U.S. patent application number 10/051345 was filed with the patent office on 2002-05-30 for methods to enhance and confine expression of genes.
This patent application is currently assigned to Research Development Foundation. Invention is credited to Fung, Yuen Kai, Gomer, Charles, T' Ang, Anne.
Application Number | 20020065243 10/051345 |
Document ID | / |
Family ID | 26792233 |
Filed Date | 2002-05-30 |
United States Patent
Application |
20020065243 |
Kind Code |
A1 |
Fung, Yuen Kai ; et
al. |
May 30, 2002 |
Methods to enhance and confine expression of genes
Abstract
The present invention provides a novel approach to gene therapy
of restricted areas such as tumors. The methods introduced here
comprise: (a) placing a gene of interest in a plasmid vector driven
by a heat or light inducible promoter; (b) modifying this vector by
including a tetracycline responsive fusion protein which acts as a
transcriptional activator, thus permitting regulation of gene
expression by varying the levels of drug and; (c) modifying this
vector by including DNA sequences that reduce or eliminate
expression of genes in normal bystander cells. Also provided are a
set of vectors for both sustained and regulable expression. There
is also presented novel vectors for the gene therapy treatment of
local and metastatic breast, ovarian and prostate cancer.
Inventors: |
Fung, Yuen Kai; (Los
Angeles, CA) ; Gomer, Charles; (Glendora, CA)
; T' Ang, Anne; (Los Angeles, CA) |
Correspondence
Address: |
Dr. Benjamin Adler
Adler & Associates
8011 Candle Lane
Houston
TX
77071
US
|
Assignee: |
Research Development
Foundation
|
Family ID: |
26792233 |
Appl. No.: |
10/051345 |
Filed: |
January 18, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10051345 |
Jan 18, 2002 |
|
|
|
09376774 |
Aug 17, 1999 |
|
|
|
60096947 |
Aug 18, 1998 |
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Current U.S.
Class: |
514/44A ;
435/320.1 |
Current CPC
Class: |
A61K 38/191 20130101;
C12N 15/635 20130101; C12N 2830/003 20130101; C12N 15/85 20130101;
C12N 2840/203 20130101; C12N 2830/002 20130101; C12N 2830/008
20130101; A61K 38/1709 20130101; A61K 48/00 20130101; C12N 2840/206
20130101 |
Class at
Publication: |
514/44 ;
435/320.1 |
International
Class: |
A61K 048/00; C12N
015/00 |
Claims
What is claimed is:
1. A method of achieving localized, temporal expression of a gene
under control of a heat inducible promoter, comprising the steps
of: inserting said gene into a cloning site of a pDATH-X (Dominant
negative, Antisense, TET-ON controllable Heat shock promoter
plasmid) vector, said vector comprising: a) cassette 1 comprising
TET-ON expressed under the control of a heat shock promoter and a
tet operator, wherein said TET-ON consists of a fusion of the
coding sequences for amino acids 1-207 of tetracycline repressor
and the C-terminus last 130 amino acid transcription activation
domain of VP16 protein of the herpes simplex virus, wherein said
heat shock promoter consists of heat shock response elements (-260
to 30) of the human heat shock 70 gene promoter linked to a minimal
cytomegalovirus promoter, pCMV; wherein said tet operator consists
of 19 base pair inverted repeats of operator O2 of TN10 to which
said tet repressor and TET-ON bind; b) cassette 2 comprising a
cloning site for a therapeutic gene downstream of a tetp-CMV
promoter consisting of a tet operator linked to a minimal
cytomegalovirus promoter, pCMV, wherein said tet operator consists
of 19 base pair inverted repeats of operator O2 of TN10 to which
said tet repressor and TET-ON bind; c) cassette 3 comprising
antisense TET-ON under the control of pCMV promoter, wherein said
antisense TET-ON consists of an antisense sequence complementary to
the first 80 nucleotides of the TET-ON sequence including the ATG
start codon; and d) cassette 4 comprising a dominant negative
TET-ON under the control of pCMV promoter, wherein said dominant
negative TET-ON consists of a tet repressor without a VP16
transactivation domain; introducing the vector containing said gene
into the host organism; and applying heat energy to a location on
said host organism where expression of said gene is desired.
2. The method of claim 1, where said host organism is a human.
3. A recombinant vector, pDATE-X (Dominant negative, Antisense,
TET-ON controllable EGR promoter expression plasmid), said vector
comprising the cassettes: (a) cassette 1 comprising the TET-ON
sequence under the control of the EGRp, the tetracycline operator
binding site and pCMV; (b) cassette 2 comprising a therapeutic gene
X under the control of the tetp-pCMV promoter; (c) cassette 3
comprising antisense TET-ON under the control of the pCMV promoter;
and (d) cassette 4 comprising dominant negative TET-ON under the
control of the pCMV promoter.
4. A recombinant vector, pRIBs-X, (Radiation-Inducible,
Breast-specific Promoter) expression vector, said vector comprising
the cassettes: (a) cassette 1 comprising "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 poly A, wherein the resulting fusion
gene GAL-DBD-mx is controlled by the radiation inducible Egr-1
promoter; (b) cassette 2 comprising the minimal CMV promoter,
"antisense Gal-DBD-mx", which is an antisense construct
complementary to the Gal-DBD-mx sequence, an internal ribosomal
entry site (IRES) and "Gal-DBD" which competes with the Gal-DBD-mx
for the pGAL binding site; (c) cassette 3 comprising "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 basic helix-loop-helix-leucine zipper domain of
c-Myc (amino acids 350-439), followed by SV40 polyA, wherein the
resulting fusion gene, VP16-TA-mc, is under the control of the
c-erbB2 promoter "perB2" up to the first ATG; (d) cassette 4
comprising "Galp", five copies of a 17-mer DNA-binding site for
Gal4, wherein a TET-ON sequence is placed under the control of the
GAPp-ptet promoter and a therapeutic gene X is linked to the TET-IN
via an IRES; (e) cassette comprising 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 under the control of
the pCMV promoter; and (f) cassette 6 comprising a dominant
negative TET-ON consisting of the coding sequences for amino acids
1-207.
5. The recombinant vector of claim 4, wherein the perbB2 promoter
of cassette 3 is replaced with the whey acidic protein
promoter.
6. The recombinant vector of claim 4, wherein the perbB2 promoter
of cassette 3 is replaced with the stromelysin 3 promoter.
7. The recombinant vector of claim 4, wherein said gene X is a gene
encoding tumor necrosis factor alpha.
8. A method of treating local and metastatic breast and ovarian
cancer comprising the step of: administering the expression vector
of claim 4 to an individual in need of such treatment.
9. A method of treating local and metastatic breast and ovarian
cancer comprising the step of: administering the expression vector
of claim 4 to an individual in need of such treatment.
10. A method of treating local and metastatic breast and ovarian
cancer comprising the step of: administering the expression vector
of claim 6 to an individual in need of such treatment.
11. A recombinant pRIPs-X (Radiation-Inducible, Prostate-specific
Promoter) expression vector, said vector comprising the cassettes:
(a) cassette 1 comprising "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, wherein the resulting fusion gene
GAL-DBD-mx is controlled by the radiation inducible Egr-1 promoter;
(b) cassette 2 comprising 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 comprising "VP16-TA-mc", 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, wherein the resulting fusion
gene, VP16-TA-mc, is under the control of the probasin gene
promoter "pProbasin" up to the first ATG; (d) cassette 4 comprising
GALp, five copies of the 17-mer DNA-binding site for Gal4, wherein
the TET-ON sequence is under the control of the GALp-ptet promoter
and a therapeutic gene X is linked to the TET-ON via an internal
ribosomal entry site; (e) cassette 5 comprising 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, under the
control of the pCMV promoter; and (f) cassette 6 comprising a
dominant negative TET-ON consisting of the coding sequence for
amino acids 1-207.
12. The recombinant vector of claim 11, wherein said probasin
promoter of cassette 3 is replaced with the prostate specific
antigen promoter.
13. The recombinant vector of claim 11, wherein said gene X is
tumor necrosis factor alpha.
14. A method of treating local and metastatic prostate cancer
comprising the step of: administering the expression vector of
claim 11 to an individual in need of such treatment.
15. A method of treating local and metastatic prostate cancer
comprising the step of: administering the expression vector of
claim 12 to an individual in need of such treatment.
16. A recombinant expression vector, pHIBs-X (Heat Inducible,
Breast-specific promoter), said vector comprising the cassettes:
(a) cassette 1 comprising 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, wherein the resulting fusion gene
GAL-DBD-mx is controlled by the heat inducible heat shock protein
promoter; (b) cassette 2 comprising 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 the pGAL binding site; (c)
cassette 3 comprising "VP16-TA-mc" which is a fusion open reading
frame encoding at the N-terminus the first 11 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
350-439), followed by SV40 polyA, wherein the resulting fusion gene
VP16-TA-mc is under the control of the c-erbB2 gene promoter
"perbB2" up to the first ATG; (d) cassette 4 contains GALp, five
copies of a 17-mer DNA-binding site for Gal4, wherein the TET-ON
sequence is under the control of the GALp-ptet promoter and a
therapeutic gene, X, is linked to the TET-ON via an internal
ribosomal entry site; (e) cassette 5 comprising 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, under the
control of the pCMV promoter; and (f) cassette 6 comprising a
dominant negative TET-ON consisting of the coding sequences for
amino acids 1-207.
17. The recombinant vector of claim 16, wherein the perbB2 promoter
of cassette 3 is replaced with the whey acidic protein
promoter.
18. The recombinant vector of claim 16, wherein the perbB2 promoter
of cassette 3 is replaced with the stromelysin 3 promoter.
19. The method of claim 16, wherein said therapeutic gene is tumor
necrosis factor alpha.
20. A method of treating local and metastatic breast and ovarian
cancer comprising the step of: administering the expression vector
of claim 16 to an individual in need of such treatment.
21. A method of treating local and metastatic breast and ovarian
cancer comprising the step of: administering the expression vector
of claim 17 to an individual in need of such treatment.
22. A method of treating local and metastatic breast and ovarian
cancer comprising the step of: administering the expression vector
of claim 18 to an individual in need of such treatment.
23. A recombinant vector, pHIPs-X (Heat-Inducible,
Prostate-specific Promoter), said vector comprising the cassettes:
(a) cassette 1 comprising 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, wherein the resulting fusion gene
GAL-DBD-mx is controlled by the heat inducible heat shock protein
promoter; (b) cassette 2 comprising the minimal CMV 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 comprising "VP16-TA-mc", 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, wherein the resulting fusion gene, VP16-TA-mc, is under
the control of the probasin gene promoter "pProbasin" up to the
first ATG; (d) cassette 4 comprising GALp, five copies of a 17-mer
DNA-binding site for Gal4, wherein the TET-ON sequence is under the
control of the GALp-ptet promoter and a therapeutic gene, X, is
linked to the TET-ON via an internal ribosomal entry site; (e)
cassette 5 comprising 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, under the control of the
pCMV promoter; and (f) cassette 6 comprising a dominant negative
TET-ON consisting of the coding sequences for amino acids
1-207.
24. The recombinant vector in claim 23, wherein the probasin
promoter is replaced with the prostate-specific antigen
promoter.
25. The recombinant vector of claim 23, wherein said therapeutic
gene is tumor necrosis alpha.
26. A method of treating local and metastatic prostate cancer
comprising the step of: administering the expression vector of
claim 23 to an individual in need of such treatment.
27. A method of treating local and metastatic prostate cancer
comprising the step of: administering the expression vector of
claim 25 to an to individual in need of such treatment.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This is a divisional application of U.S. Ser. No.
09/376,774, filed on Aug. 17, 1999, which claims benefit of
priority of provisional U.S. S. No. 60/096,947, filed Aug. 18,
1998, now abandoned.
BACKGROUND OF THE INVENTION
[0002] 1. Field of the Invention
[0003] 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, the present invention provides a method
whereby the background gene expression in non-targeted cells is
reduced or eliminated.
[0004] 2. Description of the Related Art
[0005] One of the major obstacles to the success of chemotherapy
and radiation therapy for cancer is the difficulty in achieving
tumor-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.
[0006] The use of gene therapy in cancer treatment presents many 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
(TNF.alpha.) induces systemic toxicity with such clinical
manifestations as fever and hypertension.
[0007] 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: 567-574 (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 as
the cytokine IL-2.
[0008] Weichselbaum and colleagues were the first to discover the
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-.alpha. to tumor cells to
enhance radiation cell killing by means of this promoter.
Previously, systemic administration of the cytokine TNF-.alpha. 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
TNF.alpha. appears to be dosage-dependent, as its tumor-killing
effect correlates with its serum concentration. However, systemic
toxicity of TNF.alpha. restricts the dosage that can be applied and
thus limits the usefulness of the treatment regimen. Attempts have
also been made to deliver the TNF.alpha. gene to tumor cells via
adenoviral vector and/or liposomes. Unfortunately, expression of
the TNF.alpha. gene is not restricted to the tumor sites due to the
`leakiness` of the promoter.
[0009] In an attempt to localize the level of TNF.alpha. to the
general area of radiation exposure and thereby reduce systemic
toxicity, Weichselbaum and colleagues employed the radiation
inducible Egr-1 promoter to activate the TNF.alpha. 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 TNF.alpha. gene under the control of the Egr1 promoter (EGRp),
the expression of the TNF.alpha. is enhanced in those cells
harboring an EGRp-TNF.alpha. plasmid when exposed to ionizing
radiation. In vivo, the serum level of TNF.alpha. 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 TNF.alpha. dropped precipitously within 24
hours; further decreases in serum level of TNF.alpha. coincided
with regrowth of the tumors.
[0010] There are several possible reasons for the recurrence of the
tumor upon cessation of therapy. The most obvious reason is
probably the same limitation seen with chemotherapy or radiation
therapy in general, viz., insufficient dosage levels. A major
problem, which limits the amount of TNF.alpha. 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
TNF.alpha..
[0011] Another factor that limits the production of sufficient
dosage of TNF.alpha., is that not every tumor cell will have taken
up the TNF.alpha. 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 TNF.alpha. 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 of ionizing radiation (Weichselbaum, et al.,
supra). This is not surprising, as the radiation response element,
a CArG box, is part of the serum response element.
[0012] 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.
[0013] 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 and transient nature of
gene expression driven by these promoters must be improved.
[0014] It is important to note that even when an improved inducible
vector system which can restrict the expression of a therapeutic
gene to the area of external stimuli is developed, there is still
the problem of expression in normal heated or irradiated bystander
cells. Thus, it is critical to be able to further restrict the
expression of therapeutic genes only to the intended targets, e.g.,
tumor cells.
[0015] 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
[0016] The current invention provides the composition and 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 be 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.
[0017] 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.
[0018] 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, but 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.
[0019] In yet another embodiment of the present invention, there is
provided a method of constructing the vectors for gene therapy
activation modalities.
[0020] In another embodiment of the present invention, there are
provided improved vectors for reducing background expression in
unheated and unirradiated cells.
[0021] In another embodiment of the present invention, there are
provided improved vectors for reducing expression in heated and
irradiated normal bystander cells.
[0022] 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.
[0023] In another embodiment of the present invention, there are
provided expression vectors for use in gene therapy treatment of
local and metastatic prostate cancer.
[0024] Other and further aspects, features, and advantages of the
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
[0025] 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 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.
[0026] FIG. 1A 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 as
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) tetp is the tet operator consisting of the 19 base
pair (bp) inverted repeats of the operator O2 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 the pCMV promoter. (4)
Antisense TET-ON is an antisense sequence consisting of the
complementary sequence to the first 80 bases of the TET-ON sequence
including the ATG. In cassette 4, dominant negative TET-ON is
placed under the control of the pCMV promoter. The 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 the
leakiness of the minimal promoter pCMV.
[0027] FIG. 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, the 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 bp inverted repeats of the operator O2 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.
[0028] FIG. 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 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 SV40 polyA. The
resulting fusion gene, VP16TA-mc, is placed under the control of
the c-erbB-2 promoter "perbB2" up to the first ATG. Gene cassette 4
contains "GALp", consisting of five copies of a 17-mer DNA-binding
site for Gal4. The TET-ON sequence is placed under the control of
the GALp-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 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.
[0029] FIG. 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 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 "GALp", consisting of five copies of
a 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed
under the control of the GALp-ptet promoter and the therapeutic
gene, X, is linked to the TET-ON via an IRES; Gene 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 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.
[0030] FIG. 5 is a schematic representation of the mode of action
of pRIBS-GFP.
[0031] FIG. 6 illustrates the leakiness of the HSP promoter. It
summarizes the results of testing the heat inducible system
containing the hsp70 promoter in the expression of therapeutic
genes, p53 and TNF.alpha..
[0032] FIG. 6A shows the plasmid construct for the two genes, p53
and TNF.alpha..
[0033] FIG. 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 line 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 pHSP.3 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 24 hrs after heating (lane 6).
However, even without heating (lane 5), there is a substantial
level of p53 expression (about 25%).
[0034] FIG. 7 depicts the induction of TNF.alpha. by heat or
photodynamic therapy (PDT). The coding sequence of TNF.alpha. was
subcloned into the plasmid pHSP.3 and transfected into SKOV3 cells.
Stable colonies were isolated by selection in G418. Cells were
either heated at 45.degree. C. or untreated. At 6 hours after
treatment, the level of TNF.alpha. in the medium was measured with
a Genzyme TNF.alpha. ELISA kit. TNF.alpha. shown to be induced
four-fold by heat and three-fold by PDT and secreted. However,
background expression was substantial (27%).
[0035] FIG. 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
e 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 .mu.g/ml doxycycline.
[0036] FIG. 9 depicts the pHIBS-X (Heat-Inducible, 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 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 SV40 polyA. The
resulting fusion gene, VP16TA-mc, is placed under the control of
the c-erbB-2 promoter "perbB2" up to the first ATG. Gene cassette 4
contains "GALp", consisting of five copies of a 17-mer DNA-binding
site for Gal4. The TET-ON sequence is placed under the control of
the GALp-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 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.
[0037] FIG. 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 (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 SV40
polyA. The resulting fusion gene, VP16TA-mc, is placed under the
control of the probasin gene promoter (pProbasin) up to the first
ATG. Gene cassette 4 contains "GALp", consisting of five copies of
a 17-mer DNA-binding site for Gal4. The TET-ON sequence is placed
under the control of the GALp-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 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
[0038] As used herein, the term "heat" is to mean heat energy
generated by any means, including microwaves.
[0039] 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.
[0040] In accordance with the present invention, there may be
employed conventional molecular biology, microbiology, and
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,
(1986)]; B. Perbal, "A Practical Guide To Molecular Cloning"
(1984).
[0041] 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 and
responsive to presence and concentration of antibiotic
(tetracycline and 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 level and duration of the gene expression.
[0042] For the confinement of gene expression to tumor cells, there
are provided two mechanisms for 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 is
therefore found only in cells that have been both exposed to heat
or light and that express tissue-specific transcription
factors.
[0043] 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 VP16 protein of the herpes simplex virus; (b) a heat shock
promoter consisting of heat shock response elements (-260 to 30) of
the human heat shock 70 gene promoter linked to the minimal
cytomegalovirus promoter, pCMV; (c) a tet operator consisting of
the 19 bp inverted repeats of the operator O2 of TN10 to which the
tet repressor and TET-On bind; and (d) an antisense sequence
consisting of the complementary sequence to the first 80 bases of
the TET-On sequence including the ATG.
[0044] 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.
[0045] 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 1, 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.
[0046] 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 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 "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 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 a17-mer DNA-binding site for Gal4. The
TET-ON sequence is placed under the control of the GAPp-ptet
promoter and 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; and (f) cassette 6 contains a
dominant negative TET-ON consisting of the coding sequences for
amino acids 1-207.
[0047] 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.
[0048] 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 (or a variant thereof) containing a cytotoxic gene. A
representative cytotoxic gene is tumor necrosis factor alpha.
[0049] 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-mc", 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
GALp, five copies of the 17-mer DNA-binding site for Gal4. The
TET-ON sequence is placed under the control of the GALp-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 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.
[0050] 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.
[0051] 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 the 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 11 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
350-439), followed by SV40 polyA--the resulting fusion gene
VP16-TA-mc is placed under the control of the c-erbB2 gene promoter
"perbB2" up to the first ATG; (d) cassette 4 contains GALp, five
copies of a 17-mer DNA-binding site for Gal4. The TET-ON sequence
is placed under the control of the GALp-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.
[0052] 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.
[0053] 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 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-mc", 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 GALp, five copies of a 17-mer
DNA-binding site for Gal4. The TET-ON sequence is placed under the
control of the GALp-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. A
variant of the preceding vector is contemplated, wherein the
probasin promoter is replaced with the prostate-specific antigen
promoter.
[0054] 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.
[0055] 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 undue
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.
[0056] The level of ordinary skill of the average scientist in the
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.
[0057] 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
[0058] The pDATE Vector: Structure and Mode of Action
[0059] FIG. 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-through translated TET-ON protein is inactive without
tetracycline. In 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.
[0060] 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 be unnecessary when the antisense and
dominant negative TET-ON expressions are driven by the minimal CMV
promoter, MARs may be needed if stronger promoters like the human 3
actin promoter are to drive their expression.
[0061] 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 the 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.
[0062] 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 in heated and unheated cells. It is thus possible to
adjust the difference in the level of amplified TET-ON in
irradiated and 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 leak 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.
[0063] 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 .beta. 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.
[0064] 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 The 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).
EXAMPLE 2
[0065] The pDATH Vector: Structure and Mode of Action
[0066] FIG. 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
[0067] Verification of the Concept of Amplifiable and Sustained
Expression of TET-On and p53 With the Feed-forward Inducible
Promoter
[0068] 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 300 bp of the
human heat shock protein promoter and the tetp promoter.
[0069] H358, a non-small cell lung carcinoma cell line with a
homozygous deletion of p53, was grown in RPMI+10% fetal calf serum.
10.sup.7 exponentially growing cells were cotransfected with 50
.mu.g of "ptet-splice p53wt" and 10 .mu.g of "HSP-tetp-TET-ON" by
electroporation using a BRL cell-Porator at 1180 .mu.F and 240 V in
0.8 ml RPMI+6 mM glucose. Transfected cells were plated out at 25%
confluence for 36 hours and then half of them were heat-shocked at
45.degree. C. for twenty minutes. Six hours after heat shock, cells
were treated with different doses of doxycycline. At various time
points after the 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 .mu.g/ml of doxycycline
are shown in FIG. 8.
[0070] When 0.1 .mu.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, FIG. 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 started off from a lower level, the amplified level
of TET-ON at 10 hours reached only a low level (curves c and d,
FIG. 8).
[0071] 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 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 TNF.alpha. and p53 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.
[0072] 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 the desired level and
duration of the expression of the therapeutic genes and not by the
need to suppress the level of background expression in normal
unirradiated cells.
EXAMPLE 4
[0073] Reduction in Background Levels of Expression
[0074] Employing the 300 bp 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.
[0075] To further overcome the problem of background expression,
two cassettes in the plasmid pDATH are introduced. An antisense to
TET-On is placed under the control of the pCMV promoter. The
constitutively produced antisense binds to any TET-On 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 TET-On 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 TET-On and dominant
negative TET-On, which then compete for the ptet binding site.
EXAMPLE 5
[0076] Monitoring of p53 Expression Levels
[0077] To ensure that a suitable level of antisense TET-On RNA and
dominant negative TET-On protein is produced, levels of p53
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 the copy number of antisense TET-On and dominant negative
TET-On that needs to be incorporated into pDATH to reduce
background expression.
EXAMPLE 6
[0078] The Expression Vector pRIBs for Treatment of Local and
Metastatic Breast and Ovarian Cancer
[0079] 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.
[0080] 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 (FIG. 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
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
VP16 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-mc proteins.
[0081] 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 GALp in cassette 4,
blocking Tet-On expression. Upon irradiation, GAL-DBD-mx is
transiently induced 3-4 fold and temporarily overcomes 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
GALp and activates a low level of Tet-On transcription starting the
feed-forward reaction.
[0082] In a treatment scheme using pRIBs-TNF.alpha., 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.alpha. is not expressed.
Oxytetracycline is then administered systemically followed by X-ray
irradiation of known metastatic tumor sites. As a result,
TNF.alpha. expression is induced in the tumor sites by the X-ray
and amplified and maintained by oxytetracycline. Even though not
all tumor cells may take up pRIBs-TNF.alpha., tumor cells in the
vicinity of those that do are exposed to the very high local
concentration of TNF.alpha. secreted. The design of
pRIBs-TNF.alpha. confers TNF.alpha. expression in the breast tumor
cells only and not in the 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 TNF.alpha. diffused from the tumor cells. In
addition to, or instead of, TNF.alpha., another therapeutic gene,
designated X, can be used with the pRIBS vector.
[0083] The structure of pRIBs-GFP-1 is shown in FIG. 3 and the mode
of action summarized in FIG. 5. In unirradiated cells, background
GAL-DBD-mx expression and function are suppressed by 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 GALp and
activates the transient expression of the transactivator TET-ON. In
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
[0084] Generation of Cell Lines and Xenografts Stably Expressing
pRIBs-GFP
[0085] 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 fibrosarcoma cell line HTB152 and the breast tumor cell
lines SK-BR-3 and MDAMB231 for in vitro analysis. 5.times.10.sup.6
cells are xenografted into SCID 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-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)).
[0086] 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 VP16-TA-mc 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.
[0087] The pRIBs-GFP plasmids are assembled as shown in FIG. 3. The
GAL-DBA-mx and the VP16-TA-mc 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.
[0088] 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 G418. For in vivo
analysis, 5.times.10.sup.6 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 to grow to 0.5 cm in
diameter. The expression of GFP in vitro and in the xenografts
without radiation or oxytetracycline is analyzed by extracting the
proteins into EBC buffer from the pulverized tumor and the amount
of protein is quantitated by RIA.
[0089] 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 .mu.g/ml of oxytetracycline. Data using HSPp
showed that the feed-forward reaction is very efficient and 0.01
.mu.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 .mu.g/g of
oxytetracycline is injected intraperitoneally. At 3 hour intervals
(for 24 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
[0090] Targeting Metastatic Breast Tumors With WAPp or ST3p
[0091] 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)) or 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.
[0092] 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.
[0093] The WAP promoter has been shown to be very specific for
lactating mammary epithelial cells in transgenic animals (Tzeng Y
J., et 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-mc 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
[0094] The Expression Vector pRIPs for Treatment of Local and
Metastatic Prostate Cancer
[0095] 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 pRIPs-X (Radiation-Inducible,
Prostate-specific Promoter) was designed.
[0096] 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 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 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
"GALp", consisting of five copies of a 17-mer DNA-binding site for
Gal4. The TET-ON sequence is placed under the control of the
GALp-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
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.
EXAMPLE 10
[0097] The Expression Vector pHIBs-X for Treatment of Local and
Metastatic Breast and Ovarian Cancer
[0098] 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 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 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 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,
VP-16TA-mc, is placed under the control of the c-erbB-2 promoter
"perbB2" up to the first ATG. Gene cassette 4 contains "GALp",
consisting of five copies of a 17-mer DNA-binding site for Gal4.
The TET-ON sequence is placed under the control of the GALp-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 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.
[0099] 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
[0100] The Expression Vector pHIPs-X for Treatment of Local and
Metastatic Prostate Cancer
[0101] FIG. 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 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 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 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
"GALp", consisting of five copies of a 17-mer DNA-binding site for
Gal4. The TET-ON sequence is placed under the control of the
GALp-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
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.
[0102] 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.
[0103] 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 are herein
incorporated by reference to the same extent as if each individual
publication was specifically and individually indicated to be
incorporated by reference.
[0104] One skilled in the art will readily appreciate that the
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.
* * * * *