Note: Descriptions are shown in the official language in which they were submitted.
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METHODS TO ENHANCE AND CONFINE EXPRESSION OF GENES
BACKGROUND OF THE INVENTION
Field of the Invention
The present invention relates generally to the field of gene
therapy for cancer. More specifically, the present invention presents a
method of controlling the expression of therapeutically valuable gene
products via inducible promoters. The present invention provides a
method whereby induced gene expression in the intended cell targets
is enhanced and prolonged in a spatially and temporally regulable
manner by means of heat or light inducible promoters. Moreover, t h a
present invention provides a method whereby the background gene
expression in non-targeted cells is reduced or eliminated.
pescription of the Related ,art
One of the major obstacles to the success of chemotherapy
and radiation therapy for cancer is the difficulty in achieving tumor-
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specific cell killing. The inability of radiation or cytotoxic
chemotherapeutic agents to distinguish between tumor cells and
normal cells necessarily limits the dosage that can be applied. As a
result, disease relapse due to residual surviving tumor cells is
frequently observed.
The use of gene therapy in cancer treatment presents m a n y
of the same disadvantages as chemotherapy and radiation therapy.
Problems with current state-of-the-art gene therapy strategies include
the inability to deliver the therapeutic gene specifically to the target
cells. This leads to toxicity in cells that are not the intended targets.
For example, manipulation of the p53 gene suppresses the growth of
both tumor cells and normal cells, and intravenous administration of
tumor necrosis factor alpha (TNFa) induces systemic toxicity with such
clinical manifestations as fever and hypertension.
Attempts have been made to overcome these problems.
These include such strategies as: the use of tissue-specific receptors to
direct the genes to the desired tissues (Kasahara, N., et al., Science,
266:1373-1376 (1994)), the use of tissue-specific promoters to limit
gene expression to specific tissues (e.g. use of the prostate specific
antigen promoter) and the use of heat (Voellmy R., et al., Proc. Natl.
Acad. Sci. USA, 82:4949-4953 (1985)} or ionizing radiation inducible
enhancers and promoters (Trainman, R.H., et al., Cell 46: 5 6 7 - 5 7 4
(1986); Prowess, R., et al., Proc. Natl. Acad. Sci. USA 85, 7206-7210
(1988)) to enhance expression of the therapeutic gene in a temporally
and spatially controlled manner. The heat inducible heat shock protein
(HSP) promoter has been used to direct the expression of genes such a s
the cytokine IL-2.
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Weichselbaum and colleagues were the first to discover t h a
radiation inducible response of the early growth response (Egr-1) gene
promoter. Accordingly, they have attempted to direct expression of
such cytotoxic genes as TNF-a to tumor cells to enhance radiation cell
killing by means of this promoter. Previously, systemic administration
of the cytokine TNF-a as an adjuvant to ionizing radiation was initially
reported to result in enhanced killing in a mouse xenograft tumor
system. It has since been shown partially effective in human tumors.
The effect of TNFa appears to be dosage-dependent, as its tumor-
killing effect correlates with its serum concentration. However,
systemic toxicity of TNFa restricts the dosage that can be applied and
thus limits the usefulness of the treatment regimen. Attempts have
also been made to deliver the TNFa gene to tumor cells via adenoviral
vector and/or liposomes. Unfortunately, expression of the TNFa gene
is not restricted to the tumor sites due to the 'leakiness' of the
promoter.
In an attempt to localize the level of TNFa to the general
area of radiation exposure and thereby reduce systemic toxicity,
Weichselbaum and colleaguesemployed radiation inducible
the Egr-1
promoter to activatethe TNFa gene in situ.Earlier studies showed
that
the expression of certain immediate-early genes such as jun/fos and
Egr-1 are activated in cells exposed to ionizing radiation (Sherman,
M.L., et al., Proc. Natl. Acad. Sci. USA, 87: 5663-5666 {1997); Hallahan,
D.E., et al., Proc. Natl. Acad. Sci. USA, 88: 2156-2160 (1991)). By
placing the TNFa gene under the control of the Egrl promoter (EGRp),
the expression of the TNFa is enhanced in those cells harboring a n
EGRp-TNFa plasmid when exposed to ionizing radiation. In vivo, the
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serum level of TNFa is greatly enhanced (Weichselbaum R.R., et al.,
Cancer Res. 54: 4266-4269 (1994)) within a few hours after
irradiation. The combined treatment with this plasmid and radiation
leads to a partial regression of a xenografted tumor during the course
of the treatment. The level of TNFa dropped precipitously within 2 4
hours; further decreases in serum level of TNFa coincided with
regrowth of the tumors.
There are several possible reasons far the recurrence of the
tumor upon cessation of therapy. The most obvious reason is probably
the same limitation seen with chemotherapy ar radiation therapy in
general, viz., insufficient dosage levels. A major problem, which limits
the amount of TNFa produced, is the weak and transient nature of the
Egr-1 promoter. This promoter is intrinsically weak, with a maximum
of less than three-fold increase in expression upon induction.
Moreover, the induced expression is of necessity transient. This,
coupled with the weakness of the promoter, permits only a brief
exposure of the tumor cells to the TNFa.
Another factor that limits the production of sufficient
dosage of TNFa is that not every tumor cell will have taken up the
TNFa plasmid. While it has been suggested that repeated
administration may help to improve the treatment outcome, it is not
clear if the repeated delivery of a suboptimal low dosage of TNFa will
be useful, the problems posed by an immune response
notwithstanding. Although it might be conceivable to deliver larger
doses of plasmids, the problem of promoter leakiness has hindered
such an approach. It is known that a substantial basal level of activity
(20-30%) can be detected with the Egr-1 promoter even in the absence
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of ionizing radiation (Weichselbaum, et al., supra). This is not
surprising, as the radiation response element, a CArG box, is part of t h a
serum response element.
The HSP promoter is also rather leaky. In the absence of
heat, this promoter exhibits a 25-30% background level of expression,
not suitable for most cytotoxic genes. As this level of expression will
be harmful to unirradiated normal cells that take up the gene. Hence,
administration of this plasmid has been restricted to small doses of
intra-tumoral injections to minimize systemic toxicity.
Therefore, while it may be advantageous to employ a
spatially and temporally regulated promoter such as the HSP and Egr-1
promoters to enhance specificity of gene expression at the site of heat
or radiation treatment, current versions of those promoters have
serious problems that restrict their applicability. In order to apply
these promoters for use in cancer therapy, it is necessary to eliminate
or greatly reduce background expression in unheated or unirradiated
cells. Ideally, the expression of cytotoxic genes should be limited to
the area of external stimuli (heat or radiation). Additionally, to ensure
a sufficient Level of expression of therapeutic genes, the weak a n d
transient nature of gene expression driven by these promoters must b a
improved.
It is important to note when an improved
that even
inducible vector system which can restrictthe expression of
a
therapeutic gene to the area of externalstimuliis developed, there
i s
still the problem of expression normal heated or irradiated
in
bystander cells. Thus, it is criticalbe ableto further restrict
to the
expression of therapeutic genes onlyto the intended targets,
e.g.,
tumor cell s.
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The prior art is deficient in the lack of effective means of
inhibiting unwanted toxic side effects of gene therapy treatments for
cancer, as well as providing a method for enhancing and sustaining
gene expression in targeted tumor cells in a controllable manner. The
present invention fulfills this longstanding need and desire in the art.
SUMMARY OF THE INVENTION
The current invention provides the composition a n d
methods for the controlled activation of DNA molecules for gene
therapy. Activation of these DNA molecules leads to the production of
protein products which then may provide opportunities for therapeutic
manipulation of cells containing said DNA molecules. This may b a
achieved via alterations in cell growth and metabolism of the targeted
cells and may include effects on neighboring cells via secretion of
therapeutic products. The invention offers the options of sustained
activation or activation regulable by the application of antibiotics. The
invention further provides novel expression vectors for use in gene
therapy of local and metastatic breast, ovarian and prostate cancer.
An original strategy to confine and enhance therapeutic
gene expression to tumors spatially and temporally is also presented,
in the form of an expression vector designed for use in local and
metastatic breast, ovarian and prostate cancer.
In one embodiment of the present invention, there is
provided a method for sustained and enhanced expression of a gene
via activation of a heat or light inducible promoter. In a modification
of this method, heat or light is used to activate the promoter, b a t
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continued levels of gene expression are modulated by concentrations of
an antibiotic (tetracycline or its derivatives), acting on a fusion protein
with a tetracycline-responsive element.
In yet another embodiment of the present invention, there
is provided a method of constructing the vectors for gene therapy
activation modalities.
In another embodiment of the present invention, there are
provided improved vectors for reducing background expression i n
unheated and unirradiated cells.
In another embodiment of the present invention, there are
provided improved vectors for reducing expression in heated a n d
irradiated normal bystander cells.
In another embodiment of the present invention, there are
provided expression vectors for use in gene therapy treatment of local
and metastatic breast and ovarian cancer.
In another embodiment of the present invention, there are
provided expression vectors for use in gene therapy treatment of local
and metastatic prostate cancer.
Other and further aspects, features, and advantages of t h a
present invention will be apparent from the following description of
the presently preferred embodiments of the invention given for the
purpose of disclosure.
BRIEF DESCRIPTION OF THE DRAWINGS
So that the matter in which the above-recited features,
advantages and objects of the invention, as well as others which will
become clear, are attained and can be understood in detail, more
particular descriptions of the invention briefly summarized above may
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be had by reference to certain embodiments thereof which are
illustrated in the appended drawings. These drawings form a part of
the specification. It is to be noted, however, that the appended
drawings illustrate preferred embodiments of the invention and
therefore are not to be considered limiting in their scope.
Figure I shows a schematic representation of the plasmid,
pDATH-X (Dominant negative, Antisense, TET-ON controllable Heat
shock promoter plasmid) -p53, which consists of four cassettes a s
follows. (1) TET-ON is a fusion of the coding sequences for amino acids
1-207 of the tetracycline (tet) repressor and the C-terminus last 130
amino acid transcription activation domain of the VP16 protein of the
herpes simplex virus (Gossen M., et al., Science, 268:1766-1769
(1995)). In Cassette 1, the TET-ON sequence is placed under the
control of the HSP and the tet operator binding site and pCMV. (2) HSP
is the heat shock promoter consisting of the heat shock response
element (-260 to 30) of the human heat shock 70 gene promoter
(Voellmy R., et al., Proc. Natl. Acad. Sci. USA 82: 4949-4953 (1985))
linked to the minimal CMV promoter, pCMV (Gossen M., et al., Science,
268:1766-1769 (1995)). In cassette 2, the therapeutic gene, X, is
placed under the control of the tetp-pCMV promoter. (3) ptet is the tet
operator consisting of the 19 base pair (bp) inverted repeats of the
operator 02 of TN10 (Gossen M, and Bujard H., Proc. Natl. Acad. Sci.
USA 89:5547-5551 (1992)) to which the tet repressor and TET-ON
bind. In cassette 3, antisense TET-ON is placed under the control of t h a
pCMV promoter. (4) Antisense TET-ON is an antisense sequence
consisting of the complementary sequence to the first 80 bases of t h a
TET-ON sequence including the ATG. In cassette 4, dominant negative
TET-ON is placed under the control of the pCMV promoter. The
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Dominant negative TET-ON consists of the tet-repressor but without
the VP16 transactivation domain, and it is placed under the control of
the pCMV promoter. In the absence of heat or light, a background
level of expression of the TET-ON sequence will result due to t h a
leakiness of the minimal promoter pCMV.
Figure 2 depicts the pDATE vector. The plasmid, pDATE-X
(Dominant negative, Antisense, TET-ON controllable EGR promoter
expression plasmid) consists of four cassettes as follows: 1) in cassette
1, the TET-ON sequence is placed under the control of the EGRp, t h a
tetracycline operator binding site and pCMV; 2) in cassette 2, the
therapeutic gene, X, is placed under the control of the tetp-pCMV
promoter; 3) in cassette 3, antisense TET-ON is placed under the control
of the pCMV promoter; and 4) in cassette 4, dominant negative TET-ON
is placed under the control of the pCMV promoter. "TET-ON" is a fusion
of the coding sequences for amino acids 1-207 of the tet repressor and
the C-terminus 130 amino acid transcription activation domain of the
VP16 protein of the herpes simplex virus. "EGRp" is the radiation
inducible promoter consisting of fragment -425 to +65 of the EGR-1
promoter containing four copies of the CArG domain. "ptet" is the tet
operator consisting of the 19 by inverted repeats of the operator 02 of
TN10 to which the tet repressor and TET-ON bind, linked to the
minimal CMV promoter, pCMV. "Antisense Tet-On" is a sequence
consisting of the complementary sequence to the first 80 bases of the
TET-ON sequence including the ATG. "Dominant negative TET-ON"
consists of the coding sequences for amino acids 1-207 of the tet
repressor placed under the control of the pCMV promoter. "M" is the
chicken lysosomal matrix attachment site to isolate the position effects
of each of the cassettes.
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Figure 3 depicts the structure of the pRIBs-X (Radiation-
Inducible, Breast-specific Promoter) expression vector. The pRIBS
vector is comprised of four cassettes. Gene cassette 1 differs from
previously described vectors only in that it contains "Gal-DBD-mx"
which is a fusion open reading frame (ORF) encoding the N-terminus
(amino acids 1-147) DNA-binding domain of the yeast GAL4 protein
(Gal-DBD) fused to the basis helix-loop-helix-leucine zipper (bHLHLZ)
domain of Max (mx, amino acids 8-112) followed by SV40 poly A.
Gene cassette 2 is comprised of the minimal CMV promoter (mCMVp),
"antisense Gal-DBD-mx", which is an antisense construct
complementary to the Gal-DBD-mx sequence, "IRES", which is a n
internal ribosomal entry site and "Gal-DBD" which competes with the
Gal-DBD-mx for the pGAL binding site. Gene cassette 3 is comprised of
"VP16-TA-mc" which is a fusion open reading frame encoding at the N-
terminus the first 11 amino acids of Gal4 (amino acids 1-147), followed
by the nuclear localization signal of the S V40 large T antigen, the 1 3 0
amino acid C-terminus transactivation domain of the herpes simplex
viral protein VP16, the bHLHLZ domain of c-Myc (amino acids 3 5 0-
439), followed by SV40 polyA. The resulting fusion gene, VP-16TA-
mc, is placed under the control of the c-erbB-2 promoter "perbB2" a p
to the first ATG. Gene cassette 4 contains "GALA", consisting of five
copies of a 17-mer DNA-binding site for Gal4. The TET-ON sequence i s
placed under the control of the GALA-ptet promoter and t h a
therapeutic gene, X, is linked to the TET-ON via an IRES; Gene cassette
5 contains an antisense TET-ON which is a sequence consisting of the
complementary sequence to the first 80 bases of the TET-ON sequence
including the ATG, placed under the control of the pCMV promoter.
Gene cassette 6 contains a dominant negative TET-ON consisting of the
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coding sequences for amino acids 1-207 of the tet repressor placed
under the control of the pCMV promoter.
Figure 4 shows the structure of the pRIPS-GFP (Radiation-
Inducible, Prostate-specific Promoter) expression vector. The pRIPS
vector is comprised of six cassettes. Gene cassette 1 differs from
previously described vectors only in that it contains "Gal-DBD-mx"
which is a fusion open reading frame encoding the N-terminus (amino
acids 1-147) DNA-binding domain of the yeast GAL4 protein (Gal-DBD)
fused to the basis helix-loop-helix-leucine zipper (bHLHLZ) domain of
Max (mx, amino acids 8-112) followed by SV40 poly A. Gene cassette
2 is comprised of the minimal CMV promoter (mCMVp), "antisense Gal-
DBD-mx", which is an antisense construct complementary to the Gal-
DBD-mx sequence, "IRES", which is an internal ribosomal entry site and
"Gal-DBD" which competes with the Gal-DBD-mx for the pGAL binding
site. Gene cassette 3 is comprised of "VP16-TA-mc" which is a fusion
open reading frame encoding at the N-terminus the first 11 amino
acids of Gal4 (amino acids 1-147), followed by the nuclear localization
signal of the SV40 large T antigen, the 130 amino acid C-terminus
transactivation domain of the herpes simplex viral protein VP16, the
bHLHLZ domain of c-Myc (amino acids 350-439), followed by S V40
polyA. The resulting fusion gene, VP16-TA-mc, is placed under the
control of the probasin gene promoter "pProbasin" up to the first ATG.
Gene cassette 4 contains "GALA", consisting of five copies of a 17-mer
DNA-binding site for Gal4. The TET-ON sequence is placed under t h a
control of the GALA-ptet promoter and the therapeutic gene, X, is
linked to the TET-ON via an IRES; Gene cassette 5 contains an antisense
TET-ON which is a sequence consisting of the complementary sequence
to the first 80 bases of the TET-ON sequence including the ATG, placed
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under the control of the pCMV promoter. Gene cassette 6 contains a
dominant negative TET-ON consisting of the coding sequences for
amino acids 1-207 of the tet repressor placed under the control of the
pCMV promoter.
Figure 5 is a schematic representation of the mode of
action of pRIBS-GFP.
Figure 6 illustrates the leakiness of the HSP promoter. I t
summarizes the results of testing the heat inducible system containing
the hsp70 promoter in the expression of therapeutic genes, p53 a n d
TNFa. Figure 6A shows the plasmid construct for the two genes, p 5 3
and TNFa. Figure 6B depicts p53 transcriptional activity. To analyze
the inducibility of the hsp promoter, the plasmid pHSP.3p53CD1 or the
control pHSP.3 vector alone was cotransfected with Post-2-CAT
(containing a CAT coding sequence linked to a consensus p53 binding
site) into the human ovarian carcinoma cell tine SKOV3 which has a
homozygous deletion of p53. At 36 hours after transfection, cells were
either heated or unheated. CAT activity was measured 24 hours later.
Little or no activity is seen with the SKOV3 parental untransfected cells
(lane 2, heated; lane 1, unheated). Similarly, with the pHSY.~ vector
alone, there is no activity with or without heat (lanes 3 and 4). With
the pHSP.3p53 plasmid, there is a high level of CAT activity seen at 2 4
hrs after heating (lane 6). However, even without heating (lane 5),
there is a substantial level of p53 expression (about 25%).
Figure 7 depicts the induction of TNFa by heat or
photodynamic therapy (PDT). The coding sequence of TNFa was
subcloned into the plasmid pHSP.3 and transfected into SKOV3 cells.
Stable colonies were isolated by selection in 6418. Cells were either
heated at 45° C or untreated. At 6 hours after treatment, the level of
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TNFa in the medium was measured with a Genzyme TNFa ELISA kit.
T N F a shown to be induced four-fold by heat and three-fold by PDT a n d
secreted. However, background expression was substantial (27°!0).
Figure 8 shows the expression kinetics of p53 in the
H358 lung carcinoma cell line by the feed-forward reaction, where
a,b,c,d and a represent the levels of p53 reached at 10 hours after the
feed-forward reaction. Six hours after heat shock, transfected cells
were treated with different doses of doxycycline. At various time
points after the addition of doxycycline, the cells were stained with a
p53 antibody. For each point, the digital images of fifty
immunostained cells were captured using a Nikon microscope. The
amount of protein expressed in each cell is proportional to the
intensity of staining, expressed as I= 1/T (where T is a measure of the
transmitted light/unit area. This plot shows the results of one such
experiment using 0.01-0.1 ~.g/ml doxycycline.
Figure 9 depicts the pHIBS-X (Heat-Inducibie, Breast-
specific Promoter) expression vector. The pHIBS vector is comprised of
six cassettes. Gene cassette 1 differs from the vectors described above
only in that it contains "Gal-DBD-mx" which is a fusion open reading
frame encoding the N-terminus (amino acids 1-147) DNA-binding
domain of the yeast GAL4 protein (Gal-DBD) fused to the basis helix-
loop-helix-leucine zipper (bHLHLZ) domain of Max (mx, amino acids 8 -
112) followed by SV40 poly A. The resulting fusion gene GAL-DBD-mx
is controlled by the heat inducible HSP promoter. Gene cassette 2 is
comprised of the minimal CMV promoter (mCMVp), "antisense Gal-
DBD-mx", which is an antisense construct complementary to the Gal-
DBD-mx sequence, "IRES", which is an internal ribosomal entry site a n d
"Gal-DBD" which competes with the Gal-DBD-mx for the pGAL binding
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site. Gene cassette 3 is comprised of "VP16-TA-mc" which is a fusion
open reading frame encoding at the N-terminus the first 11 amino
acids of Gal4 (amino acids 1-147}, followed by the nuclear localization
signal of the SV40 large T antigen, the 130 amino acid C-terminus
transactivation domain of the herpes simplex viral protein VP16, the
bHLHLZ domain of c-Myc (amino acids 350-439), followed by S V40
polyA. The resulting fusion gene, VP-16TA-mc, is placed under the
control of the c-erbB-2 promoter "perbB2" up to the first ATG. Gene
cassette 4 contains "GALA", consisting of five copies of a 17-mer DNA-
binding site for Gal4. The TET-ON sequence is placed under the control
of the GALA-ptet promoter and the therapeutic gene, X, is linked to t h a
TET-ON via an IRES; Gene cassette 5 contains an antisense TET-ON
which is a sequence consisting of the complementary sequence to t h a
first 80 bases of the TET-ON sequence including the ATG, placed a n d a r
the control of the pCMV promoter. Gene cassette 6 contains a
dominant negative TET-ON consisting of the coding sequences f or
amino acids 1-207 of the tet repressor placed under the control of the
pCMV promoter.
Figure 10 illustrates the structure of the pHIPs-GFP
(Heat-Inducible, Prostate-specific Promoter) expression vector. The
pHIPS vector is comprised of six cassettes. Gene cassette 1 differs from
previously described vectors only in that it contains "Gal-DBD-mx"
which is a fusion open reading frame encoding the N-terminus (amino
acids 1-147) DNA-binding domain of the yeast GAL4 protein (Gal-DBD)
fused to the basis helix-loop-helix-leucine zipper {bHLHLZ) domain of
Max (mx, amino acids 8-112) followed by SV40 poly A. The resulting
fusion gene GAL-DBD-mx is controlled by the heat inducible HSP
promoter. Gene cassette 2 is comprised of the minimal CMV promoter
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(mCMVp), "antisense Gal-DBD-mx", which is an antisense construct
complementary to the Gal-DBD-mx sequence, "IRES", which is a n
internal ribosomal entry site and "Gal-DBD" which competes with the
Gal-DBD-mx for the pGAL binding site. Gene cassette 3 is comprised of
"VP16-TA-mc" which is a fusion open reading frame encoding at the N-
terminus the first 11 amino acids of Gal4 (amino acids 1-147), followed
by the nuclear localization signal of the SV40 large T antigen, the 130
amino acid C-terminus transactivation domain of the herpes simplex
viral protein VP16, the bHLHLZ domain of c-Myc (amino acids 3 5 0-
439), followed by SV40 polyA. The resulting fusion gene, VP16-TA-
mc, is placed under the control of the probasin gene promoter
(pProbasin) up to the first ATG. Gene cassette 4 contains "GALA",
consisting of five copies of a 17-mer DNA-binding site for Gal4. The
TET-ON sequence is placed under the control of the GALA-ptet
promoter and the therapeutic gene, X, is linked to the TET-ON via a n
IRES; Gene cassette 5 contains an antisense TET-ON which is a sequence
consisting of the complementary sequence to the first 80 bases of the
TET-ON sequence including the ATG, placed under the control of the
pCMV promoter. Gene cassette 6 contains a dominant negative TET-ON
consisting of the coding sequences for amino acids 1-207 of the tet
repressor placed under the control of the pCMV promoter.
DETAILED DESCRIPTION OF THE INVENTION
As used herein, the term "heat" is to mean heat energy
generated by any means, including microwaves.
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As used herein, the term "light" is to mean light energy
with frequencies in the visible as well as the invisible spectrum,
including ionizing radiation generated by any means. This would
include a radiation source such as radionuclides capable of emitting
gamma and or beta particles, or by a linear accelerator.
In accordance with the present invention, there may b a
employed conventional molecular biology, microbiology, a n d
recombinant DNA techniques within the skill of the art. Such
techniques are explained fully in the literature. See, e.g., Maniatis,
Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual ( 1982);
"DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed.
1985); "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid
Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)]; "Transcription
and Translation" [B.D. Hames & S.J. Higgins eds. (1984)]; "Animal Cell
Culture" [R.I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes"
[IRL Press, ( I986)]; B. Perbal, "A Practical Guide To Molecular Cloning"
(1984).
The present invention is directed towards a new method of
gene therapy for confined areas such as tumors. In accordance with
the above-mentioned object there is provided a mechanism for both
constitutively active and regulable gene expression via plasmids
containing elements which are heat and or light activated a n d
responsive to presence and concentration of antibiotic (tetracycline a n d
its derivatives). In regulating gene expression, heat or light initiates
the expression, but the gene is constitutively expressed only in the
presence of the antibiotic {tetracycline and its derivatives).
Concentration of the antibiotic controls the Ieve1 and duration of t h a
gene expression.
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For the confinement of gene expression to tumor cells,
there are provided two mechanisms far the suppression of gene
expression in normal cells that are bystander targets of heat or
radiation. In the instance of normal cells not exposed to heat or light,
which inadvertently take up the plasmid, expression of the therapeutic
gene due to background activity of the promoter is suppressed by the
constitutive expression of antisense and dominant negative DNA
sequences to the heat or light inducible, antibiotic dependent
transcriptional activator built into the plasmid. In the instance
whereby normal cells that take up the plasmid are then exposed to
heat or light, there is an additional mechanism for preventing the
expression of the therapeutic gene. This is achieved by the use of a
modified 'two hybrid' system where the antibiotic dependent
transcriptional activator is itself under the control of both the
expression of tissue-specific transcriptional activators and the
exposure to heat or light. Expression of the therapeutic gene i s
therefore found only in cells that have been both exposed to heat or
light and that express tissue-specific transcription factors.
In one embodiment of the present invention, there is
provided a recombinant vector, pDATH-X (Dominant negative,
Antisense, TET-ON controllable Heat shock promoter plasmid), for the
purpose of reducing background levels of expression. This vector is
comprised of the cassettes: (a) a fusion of the coding sequences for
amino acids 1-207 of the tetracycline repressor and the C-terminus
last 130 amino acid transcription activation domain of the V P 16
protein of the herpes simplex virus; (b) a heat shock promoter
consisting of heat shock response elements (-260 to 30) of the h a m a n
heat shock 70 gene promoter linked to the minimal cytomegalovirus
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promoter, pCMV; (c) a tet operator consisting of the 19 by inverted
repeats of the operator 02 of TN10 to which the tet repressor and
TAKON bind; and (d} an antisense sequence consisting of t h a
complementary sequence to the first 80 bases of the TAKON sequence
including the ATG.
In another embodiment of the present invention provides a
method of achieving sustained expression of a gene under control of a
heat or light inducible promoter, comprising the step of: introducing
the vector containing said gene into the host organism; and applying
heat or light energy. In another embodiment of the invention, said
host organism is a human.
In yet another embodiment of the invention, there is
provided a recombinant vector, pDATE-X (Dominant negative,
Antisense, TET-ON controllable EGR promoter expression plasmid), said
vector comprising the cassettes: (a) in cassette l, the TET-ON
sequence is placed under the control of the EGRp, the tetracycline
operator binding site and pCMV; (b) in cassette 2, the therapeutic
gene X, is placed under the control of the tetp-pCMV promoter; (c) in
cassette 3, antisense TET-ON is placed under the control of the pCMV
promoter; and (d) in cassette 4, dominant negative TET-ON is placed
under the control of the pCMV promoter.
Another embodiment of the present invention provides a
recombinant vector, pRIBs-X, (Radiation-Inducible, Breast-specific
Promoter) expression vector, said vector comprising the cassettes: (a)
cassette 1 contains "Gal-DBD-mx" which is a fusion open reading frame
encoding the N-terminus (amino acids 1-147) DNA-binding domain of
the yeast GAL4 protein (Gal-DBD) fused to the basic helix-loop-helix-
leucine zipper domain of Max (amino acids 8-112) followed by SV40
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poly A- the resulting fusion gene GAL-DBD-mx is controlled by the
radiation inducible Egr-1 promoter; (b) cassette 2 is comprised of the
minimal CMV promoter, "antisense Gal-DBD-mx", which is an antisense
construct complementary to the Gal-DBD-mx sequence, "IRES", which is
an internal ribosomal entry site and "Gal-DBD" which competes with
the Gal-DBD-mx for the pGAL binding site; (c) cassette 3 is comprised
of "VP 16-TA-mc" which is a fusion open reading frame encoding at t h a
N-terminus the first 11 amino acids of Gal4 (amino acids 1-147 ) ,
followed by the nuclear localization signal of the SV40 large T antigen,
the 130 amino acid C-terminus transactivation domain of the herpes
simplex viral protein VP16, the basic helix-loop-helix-leucine zipper
domain of c-Myc (amino acids 350-439), followed by SV40 polyA - the
resulting fusion gene, VP16-TA-mc, placed under the control of the c-
erbB2 promoter "perB2" up to the first ATG; (d) cassette 4 contains
"Galp", five copies of all-mer DNA-binding site for Gal4. The TET-ON
sequence is placed under the control of the GAPp-ptet promoter a n d
the therapeutic gene, X, is linked to the TET-IN via an IRES; (e) cassette
contains an antisense TET-ON which is a sequence consisting of the
complementary sequence to the first 80 bases of the TET-ON sequence
including the ATG, placed under the control of the pCMV promoter; a n d
(f) cassette 6 contains a dominant negative TET-ON consisting of the
coding sequences for amino acids 1-207.
There are further provided variants of the preceding
vectors, wherein the perbB2 promoter is replaced with the whey acidic
protein promoter or the stromelysin 3 promoter.
Another embodiment of the invention provides a method
for the treatment of local and metastatic breast and ovarian cancer
comprising: administration to the patient a pRIBs-X expression vector
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(or a variant thereof} containing a cytotoxic gene. A representative
cytotoxic gene is tumor necrosis factor alpha.
The present invention is also directed to a recombinant
pRIPs-X (Radiation-Inducible, Prostate-specific Promoter) expression
vector, said vector comprising the cassettes: (a) cassette 1 contains
"Gal-DBD-mx" which is a fusion open reading frame encoding the N-
terminus (amino acids 1-147) DNA-binding domain of the yeast GAL4
protein fused to the basic helix-loop-helix leucine zipper domain of
Max (amino acids 8-112) followed by SV40 polyA - the resulting
fusion gene GAL-DBD-mx is controlled by the radiation inducible Egr-1
promoter; (b) cassette 2 is comprised of the minimal CMV promoter,
antisense Gal-DBD-mx, which is an antisense construct complementary
to the Gal-DBD-mx sequence, IRES, which is an internal ribosomal entry
site and Gal-DBD which competes with the Gal-DBD-mx for the pGAL
binding site; (c) cassette 3 is comprised of '"VP16-TA-me", a fusion
open reading frame encoding at the N-terminus the first 11 amino
acids of GaI4, followed by the nuclear localization signal of the S V40
large T antigen, the 130 amino acid C-terminus transactivation domain
of the herpes simplex viral protein VP16, the basic helix-loop-helix
leucine zipper domain of c-Myc (amino acids 350-439}, followed b y
SV40 polyA - the resulting fusion gene, VP16-TA-mc, is placed under
the control of the probasin gene promoter "pPrabasin" up to the first
ATG; (d) cassette 4 contains GALA, five copies of the 17-mer DNA-
binding site for Gal4. The TET-ON sequence is placed under the control
of the GALA-ptet promoter and the therapeutic gene, X, is linked to t h a
TET-ON via an internal ribosomal entry site; (e) cassette 5 contains a n
antisense TET-ON which is a sequence consisting of the complementary
sequence to the first 80 bases of the TET-ON sequence including the
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ATG, placed under the control of the pCMV promoter; and (f) cassette
6 contains a dominant negative TET-ON consisting of the coding
sequence for amino acids 1-207. A variant of the preceding vector is
also contemplated, wherein the probasin promoter is replaced with the
prostate specific antigen promoter.
Another embodiment of the invention provides a method
for the treatment of local and metastatic prostate cancer comprising:
administration to the patient a pRIPs-X expression vector (or a variant
thereof) containing a cytotoxic gene. A representative cytotoxic gene is
tumor necrosis factor alpha.
In yet another embodiment of the present invention, there
is provided a recombinant expression vector, pHIBs-X (Heat Inducible,
Breast-specific promoter), said vector comprising the cassettes: (a)
cassette 1 contains Gal-DBD-mx which is a fusion open reading frame
encoding the N-terminus (amino acids 1-147) DNA-binding domain of
the yeast GAL4 protein fused to the basic helix-loop-helix leucine
zipper domain of Max (amino acids 8-112) followed by SV40 polyA -
the resulting fusion gene GAL-DBD-mx is controlled by the heat
inducible heat shock protein promoter; (b) cassette 2 is comprised of
the minimal CMV promoter, antisense Gal-DBD-mx, a construct
complementary to the Gal-DBD-mx sequence, an internal ribosomal
entry site and Gal-DBD, which competes with the Gal-DBD-mx for t h a
pGAL binding site; (c) cassette 3 is comprised of "VP16-TA-mc" which
is a fusion open reading frame encoding at the N-terminus the first 1 1
amino acids (amino acids 1-147), followed by the nuclear localization
signal of the SV40 large T antigen, the 130 amino acid C-terminus
transactivation domain of the herpes simplex viral protein VP16, the
basic helix-loop-helix leucine zipper domain of c-Myc (amino acids
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350-439), followed by SV40 polyA - the resulting fusion gene VP 16-
TA-me is placed under the control of the c-erbB2 gene promoter
"perbB2" up to the first ATG; (d) cassette 4 contains GALA, five copies
of a 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed
under the control of the GALA-ptet promoter and the therapeutic gene,
X, is linked to the TET-ON via an internal ribosomal entry site; (e)
cassette 5 contains an antisense TET-ON which is a sequence consisting
of the complementary sequence to the first 80 bases of the TET-ON
sequence including the ATG, placed under the control of the pCMV
promoter; and (f) cassette 6 contains a dominant negative TET-ON
consisting of the coding sequences for amino acids 1-207. Variants of
the preceding vector are contemplated, wherein the perbB2 promoter
is replaced with the whey acidic protein promoter or the stromelysin 3
promoter.
The present invention is further directed to a method for
the treatment of local and metastatic breast and ovarian cancer
comprising: administration to the patient a pHIBs-X expression vector
(or a variant thereof) containing a therapeutic gene. A representative
therapeutic gene is tumor necrosis factor alpha.
Another embodiment of the invention provides a
recombinant vector, pHIPs-X (Heat-Inducible, Prostate-specific
Promoter), said vector comprising the cassettes: (a) cassette 1 contains
Gal-DBD-mx which is a fusion open reading frame encoding the N-
terminus (amino acids 1-147) DNA-binding domain of the yeast GAL4
protein fused to the basic helix-loop-helix leucine zipper domain of
Max (amino acids 8-112) followed by SV40 polyA - the resulting
fusion gene GAL-DBD-mx is controlled by the heat inducible heat shock
protein promoter; (b) cassette 2 is comprised of the minimal CMV
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promoter (mCMVp), antisense Gal-DBD-mx, a construct complementary
to the Gal-DBD-mx sequence, an internal ribosomal entry site and Gal-
DBD, which competes with the Gal-DBD-mx for the pGAL binding site;
(c) cassette 3 is comprised of "VP16-TA-me", a fusion open reading
frame encoding at the N-terminus the first 11 amino acids of Gal4,
followed by the nuclear localization signal of the SV40 large T antigen,
the 130 amino acid C-terminus transactivation domain of the herpes
simplex viral protein VP16, the basic helix-loop-helix leucine zipper
domain of c-Myc (amino acids 350-439), followed by SV40 polyA - the
resulting fusion gene, VP16-TA-mc, is placed under the control of the
probasin gene promoter "pProbasin" up to the first ATG; (d) cassette 4
contains GALA, five copies of a 17-mer DNA-binding site for Gal4. The
TET-ON sequence is placed under the control of the GALA-ptet
promoter and the therapeutic gene, X, is linked to the TET-ON via a n
internal ribosomal entry site; (e) cassette 5 contains an antisense TET-
ON which is a sequence consisting of the complementary sequence to
the first 80 bases of the TET-ON sequence including the ATG, placed
under the control of the pCMV promoter; and (f) cassette 6 contains a
dominant negative TET-ON consisting of the coding sequences for
amino acids 1-207. A variant of the preceding vector is contemplated,
wherein the probasin promoter is replaced with the prostate-specific
antigen promoter.
In another embodiment of the invention, there is provided
a method for the treatment of local and metastatic prostate cancer
comprising: administration to the patient a pHIPs-X vector (or a
variant thereof) containing a therapeutic gene. representative
therapeutic gene is tumor necrosis alpha.
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It is specifically contemplated that pharmaceutical
compositions of the present invention may be prepared for the
purpose of gene therapy. In such a case, the composition comprises a
vector of the present invention and a pharmaceutically acceptable
carrier. A person having ordinary skill in the art of cancer
chemotherapy would readily be able to determine, without a n d a a
experimentation, appropriate dosages and routes of administration.
For gene therapy, the gene of interest contained in one of the plasmid
vectors of the present invention, could be delivered to the target cell
via a viral vector or liposome.
The level of ordinary skill of the average scientist in t h a
area of molecular cancer biology has increased substantially in recent
years. A person having ordinary skill in this art would readily be able
to construct and utilize the plasmids for this novel approach to gene
therapy given the teachings of the present specification.
The following examples are given for the purpose of
illustrating various embodiments of the invention and are not meant to
limit the present invention in any fashion.
EXAMPLE 1
The nDATE vector' Structure and mode of action
Figure 2 is a schematic depiction of the pDATE vector. The
pDATE-X plasmid functions via a feed-forward reaction to amplify the
expression of TET-ON and X. In the absence of radiation, background
expression due to leakiness of the EGRp will result in the synthesis of
TET-ON mRNA. Translation of this mRNA is reduced by the
concomitant expression of antisense TET-ON RNA. Moreover, leaked-
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through translated TET-ON protein is inactive without tetracycline. I n
the presence of tetracycline, the leaked (translated) TET-ON protein
becomes active, but the feed-forward reaction is prevented by the
constitutively expressed dominant negative TET-ON protein which
competes for the same DNA binding site of the ptet promoter.
Two chicken lysosomal matrix attachment sites (MAR) are
inserted to isolate the position effects of the cassettes (McKnight, R.A.,
et al., Mol. Reprod. & Dev., 44:179-184 ( 1996)}. While they may b a
unnecessary when the antisense and dominant negative TET-ON
expressions are driven by the minimal CMV promoter, MARs may b a
needed if stronger promoters like the human l3 actin promoter are to
drive their expression.
When cells harboring the pDATE-X are exposed to radiation,
an initial burst of TET-ON transcription occurs, leading to the synthesis
of 2-4-fold above background level of TET-ON in greater excess than
the dominant negative TET-ON. This excess TET-ON protein, in the
presence of tetracycline, then binds to the tetp promoters to which t h a
coding sequence of both TET-ON and X are linked and engages in a
feed-forward reaction. This reaction is controlled by the level of
tetracycline. As such, ,X expression is elevated and the duration
lengthened until tetracycline is removed, at which point the half-life of
the TET-ON protein will determine how long the feed-forward reaction
can be restarted using tetracycline without further radiation exposure.
This vector makes use of a feed-forward reaction to
achieve and maintain a high level of inducible gene expression. This
feed-forward feature overcomes the transient nature and weakness of
the inducible promoter. When the feed-forward reaction is limited to
a few hours, there is a large difference in the level of TET-ON achieved
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in heated and unheated cells. It is thus possible to adjust t h a
difference in the level of amplified TET-ON in irradiated a n d
unirradiated cells by enhancing the former with the alternate addition
and removal of tetracycline. However, while the addition and removal
of tetracycline can be precisely controlled in cell culture, it is difficult
to do so in vivo due to the heterogeneity of tetracycline level in tissues
and the variation in the absorption and removal of tetracycline in vivo
in different individuals. Thus, it is critical to minimize the Ieak
through expression of TET-ON with antisense and dominant negative
cassettes so that the feed-forward reaction does not significantly
amplify its level in unirradiated cells.
If necessary, the action of this vector can be further fine-
tuned by replacing the pCMV minimal promoter with a much stronger
promoter such as the human !3 actin promoter to drive the expression
of the antisense and the dominant negative TET-ON. In addition, the
copy numbers of the antisense and the dominant negative coding
sequences can be increased.
For in vivo induction of TET-ON expression, oxytetracycline
will be used because of its short in vivo half-life. In humans, after a
single oral dose peak plasma concentration of oxytetracycline is
reached at 2-4 hours (see, e.g., Goodman & Gilman's Th a
Pharmacological Basis of Therapeutics). The level of TET-ON expression
as a function of oxytetracycline concentration can thus be monitored.
Oxytetracycline is short acting with an in vivo half-life of only 9 hours
(versus doxycycline which has a half-life of 18 hours). At the end of
24 hours, the oxytetracycline level is reduced to <25% of input (about
10-30 % are never absorbed and are excreted in the active form).
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The ~DATH vector: structure and mode of action
Figure 1 is a schematic depiction of the pDATH-X vector.
This vector operates in identical fashion to the pDATE-X vector, except
that the Egr-1 promoter is replaced with the HSP promoter and that
heat is used in place of light/ionizing radiation.
EXAMPLE 3
Verification of the concept of amplifiable and sustained expression of
TFT-On and p53 with the feed-forward inducible promoter
To further validate the concept of heat inducible,
tetracycline feed-forward amplification of gene expression, two
plasmids were constructed. The plasmid "ptet-splice p53wt" was
constructed by subcloning a wild-type p53 cDNA into the ptet-splice
vector (Gibco BRL) which places p53 under the control of the tetp
promoter (consists of the regulatory sequences from the tetracycline-
resistance operon upstream of a minimal hCMV promoter). The
plasmid "HSP-tetp-TET-ON" was constructed by replacing the CMV
promoter in ptet-on (Clontech) with 300bp of the human heat shock
protein promoter and the tetp promoter.
H358, a non-small cell lung carcinoma cell line with a
homozygous deletion of p53, was grown in RPMI + 10% fetal calf
serum. 10' exponentially growing cells were cotransfected with SO ~g
of "ptet-splice p53wt" and 10 ~.g of "HSP-tetp-TET-ON" b y
electroporation using a BRL cell-Porator at 1180 ~.F and 240 V in 0.8
ml RPMI + 6 mM glucose. Transfected cells were plated out at 25%
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confluence for 36 hours and then half of them were heat-shocked a t
45°C for twenty minutes. Six hours after heat shock, cells were treated
with different doses of doxycycline. At various time points after t h a
addition of doxycycline, cells were stained immunohistochemically
with the monoclonal p53 antibody DO-1 (Santa Cruz Biologicals) using
an immunoperoxidase cell staining kit (Vector) and diaminobenzidine
(DAB). For each point, the digital images of fifty immunostained cells
were captured using a Nikon microscope. The amount of protein
expressed in each cell is proportional to the intensity of staining which
was expressed as I=1/T, where T is a measure of the transmitted
light/unit area. Results of one such experiment at 0.01-0.1 ~.g/ml of
doxycycline are shown in Fig. 8.
When 0.1 ~g/ml of doxycycline was added at 6 hours after
heating (when the level of induced TET-ON should have been at its
peak), more than 12 fold amplification of p53 was reached in 10 hours
(curves a and b, Figure 8). During this time, doxycycline also started a
feed-forward reaction in the unheated cells as indicated by the
substantial level of TET-ON. However, since the amplification s tarted
off from a lower level, the amplified level of TET-ON at 10 hours
reached only a low level (curves c and d, Fig.B).
It is possible to regulate the level of induced p53 in the
feed-forward system with an alternate regimen of tetracycline
addition and removal. In the time it takes for TET-ON (e.g. Fig.8 level
[c]) in the unheated cells to decline back to background level [e] after
removal, the level of TET-ON in the heated cells, [a], would have
declined by a similar proportion (which is equal to [c]-[e]). However,
since this level ([a]-[c]-[e]) is much higher than in the unheated cells
[e], the addition of tetracycline will re-start the feed-forward reaction
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for the heated cells from a much higher level ([a]-([c]-[e])). As such,
the level of background p53 in unheated cells can be kept at or below
the low level reached at 10 hours ([c]) whereas the p53 level in heated
cells will continue to escalate. Thus, while the TNFa and p S 3
expression driven by the HSP directly is transient, the expression
driven by the feed-forward system is on for as long as tetracycline is
available. Since the regimen of tetracycline addition in vivo will be
determined by the decay rate of tetracycline in vivo, it is important to
know the half-life of the TET-ON in tumor cells.
In vivo, the pharmacokinetics of tetracycline is
heterogeneous for different tissues. Preferential concentration of
tetracycline in specific tissues will lead to higher background
expression of TET-ON in some tissues. For example, in humans, 10-35%
of oxytetracycline is removed via the kidney, a substantial amount of
which is excreted in the active form. Therefore, it is desirable to
minimize the background expression levels at the onset to prevent run
away amplification in the unintended tissues. The pDATE and pDATH
inducible systems use a constitutively expressed antisense TET-ON to
suppress the background level of TET-ON translation and a dominant
negative TET-ON to compete with leak-through expressed TET-ON to
suppress the background expression. With the suppressed
background, the timing of tetracycline addition is only affected by t h a
desired level and duration of the expression of the therapeutic genes
and not by the need to suppress the level of background expression i n
normal unirradiated cells.
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EXAMPLE 4
Reduction in background levels of expression
Employing the 300 by HSP promoter, the background level
of expression without heat or light is about 25% of the level seen with
heat or light. To reduce this, the HSP was linked from -80 to +30 to the
minimal pCMV promoter. The pCMV promoter is preferred due to its
lower background expression. Additionally, it permits greater
amplification of the expression of the therapeutic gene, independent of
the constraints of the weaker HSP promoter, which is used to initiate
the reaction with a burst of heat or light.
To further overcome the problem of background
expression, two cassettes in the plasmid pDATH are introduced. An
antisense to TAKON is placed under the control of the pCMV promoter.
The constitutively produced antisense binds to any TAKON sense
mRNA from the background transcription and prevents its being
translated. An additional block on background transcription is
provided in cassette #4 in which a dominant negative TAKON with the
DNA binding site, but not the transcription activation domain, is placed
under the control of the pCMV. This results in background
transcription driving the production of TAKON and dominant negative
TAKON, which then compete for the ptet binding site.
EXAMPLE 5
Monitoring- of Sp 3 expression levels
To ensure that a suitable level of antisense TAKON RNA a n d
dominant negative TAKON protein is produced, levels of p 5 3
CA 02340929 2001-02-16
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expression are monitored to calibrate copy number and strength of the
promoter needed in order to reduce background. First, cell lines
harboring pDATH are isolated in the absence of tetracycline. The level
of p53 or a cotransfected ptet-EGFP is then monitored to determine t h a
copy number of antisense TAKON and dominant negative TAKON that
needs to be incorporated into pDATH to reduce background expression.
EXAMPLE 6
The expression vector DRIBs for treatment of local and metastatic
breast and ovarian cancer
As mentioned supra, genes placed under the control of such
promoters as the radiation inducible promoter of the Egr-1 gene are
often expressed only transiently and at low levels. This renders them
unsuitable for use in cancer therapy. To overcome these problems, the
expression vector pRIBs-X (Radiation-Inducible, Breast-specific
Promoter) was designed.
Gene expression levels were optimized using a feed-forward
reaction with the tetracycline-dependent transactivator, Tet-On, placed
under the control of a tetracycline promoter (tetp), followed by the GAL
4 promoter (pGAL). Transient transcription initiated at pGAL leads to
synthesis of a low level of Tet-On, which then binds to tetp in the
presence of tetracycline. Tet-On then amplifies its own expression and
that of the therapeutic gene linked to it via a feed-forward reaction. The
expression of therapeutic genes is controlled by six gene cassettes in the
pRIBs vector (Figure 3). In cassette 1, the fusion gene GAL-DBD-mx
(HLH-LZ domain of max fused to the DNA-binding domain of GAL-4) is
regulated by EGRp. Background expression of GAL-DBD-mx is
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suppressed by a constitutively expressed antisense GAL-DBD-mx and a
dominant negative GAL-DBD in cassette 2. In cassette 3, the
transcription activation domain of the herpes simplex viral protein V P 16
is fused to the HLH-LZ domain of c-Myc. The resulting fusion gene,
VP16-TA-mc, placed under the control of the c-erbB-2 promoter, is
expressed in breast tumor cells overexpressing c-erbB-2. GAL-DBD-mx
fusion protein binds to and activates transcription from the pGAL
promoter (cassette 4) by recruiting the VP16-TA-me proteins.
In unirradiated cells, the translation of the background
GAL-DBD-mx mRNA is reduced and the dominant negative GAL-DBD
{without mx) competitively occupies the GALA in cassette 4, blocking
Tet-On expression. Upon irradiation, GAL-DBD-mx is transiently
induced 3-4 fold and temporarily overcomes the suppression b y
cassette 2. The GAL-DBD-mx recruits the VP-16-TA-me (a fusion gene
of the VP16 transactivation domain and the leucine zipper of myc
under the control of the c-erbB-2 promoter) to the GALA and activates
a low level of Tet-On transcription starting the feed-forward reaction.
In a treatment scheme using pRIBs-TNFa, for example, can
be delivered systemically in a liposome complex or as a recombinant
virus to tumor and normal cells alike. Without radiation and
tetracycline, TNF a is not expressed. Oxytetracycline is then
administered systemically followed by X-ray irradiation of known
metastatic tumor sites. As a result, TNFa expression is induced in the
tumor sites by the X-ray and amplified and maintained b y
oxytetracycline. Even though not all tumor cells may take up pRIBs-
TNFa, tumor cells in the vicinity of those that do are exposed to the very
high local concentration of TNFa secreted. The design of pRIBs-TNFa
confers TNFa expression in the breast tumor cells only and not in the
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irradiated normal cells that were in the path of the X-ray. As such,
systemic toxicity, if any, is limited to the low level of TNFa diffused from
the tumor cells. In addition to, or instead of, TNF a, another therapeutic
gene, designated X, can be used with the pRIBS vector.
The structure of pRIBs-GFP-1 is shown in Figure 3 and the
mode of action summarized in Figure 5. In unirradiated cells,
background GAL-DBD-mx expression and function are suppressed b y
cassette 2 in two ways. The antisense to GAL-DBD-mx suppresses the
translation of background GAL-DBD-mx mRNA whereas the GAL-DBD
protein acts as a dominant negative inhibitor by competing with GAL-
DBD-mx for the pGAL promoter. In irradiated cells, GAL-DBD-mx
expression is transiently induced three to 4 fold, overcoming the
suppression by cassette 2. The GAL-DBD-mx recruits the VP-16-TA-
mc (a fusion gene of the VP16 transactivation domain and the leucine
zipper of Myc under the control of the c-erbB-2 promoter) to the GALA
and activates the transient expression of the transactivator TET-ON. I n
the presence of tetracycline, Tet-ON is activated and it binds to and
transactivates the tetp promoter (Gossen, M., et al., Science, 268:1766-
1769 (1995)), amplifying its own level and GFP in a feed-forward
reaction. Background expression of TET-ON and GFP is null in the
absence of radiation or tetracycline.
EXAMPLE 7
Generation of cell lines and xenografts stabl,~ ex ressing oRIBs-GFP
Two pRIBs-GFP plasmids, pRIBs-GFP-1 and pRIBs-GFP-4,
with one and four copies of antisense and dominant negative gene
cassettes, respectively, were constructed and stably transfected into the
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fibrosarcoma cell line HTB152 and the breast tumor cell lines SK-BR-3
and MDAMB231 for in vitro analysis. Sx106 cells are xenografted into
SC>D mice. While all three human cell lines form poorly differentiated
tumors, only SK-BR-3 expresses a high level of c-erbB-2. ' Indeed, anti-
s erbB-2 intracellular single-chain antibody which down-regulates cell
surface erbB-2, induces apoptosis only in SK-BR-3 but not MDA-MB-231
(Chumakov A.M., et al., Oncogene 8:3005-3011 (1993)).
The pRIBs-GFP-1 and -4 plasmids are thus used as models to
optimize the conditions for testing treatment of metastatic breast tumor
xenografts in nude mice with cytotoxic genes. As cytotoxic genes linked
to EGRp are induced only in irradiated cells, toxicity to unirradiated cells
is eliminated. However, it is important to prevent expression of cytotoxic
genes in normal cells that are in the pathway of the X-ray. The three cell
lines, which differ in c-erbB-2 expression, show that controlling V P 16-
TA-me expression with a tissue- or tumor-specific promoter confines
expression to irradiated breast tumor cells only and not the irradiated
normal cells of the vital organs where the metastatic tumor cells reside.
The pRIBs-GFP plasmids are assembled as shown in Figure 3.
The GAL-DBA-mx and the VP16-TA-me are modified from the
mammalian two hybrid system (Fearon, E. R., et al., Proc. Natl. Acad. Sci.
USA, 89:7958-7962 (1992)). Two plasmids, pRIBs-GFP-1 and pRIBs-
GFP-4, with 1 and 4 copies of antisense and dominant negative GAL-DBD
driven by the minimal CMV promoter are tested.
All three cell lines are cotransfected with pRIBs-GFP and a
SVneo plasmid. Cell lines stably expressing pRIBs-GFP-1 and pRIBs
GFP-4 are isolated by selection in 6418. For in vivo analysis, Sx106
cells of each of the cell lines stably expressing the pRIBs-GFP plasmids
are implanted into the flank of SCID mice (four per group) and allowed
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WO 00/10612 PCT/US99/19095
to grow to 0.5 cm in diameter. The expression of GFP ire vitro and in
the xenografts without radiation or oxytetracycline is analyzed b y
extracting the proteins into EBC buffer - from the pulverized tumor a n d
the amount of protein is quantitated by RIA.
The inducible level of GFP in vitro is measured by Western
analysis and quantitated by RIA after irradiating the cells at 0-4 Gy with
a Varian Clinac 2000 X-ray generator followed by administration of 0-2
p.g/ml of oxytetracycline. Data using HSPp showed that the feed-forward
reaction is very efficient and 0.01 p.g/ml is sufficient to induce a nine-
fold increase of p53 expression in 10 hours. For in vivo analysis, tumors
are exposed to 0-4 Gy/X-ray. Six hours after radiation, 0-15 p.g/g of
oxytetracycline is injected intraperitoneally. At 3 hour intervals (for 2 4
hours) after an injection, tumor mass is removed and the amount of TET-
ON and GFP measured relative to the total amount of actin proteins. To
achieve a higher or lower level of GFP, the experiments are repeated
with the level of TET-ON modified by adjusting the dose of
oxytetracycline. The rate of oxytetracycline removal by excretion is
monitored by analyzing plasma concentration at three hour intervals.
EXAMPLE 8
~geting, metastatic breast tumors with WAPp or ST3p
The c-erbB-2 promoter had been chosen to initially validate
the pRIB-X concept because human cancers overexpressing c-erbB-2 are
associated with poor prognosis. It is unlikely, however, that one
particular promoter will address the problem of treating different breast
tumors. Therefore it is also important to target GAL-DBD-mx expression
to metastatic breast tumors with the whey acidic protein promoter,
WAPp (McKnight, R.A., et al., Mol. Reprod. & Dev., 44:179-184 ( 1996)) o r
CA 02340929 2001-02-16
WO 00/10612 PCT/US99/19095
the stromelysin 3 promoter, ST3p (Ahmad, A., et al., Int. J. of Cancer,
73:290-296 ( 1997)). WAPp targets expression to breast epithelial cells
while ST3p targets expression to matrix-metalloproteinase-secreting
stromal cells adjacent to tumors.
pRIBs is reconstructed by replacing the c-erbB-2 promoter
with either WAPp or ST3p. Breast and other tumor cell lines are
screened for high and low expression of WAP and ST3. Cell lines
differing in their expression of WAP and/or ST3 are used to test the
expression of GFP.
The WAP promoter has been shown to be very specific for
lactating mammary epithelial cells in transgenic animals (Tzeng YJ., a t
al., Oncogene 16(16):2103-2114 (1998)) and the stromelysin 3
promoter, ST3p, has been shown to be expressed only in stromal
fibroblasts adjacent to cancer cells. Evidence suggests that production
in stromal cell of matrix-metalloproteinases (including ST3), implicated
in the process of tumor metastasis, is stimulated by the cancer cells.
Thus, the targeting of VP16-TA-me to the stromal cells will lead to the
expression and release of therapeutic gene products in the vicinity of
the metastatic tumor cells. It must be noted that additional treatment
specificity is attained by delivering pRIBs-X with liposomes coated
with antibodies to c-erbB2.
EXAMPLE 9
The expression vector pRIPs for treatment of local and metastatic
p~tate cancer
As mentioned supra, genes placed under the control of such
promoters as the radiation inducible promoter of the Egr-1 gene are
36
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WO 00/10612 PCT/US99/19095
often expressed only transiently and at low levels. This renders them
unsuitable for use in cancer therapy. To overcame these problems, the
expression vector pRIPs-X (Radiation-Inducible, Prostate-specific
Promoter) was designed.
The pRIPS vector is comprised of six cassettes. Gene
cassette 1 differs from previously described vectors only in that it
contains "Gal-DBD-mx" which is a fusion ORF encoding the N-terminus
(amino acids 1-147) DNA-binding domain of the yeast GAL4 protein
(Gal-DBD) fused to the basis helix-loop-helix-leucine zipper (bHLHLZ)
domain of Max (mx, amino acids 8-112) followed by SV40 poly A.
Gene cassette 2 is comprised of the minimal CMV promoter (mCMVp),
"antisense Gal-DBD-mx", which is an antisense construct
complementary to the Gal-DBD-mx sequence, "IRES", which is a n
internal ribosomal entry site and "Gal-DBD" which competes with t h a
Gal-DBD-mx for the pGAL binding site. Gene cassette 3 is comprised of
"VP16-TA-mc" which is a fusion ORF encoding at the N-terminus the
first 11 amino acids of Gal4 {amino acids 1-147), followed by the
nuclear localization signal of the SV40 large T antigen, the 130 amino
acid C-terminus transactivation domain of the herpes simplex viral
protein VP16, the bHLHLZ domain of c-Myc (amino acids 350-439),
followed by SV40 polyA. The resulting fusion gene, VP16-TA-mc, is
placed under the control of the probasin gene promoter "pProbasin" a p
to the first ATG. Gene cassette 4 contains "GALA", consisting of five
copies of a 17-mer DNA-binding site for Gal4. The TET-ON sequence is
placed under the control of the GALA-ptet promoter and t h a
therapeutic gene, X, is linked to the TET-ON via an IRES; Gene cassette
5 contains an antisense TET-ON which is a sequence consisting of the
complementary sequence to the first 80 bases of the TET-ON sequence
37
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WO 00/10612 PCT/US99/I9095
including the ATG, placed under the control of the pCMV promoter.
Gene cassette 6 contains a dominant negative TET-ON consisting of the
coding sequences for amino acids 1-207 of the tet repressor placed
under the control of the pCMV promoter. In other variants of pRIPs-X,
pProbasin is replaced by PSA, the promoter region of the prostate
specific antigen, or other prostate-specific genes.
The expression vector pHIBs-X treatment of local and metastatic
for
breast and ovarian cancer
The expression vector pHIBs-X was designed and is
comprised of six cassettes. Gene cassette 1 differs from previously
described vectors only in that it contains "Gal-DBD-mx" which
is a
fusion ORF encoding the N-terminus(amino acids 1-147) DNA-binding
domain of the yeast GAL4 protein (Gal-DBD) fused to the basis
helix-
loop-helix-leucine zipper (bHLHLZ) domain of Max (mx, amino acids
8 -
112) followed by SV40 poly A. The resulting fusion gene GAL-DBD-mx
is controlled by the heat inducibleHSP promoter. Gene cassette
2 is
comprised of the minimal CMV
promoter (mCMVp}, "antisense Gal-
DBD-mx", which is an antisense co nstruct complementary to the
Gal-
DBD-mx sequence, "IRES", which is an internal ribosomal entry
site a n d
"GaI-DBD" which competes with the GaI-DBD-mx for the pGAL binding
site. Gene cassette 3 is comprised of "VP16-TA-mc" which is a
fusion
ORF encoding at the N-terminus first 11 amino acids of Gal4
the (amino
acids 1-147), followed by the nucl ear localization signal of
the SV40
large T antigen, the 130 amino acidC-terminus transactivation
domain
of the herpes simplex viral protein
VP16, the bHLHLZ domain of c-Myc
(amino acids 350-439), followed
by SV40 polyA. The resulting fusion
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WO 00/10612 PCT/CJS99/19095
gene, VP-16TA-mc, is placed under the control of the c-erbB-2
promoter "perbB2" up to the first ATG. Gene cassette 4 contains
"GALA", consisting of five copies of a 17-mer DNA-binding site for Gal4.
The TET-ON sequence is placed under the control of the GALA-ptet
promoter and the therapeutic gene, X, is linked to the TET-ON via a n
IRES; Gene cassette 5 contains an antisense TET-ON which is a sequence
consisting of the complementary sequence to the first 80 bases of the
TET-ON sequence including the ATG, placed under the control of t h a
pCMV promoter. Gene cassette 6 contains a dominant negative TET-ON
consisting of the coding sequences for amino acids 1-207 of the tet
repressor placed under the control of the pCMV promoter.
The pHIBs-X expression vector is identical to the pRIBs-X
plasmid except for gene cassette 1 where the Egr-1 promoter in pRIBs-
X is replaced by the HSP 70 promoter. pHIBs-X specifically targets
local and metastatic breast and ovarian tumors when the tumors are
exposed to heat.
EXAMPLE 11
The expression vector pHIPs-X for treatment of local and metastatic
prostate ca~~er
Figure 10 illustrates the structure of the pHIPs-GFP (Heat-
Inducible, Prostate-specific Promoter) expression vector. This vector is
comprised of six cassettes. Gene cassette 1 differs from previously
described vectors only in that it contains "Gal-DBD-mx" which is a
fusion ORF encoding the N-terminus (amino acids 1-147) DNA-binding
domain of the yeast GAL4 protein (Gal-DBD) fused to the basis helix-
loop-helix-leucine zipper (bHLHLZ) domain of Max (mx, amino acids 8 -
112) followed by SV40 poly A. The resulting fusion gene GAL-DBD-mx
39
CA 02340929 2001-02-16
WO 00/10612 PCTNS99/19095
is controlled by the heat inducible HSP promoter. Gene cassette 2 is
comprised of the minimal CMV promoter (mCMVp), "antisense Gal-
DBD-mx", which is an antisense construct complementary to the Gal-
DBD-mx sequence, "IRES", which is an internal ribosomal entry site a n d
"Gal-DBD" which competes with the Gal-DBD-mx for the pGAL binding
site. Gene cassette 3 is comprised of "VP16-TA-mc" which is a fusion
ORF encoding at the N-terminus the first 11 amino acids of Gal4 (amino
acids 1-147), followed by the nuclear localization signal of the SV40
large T antigen, the 130 amino acid C-terminus transactivation domain
of the herpes simplex viral protein VP16, the bHLHLZ domain of c-Myc
(amino acids 350-439), followed by SV40 polyA. The resulting fusion
gene, VP16-TA-mc, is placed under the control of the probasin gene
promoter (pProbasin) up to the first ATG. Gene cassette 4 contains
"GALA", consisting of five copies of a 17-mer DNA-binding site for Gal4.
The TET-ON sequence is placed under the control of the GALA-ptet
promoter and the therapeutic gene, X, is linked to the TET-ON via a n
IRES; Gene cassette S contains an antisense TET-ON which is a sequence
consisting of the complementary sequence to the first 80 bases of the
TET-ON sequence including the ATG, placed under the control of the
pCMV promoter. Gene cassette 6 contains a dominant negative TET-ON
consisting of the coding sequences for amino acids 1-207 of the t a t
repressor placed under the control of the pCMV promoter.
The pHIPs-X expression vector is identical to the pRIPs-X
plasmid except for gene cassette 1 where the Egr-1 promoter in pRIBs-
X and pRIPs-X is replaced by the HSP 70 promoter. pHIPs-X
specifically targets local and metastatic prostate tumors when the
tumors are exposed to heat.
CA 02340929 2001-02-16
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Any patents or publications mentioned in this specification
are indicative of the levels of those skilled in the art to which the
invention pertains. These patents and publications axe herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to b a
incorporated by reference.
One skilled in the art will readily appreciate that t h a
present invention is well adapted to carry out the objects and obtain
the ends and advantages mentioned, as well as those inherent therein.
The present examples along with the methods, procedures, treatments,
molecules, and specific compounds described herein are presently
representative of preferred embodiments, are exemplary, and are not
intended as limitations on the scope of the invention. Changes therein
and other uses will occur to those skilled in the art which are
encompassed within the spirit of the invention as defined by the scope
of the claims.
41
