctccccgcc tggaattcga 600gctcggtacg ggctcgacta
gagtcggggc ggccggccgc ttcgag 64630819DNAArtificial
SequenceDescription of Artificial Sequence Synthetic Primer
30ttatgtcaca ccacagaagt aaggttcctt cacaaagatc ccaagctgtc gatcgacatt
60tctagaggat ctcggacccg gggaatcccc gtcccccaac atgtccagat cgaaatcgtc
120tagcgcgtcg gcatgcgcca tcgccacgtc ctcgccgtct aagtggagct
cgtcccccag 180gctgacatcg gtcggggggg cggatctcgg acccggggaa
tccccgtccc ccaacatgtc 240cagatcgaaa tcgtctagcg cgtcggcatg
cgccatcgcc acgtcctcgc cgtctaagtg 300gagctcgtcc cccaggctga
catcggtcgg gggggcggat cccccgggct gcaggaattc 360cggcgataca
gtcaactgtc tttgaccttt gttactactc tcttccgatg atgatgtcgc
420acttattcta tgctgtctca atgttagagg catatcagtc tccactgaag
ccaatctatc 480tgtgacggca tctttattca cattatcttg tacaaataat
cctgttaaca atgcttttat 540atcctgtaaa gaatccattt tcaaaatcat
gtcaaggtct tctcgaggaa aaatcagtag 600aaatagctgt tccagtcttt
ctagccttga ttccacttct gtcagatgtg ccctagtcag 660cggagacctt
ttggttttgg gagagtagcg acactcccag ttgttcttca gacacttggc
720gcacttcggt ttttctttgg agcacttgag ctttttaagt cggcaaatat
cgcatgcttg 780ttcgatagaa gacagtagct tcatctttca ggaggctag
819311352DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 31acagacattg acattgtgtc atctagtata caaataggtt
cttggagtac tttactaggc 60atggacaatg cccaatgcct gtcccattct tcaggcatat
ttttatttgt gggctttatg 120tccctattaa gaaaaagact aagaacaaga
tgctatcata ttttcttaac tggaatggta 180gatgtttaaa catgatgact
accaagcttg gctagaacat tgtgtcatct agtatacaaa 240taggttcttg
gagtacttta ctaggcatgg acaatgccca atgcctgtcc cattcttcag
300gcatattttt atttgtgggc tttatgtccc tattaagaaa aagactaaga
acaagatgct 360atcataagct ccaagcttat cgccagctgg gaatagagat
aggaggggac ccagctggat 420gcagtgggca gtgggggtca tagagtcaag
agggtacaga atacaatggg gtcctagtat 480catggtggag gtcagaaaga
gccctaaaag agagggtcaa ggtaggaggt tagtgaaggt 540ccacctccac
cctctccagg acagggacat caggccacaa ttaatttctc tgcagttggt
600gagtggtcat ggtctctgga gtccccagca tccagagtgt ccctggtcta
gtggtccccc 660ctttctgagc cacagccact ttctccatca aatgaggcca
gtaataccca tcccatagtg 720atgctgtgag gatgagatga gcatctgtaa
gtgctgaaga taatccctga cacatcccaa 780gcattcagca gtgcaagcat
acacttacac ggcactcccc agagccaggc atgtgctggt 840gcctcataca
cgtgaccaca tttgatcgtc acaatgaccc tgtgagggag actgtgcaac
900agaggactga ccttgctcaa agacctcagg cgtttcccct cagagcctga
gaggtcatct 960cttttttttt ttttttttcc tttctttctt tttcttttcc
atttcttttt ctttgcaaga 1020ggtcatctct aatgctttgg aatatcctgc
cagattagag tccctttgtt cacctgaagg 1080tttgggccac accagatagt
ctaacggtgt gatttgtgct gaaggttttg agccacacta 1140tatcagctag
atttctagag cggccggccg caataaaata tctttatttt cattacatct
1200gtgtgttggt tttttgtgtg aatcgatagt actaacatac gctctccatc
aaaacaaaac 1260gaaacaaaac aaactagcaa aataggctgt ccccagtgca
agtgcaggtg ccagaacatt 1320tctctatcga taggtaccga gctcatttag gt
135232496DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 32ggccgcccca cgtgcgcagc aggacgcagc gctgcctgaa
actcgcgccg cgaggagagg 60gcggggccgc ggaaaggaaa ggggggggct gggaggcccg
gagggggctg ggccggggac 120ccgggagggg tcgggacggg gcggggtccg
cgcggaggag gcggagctgg aaggtgaagg 180ggcaggacgg gtgcccgggt
ccccagtccc tccgccacgt ggggagcgcg gtcctgggcg 240tctgtgcccg
cgaatccact gggagcccgg cctggccccg acagcgcagc tgctccgggc
300ggacccgggg gtctgggccg cgcttccccg cccgcgcgcc gctcgcgctc
ccagggtgca 360gggacgccag cgagggcccc agcggagaga ggtcgaatcg
gcctaggctg tggggtaacc 420cgagggaggg gcctctagat ataagggcga
attccagcac actggcggcc gttactagtg 480gatccgagct cggtac
49633857DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 33ttatgtcaca ccacagaagt aaggttcctt cacaaagatc
ccaagctgtc gatcgacatt 60tctagaggat ctcggacccg gggaatcccc gtcccccaac
atgtccagat cgaaatcgtc 120tagcgcgtcg gcatgcgcca tcgccacgtc
ctcgccgtct aagtggagct cgtcccccag 180gctgacatcg gtcggggggg
cggatctcgg acccggggaa tccccgtccc ccaacatgtc 240cagatcgaaa
tcgtctagcg cgtcggcatg cgccatcgcc acgtcctcgc cgtctaagtg
300gagctcgtcc cccaggctga catcggtcgg gggggcggat cccccgggct
gcaggaattc 360cggcgataca gtcaactgtc tttgaccttt gttactactc
tcttccgatg atgatgtcgc 420acttattcta tgctgtctca atgttagagg
catatcagtc tccactgaag ccaatctatc 480tgtgacggca tctttattca
cattatcttg tacaaataat cctgttaaca atgcttttat 540atcctgtaaa
gaatccattt tcaaaatcat gtcaaggtct tctcgaggaa aaatcagtag
600aaatagctgt tccagtcttt ctagccttga ttccacttct gtcagatgtg
ccctagtcag 660cggagacctt ttggttttgg gagagtagcg acactcccag
ttgttcttca gacacttggc 720gcacttcggt ttttctttgg agcacttgag
ctttttaagt cggcaaatat cgcatgcttg 780ttcgatagaa gacagtagct
tcatctttca ggaggctagg gccgccagtg tgatggatat 840ctgcagaatt cgccctt
857348512DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 34ggtacgggag gtacttggag cggccgcgat ccagacatga
taagatacat tgatgagttt 60ggacaaacca caactagaat gcagtgaaaa aaatgcttta
tttgtgaaat ttgtgatgct 120attgctttat ttgtaaccat tataagctgc
aataaacaag ttaacaacaa caattgcatt 180cattttatgt ttcaggttca
gggggaggtg tgggaggttt tttaaagcaa gtaaaacctc 240tacaaatgtg
gtatggctga ttatgatcat gaacagactg tgaggactga ggggcctgaa
300atgagccttg ggactgtgaa tttaaaatac acaaacaatt agaatcagta
gtttaacaca 360ttatacactt aaaaatttta tatttacctt agagctttaa
atctctgtag gtagtttgtc 420caattatgtc acaccacaga agtaaggttc
cttcacaaag atcccaagct gtcgatcgac 480atttctagag gatctcggac
ccggggaatc cccgtccccc aacatgtcca gatcgaaatc 540gtctagcgcg
tcggcatgcg ccatcgccac gtcctcgccg tctaagtgga gctcgtcccc
600caggctgaca tcggtcgggg gggcggatct cggacccggg gaatccccgt
cccccaacat 660gtccagatcg aaatcgtcta gcgcgtcggc atgcgccatc
gccacgtcct cgccgtctaa 720gtggagctcg tcccccaggc tgacatcggt
cgggggggcg gatcccccgg gctgcaggaa 780ttccggcgat acagtcaact
gtctttgacc tttgttacta ctctcttccg atgatgatgt 840cgcacttatt
ctatgctgtc tcaatgttag aggcatatca gtctccactg aagccaatct
900atctgtgacg gcatctttat tcacattatc ttgtacaaat aatcctgtta
acaatgcttt 960tatatcctgt aaagaatcca ttttcaaaat catgtcaagg
tcttctcgag gaaaaatcag 1020tagaaatagc tgttccagtc tttctagcct
tgattccact tctgtcagat gtgccctagt 1080cagcggagac cttttggttt
tgggagagta gcgacactcc cagttgttct tcagacactt 1140ggcgcacttc
ggtttttctt tggagcactt gagcttttta agtcggcaaa tatcgcatgc
1200ttgttcgata gaagacagta gcttcatctt tcaggaggct agggccgcca
gtgtgatgga 1260tatctgcaga attcgccctt ccttgcctgc tgctttccac
caagtgctgg agagctggtg 1320aattgctcac tcccggctca ttcctctaat
gaccgaagcg tctcgcagat gcaacctgcc 1380gtggaggagc agggagggag
tgatttccag gtgtgggctt tttcagccat tcctaaaggc 1440gacttgagtt
cacctcactc actccagcat ttgtactcct gttgtggaaa aggcagtgag
1500cacaagccaa gcccgctcca ccttcacccc gccccacctc ccccggccct
ttcctgggcc 1560agtcttaggg ccctgagtac agacagcctg gctacccgtt
aaccattctc agcgtgtggc 1620tgctttttac acacatgtgt acatatgcac
ggacacacac acacacacag aggcttcccc 1680agtactcctc tatataggaa
cccgtcacca tcccagacat atgcagaaga aagcccaaac 1740cggctgtgtg
agacaggaac aattaacacg gtaacagatc cgataatgca gaccatcagg
1800cctaaagaac acggagggac tgtgttctac ctccttatag aaaagcaatt
agtgcctttt 1860tagctttgga accatgcccg gtggtgtgtg tgtggacaga
actgctggct ggttgttaag 1920ttgctactaa acacagtgtt gtttctcgtg
gtctctgccc ttgttaacta ggattgaggc 1980acttttgaac ataggtacct
gggtcctaag ggcgaattcc agcacactgg cggccgttac 2040tagtggatcc
gagctcggta ccaagctcca gctgggaata gagataggag gggacccagc
2100tggatgcagt gggcagtggg ggtcatagag tcaagagggt acagaataca
atggggtcct 2160agtatcatgg tggaggtcag aaagagccct aaaagagagg
gtcaaggtag gaggttagtg 2220aaggtccacc tccaccctct ccaggacagg
gacatcaggc cacaattaat ttctctgcag 2280ttggtgagtg gtcatggtct
ctggagtccc cagcatccag agtgtccctg gtctagtggt 2340cccccctttc
tgagccacag ccactttctc catcaaatga ggccagtaat acccatccca
2400tagtgatgct gtgaggatga gatgagcatc tgtaagtgct gaagataatc
cctgacacat 2460cccaagcatt cagcagtgca agcatacact tacacggcac
tccccagagc caggcatgtg 2520ctggtgcctc atacacgtga ccacatttga
tcgtcacaat gaccctgtga gggagactgt 2580gcaacagagg actgaccttg
ctcaaagacc tcaggcgttt cccctcagag cctgagaggt 2640catctctttt
tttttttttt tttcctttct ttctttttct tttccatttc tttttctttg
2700caagaggtca tctctaatgc tttggaatat cctgccagat tagagtccct
ttgttcacct 2760gaaggtttgg gccacaccag atagtctaac ggtgtgattt
gtgctgaagg ttttgagcca 2820cactatatca gctagatttc tagagcggcc
ggccgcaata aaatatcttt attttcatta 2880catctgtgtg ttggtttttt
gtgtgaatcg atagtactaa catacgctct ccatcaaaac 2940aaaacgaaac
aaaacaaact agcaaaatag gctgtcccca gtgcaagtgc aggtgccaga
3000acatttctct atcgataggt accgagctca tttaggtgac actatagaat
acaagcttgc 3060atgcctgcag gtccggagga cagtactccg ctcggaggac
agtactccgc tcggaggaca 3120gtactccgct cggaggacag tactccgctc
ggaggacagt actccgactc tagaggatcc 3180ccagtcctat atatactcgc
tctgcacttg gccctttttt acactgtgac tgattgagct 3240ggtgccgtgt
cgagtggtgt ctcgagatct gcgatctaag taagcttggc attccggtac
3300tgttggtaaa gccaccatgg aagacgccaa aaacataaag aaaggcccgg
cgccattcta 3360tccgctggaa gatggaaccg ctggagagca actgcataag
gctatgaaga gatacgccct 3420ggttcctgga acaattgctt ttacagatgc
acatatcgag gtggacatca cttacgctga 3480gtacttcgaa atgtccgttc
ggttggcaga agctatgaaa cgatatgggc tgaatacaaa 3540tcacagaatc
gtcgtatgca gtgaaaactc tcttcaattc tttatgccgg tgttgggcgc
3600gttatttatc ggagttgcag ttgcgcccgc gaacgacatt tataatgaac
gtgaattgct 3660caacagtatg ggcatttcgc agcctaccgt ggtgttcgtt
tccaaaaagg ggttgcaaaa 3720aattttgaac gtgcaaaaaa agctcccaat
catccaaaaa attattatca tggattctaa 3780aacggattac cagggatttc
agtcgatgta cacgttcgtc acatctcatc tacctcccgg 3840ttttaatgaa
tacgattttg tgccagagtc cttcgatagg gacaagacaa ttgcactgat
3900catgaactcc tctggatcta ctggtctgcc taaaggtgtc gctctgcctc
atagaactgc 3960ctgcgtgaga ttctcgcatg ccagagatcc tatttttggc
aatcaaatca ttccggatac 4020tgcgatttta agtgttgttc cattccatca
cggttttgga atgtttacta cactcggata 4080tttgatatgt ggatttcgag
tcgtcttaat gtatagattt gaagaagagc tgtttctgag 4140gagccttcag
gattacaaga ttcaaagtgc gctgctggtg ccaaccctat tctccttctt
4200cgccaaaagc actctgattg acaaatacga tttatctaat ttacacgaaa
ttgcttctgg 4260tggcgctccc ctctctaagg aagtcgggga agcggttgcc
aagaggttcc atctgccagg 4320tatcaggcaa ggatatgggc tcactgagac
tacatcagct attctgatta cacccgaggg 4380ggatgataaa ccgggcgcgg
tcggtaaagt tgttccattt tttgaagcga aggttgtgga 4440tctggatacc
gggaaaacgc tgggcgttaa tcaaagaggc gaactgtgtg tgagaggtcc
4500tatgattatg tccggttatg taaacaatcc ggaagcgacc aacgccttga
ttgacaagga 4560tggatggcta cattctggag acatagctta ctgggacgaa
gacgaacact tcttcatcgt 4620tgaccgcctg aagtctctga ttaagtacaa
aggctatcag gtggctcccg ctgaattgga 4680atccatcttg ctccaacacc
ccaacatctt cgacgcaggt gtcgcaggtc ttcccgacga 4740tgacgccggt
gaacttcccg ccgccgttgt tgttttggag cacggaaaga cgatgacgga
4800aaaagagatc gtggattacg tcgccagtca agtaacaacc gcgaaaaagt
tgcgcggagg 4860agttgtgttt gtggacgaag taccgaaagg tcttaccgga
aaactcgacg caagaaaaat 4920cagagagatc ctcataaagg ccaagaaggg
cggaaagatc gccgtgtaat tctaggtacc 4980gagctcttac gcgtgctagc
cctcgacaat caacctctgg attacaaaat ttgtgaaaga 5040ttgactggta
ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
5100cctttgtatc atgctattgc ttcccgtatg gctttcattt tctcctcctt
gtataaatcc 5160tggttgctgt ctctttatga ggagttgtgg cccgttgtca
ggcaacgtgg cgtggtgtgc 5220actgtgtttg ctgacgcaac ccccactggt
tggggcattg ccaccacctg tcagctcctt 5280tccgggactt tcgctttccc
cctccctatt gccacggcgg aactcatcgc cgcctgcctt 5340gcccgctgct
ggacaggggc tcggctgttg ggcactgaca attccgtggt gttgtcgggg
5400aagctgacgt cctttccatg gctgctcgcc tgtgttgcca cctggattct
gcgcgggacg 5460tccttctgct acgtcccttc ggccctcaat ccagcggacc
ttccttcccg cggcctgctg 5520ccggctctgc ggcctcttcc gcgtcttcgc
cttcgccctc agacgagtcg gatctccctt 5580tgggccgcct ccccgcctgg
aattcgagct cggtacgggc tcgactagag tcggggcggc 5640cggccgcttc
gagcagacat gataagatac attgatgagt ttggacaaac cacaactaga
5700atgcagtgaa aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt
atttgtaacc 5760attataagct gcaataaaca agttaacaac aacaattgca
ttcattttat gtttcaggtt 5820cagggggagg tgtgggaggt tttttaaagc
aagtaaaacc tctacaaatg tggtaaaatc 5880gataaggatc cgtcgaccga
tgcccttgag agccttcaac ccagtcagct ccttccggtg 5940ggcgcggggc
atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt
6000aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg
cgctcggtcg 6060ttcggctgcg gcgagcggta tcagctcact caaaggcggt
aatacggtta tccacagaat 6120caggggataa cgcaggaaag aacatgtgag
caaaaggcca gcaaaaggcc aggaaccgta 6180aaaaggccgc gttgctggcg
tttttccata ggctccgccc ccctgacgag catcacaaaa 6240atcgacgctc
aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
6300cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
ggatacctgt 6360ccgcctttct cccttcggga agcgtggcgc tttctcaatg
ctcacgctgt aggtatctca 6420gttcggtgta ggtcgttcgc tccaagctgg
gctgtgtgca cgaacccccc gttcagcccg 6480accgctgcgc cttatccggt
aactatcgtc ttgagtccaa cccggtaaga cacgacttat 6540cgccactggc
agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
6600cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta
tttggtatct 6660gcgctctgct gaagccagtt accttcggaa aaagagttgg
tagctcttga tccggcaaac 6720aaaccaccgc tggtagcggt ggtttttttg
tttgcaagca gcagattacg cgcagaaaaa 6780aaggatctca agaagatcct
ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 6840actcacgtta
agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
6900taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
tggtctgaca
6960gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
cgttcatcca 7020tagttgcctg actccccgtc gtgtagataa ctacgatacg
ggagggctta ccatctggcc 7080ccagtgctgc aatgataccg cgagacccac
gctcaccggc tccagattta tcagcaataa 7140accagccagc cggaagggcc
gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 7200agtctattaa
ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
7260acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
atggcttcat 7320tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
ccccatgttg tgcaaaaaag 7380cggttagctc cttcggtcct ccgatcgttg
tcagaagtaa gttggccgca gtgttatcac 7440tcatggttat ggcagcactg
cataattctc ttactgtcat gccatccgta agatgctttt 7500ctgtgactgg
tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
7560gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
ttaaaagtgc 7620tcatcattgg aaaacgttct tcggggcgaa aactctcaag
gatcttaccg ctgttgagat 7680ccagttcgat gtaacccact cgtgcaccca
actgatcttc agcatctttt actttcacca 7740gcgtttctgg gtgagcaaaa
acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 7800cacggaaatg
ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
7860gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
caaatagggg 7920ttccgcgcac atttccccga aaagtgccac ctgacgcgcc
ctgtagcggc gcattaagcg 7980cggcgggtgt ggtggttacg cgcagcgtga
ccgctacact tgccagcgcc ctagcgcccg 8040ctcctttcgc tttcttccct
tcctttctcg ccacgttcgc cggctttccc cgtcaagctc 8100taaatcgggg
gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa
8160aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
gtttttcgcc 8220ctttgacgtt ggagtccacg ttctttaata gtggactctt
gttccaaact ggaacaacac 8280tcaaccctat ctcggtctat tcttttgatt
tataagggat tttgccgatt tcggcctatt 8340ggttaaaaaa tgagctgatt
taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 8400ttacaatttc
ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
8460cctcttcgct attacgccag cccaagctac catgataagt aagtaatatt aa
8512358565DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 35cgccattcag gctgcgcaac tgttgggaag ggcgatcggt
gcgggcctct tcgctattac 60gccagcccaa gctaccatga taagtaagta atattaaggt
acgggaggta cttggagcgg 120ccgcgatcca gacatgataa gatacattga
tgagtttgga caaaccacaa ctagaatgca 180gtgaaaaaaa tgctttattt
gtgaaatttg tgatgctatt gctttatttg taaccattat 240aagctgcaat
aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
300ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta
tggctgatta 360tgatcatgaa cagactgtga ggactgaggg gcctgaaatg
agccttggga ctgtgaattt 420aaaatacaca aacaattaga atcagtagtt
taacacatta tacacttaaa aattttatat 480ttaccttaga gctttaaatc
tctgtaggta gtttgtccaa ttatgtcaca ccacagaagt 540aaggttcctt
cacaaagatc ccaagctgtc gatcgacatt tctagaggat ctcggacccg
600gggaatcccc gtcccccaac atgtccagat cgaaatcgtc tagcgcgtcg
gcatgcgcca 660tcgccacgtc ctcgccgtct aagtggagct cgtcccccag
gctgacatcg gtcggggggg 720cggatctcgg acccggggaa tccccgtccc
ccaacatgtc cagatcgaaa tcgtctagcg 780cgtcggcatg cgccatcgcc
acgtcctcgc cgtctaagtg gagctcgtcc cccaggctga 840catcggtcgg
gggggcggat cccccgggct gcaggaattc cggcgataca gtcaactgtc
900tttgaccttt gttactactc tcttccgatg atgatgtcgc acttattcta
tgctgtctca 960atgttagagg catatcagtc tccactgaag ccaatctatc
tgtgacggca tctttattca 1020cattatcttg tacaaataat cctgttaaca
atgcttttat atcctgtaaa gaatccattt 1080tcaaaatcat gtcaaggtct
tctcgaggaa aaatcagtag aaatagctgt tccagtcttt 1140ctagccttga
ttccacttct gtcagatgtg ccctagtcag cggagacctt ttggttttgg
1200gagagtagcg acactcccag ttgttcttca gacacttggc gcacttcggt
ttttctttgg 1260agcacttgag ctttttaagt cggcaaatat cgcatgcttg
ttcgatagaa gacagtagct 1320tcatctttca ggaggctagg gccgccccac
gtgcgcagca ggacgcagcg ctgcctgaaa 1380ctcgcgccgc gaggagaggg
cggggccgcg gaaaggaaag gggggggctg ggaggcccgg 1440agggggctgg
gccggggacc cgggaggggt cgggacgggg cggggtccgc gcggaggagg
1500cggagctgga aggtgaaggg gcaggacggg tgcccgggtc cccagtccct
ccgccacgtg 1560gggagcgcgg tcctgggcgt ctgtgcccgc gaatccactg
ggagcccggc ctggccccga 1620cagcgcagct gctccgggcg gacccggggg
tctgggccgc gcttccccgc ccgcgcgccg 1680ctcgcgctcc cagggtgcag
ggacgccagc gagggcccca gcggagagag gtcgaatcgg 1740cctaggctgt
ggggtaaccc gagggagggg cctctagata taagggcgaa ttccagcaca
1800ctggcggccg ttactagtgg atccgagctc ggtacacaga cattgacatt
gtgtcatcta 1860gtatacaaat aggttcttgg agtactttac taggcatgga
caatgcccaa tgcctgtccc 1920attcttcagg catattttta tttgtgggct
ttatgtccct attaagaaaa agactaagaa 1980caagatgcta tcatattttc
ttaactggaa tggtagatgt ttaaacatga tgactaccaa 2040gcttggctag
aacattgtgt catctagtat acaaataggt tcttggagta ctttactagg
2100catggacaat gcccaatgcc tgtcccattc ttcaggcata tttttatttg
tgggctttat 2160gtccctatta agaaaaagac taagaacaag atgctatcat
aagctccaag cttatcgcca 2220gctgggaata gagataggag gggacccagc
tggatgcagt gggcagtggg ggtcatagag 2280tcaagagggt acagaataca
atggggtcct agtatcatgg tggaggtcag aaagagccct 2340aaaagagagg
gtcaaggtag gaggttagtg aaggtccacc tccaccctct ccaggacagg
2400gacatcaggc cacaattaat ttctctgcag ttggtgagtg gtcatggtct
ctggagtccc 2460cagcatccag agtgtccctg gtctagtggt cccccctttc
tgagccacag ccactttctc 2520catcaaatga ggccagtaat acccatccca
tagtgatgct gtgaggatga gatgagcatc 2580tgtaagtgct gaagataatc
cctgacacat cccaagcatt cagcagtgca agcatacact 2640tacacggcac
tccccagagc caggcatgtg ctggtgcctc atacacgtga ccacatttga
2700tcgtcacaat gaccctgtga gggagactgt gcaacagagg actgaccttg
ctcaaagacc 2760tcaggcgttt cccctcagag cctgagaggt catctctttt
tttttttttt tttcctttct 2820ttctttttct tttccatttc tttttctttg
caagaggtca tctctaatgc tttggaatat 2880cctgccagat tagagtccct
ttgttcacct gaaggtttgg gccacaccag atagtctaac 2940ggtgtgattt
gtgctgaagg ttttgagcca cactatatca gctagatttc tagagcggcc
3000ggccgcaata aaatatcttt attttcatta catctgtgtg ttggtttttt
gtgtgaatcg 3060atagtactaa catacgctct ccatcaaaac aaaacgaaac
aaaacaaact agcaaaatag 3120gctgtcccca gtgcaagtgc aggtgccaga
acatttctct atcgataggt accgagctca 3180tttaggtgac actatagaat
acaagcttgc atgcctgcag gtccggagga cagtactccg 3240ctcggaggac
agtactccgc tcggaggaca gtactccgct cggaggacag tactccgctc
3300ggaggacagt actccgactc tagaggatcc ccagtcctat atatactcgc
tctgcacttg 3360gccctttttt acactgtgac tgattgagct ggtgccgtgt
cgagtggtgt ctcgagatct 3420gcgatctaag taagcttggc attccggtac
tgttggtaaa gccaccatgg aagacgccaa 3480aaacataaag aaaggcccgg
cgccattcta tccgctggaa gatggaaccg ctggagagca 3540actgcataag
gctatgaaga gatacgccct ggttcctgga acaattgctt ttacagatgc
3600acatatcgag gtggacatca cttacgctga gtacttcgaa atgtccgttc
ggttggcaga 3660agctatgaaa cgatatgggc tgaatacaaa tcacagaatc
gtcgtatgca gtgaaaactc 3720tcttcaattc tttatgccgg tgttgggcgc
gttatttatc ggagttgcag ttgcgcccgc 3780gaacgacatt tataatgaac
gtgaattgct caacagtatg ggcatttcgc agcctaccgt 3840ggtgttcgtt
tccaaaaagg ggttgcaaaa aattttgaac gtgcaaaaaa agctcccaat
3900catccaaaaa attattatca tggattctaa aacggattac cagggatttc
agtcgatgta 3960cacgttcgtc acatctcatc tacctcccgg ttttaatgaa
tacgattttg tgccagagtc 4020cttcgatagg gacaagacaa ttgcactgat
catgaactcc tctggatcta ctggtctgcc 4080taaaggtgtc gctctgcctc
atagaactgc ctgcgtgaga ttctcgcatg ccagagatcc 4140tatttttggc
aatcaaatca ttccggatac tgcgatttta agtgttgttc cattccatca
4200cggttttgga atgtttacta cactcggata tttgatatgt ggatttcgag
tcgtcttaat 4260gtatagattt gaagaagagc tgtttctgag gagccttcag
gattacaaga ttcaaagtgc 4320gctgctggtg ccaaccctat tctccttctt
cgccaaaagc actctgattg acaaatacga 4380tttatctaat ttacacgaaa
ttgcttctgg tggcgctccc ctctctaagg aagtcgggga 4440agcggttgcc
aagaggttcc atctgccagg tatcaggcaa ggatatgggc tcactgagac
4500tacatcagct attctgatta cacccgaggg ggatgataaa ccgggcgcgg
tcggtaaagt 4560tgttccattt tttgaagcga aggttgtgga tctggatacc
gggaaaacgc tgggcgttaa 4620tcaaagaggc gaactgtgtg tgagaggtcc
tatgattatg tccggttatg taaacaatcc 4680ggaagcgacc aacgccttga
ttgacaagga tggatggcta cattctggag acatagctta 4740ctgggacgaa
gacgaacact tcttcatcgt tgaccgcctg aagtctctga ttaagtacaa
4800aggctatcag gtggctcccg ctgaattgga atccatcttg ctccaacacc
ccaacatctt 4860cgacgcaggt gtcgcaggtc ttcccgacga tgacgccggt
gaacttcccg ccgccgttgt 4920tgttttggag cacggaaaga cgatgacgga
aaaagagatc gtggattacg tcgccagtca 4980agtaacaacc gcgaaaaagt
tgcgcggagg agttgtgttt gtggacgaag taccgaaagg 5040tcttaccgga
aaactcgacg caagaaaaat cagagagatc ctcataaagg ccaagaaggg
5100cggaaagatc gccgtgtaat tctaggtacc gagctcttac gcgtgctagc
cctcgacaat 5160caacctctgg attacaaaat ttgtgaaaga ttgactggta
ttcttaacta tgttgctcct 5220tttacgctat gtggatacgc tgctttaatg
cctttgtatc atgctattgc ttcccgtatg 5280gctttcattt tctcctcctt
gtataaatcc tggttgctgt ctctttatga ggagttgtgg 5340cccgttgtca
ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac ccccactggt
5400tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
cctccctatt 5460gccacggcgg aactcatcgc cgcctgcctt gcccgctgct
ggacaggggc tcggctgttg 5520ggcactgaca attccgtggt gttgtcgggg
aagctgacgt cctttccatg gctgctcgcc 5580tgtgttgcca cctggattct
gcgcgggacg tccttctgct acgtcccttc ggccctcaat 5640ccagcggacc
ttccttcccg cggcctgctg ccggctctgc ggcctcttcc gcgtcttcgc
5700cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctgg
aattcgagct 5760cggtacgggc tcgactagag tcggggcggc cggccgcttc
gagcagacat gataagatac 5820attgatgagt ttggacaaac cacaactaga
atgcagtgaa aaaaatgctt tatttgtgaa 5880atttgtgatg ctattgcttt
atttgtaacc attataagct gcaataaaca agttaacaac 5940aacaattgca
ttcattttat gtttcaggtt cagggggagg tgtgggaggt tttttaaagc
6000aagtaaaacc tctacaaatg tggtaaaatc gataaggatc cgtcgaccga
tgcccttgag 6060agccttcaac ccagtcagct ccttccggtg ggcgcggggc
atgactatcg tcgccgcact 6120tatgactgtc ttctttatca tgcaactcgt
aggacaggtg ccggcagcgc tcttccgctt 6180cctcgctcac tgactcgctg
cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 6240caaaggcggt
aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
6300caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
tttttccata 6360ggctccgccc ccctgacgag catcacaaaa atcgacgctc
aagtcagagg tggcgaaacc 6420cgacaggact ataaagatac caggcgtttc
cccctggaag ctccctcgtg cgctctcctg 6480ttccgaccct gccgcttacc
ggatacctgt ccgcctttct cccttcggga agcgtggcgc 6540tttctcaatg
ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
6600gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
aactatcgtc 6660ttgagtccaa cccggtaaga cacgacttat cgccactggc
agcagccact ggtaacagga 6720ttagcagagc gaggtatgta ggcggtgcta
cagagttctt gaagtggtgg cctaactacg 6780gctacactag aaggacagta
tttggtatct gcgctctgct gaagccagtt accttcggaa 6840aaagagttgg
tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
6900tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
ttgatctttt 6960ctacggggtc tgacgctcag tggaacgaaa actcacgtta
agggattttg gtcatgagat 7020tatcaaaaag gatcttcacc tagatccttt
taaattaaaa atgaagtttt aaatcaatct 7080aaagtatata tgagtaaact
tggtctgaca gttaccaatg cttaatcagt gaggcaccta 7140tctcagcgat
ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
7200ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
cgagacccac 7260gctcaccggc tccagattta tcagcaataa accagccagc
cggaagggcc gagcgcagaa 7320gtggtcctgc aactttatcc gcctccatcc
agtctattaa ttgttgccgg gaagctagag 7380taagtagttc gccagttaat
agtttgcgca acgttgttgc cattgctaca ggcatcgtgg 7440tgtcacgctc
gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
7500ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
ccgatcgttg 7560tcagaagtaa gttggccgca gtgttatcac tcatggttat
ggcagcactg cataattctc 7620ttactgtcat gccatccgta agatgctttt
ctgtgactgg tgagtactca accaagtcat 7680tctgagaata gtgtatgcgg
cgaccgagtt gctcttgccc ggcgtcaata cgggataata 7740ccgcgccaca
tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
7800aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
cgtgcaccca 7860actgatcttc agcatctttt actttcacca gcgtttctgg
gtgagcaaaa acaggaaggc 7920aaaatgccgc aaaaaaggga ataagggcga
cacggaaatg ttgaatactc atactcttcc 7980tttttcaata ttattgaagc
atttatcagg gttattgtct catgagcgga tacatatttg 8040aatgtattta
gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
8100ctgacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
cgcagcgtga 8160ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
tttcttccct tcctttctcg 8220ccacgttcgc cggctttccc cgtcaagctc
taaatcgggg gctcccttta gggttccgat 8280ttagtgcttt acggcacctc
gaccccaaaa aacttgatta gggtgatggt tcacgtagtg 8340ggccatcgcc
ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata
8400gtggactctt gttccaaact ggaacaacac tcaaccctat ctcggtctat
tcttttgatt 8460tataagggat tttgccgatt tcggcctatt ggttaaaaaa
tgagctgatt taacaaaaat 8520ttaacgcgaa ttttaacaaa atattaacgt
ttacaatttc ccatt 856536279DNAArtificial SequenceDescription of
Artificial Sequence Synthetic Primer 36gacactatag aatacaagct
tgcatgcctg caggtccgga ggacagtact ccgctcggag 60gacagtactc cgctcggagg
acagtactcc gctcggagga cagtactccg ctcggaggac 120agtactccga
ctctagagga tccccagtcc tatatatact cgctctgcac ttggcccttt
180tttacactgt gactgattga gctggtgccg tgtcgagtgg tgtctcgaga
tctgcgatct 240aagtaagctt ggcattccgg tactgttggt aaagccacc
27937650DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 37gctagctacg ggccagatat acgcgttgac attgattatt
gactagttat taatagtaat 60caattacggg gtcattagtt catagcccat atatggagtt
ccgcgttaca taacttacgg 120taaatggccc gcctggctga ccgcccaacg
acccccgccc attgacgtca ataatgacgt 180atgttcccat agtaacgcca
atagggactt tccattgacg tcaatgggtg gagtatttac 240ggtaaactgc
ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
300acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
ttatgggact 360ttcctacttg gcagtacatc tacgtattag tcatcgctat
taccatggtg atgcggtttt 420ggcagtacat caatgggcgt ggatagcggt
ttgactcact cgagagacgg tgagagcgag 480tcagggattg gctggtctgc
ttcgggcggg ctaaaggaag gttcaagtgg agctctccta 540accgacgcgc
gtctgtggag aagcggcttg gtcgggggtg gtctcgtggg gtcctgcctg
600tttagtcgct ttcagggttc ttgagcccct tcacgaccgt caccaagctt
65038701DNAArtificial SequenceDescription of Artificial Sequence
Synthetic Primer 38gctagctacg ggccagatat acgcgttgac attgattatt
gactagttat taatagtaat 60caattacggg gtcattagtt catagcccat atatggagtt
ccgcgttaca taacttacgg 120taaatggccc gcctggctga ccgcccaacg
acccccgccc attgacgtca ataatgacgt 180atgttcccat agtaacgcca
atagggactt tccattgacg tcaatgggtg gagtatttac 240ggtaaactgc
ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
300acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
ttatgggact 360ttcctacttg gcagtacatc tacgtattag tcatcgctat
taccatggtg atgcggtttt 420ggcagtacat caatgggcgt ggatagcggt
ttgactcact cgagcggccg ccagtgtgat 480ggatatctgc agaattcgcc
cttgcgatct gtcagagcac ctcgcgagcg tacgtgcctc 540aggaagtgac
gcacagcccc cctgggggcc gggggcgggg ccaggctata aaccgccggt
600taggggccgc catcccctca gagcgtcggg atatcgggtg aagggcgaat
tccagcacac 660tggcggccgt tactagtgga tccgagctcg gtaccaagct t 701
* * * * *