Note: Descriptions are shown in the official language in which they were submitted.
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METHOD COMPOSITION FOR TREATING TUMORS
BY SELECTIVE INDUCTION OF APOPTOSIS
BACKGROUND OF THE INVENTION
FIELD OF THE INVENTION
The present invention relates to compositions and methods for
inducing programmed cell death (apoptosis) in cancer cells, and more
particularly, relates to compositions and methods for treating tumors by
using expression vectors that expresses an apoptosis-signaling ligand
such as Fas ligand (Apo-1 ligand) and TRAIL (Apo-2 ligand).
Expression of the apoptosis-signaling iigand induces apoptosis in cells
expressing an apoptosis-mediating receptor such as Fas (receptor for
Fas ligand), and DR4 or DR5 (receptor for TRAIL).
BACKGROUND OF THE INVENTION
Currently, a major treatment for cancerous tumors is surgical
removal of the affected areas of the tissue, organ, or gland. For
example, the current treatment for breast cancer is focused on removal
of the diseased mammary gland, followed by combination of chemo-
and radiation therapy. However, high recurrence rates are a major
obstacle to the complete eradication of cancerous cells. It is believed
that although the cancer cells in the malignant tumors can be removed
surgically, cancerous cells that have invaded the surrounding tissue or
lymph nodes frequently cause tumor recurrence. One reason for
frequent tumor recurrence may be that during the development of the
primary cancer, complete removal of all the cancer cells by surgical
procedures is extremely difficult. The remaining cancer cells often
remain quiescent for extended periods of time, which is termed tumor
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dormancy. Meltzer et al. (1990) "Dormancy and breast cancer" J. Surg.
Oncol. 43:181-188. Once the primary tissue is surgically removed, the
surgical injury can stimulate rapid tissue and blood vessel regeneration
at the wound. These regeneration processes send out positive signals
to the surrounding tissue, for example by tissue and vessel growth
factors. These factors and the rapid proliferative environment induce
the transition of the remaining tumor cells from dormancy to rapid
proliferation, and thereby cause reoccurrence of the cancer.
Two basic features are shared by all cancer cells: the
uncontrolled cell cycling; and the inability to enter the pathway of
programmed cell death, apoptosis. Apoptosis, or programmed cell
death (PCD), is a genetically controlled response for cells to commit
suicide. The symptoms of apoptosis are viability loss accompanied by
cytotoxic boiling, chromatin condensation, and DNA fragmentation.
Wyllie et al. (1980) "Cell death: the significance of apoptosis" Int. Rev.
Cytol. 68:251-306. The apoptotic process has important roles in
regulating the development of tissues, the sizes and shapes of organs,
and the life span of cells. In the process of tissue and organ
development apoptosis accounts for most or all of the PCD responsible
for tissue modeling in vertebrate development for the physiological cell
death in the course of normal tissue turn over. Apoptosis is also
responsible for the extensive elimination of cells of the B and T cell
lineages during negative selection in the immune response.
Apoptosis acts as a safeguard to prevent overgrowth of cells and
tissues. The development of defects in PCD mechanisms can extend
the life span of a cell and can contribute to neoplastic cell expransion.
Also, defects in PCD can contribute to carcinogenesis by permitting
genetic instability and accumulation of gene mutations promoting
resistance to immune-based destruction and conferring resistance to
cytotoxic drugs and radiation. These manifestations indeed are seen in
malignant cells not responding to these therapies. Although irradiation,
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chemotherapy and the appropriate hormone therapy all induce
apoptosis to some extent in tumor cells, higher doses of the drugs or
radiation may be required for suppressing the growth of cancer cells,
which, in turn, can cause severe side effects on patients.
SUMMARY OF THE INVENTION
The present invention provides novel methods and compositions
for treating cancer, in particular, solid tumors, by expressing apoptosis
signaling ligands such as Fast and TRAIL in a site-specific and
controlled manner.
In one aspect, the present invention provides a method for
inducing death in cells that express an apoptosis-mediating receptor.
In one, the method comprises: introducing an expression vector
into a group of cells comprising cells that express an apoptosis-
mediating receptor. The expression vector comprises a polynucleotide
sequence encoding an apoptosis-signaling ligand whose expression is
preferably regulated by a conditional promoter in the vector. The cells
into which the expression vector is introduced express the apoptosis-
signaling ligand when conditions are suitable to activate the conditional
promoter. The expressed apoptosis-signaling ligand induces cell death
in those cells which express the apoptosis-mediating receptor through
interaction between the apoptosis-signaling ligand and the apoptosis-
mediating receptor.
According to the embodiment, the apoptosis-mediating receptor
may be a membrane-bound receptor such as the receptor for Fas
ligand, Fas, and the receptors of TRAIL, DR4 and DRS. Optionally, the
apoptosis-mediating receptor may be a receptor for tumor necrosis
factor (TNF) although TNF may have higher systemic toxicity than Fas
and TRAIL.
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Also according to the embodiment, the apoptosis-signaling ligand
can be any protein that is capable of binding to the apoptosis-mediating
receptor. For example, the apoptosis-signaling ligand is an antibody
that is capable of binding to Fas (or DR4/DR5) and signals Fas (or
DR4/DR5)-mediated apoptosis in cells expressing Fas (or DR4/DR5).
The antibody may be expressed as a single-chain antibody by an
expression vector of the present invention and binds to its cognate
antigen on the apoptosis-mediating receptor.
Preferably, the apoptosis-signaling ligand is a membrane protein
such as Fast and TRAIL. Optionally, the apoptosis-signaling ligand
may be TNF although TNF may have higher systemic toxicity than Fas
and TRAIL.
Also optionally, the apoptosis-signaling ligand may be a non-
membrane-bound protein that can induce apoptosis when expressed
intracellularly. Examples of such an intracellular apoptosis-signaling
ligand include, but are not limited to, Bax, Bad, Bak, and Bik.
Also according to the embodiment, the expression vector may be
a plasmid. The plasmid can be transfected into cancer cells via
liposome-mediated delivery or other methods of transfection.
Preferably, the expression vector is a viral vector. The viral
vector may be an adenovirus, adeno-associated virus, vaccinia,
retrovirus, or herpes simplex virus vector.
Most preferably, the expression vector is an adenoviral vector.
The adenoviral vector may be replication competent or replication
incompetent, depending on the dosage of the apoptosis-signaling ligand
to be administered into the tumor site.
The expression of the apoptosis-signaling ligand is regulated by a
conditional promoter in the expression vector. The conditional promoter
may be a tissue-specific promoter such as a prostate-specific promoter,
a breast-specific promoter, a pancreas-specific promoter, a colon-
specific promoter, a brain-specific promoter, a kidney-specific promoter,
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a bladder-specific promoter, a lung-specific promoter, a liver-specific
promoter, a thyroid-specific promoter, a stomach-specific promoter, an
ovary-specific promoter, and a cervix-specific promoter.
Examples of the prostate-specific promoter include, but are not
limited to, prostate specific antigen (PSA) promoter and its mutants
~PSA, ARR2PB and probasin (PB) promoters, gp91-phox gene
promoter, and prostate-specific kallikrein (hKLK2) promoter.
Examples of the liver-specific promoter include, but are not
limited to,
liver albumin promoter, alpha-fetoprotein promoter, a,-antitrypsin
promoter, and transferrin transthyretin promoter.
Examples of the colon-specific promoter include, but are not
limited to,
carbonic anhydrase I promoter and carcinoembrogen's antigen
promoter.
Examples of the ovary- or placenta-specific promoter include, but
are not limited to, estrogen-responsive promoter, aromatase cytochrome
P450 promoter, cholesterol side chain cleavage P450 promoter, 17
alpha-hydroxylase P450 promoter.
Examples of the breast-specific promoter include, but are not
limited to, G.I. erb-B2 promoter, erb-B3 promoter, ~-casein, ~-lacto-
globulin, and WAB (whey acidic protein) promoter.
Examples of the lung-specific promoter include, but are not
limited to, surfactant protein C Uroglobin (cc-10, Cllacell 10 kd protein)
promoter.
Examples of the skin-specific promoter include, but are not limited
to,
K-14-keratin promoter, human keratin 1 or 6 promoter, and loicrin
promoter.
Examples of the brain-specific promoter include, but are not
limited to, glial fibrillary acidic protein promoter, mature astrocyte
specific
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protein promoter, myelin promoter, and tyrosine hydroxylase promoter.
Examples of the pancreas-specific promoter include, but are not
limited villin promoter, glucagon promoter, and Insulin Islet amyloid
polypeptide (amylin) promoter.
Examples of the thyroid-specific promoter include, but are not
limited to, thyroglobulin promoter, and calcitonin promoter.
Examples of the bone-specific promoter include, but are not
limited to, Alpha 1 (I) collagen promoter, osteocalcin promoter, and bone
sialoglycoprotein promoter.
Examples of the kidney-specific promoter include, but are not
limited to, renin promoter, liver/bone/kidney alkaline phosphatase
promoter, and erythropoietin (epo) promoter.
Alternatively, the conditional promoter may be an inducible
promoter which is activated or suppressed in the presence of an
inducing agent, such as tetracycline and its derivatives or analogs (e.g.
doxycycline), steroid such as glucocorticoid, estrogen, androgen, and
progestrone.
Also according to embodiment, the method further comprises
creating the conditions suitable to activate the conditional promoter,
such as delivering to the group of cells tetracycline or deoxycycline, and
delivering to the group of cells a steroid selected from the group
consisting of glucocorticoid, estrogen, androgen, and progestrone .
Also according to embodiment the expression vector further
comprises a reporter gene. The expression vector may express the
reporter gene as a fusion protein with the apoptosis-signaling ligand.
Alternatively, the expression vector may express the reporter gene as a
single protein bicistronically with the apoptosis-signaling ligand via a
mechanism of internal ribosome entry site (IRES) or splicing
donor/acceptor sites.
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The reporter gene preferably encodes a fluorescent protein such
as green, yellow and blue fluorescent proteins, and more preferably
green fluorescent protein (GFP).
Also according to the embodiment, the expression vector further
comprises a polynucleotide sequence encoding a regulatory protein.
The regulatory protein may be expressed as a fusion protein with the
apoptosis-signaling ligand, or expressed as a single protein from a
different promoter on the expression vector. Optionally, the regulatory
protein may be expressed as a single protein bicistronically with the
apoptosis-signaling ligand via a mechanism of internal ribosome entry
site (IRES) or splicing donor/acceptor sites.
For example, the regulatory protein may be a protein that causes
tissue-specific localization of the apoptosis-signaling ligand.
The method of present invention can be used to treat tumors.
Accordingly, the group of cells to be induced to undergo apoptosis are
contained in a solid tumor. Examples of solid tumors include, but are
not limited to, breast, prostate, brain, bladder, pancreas, rectum,
parathyroid, thyroid, adrenal, head and neck, colon, stomach, bronchi
and kidney tumors.
The expression vector may be introduced into a tumor by using
any pharmaceutically acceptable routes of administration. For example,
the expression vector may be administered into the group of tumor cells
parenterally, intraperitoneally, intravenously, intraartierally,
transdermally, sublingually, intramuscularly, rectally, transbuccally,
intranasally, liposomally, via inhalation, vaginally, intraoccularly, via
local
delivery by catheter or stent, subcutaneously, intraadiposally,
intraarticularly, intrathecally, or in a slow release dosage form.
Preferably, the expression vector is introduced into the tumor by
direct injection of the expression vector into the tumor loci.
Optionally, the method can be performed ex vivo where the group
of cells into which the expression vector is introduced are contained in a
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sample taken from a patient having cancer, or contained in contained in
a cell culture.
The expression vector may be introduced into a mixture of cells
which express Fas and cells which do not express Fas.
Optionally, the expression vector may be introduced into cells
which do not express Fas.
Also optionally, the expression vector may be introduced into
cells which do express Fas.
Also optionally, the expression vector may be introduced into
cells which cells which do not express Fas. By a "bystander effect",
those cancer cells expressing Fas near those cells transduced by the
expression vector are killed via Fas-Fast interactions.
In another aspect, the present invention provides an adenoviral
expression vector that can be used to induce apoptosis of cancer cells.
The adenoviral vector comprises: a conditional promoter, and a
polynucleotide sequence encoding a membrane-bound ligand whose
expression is regulated by the conditional promoter in the vector, the
ligand signaling apoptosis in cells that express an apoptosis-mediating
receptor.
Also according to the embodiment, the membrane-bound ligand
can be any protein that is capable of binding to an apoptosis-mediating
receptor on the surface of cancer cells. Preferably, the membrane-
bound protein is Fast or TRAIL. Optionally, the membrane-bound
protein may be TNF although TNF may have higher systemic toxicity
than Fas and TRAIL.
Also according to the embodiment, the adenoviral vector may be
replication competent or replication incompetent, depending on the
dosage of the the ligand to be administered into the tumor site.
The expression of the ligand is regulated by a conditional
promoter in the adenovirai expression vector. The conditional promoter
may be a tissue-specific promoter such as a prostate-specific promoter,
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a breast-specific promoter, a pancreas-specific promoter, a colon-
specific promoter, a brain-specific promoter, a kidney-specific promoter,
a bladder-specific promoter, a lung-specific promoter, a liver-specific
promoter, a thyroid-specific promoter, a stomach-specific promoter, an
ovary-specific promoter, and a cervix-specific promoter.
In yet another, the present invention provides an adenoviral
expression vector for tight controlling expression of a target protein in
response to tetracycline. The adenoviral expression vector comprises: a
tetracycline-responsive element; a polynucleotide sequence encoding a
transactivator protein which is capable of binding to the tetracycline-
responsive element; and a polynucleotide sequence encoding a target
protein whose expression is regulated by the binding of the
transactivator protein to the tetracycline-responsive element.
According to this embodiment, the tetracycline-responsive
element and the polynucleotide sequence encoding the transactivator
protein are positioned at opposite ends of the adenoviral vector. For
example, the tetracycline-responsive element is positioned in the E4
region of the adenoviral vector and the polynucleotide sequence
encoding the transactivator protein is positioned in the E1 of the
adenoviral vector.
Optionally, the adenoviral vector does not include the E3 region
of adenovirus.
Also optionally, the adenoviral vector does not include the E4
region of adenovirus except for the Orf6 of the E4 region.
The expression of the target protein may be repressed in the
presence of tetracycline or doxycycline. Alternatively, expression of the
target protein may be activated in the presence of doxycycline.
Also according to the embodiment, the target protein may be
membrane-bound apoptosis signaling protein such as Fast and TRAIL.
Also according to the embodiment, the viral expression vector
may further comprise a polynucleotide sequence encoding a reporter
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protein. The reporter protein and the target protein may be encoded as
a fusion protein or expressed as a single protein bicistronically with the
target protein via a mechanism of internal ribosome entry site (IRES) or
splicing donor/acceptor sites. .
The reporter gene preferably encodes a fluorescent protein such
as green, yellow and blue fluorescent proteins, and more preferably
green fluorescent protein (GFP).
Also according to the embodiment, the expression vector further
comprises a polynucleotide sequence encoding a regulatory protein.
The regulatory protein may be expressed as a fusion protein with the
apoptosis-signaling ligand, or expressed as a single protein from a
different promoter on the expression vector. Optionally, the regulatory
protein may be expressed as a single protein bicistronically with the
apoptosis-signaling ligand via a mechanism of internal ribosome entry
site (IRES) or splicing donor/acceptor sites.
For example, the regulatory protein may be a protein that causes
tissue-specific localization of the apoptosis-signaling ligand.
Examples of the adenoviral vector according to the embodiment,
include, but are not limited to, pAdTEr and Ad/FasL-GFPTET.
The expression vectors of the present invention can also be used
in combination with other anti=cancer agents such as chemotherapeutics
(e.g. alkylating agents, antibiotic agents, antimetabolic agents, hormonal
agents and plant-derived agents) and biologic agents (e.g. cytokines,
cancer vaccines, and gene therapy delivering tumor suppressing
genes). For example, co-administering to the cancer patient the
expression vector encoding TRAIL and an anti-cancer drug such as
doxorubicin should overcome the resistance by synergistically
sensitizing the cancer cells to TRAIL-mediated apoptosis through
suppression of apoptosis-inhibiting molecules or upregulation of pro-
apoptosis molecules by the drug. Therefore, by using the combination
therapy of the present invention, cancer patients may be treated with
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subtoxic amount of chemotherapeutics and yet achieve a better clinical
efficacy without suffering from severe side effects associated with using
high dosages of chemotherapeutics.
BRIEF DESCRIPTION OF THE FIGURES
Figures 1A, 1 B, and 1 C schematically show the pLAd-C.tTA
vector, the pRAd.T.GFsL vector, and the rAd/FasL-GFPTET vector,
respectively. In Figure 1A, the pLAd-C.tTA vector is shown. This
plasmid contains the leftmost 450 by of Ad5 genome, followed by a
strong CMVie enhancer/promoter and a tTA gene from pUHD15-1
inserted into the MCS. Adapter contains restriction sites Xba1, Avr2 and
Spe1, all of which generate cohesive ends compatible with Xba1. After
assembly into rAd vectors, E1A poly A is utilized for efficient tTA
expression. A similar strategy was used to construct pLAd vectors
containing other transgenes. In Figure 1 B, the pRAd.T.GFsL vector is
shown. This plasmid contains Ad5 (sub360) sequences from the unique
EcoR1 site (27333 bp) to the right ITR (35935 bp), with E3 and E4
deletions (the Orf6 of E4 is retained). The diagram shows the structure
of the regulatable Fast-GFP expression cassette, consisting of the THE
promoter, Fast-GFP fusion protein and bovine groth hormone (BGH)
poly A. This cassette was inserted into a MCS at 35810 bp. In vitro
assembly of the rAd/FasL-GFPTErvector is shown in Figure 1 C. The
region of the junction between the GFP and Fast reading frames is
expanded. Other rAd vectors were generated using a similar strategy.
Figure 2 is a graph showing a comparison of titers of rAd vectors
with Fast activity in 293 and 293CrmA cells. Twelve-well plates were
seeded with 104 293 or 293CrmA cells and infected with r-Ad/FasL,
rAdFasL-GFPTEr, or rAd/LacZ at MOI of 5 one day later. Fourty-eight
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hours post-transduction, cells were collected and lysed. Lysates were
titrated and PFU/ml determined on 293CrmA cells. Results represent
means and average errors of 2 sets of independent experiments.
Figure 3 illustrates the construction of the TRAIL expression
vector Ad.TRAIL/GFPTer which was constructed by using similar
methods described in the legend of Figure 1 except that TRAIL and GFP
genes are separated by an IRES which facilitates bicistronical
expression of these two genes.
Figure 4 shows different sensitivities of cancer cells to Fast- and
TRAIL-induced apoptosis. Cancer cells, A459, HeLa, LnCP, and C3A,
were analyzed for susceptbility to adenovirus infection and sensitivity to
Fast- and TRAIL-induced apoptosis. Cells were infected at MOI 10 with
AdGFP (pannels in the first and the second columns from left), Ad/FasL-
GFPTEr (the third column) and Ad.TRAIL/GFPTET (the forth column). The
susceptibility of adenovirus infection of the cells are represented by the
number of GFP expression cells (the first collum), the morphology of the
cells are shown in the bright-field view (second column). Morphology of
the cells infected with Ad/FasL-GFPTET and Ad.TRAIL/GFPTET are shown
in panels in the third and the forth colum, respectively.
Figure 5 shows that TRAIL expression does not induce apoptosis
in untransformed fibroblasts. To determine that if TRAIL expression will
induce apoptosis in normal cells, low-passage human foreskin
fibroblasts were infected with AdGFP, Ad/FasL-GFPTET, and
Ad.TRAIL/GFPTET at MOI about 10. The bright-field veiw shows the
normal morphology of fibroblasts transduced with AdGFP ( panel GFP
hFF). Fibroblasts demonstrated poor infectability by adenovirus as
shown by the low number of GFP expression cells (panel GFP).
However, these cells are highly sensitive to Fast induced apoptosis
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(panel FasL). In contrast, no apparent apoptosis can be observed in
TRAIL transduced cells (panel TRAIL), even at five folds of the MOI
(panel TRAIL x5).
Figure 6 shows suppression of the growth of human breast
turmors implanted in nude mice by injection of an adenoviral vector of
the present invention (Ad/FasL-GFPTET vector) which comprises Fas
ligand. Equal numbers of breast cancer cells were implanted in each
side of six mice. Tumors on the right side of the mice were injected with
the Ad/FasL-GFPTEr vector, and tumors on the left side of the same
mice were injected with a control vector, Ad/LacZ. In four of the six
mice, most of the tumor masses disappeared after one injection
(indicated by yellow arrows). In two of the mice, suppression of tumor
growth was greater than 80% (black arrows) in comparison to tumors on
the control side of the same mice.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides novel methods and expression
vectors for treating cancer, in particular, solid tumors, by expressing
apoptosis-signaling ligands such as Fast and TRAIL in a site-specific
and controlled manner. The controlled expression of these apoptosis-
signaling ligands should significantly reduce cytotoxicity associated with
uncontrolled, systemic administration of these ligands.
According to the present invention, an expression vector such as
an adenoviral vector carrying genes encoding the apoptosis-signaling
ligand (e.g. FasL and TRAIL) can be introduced into the tumor site via
many pharmaceutically acceptable routes of administration. The cells
transduced by the adenovirus expresses the ligand, preferably, as a
membrane-bound protein. Through interactions between the apoptosis-
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signaling ligand (e.g. TRAIL) and an apoptosis-mediating receptor (e.g.
DR4 and DR5) in the cell, a cascade of signal transduction occurs. The
event triggers multiple apoptosis pathways in which the apoptosis signal
is amplified by expression of multiple apoptotic enzymes such as
proteases and endonucleases. Since the interactions between the
ligand and the receptor can occur between two cells, the tumor cells that
are not transduced by adenovirus can be induced to undergo apoptosis
due to a "bystander effect". This effect may be due to specific
interactions between the apoptosis-signaling ligand expressed in cells
transduced by the adenovirus and the apoptosis-mediating receptor
expressed on the surface of the untransduced tumor cells. Therefore,
by using the method of the present invention the efficiency of cell killing
should be higher than those approaches involving direct injection of the
ligand as a protein or cells expressing the ligand.
One important feature of the present invention is that expression
of the apoptosis-signaling ligand is controlled by a conditional promoter,
such as a tissue-specific or an inducible promoter. By controlling the
expression of the ligand site-specifically (e.g. using a tissue-specific
promoter) and/or flexible adjustment of dosage (e.g. using an inducible
promoter), potential systemic toxicity of the ligand should be significantly
reduced.
fn particular, the adenoviral vector encoding the ligand can be
directly injected into the tumor site and locally transfers the ligand into
the tumor cells. Depending on the dosage of the ligand to be delivered,
the adenoviral vector can be replication competent or replication
incompetent. Once injected into the tumor, the adenovirus transduces
the tumor cells which, as a result, expresses high levels of the ligand
locally. Through interactions between the ligand and the receptors)
expressed on the surface of the tumor cells, the apoptosis signal is
amplified by expression of multiple proteins and enzymes along the
pathways of the ligand-induced apoptosis. Thus, massive tumor cells
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can be eradicated with minimum injuries to surrounding healthy tissues.
In a sense, this approach provided by the present invention is like a
"molecular surgery" which is more precise and safer than conventional
approaches involving undiscriminating, uncontrolled administration of
cancer therapeutics.
By using the methods of the present invention, primary tumors
can be eradicated and meanwhile, the reoccurrence of the cancer can
be prevented by activating cancer cell apoptosis at the tumor site.
In one aspect, the present invention provides a method for .
inducing death in cells that express an apoptosis-mediating receptor.
The mode of death may be necrosis, apoptosis or combination of both.
The method comprises: introducing an expression vector into a
group of cells comprising cells that express an apoptosis-mediating
receptor. The expression vector comprises a polynucleotide sequence
encoding an apoptosis-signaling ligand whose expression is preferably
regulated by a conditional promoter in the vector. The cells into which
the expression vector is introduced express the apoptosis-signaling
ligand when conditions are suitable to activate the conditional promoter.
The expressed apoptosis-signaling ligand induces cell death in those
cells which express the apoptosis-mediating receptor through interaction
between the apoptosis-signaling ligand and the apoptosis-mediating
receptor.
According to the embodiment, the apoptosis-mediating receptor
may be a membrane-bound receptor such as the receptor for Fas
ligand, Fas, and the receptors of TRAIL, DR4 and DRS. Optionally, the
apoptosis-mediating receptor may be a receptor for tumor necrosis
factor (TNF) although TNF may have higher systemic toxicity than Fas
and TRAIL.
Also according to the embodiment, the apoptosis-signaling ligand
can be any protein that is capable of binding to the apoptosis-mediating
receptor. For example, the apoptosis-signaling ligand is an antibody
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that is capable of binding to Fas (or DR4/DR5) and signals Fas (or
DR4/DR5)-mediated apoptosis in cells expressing Fas (or DR4/DR5).
The antibody may be expressed as a single-chain antibody by an
expression vector of the present invention and binds to its cognate
antigen on the apoptosis-mediating receptor.
Preferably, the apoptosis-signaling ligand is a membrane protein
such as Fast and TRAIL. Optionally, the apoptosis-signaling ligand
may be TNF although TNF may have higher systemic toxicity than Fas
and TRAIL.
1. Apoptosis-mediating receptors and apoptosis-signaling
ligands
According to the present invention, the apoptosis-mediating
receptor is death receptor that mediates programmed cells death upon
binding with an apoptosis signaling ligand. The receptor may be a cell-
surface receptor that is membrane-bound, or resides in cytoplasm or
nucleus. In a preferred embodiment, the apoptosis-mediating receptor
is a cell membrane-associated receptor. A prominent example of such
an apoptosis-mediating receptor belongs to the tumor necrosis factor
(TNF) receptor superfamily.
The TNF receptor superfamily is defined by the presence of
related, cysteine-rich, extraceliular domains. Examples of TNF
receptors include, but are not limited to NTR/GFR (p75) such as NGF,
BDNF, NT-3 and NT-4, TNF-R1 (CD120a), TNF-R2 (CD120b), Fas
(CD5/Apo-1), DR3 (TRAMP/WSL-1), DR4 (TRAIL-R1), DR5 (TRAIL-R2),
DcR1 (TRAIL-R3), DcR2 (TRAIL-R4), CD30, CD40, Cd27, 4-1 BB
(CD137), OX-40, LT-~iR, human HVEM (herpes virus early mediator),
OPG (osteoprotegerin)/OC1 F, and RANK. Ashkenazi and Dixit (1999)
"Apoptosis control by death and decoy receptors" Curr. Opin. Cell Biol.
11:255-260.
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All of the receptors are type I transmembrane proteins with an
extracellular region composed of two-six cysteine rich domains that are
about 25% identity among members and contribute to ligand binding.
Fas, TNF-R1, TRAIL-DR4, DRS, TRAMP (DR3), CAR1 have similar
cytoplasmic domains. Sequence comparison of the intracellular region
of these receptors revealed a homologous, well-conserved region of
about 80 amino acids called the death domain. Orlinck and Chao
(1998) "TNF-related ligands and their receptors" Cell Signal 10:543-551.
The death domain is required for the specific recruitment of cellular
signaling molecules (adaptor proteins) that are implicated in apoptosis.
Nagata (1997) "Apoptosis by death factor" Cell 88:355-365.
The ligands that bind to the receptors in the TNF receptor
superfamily include, but are not limited to, neorotrophins, TNF-a, Fas
ligand (FasL/CD-95L/Apo-1 L), TRAIL/Apo-2L, CD30L, CD40L, CD27L,
4-1 BBL, OX-40L, and lymphotoxin (LT) a, ~. Except for LT-oc, all
ligands are synthesized as type II membrane proteins; their N-terminus
is in the cytoplasm and their C-terminus extends into the extracellular
region. Nagata (1997) "Apoptosis by death factor" Cell 88:355-365. A
region of about 150 amino acid residues in the extracellular domain is
20-25% homologous among the TNF family members.
A common feature of the ligands is that all active ligands are
composed of three identical subunits (trimers) and activate their
respective receptors by oligomerization. Schulze-Osthoff et al. (1998)
"Apoptosis by death receptors" Eur. J. Biochem. 254;439-459. Although
most members are found as membrane-bound molecules; specific
metalloproteases are capable of generating soluble forms. The zinc-
dependent metalloprotease for TNF-a called TACE is one example of
such specific metalloproteases. Orlinck and Chao (1998) "TNF-related
ligands and their receptors" Cell Signal 10:543-551.
2. Fas ligand-mediated apoptosis
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In a preferred emobodiment, the apoptosis-signaling ligand is Fas
ligand. According to the present invention, controlled expression of Fas
ligand by an expression vector in tumor site should induce apoptosis in
cells expressing Fas through Fas-Fast interactions while minimizing
side effects associated with undiscriminating attack of Fas ligand to
those normal cells which also express Fas.
Fas (APO-1, CD95), or the Fas ligand receptor, is a 45 kDa type I
membrane protein and belongs to the TNF/nerve growth factor receptor
superfamily. Bajorath, J. and A. Aruffo. (1997) "Prediction of the three
dimensional structure of the human Fas receptor by comparative
molecular modeling" J. Comput Aided Mol Des 11:3-8; and Watanabe-
Fukunaga, R., C. 1. Brannan, N. Itoh, S. Yonehara, N. G. Copeland, N.
A. Jenkins and S. Nagata "The cDNA structure, expression, and
chromosomal assignment of the mouse Fas antigen" J. Immunol.
148:1274-9.
The ligand of Fas, Fast, is a 40-kDa type II membrane protein
belonging to the tumor necrosis factor family. Takahashi, T., M. Tanaka,
J. Inazawa, T. Abe, T. Suda and S. Nagata. (1994) "Human Fas ligand:
gene structure, chromosomal location and species specificity" lnt.
Immunol. 6:1567-74. Binding of Fast (and certain anti-Fas antibodies)
to Fas causes receptor oligomerization and sends a signal through a
caspase pathway, resulting in rapid death of receptor-bearing cells
through apoptosis. Larsen, C. P., D. Z. Alexander, R. Hendrix, S. C.
Ritchie and T. C. Pearson. (1995) "Fas-mediated cytotoxicity. An
immunoeffector or immunoregulatory pathway in T cell-mediated
immune responses?" Transplantation 60:221-4; Longthorne, V. L. and
G. T. Williams. (1997) "Caspase activity is required for commitment to
Fas-mediated apoptosis" EMBO. J. 16:3805-12; Nagata, S. and P.
Golstein. (1995) "The Fas death factor" Science 267:1449-56; and
Ogasawara, J., R. Watanabe-Fukunaga, M. Adachi, A. Matsuzawa, T.
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Kasugai, Y. Kitamura, N. Itoh, T. Suda and S. Nagata. (1993) "Lethal
effect of the anti-Fas antibody in mice" [published erratum appears in
(1993) Nature Oct 7;365(6446):568] Nature 364:806-9.
Fas is expressed in almost all cell types. When Fas binds to
Fast, it activates the genetically programmed cell death through a
cascade expression of interleukin-coupled enzymes (ICE) or caspases.
Chandler et al. (1998) "Different subcellular distribution of caspase-3
and caspase-7 following Fas-induced apoptosis in mouse liver" J. Biol.
Chem. 273:10815-10818; Jones et al. (1998) "Fas-mediated apoptosis
in mouse hepatocytes involves the processing and activation of
caspases" Hepatology 27:1632-1642.
Since both ligand and receptor are membrane proteins, Fas-
induced apoptosis is normally mediated through cell-cell contact.
However, a soluble form of Fast is also produced by some cells and
has been shown to have a somewhat altered activity, depending on the
target cell Tanaka. M., T. Itai, M. Adachi and S. Nagata (1998)
"Downregulation of Fas ligand by shedding" [see comments]. Nat. Med.
4:31-6; and Tanaka, M., T. Suda, T. Takahashi and S. Nagata (1995)
"Expression of the functional soluble form of human fas ligand in
activated lymphocytes" EMBO. J. 14:1129-35.
For example, the present invention provides a method for
inducing death of tumor cells expressing Fas (Fas+ cells) by a vector-
mediated gene transfer of a Fas ligand to the cells. In this method, the
vector-transduced cell expressing the Fas ligand induces Fas+ tumor
cells to undergo apoptosis and die. The vector may be injected into the
tumor with a syringe or a micropump, thus eliminating the need for
conventional surgery to remove the tumor.
There may be multiple mechanism by which Fast expressed by
the cancer cells transduced by the vector. The cancer cell death may
be induced in several ways: 1) Fast binds to Fas on adjacent tumor
cells and induces their apoptosis; 2) Fast induces apoptosis of
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endothelial cells and destroys the blood vessels supplying the tumor; 3)
expression of Fast on tumor cells induces apoptosis of surrounding
tissues and deprives tumor cells of any nursery support; and 4)
apoptosis prevents the release of positive factors that may reactivate
quiescent tumor cells responsible for reoccurring cancers.
A major advantage of this approach is that the Fas-Fast
interaction is the major signaling event that activates several apoptosis
pathways, following both p53-dependent and independent pathways.
Callers et al. (1998) "Fas-mediated apoptosis with normal expression of
bcl-2 and p53 in lymphocytes from aplastic anemia" Br. J. Haematol.
100:698-703. Thus, apoptosis signaling is amplified by more than one
cascade of enzyme expressions, and the apoptosis does not depend on
p53 or other cell-cycle checkpoint proteins. For example, although gene
therapy with the p53 gene has shown great promise in treating cancers,
(8oulikas (1997) "Gene therapy of prostate cancer: p53, suicidal genes,
and other targets." Anticancer Res. 17:1471-1505), p53 gene therapy
may be effective in about 50-60% of the tumor cells that have a p53
mutation. Iwaya et al. (1997) "A histologic grade and p53
immunoreaction as indicators of early recurrence of node-negative
breast cancer" Jpn J Clin Oncol 27:6-12.
Another advantage is that Fast is generally a membrane-bound
signaling protein rather than an intracellular protein, such as p53 and
caspases. Fast expression on the cell surface transmits the apoptotic
signal to surrounding cancer cells by a strong "bystander effect", and
does not require delivering the therapeutic gene into all cancer cells.
Therefore, the present invention fulfills the need for a non-surgical
method of cancer treatment that provides significant improvement over
current gene therapy methods, avoids the use of toxic drugs and helps
prevent tumor recurrence.
By expressing Fas ligand in a controlled manner, e.g. via a
control of a tissue-specific or an inducible promoter, growth of tumors
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can be suppressed by selectively promoting apoptosis in tumors and
systemic toxicity of Fas Ligand can be reduced.
3. TRAIL-mediated selective apoptosis of cancer cells
TRAIL, or Apo-2 ligand, is a 281 amino acid, type II
transmembrane protein and is most closely related to Fast (28% amino
acid homology). Like Fast, TRAIL can kill many sensitive tumor cell
lines in 4-8 h. In contrast, TNF kill tumor cell lines in more than 24 h.
Wiley et al. (1995) "Identification and characterization of a new member
of the TNF family that induces apoptosis" Immunity 3:673-682. The
TRAIL receptors, DR4 and DRS, like the full-length Fas receptors,
contain a death domain that possibly interacts with an adaptor molecule
(e.g. FADD (Fas-associated death domain)-like adaptor) in order to
mediate the apoptosis signal.
The initiation of TRAIL-mediated apoptosis involves the clustering
of three DR4 or DR5 on the target cell surface by cross-linking the
receptors with the ligand (TRAIL). Upon oligomerization of the
receptors, an adaptor molecule similar to FADD is recruited to the DR4
or DR5 receptor cluster via death domain interactions. Chinnaiyan et al.
(1996) "Signal transduction by DR3, a death-domain-containing receptor
related to TNFR-1 and CD95" Science 274:990-992.
The cross-linking of agonistic receptors DR4 and DR5 to TRAIL can be
inhibited by the decoy receptors (DcR1 and DcR2). Sheridan et al.
(1997) "Control of TRAIL-induced apoptosis by a family of signaling and
decoy receptors" Science 277:818-821. The decoy receptor are able to
inhibit TRAIL-mediated apoptosis because they lack functional death
domain to mediate the death signal and they can compete with the
binding to TRAIL by DR4 and DRS. Griffith et al. (1999) "Functional
analysis of TRAIL receptors using monoclonal antibodies" J. Immunol.
162:2597-2605.
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The TRAIL adaptor molecule similar to FADD possibly contains a
death effector domain that binds to FLICE (capase-8), the aspartate-
specific cysteine protease that initiates a caspase amplification cascade
leading to the ultimate apoptotic phenotypes. Muzio (1998) "Singling by
proteolysis: death adaptors induce apoptosis" Int. J. Clin. Lab. Res. 28:
141-147. When the adaptor is recruited to the death domain of the
TRAIL receptors DR4 or DRS, FLICE zymogen is brought together in
close proximity by the FADD-like adaptor and is activated by by FLICE
auto-cleavage. The FLICE activating complex that consists of TRAIL
receptor-adaptor-FLICE is named as DISC (death inducing signaling
complex). Kischkel et al. (1995) "Cytotoxicity-associated dependent
APO-1 (Fas/CD95)-associated protein form a death-inducing signaling
complex (DISC) with the receptor" EMBO J. 14:5579-5588. The FLICE
enzyme subsequently activates caspase-3 and other caspases by
cleaving their zymogen forms. Martinet-Lorenzo et al. (1998)
"Involvement of Apo-2 ligand/TRAIL in activation-induced death of
Jurkat and human peripheral blood T cells" Euro. J. Immunol. 28:2714-
2725. Active caspase-3 can then cleave ICAD (inhibitor of caspase-
activated deoxy-ribonuclease), resulting in the release of active
nuclease that cleaves DNA into 180-220 by fragments, a typical
hallmark of apoptosis.
TRAIL expression has been detected in a wide variety of human
tissues, with highest levels found in spleen, lung and prostate. Wiley et
al. (1995) "Identification and characterization of a new member of the
TNF family that induces apoptosis" Immunity 3:673-82. In the present
invention, it is demonstrated that compared to normal cells cancer cells
have selective sensitivities to TRAIL-induced apoptosis. For example,
while human cancer cells line, such as LNCAP (prostate), HeLa
(cervical), A549 (lung), and C3A (liver), are sensitive to TRAIL-mediated
apoptosis, primary human fibroblasts from foreskin samples are
essentially unaffected when similar levels of TRAIL are expressed in the
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cells. Thus, compared to Fast, TRAIL induces apoptosis in a more
tumor-specific manner, which, in turn, can have a less systemic toxicity
when expressed in vivo.
There may be many possible reasons why tumor cells are
particularly sensitive to TRAIL-mediated apoptosis. One possibility is
that healthy normal cells may express intracellular regulators such as
FLICE-inhibitory proteins (FLIPs) that blocks the biochemical signaling
pathways that lead to cell death. Griffith et al. (1998) "Intracellular
regulation of TRAIL-induced apoptosis in human melanoma cells" J.
Immunol. 161:2833-2840. It may also be possible that the lack of
cytotoxic effects of TRAIL on normal cells may be due to expression of
decoy receptors such as DcR1 and DcR2 which inhibit TRAIL-mediated
apoptosis by competing with DR4 or DR5 for binding to TRAIL.
By using the method of present invention, TRAIL can be
introduced into cancer cells by a conditional expresssion vector such as
an adenoviral vector and induces apoptosis of cancer cells selectively.
Since TRAIL exerts less toxicity to normal cells and its expression can
be controlled site-specifically and dose-dependently, systemic toxicity of
this ligand should be reduced.
4. Expression vectors for apoptosis-signaling ligands
The expression vector that can be used to practice the methods
of the present invention may be any gene-transferring vector. The
expression vector may be a plasmid encoding the apoptosis-signaling
ligand (e.g. TRAIL). The plasmid can be transfected into cancer cells
via liposome-mediated delivery or other methods of transfection.
Preferably, the expression vector is a viral vector. The viral
vector may be an ~adenovirus, adeno-associated virus, vaccinia,
retrovirus, or herpes simplex virus vector.
The present invention provides an adenoviral vector that is
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preferably used to induce death of cancer cells in a site-specific and
controlled manner. The expression of the apoptosis-signaling ligand
may be controlled by using a tissue-specific promoter or an inducible
promoter. Alternatively, the expression of the apoptosis-signaling ligand
may be constitutive in the transduced cells. The adeniviral
expression vector can be used for delivering the apoptosis-signaling
ligand to a wide range of cell types both in vitro and in vivo. Further, the
expression of apoptosis-signaling can be tightly regulated, which not
only facilitates production of adenoviral expression vectors encoding the
apoptosis-signaling ligand, but also provides a means for controlling
expression of the ligand in vivo to minimize systemic toxicity. In
addition, the present invention also provides means for easily and
reliably quantitating the levels and cellular localization of exogenous
apoptosis-signaling ligands.
In a preferred embodiment, the adenoviral vector comprises: a
conditional promoter, and a polynucleotide sequence encoding a
membrane-bound ligand whose expression is regulated by the
conditional promoter in the vector, the ligand signaling apoptosis in cells
that express an apoptosis-mediating receptor. The adenoviral vector
may be replication competent or replication incompetent, depending on
the dosage of the apoptosis-signaling ligand to be administered into the
tumor site.
The membrane-bound ligand can be any protein that is capable
of binding to an apoptosis-mediating receptor on the surface of cancer
cells. Preferably, the membrane-bound protein is Fast or TRAIL.
Optionally, the membrane-bound protein may be TNF although TNF
may have higher systemic toxicity than Fas and TRAIL.
Alternatively, the adenoviral vector may encode another type of
apoptosis-signaling ligand such that when that the ligand is introduced
into a cell, the transduced cell expresses the ligand intracellularly.
Interactions of the ligand with an apoptosis-mediating receptor causes
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the cell to undergo apoptosis. Examples of such intracellular apoptosis
signaling molecules include, but are not limited to, Bax, Bad, Bak, and
Bik. Adams et al. "Control of cell death" WEHI Annual Report
1996/1997.
In another embodiment of the present invention, the expression
vector encoding the apoptosis-signaling ligand can also encode another
protein such as a regulatory protein, which may be used to regulate the
expression of the ligand. For example, the regulatory protein can cause
the tissue-specific localization of the Fas ligand on the cell membrane,
or alternatively cause the premature turn-over of the Fas ligand in non-
target cells, or regulate the expression of the Fast via regulation of
transcription and/or translation.
The regulatory protein can also be encoded by another
expression vector that is delivered to the cell, either concurrently or
consecutively with the nucleic acid encoding the protein to be
expressed. In this embodiment, the two expression vectors can have
different sequences, such as different promoters, such that they can be
independently regulated, such as by the administration of a drug that
selectively regulates the expression of one or both of the promoters,
such as by the use of a steroid hormone, e.g. a glucocorticoid hormone
that can regulate a promoter that is inducible by that hormone. Other
steroid hormones fihat may be used include, but are not limited to,
estrogen, androgen, and progestrone.
The apoptosis-signaling ligand may also be expressed as a
fusion protein with another protein. This protein fused with the ligand
may be used for such purposes as localization of the protein, activation
or deactivation of the ligand, monitoring the location of the ligand,
isolation of the ligand, and quantitating the amount of the ligand.
In one embodiment, the fusion protein comprises a Fas ligand
and reporter protein such as a fluorescent protein (FP). Examples of
reporter proteins include, but are not limited, the GFP (green fluorescent
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protein) gene, the YFP (yellow fluorescent protein) gene, BFP (blue
fluorescent protein) gene, the CAT gene, the neo gene, the hygromycin
gene, and so forth. An example of a Fast-GFP fusion protein-
expressing construct is shown in Figure 1 and is further described
herein.
Alternatively, the reporter gene may be expressed as a single
protein bicistronically with the apoptosis-signaling ligand via a
mechanism of internal ribosome entry site (IRES) or splicing
donor/acceptor sites.
The expression vector may further encode a sequence that is
capable of regulating the expression of the apoptosis-signaling ligand.
For example, the vector can contain a glucocorticoid regulatory element
(GRE) such that glucocorticoid hormones can be used to regulate the
expression of the Fas ligand.
Another example of a regulatory sequence that can regulate the
expression of an adjacent gene is by cloning an RNA aptamer, such as
H10 and H19, into the promoter region whereby administration of a drug
such as Hoechst dye 33258 can block expression of the gene in vivo.
Werstuck et al. (1998) "Controlling gene expression in living cells
through small molecule-RNA interactions" Science 282:296-298.
In other embodiments of the present invention, the regulatory
sequence comprises the Tet-operon or the lac operon, or any other
operon that can function as a regulatory sequence in a eukaryotic cell.
In a preferred embodiment, expression of apoptosis-signaling
ligand is under the control of tetracycline-regulated gene expression
system, wherein expression of the ligand is controlled by a tet-
responsive element, wherein the ligand expression requires the
interaction of the tet-responsive element and a tet transactivator.
In a more preferred embodiment, tight control of the ligand
expression is achieved using an Ad vector in which the tet-responsive
element arid the transactivator element are built into the opposite ends
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of the same vector to avoid enhancer interference. Expression can be
conveniently regulated by tetracycline or any derivative thereof, which
includes, but is not limited to, doxycycline, in a dose-dependent manner.
For example, the vector efficiently delivers Fast-GFP gene to cells in
vitro, and the expression level of the fusion protein may be modulated
by the concentration of doxycycline in culture media. An example of
such a regulatory system is particularly described herein.
In one embodiment of the present invention, the promoter is a
tissue-specific promoter which one skilled in the art will appreciate can
confer tissue-specificity to the expression of the nucleic acid encoding
the apoptosis-signaling ligand such as Fast and TRAIL.
For example, the tissue-specific promoter may be a prostate-
specific, a breast tissue-specific, a colon tissue-specific, a pancreas-
specific a brain-specific, a kidney-specific, a liver-specific, a bladder-
specific, a bone-specific, a lung-specific, a thyroid-specific a stomach-
specific, an ovary-specific, or a cervix-specific promoter.
Where the tissue-specific promoter is a prostate-specific
promoter, the promoter includes, but is not limited to the PSA promoter,
the ~PSA promoter, the ARR2PB promoter, the PB promoter, gp91-
phox gene promoter, and prostate-specific kallikrein (hKLK2) promoter.
Where the tissue-specific promoter is a breast-specific promoter,
the promoter includes, but is not limited to MMTV promoter, G.I. erb-B2
promoter, erb-B3 promoter, (i-casein, ~3-lacto-globulin, and WAB (whey
acidic protein) Where the tissue-specific promoter is a liver-specific
promoter, the promoter includes, but is not limited to liver albumin
promoter, alpha-fetoprotein promoter, a~-antitrypsin promoter, and
transferrin transthyretin promoter.
Where the tissue-specific promoter is a brain-specific promoter,
the promoter includes, but is not limited to, JC virus early promoter,
tyrosine hydoxylase promoter, dopamine hydroxylase promoter, neuron
specific enolase promoter, glial fibrillary acidic protein promoter, mature
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astrocyte specific protein promoter, and myelin promoter.
Where the tissue-specific promoter is a colon-specific promoter,
the promoter includes, but is not limited to, the MUC1 promoter,
carbonic anhydrase I promoter and carcinoembrogen's antigen
promoter.
Where the tissue-specific promoter is ovary- or placenta-specific
promoter, the promoter includes, but is not limited to, estrogen-
responsive promoter, aromatase cytochrome P450 promoter,
cholesterol side chain cleavage P450 promoter, and 17 alpha-
hydroxylase P450 promoter.
Where the tissue-specific promoter is a lung-specific promoter,
the promoter includes, but is not limited to, surfactant protein C
Uroglobin (cc-10, Cllacell 10 kd protein) promoter.
Where the tissue-specific promoter is a skin-specific promoter,
the promoter includes, but is not limited to, K-14-keratin promoter,
human keratin 1 or 6 promoter, and loicrin promoter.
Where the tissue-specific promoter is a pancreas-specific
promoter, the promoter includes, but is not limited to, villin promoter,
glucagon promoter, and Insulin Islet amyloid polypeptide (amylin)
promoter.
Where the tissue-specific promoter is a thyroid-specific promoter,
the promoter includes, but is not limited to, thyroglobulin promoter, and
calcitonin promoter.
Where the tissue-specific promoter is a bone-specific promoter,
the promoter includes, but is not limited to, Alpha 1 (I) collagen
promoter, osteocalcin promoter, and bone sialoglycoprotein promoter.
Where the tissue-specific promoter is a kidney-specific promoter,
the promoter includes, but is not limited to, renin promoter,
liver/bone/kidney alkaline phosphatase promoter, and erythropoietin
(epo) promoter.
It should be noted that other tissue specific promoters will be
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revealed by the human genome project and other endeavors of human
gene discovery. These promoters will be useable as appropriate means
to direct tissue specific expression from the expression vectors of the
present invention.
Furthermore, one of ordinary skill will readily know how to identify
a promoter specific to a particular cell type. For example, by comparing
the differential expression of genes in different tissue types, e.g., using
gene chip technology, one can identify genes expressed only in one
particular tissue type. These genes can then be isolated and
sequenced, and their promoters may be isolated and tested in an animal
model for the ability to drive tissue specific expression of a heterologous
gene. Such methods are well within the ability of the one of ordinary
skill in the art. An example of a method by which a tissue specific
promoter may be identified may be found in Greenberg et al. (1994)
Molecular Endocrinology 8: 230-239.
The tissue-specificity may also be achieved by selecting an
expression vector that has a high degree of tissue specificity. For
example, a vector that selectively infects mucosal cells, such as those
associated with colon cancer, can be chosen, and then optionally, used
in combination with a specific delivery means, such as by the use of a
suppository, to selectively deliver the nucleic acid encoding the
apoptosis-signaling ligand such as Fas and TRAIL to those desired
cells.
One skilled in the art will recognize that various vectors have
more or less applicability depending on the particular host. One
example of a particular technique for introducing nucleic acids into a
particular host is the use of retroviral vector systems which can package
a recombinant retroviral genome. See e.g., Pastan et al. (1988) "A
retrovirus carrying an MDR1 cDNA confers multidrug resistance and
polarized expression of P-glycoprotein in MDCK cells." Proc. Nat. Acad.
Sci. 85:4486; and Miller et al. (1986) "Redesign of retrovirus packaging
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cell lines to avoid recombination leading to helper virus production." Mol.
Cell Biol. 6:2895. The produced recombinant retrovirus can then be
used to infect and thereby deliver to the infected cells a nucleic acid
sequence encoding the apoptosis-signaling ligand. The exact method
of introducing the nucleic acid into mammalian cells is, of course, not
limited to the use of retroviral vectors. Other techniques are widely
available for this procedure including the use of adenoviral vectors
(Mitani et al. "Transduction of human bone marrow by adenoviral
vector." Human Gene Therapy 5:941-948 (1994)), adenoassociated viral
vectors (Goodman et al. "Recombinant adenoassociated virus-mediated
gene transfer into hematopoietic progenitor cells." Blood 84:1492-1500
(1994)), lentiviral vectors (Naidini et al. "In vivo gene delivery and stable
transduction of nondividing cells by a lentiviral vector." Science 272:263-
267 (1996)), pseudotyped retroviral vectors (Agrawal et al. "Cell-cycle
kinetics and VSV-G pseudotyped retrovirus mediated gene transfer in
blood-derived CD34+ cells." Exp. Hematol. 24:738-747 (1996)), vaccinia
vectors, and physical transfection techniques (Schwarzenberger et al.
"Targeted gene transfer to human hematopoietic progenitor cell lines
through the c-kit receptor." Blood 87:472-478 (1996)). This invention
can be used in conjunction with any of these or other commonly used
gene transfer methods. In a preferred embodiment of the present
invention, the specific vector for delivering the nucleic acid encoding a
Fas ligand comprises an adenovirus vector.
5. Expression vectors encoding a trans-regulatory protein
Because it is desirable to be able to regulate expression of the
apoptosis-signaling ligand, the present invention also provides an
expression vector for the regulatable expression for tightly controlling
expression of a target protein (e.g. FasL and TRAIL).
The expression vector comprises: a transcription regulatory
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sequence; a polynucleotide sequence encoding a trans-acting regulator
protein which is capable of binding to the transcription regulatory
sequence; and a polynucleotide sequence encoding a target protein
whose expression is regulated by the binding of the traps-acting
regulator protein to the transcription regulatory sequence.
The transcription regulatory sequence and the polynucleotide
sequence encoding the traps-acting regulator protein may be positioned
at opposite ends of the adenoviral vector. For example, the transcription
regulatory sequence is positioned in the E4 region of the adenoviral
vector and the polynucleotide sequence encoding the traps-acting
protein is positioned in the E1 of the adenoviral vector.
In this vector, the nucleic acid encoding the target protein is
operatively linked to a transcription regulatory sequence. The
expression of the target protein may be inducible, e.g. expression of
Fast or a Fast fusion will not proceed unless the appropriate activator
for the particular transcription regulatory sequence is present.
Alternatively, the expression of the target protein may be
repressible, i.e., expression of Fast or a Fast fusion will proceed unless
the appropriate repressor for the particular transcription regulatory
sequence is present.
The traps-acting regulator protein interacts with the transcription
regulatory sequence to affect transcription of the target protein. Where
the transcription regulatory sequence is inducible, the traps-acting
regulator protein is a traps-activator. Where the transcription regulatory
sequence is repressible, the traps-acting factor is a traps-repressor.
In a more preferred embodiment, the transcription regulatory
sequence is a tet responsive element (TRE), and the traps-acting factor
is a tet-responsive transacting expression element (tTA).
In the most preferred embodiment, the invention utilizes the
vector Ad/FasL-GFPT~T. This is a replication-deficient adenoviral vector
that expresses a fusion of murine Fast and green fluorescent protein
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(GFP). Fast-GFP retains full activity of wild-type Fast, at the same time
allowing for easy visualization and quantification in both living and fixed
cells. The fusion protein is under the control of tetracycline-regulated
gene expression system. A tight control is achieved by creating this
novel "double recombinant" Adenoviral vector, in which the tet-
responsive element and the transactivator element are built into the
opposite ends of the same vector to avoid enhancer interference.
Expression of the Fast-GFP fusion can be conveniently
regulated by tetracycline or any derivative thereof, which includes, but is
not limited to, doxycycline, in a dose-dependent manner. The vector
efficiently delivers Fast-GFP gene to cells in vivo and in vitro, and the
expression level of the fusion protein may be modulated by the
concentration of doxycycline added to the culture media or administered
to the subject. As may be seen in the following examples, Ad/FasL-
GFPTe-r, is able to deliver Fast-GFP to transformed and primary cell
lines, with the expression of the fusion protein in those cells regulated by
varying the level of doxycycline in the media. Amounts of Fast-GFP
can be easily detected and quantified through the fluorescence of its
GFP component, and correlated with the levels of apoptosis in the target
and neighboring cells.
This vector design, which delivers an entire tet-regulated gene
expression system, is more efficient and economical than strategies
using multiple vectors, and can be applied to any situation where
regulation of protein expression is desired.
Accordingly, the present invention provides an expression vector
for tightly controlling expression of a target protein in response to
tetracycline or a tetracycline derivative. The expression vector
comprises: a tetracycline-responsive element; a polynucleotide
sequence encoding a transactivator protein which is capable of binding
to the tetracycline-responsive element; and a polynucleotide sequence
encoding a target protein whose expression is regulated by the binding
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of the transactivator protein to the tetracycline-responsive element.
In a preferred embodiment, the vector is a viral vector. In a more
preferred embodiment, the viral vector is an adenoviral vector. In this
adenoviral vector, the tetracycline-responsive element and the
polynucleotide sequence encoding the transactivator protein are
positioned at opposite ends of the adenoviral vector. For example, the
tetracycline-responsive element is positioned in the E4 region of the
adenoviral vector and the polynucleotide sequence encoding the
transactivator protein is positioned in the E1 of the adenoviral vector.
Optionally, the adenoviral vector does not include the E3 region of
adenovirus. Also optionally, the adenoviral vector does not include the
E4 region of adenovirus except for the Orf6 of the E4 region.
The expression of the target protein may be repressed in the
presence of tetracycline or doxycycline. Alternatively, expression of the
target protein may be activated in the presence of doxycycline.
It should be noted that the vector may also be any other type of
viral vector, including but not limited to an adeno-associated viral vector,
a vaccinia viral vector or a retroviral vector.
The expression vector may further comprise a polynucleotide
sequence encoding a reporter protein. The reporter protein and the
target protein may be encoded as a fusion protein or expressed as a
single protein bicistronically with the target protein via a mechanism of
internal ribosome entry site (IRES) or splicing donor/acceptor sites. .
The reporter gene preferably encodes a fluorescent protein such
as green, yellow and blue fluorescent proteins, and more preferably
green fluorescent protein (GFP).
For example, an adenoviral vector can be constructed for
expression of a fusion protein, Fast-GFP, by ligating pLAd-C.tTA and
pRAd-TGFsL to a portion of the Ad5 genome (snb 360) to produce the
vector Ad/FasL-GFPTET as described below and as shown in Figures 1A-
C.
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Expression a target protein other than the Fast-GFP fusion can
be regulated by using a similar adenoviral vector (designated pAdTeT)
with the Fast-GFP fusion sequence replaced by the polynucleotide
encoding the target sequence. The vector pAdTET can be constructed by
removing the Fast-GFP fusion sequence from vector pRAd-TGFsL,
inserting the target sequence into this site, and ligating the resulting
vector to pLAd-C.tTA, in the same way as described for the production
of the vector Ad/FasL-GFPTErin Figure 1A-C. The vector pAdTETcan be
utilized to express an unlimited variety of heterologous proteins for
which tight regulation is desired.
The expression vector may further comprise a selectable marker
which can be used to screen for those cells which contain the vector and
which express the selectable marker. In this manner, one can readily
separate those cells containing the nucleic acid or the vector and
expressing the selectable marker from those cells either containing the
nucleic acid or the vector but not expressing the selectable marker, and
from those cells not containing the nucleic acid or the vector. The
specific selectable marker used can of course be any selectable marker
which can be used to select against eukaryotic cells not containing and
expressing the selectable marker. The selection can be based on the
death of cells not containing and expressing the selectable marker, such
as where the selectable marker is a gene encoding a drug resistance
protein. An example of such a drug resistance gene for eukaryotic cells
is a neomycin resistance gene. Cells expressing a neomycin resistance
gene are able to survive in the presence of the antibiotic 6418, or
Geneticin7, whereas those eukaryotic cells not containing or not
expressing a neomycin resistance gene are selected against in the
presence of 6418. One skilled in the art will appreciate that there are
other examples of selectable markers, such as the hph gene which can
be selected for with the antibiotic Hygromycin B, or the E, coli Ecogpt
gene which can be selected for with the antibiotic Mycophenolic acid.
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The specific selectable marker used is therefore variable.
The selectable marker can also be a marker that can be used to
isolate those cells containing and expressing the selectable marker
gene from those not containing and/or not expressing the selectable
marker gene by a means other than the ability to grow in the presence
of an antibiotic. For example, the selectable marker can encode a
protein which, when expressed, allows those cells expressing the
selectable marker encoding the marker to be identified. For example,
the selectable marker can encode a luminescent protein, such as a
luciferase protein or a green fluorescent protein, and the cells
expressing the selectable marker encoding the luminescent protein can
be identified from those cells not containing or not expressing the
selectable marker encoding a luminescent protein. Alternatively, the
selectable marker can be a sequence encoding a protein such as
chloramphenicol acetyl transferase (CAT). By methods well known in
the art, those cells producing CAT can readily be identified and
distinguished from those cells not producing CAT.
6. Construction of the expression vectors of the present
invention
The expression vectors of the present invention can be
constructed by using recombinant DNA technologies. For example, the
regulatable adenoviral vector described above may be derived from
adenvirus type 5 and modified to include heterologous sequences
encoding the apoptosis-signaling ligand (e.g. Fas and TRAIL) and the
transcription regulatory sequence.
One skilled in the art will appreciate that there are numerous
techniques available by which one can obtain a nucleic acid sequence
encoding an apoptosis-signaling ligand, and optionally, additional
sequences such as one or more transcrition regulatory sequence. One
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method of obtaining the nucleic acid is by constructing the nucleic acid
by synthesizing a recombinant DNA molecule. For example,
oligonucleotide synthesis procedures are routine in the art and
oligonucleotides coding for a particular protein or regulatory region are
readily obtainable through automated DNA synthesis. A nucleic acid for
one strand of a double-stranded molecule can be synthesized and
hybridized to its complementary strand. One can design these
oligonucleotides such that the resulting double-stranded molecule has
either internal restriction sites or appropriate 5' or 3' overhangs at the
termini for cloning into an appropriate vector. Double-stranded
molecules coding for relatively large proteins or regulatory regions can
be synthesized by first constructing several different double-stranded
molecules that code for particular regions of the protein or regulatory
region, followed by ligating these DNA molecules together. For
example, Cunningham, et al. (1989) "Receptor and Antibody Epitopes in
Human Growth Hormone Identified by Homolog-Scanning Mutagenesis"
Science, Vol. 243, pp. 1330-1336, have constructed a synthetic gene
encoding the human growth hormone gene by first constructing
overlapping and complementary synthetic oligonucleotides and ligating
these fragments together. See also, Ferretti et al. (1986) Proc. Nat.
Acad. Sci. 82:599-603, wherein synthesis of a 1057 base pair synthetic
bovine rhodopsin gene from synthetic oligonucleotides is disclosed.
Once the appropriate DNA molecule is synthesized, this DNA can be
cloned downstream of an appropriate promoter. Techniques such as
this are routine in the art and are well documented.
An example of another method of obtaining a nucleic acid
encoding an apoptosis-signaling ligand is to isolate the corresponding
wild-type nucleic acid from the organism in which it is found and clone it
in an appropriate vector. For example, a DNA or cDNA library can be
constructed and screened for the presence of the nucleic acid of
interest. Methods of constructing and screening such libraries are well
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known in the art and kits for performing the construction and screening
steps are commercially available (for example, Stratagene Cloning
Systems, La Jolla, CA). Once isolated, the nucleic acid can be directly
cloned into an appropriate vector, or if necessary, be modified to
facilitate the subsequent cloning steps. Such modification steps are
routine, an example of which is the addition of oligonucleotide linkers
which contain restriction sites to the termini of the nucleic acid. General
methods are set forth in Sambrook et al., "Molecular Cloning, a
Laboratory Manual" Cold Spring Harbor Laboratory Press (1989). Once
isolated, one can alter selected codons using standard laboratory
techniques, PCR for example.
Yet another example of a method of obtaining a nucleic acid
encoding an apoptosis-signaling ligand is to amplify the corresponding
wild-type nucleic acid from the nucleic acids found within a host
organism containing the wild-type nucleic acid and clone the amplified
nucleic acid in an appropriate vector. One skilled in the art will
appreciate that the amplification step may be combined with a mutation
step, using primers not completely homologous to the target nucleic acid
for example, to simultaneously amplify the nucleic acid and alter specific
positions of the nucleic acid.
By using these recombinant DNA techniques, a replication-
incompetent adenoviral vector encoding an apoptosis-signaling ligand
can be constructed. For example, a complex adenoviral vector
encoding TRAIL can be constructed and used to infect tumor cells. The
vector that further comprises GFP which is expressed bicistronically with
TRAIL is designated Ad.TRAIL/GFPTEr.
The vector, Ad.TRAIL/GFPTET is a complex adenoviral vector that
expresses multiple genes and regulatory mechanisms. Construction of
the adenoviral vectors is diagramed in Figure 3. The sequence
encoding TRAIL and GFP separated by an IRES is cloned into the right-
end (E4 region) of the type 5 adenovirus genome using a shuttle vector,
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resulting in a shuttle vector pRAdTRE-TRAIL/GFP. The pRAdTRE-
TRAIL/GFP shuttle vector contains the right end of the adenoviral
genome including the right long terminal repeats R-TR.
Another shuttle vector, pLAd-C.tTA, contains a tetracycline
transactivator gene tTA in the E1 region of the type 5 adenovirus
genome. The vector pLAd-C.tTA also contains the left end of the
adenoviral genome including the left long terminal repeats L-TR and the
adenoviral packaging signal yr. The vectors pRAdTRE-TRAIL/GFP and
pLAd-C.tTA are both linearized and ligated to the backbone of the
adenovirus to form the recombinant adenoviral vector,
Ad.TRAIL/GFPTer.
7. Routes of.administration and formulations
The expression vector encoding the apoptosis-signaling ligand
may be introduced into a tumor by using any pharmaceutically
acceptable routes of administration. For example, the expression vector
may be administered into a group of tumor cells parenterally,
intraperitoneally, intravenously, intraartierally, transdermally,
sublingually, intramuscularly, rectally, transbuccally, intranasally,
liposomally, via inhalation, vaginally, intraoccularly, via local delivery by
catheter or stent, subcutaneously, intraadiposally, intraarticularly,
intrathecally, or in a slow release dosage form.
One skilled in the art will recognize that this aspect of the
methods can comprise either a stable or a transient introduction of the
sequence encoding the apoptosis-signaling ligand (e.g. FasL and
TRAIL) into the cell. Additionally, the stably or the transiently introduced
ligand-encoding sequence may or may not become integrated into the
genome of the host.
One skilled in the art will also recognize that the precise
procedure for introducing the expression vector into the cell may, of
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course, vary and may depend on the specific type or identity of the cell.
Examples of methods for introducing an expression vector into a cell
include, but are not limited to electroporation, cell fusion, DEAE-dextran
mediated transfection, calcium phosphate-mediated transfection,
infection with a viral vector, microinjection, lipofectin-mediated
transfection, liposome delivery, and particle bombardment techniques,
including various procedures for "naked DNA" delivery.
Optionally, the method can be performed ex vivo where the group of
cells into which the expression vector is introduced are contained in a
sample taken from a patient having cancer, or contained in contained in
a cell culture.
For example, the expression vector may be introduced into a
mixture of cells which express Fas and cells which do not express Fas.
Optionally, the expression vector may be introduced into cells which do
not express Fas. Also optionally, the expression vector may be
introduced into cells which do express Fas. Also optionally, the
expression vector may be introduced into cells which cells which do not
express Fas. By a "bystander effect", those cancer cells expressing Fas
near those cells transduced by the expression vector are killed via Fas-
Fast interactions.
The various vectors and hosts used to express the apoptosis-
signaling ligand may be used to express the ligand in cell culture or in
vitro. For example, an expression vector encoding a Fas ligand may be
introduced into a tissue culture cell line, such as COS cells, and
expressed in the cell culture. In this manner, one skilled in the art can
select a cell type that may have a limited life in the host organism such
that the host can effectively clear the cell expressing the the apoptosis-
signaling ligand in a period of time such that any possible apoptotic
effects on non-target surrounding cells or tissues can be minimized.
Alternatively, cells from a subject may be removed from the
subject, administered the expression vector encoding the apoptosis-
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signaling ligand, and then replaced into the subject. In this ex vivo
treatment procedure, the cells can be manipulated to facilitate the
uptake of the nucleic acid encoding the apoptosis-signaling ligand
without unnecessary adverse effects on the subject.
The various vectors and hosts used to express the apoptosis-
signaling ligand may be used to express the nucleic acids in vivo. For
example, an expression vector encoding Fast may be introduced into
cells of a eukaryotic host, preferably tumor cells, to treat Fas+ tumor
cells in situ.
As briefly discussed above, one skilled in the art will appreciate
that specific tissues can be treated by selectively administering the
vector to the host. For example, administering an adenovirus vector via
an aerosol such as through the use of an inhaler can selectively
administer the vector to the lungs. Optionally, the use of a
suppository can be used to selectively administer the vector to cells of
the colon.
Also optionally, delivering the vector topically such as in a cream
can selectively deliver the vector or nucleic acid to skin cells or the
cervix.
One skilled in the art will recognize the various methods that can
routinely be used to selectively deliver the expression vector to specific
organs or cells. For example, delivery of the expression vector can be
manually facilitated through such methods as injection of the vector into
the selected site. For example, direct injection can be used to deliver
the vector to specific brain and/or breast location. In one embodiment of
the present invention, direct injection of the vector encoding a Fas ligand
or TRAIL is used for delivery into breast tumor masses.
It is contemplated that using the methods and vectors of the
present invention, apoptosis-signaling ligand can be administered to a
cell or to a subject, most preferably, humans, to treat disease states,
preferably cancer. The present vector, whether alone, in combination
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with another compound or composition (e.g., a chemotherapy agent), or
as part of a vector-based delivery system, may be administered
parenterally (e.g., intravenously), by intramuscular injection, by
intraperitoneal injection, topically, transdermally, or the like, although
topical administration is typically preferred.
The exact amount of such nucleic acids, compositions, vectors,
etc., required may vary from subject to subject, depending on the
species, age, weight and general condition of the subject, the severity of
the disease or condition that is being treated, the particular compound or
composition used, its mode of administration, and the like. Thus, it is
not possible or necessary to specify an exact amount. However, an
appropriate amount may be determined by one of ordinary skill in the art
using methods well known in the art (see, e.g., Martin et al., 1989).
For topical administration, the composition of the present
invention may be in pharmaceutical compositions in the form of solid,
semi-solid or liquid dosage forms, such as, for example powders, liquids,
suspension, lotions, creams, gels or the like, preferably in unit dosage
form suitable for single administration of a precise dosage. The
compositions can typically include an effective amount of the selected
nucleic acid, composition, or vector in combination with a
pharmaceutically acceptable carrier and, in addition, may include other
medicinal agents, pharmaceutical agents, carriers, adjuvants, diluents,
etc.
By "pharmaceutically acceptable" is meant a material that is not
biologically or otherwise undesirable, i.e., the material may be
administered to an individual along with the selected nucleic acid,
composition thereof, or vector without causing any undesirable
biological effects or interacting in a deleterious manner with any of the
other components of the pharmaceutical composition in which it is
contained.
Alternatively or additionally, parenteral administration, if used, is
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generally characterized by injection e.g., by intravenous injection
including regional perfusion through a blood vessel supplying the
tissues(s) or organs) having the target cell(s). Injectables can be,
prepared in conventional forms, either as liquid solutions or
suspensions, solid forms suitable for solution or suspension in liquid
prior to injection, or as emulsions. Parenteral administration can also
employ the use of a slow release or sustained release system, such that
a constant level of dosage is maintained (See, for example, U.S. Patent
No. 3,710,795). The compound can be injected directly to the site of
cells or tissues expressing a Fas+ phenotype, or they can be injected
such that they diffuse or circulate to the site of the Fas+ phenotypic cells.
Dosages will depend upon the mode of administration, the
disease or condition to be treated, and the individual subject's condition.
Dosages will also depend upon the material being administered, e.g., a
nucleic acid, a vector comprising a nucleic acid, or another type of
compound or composition. Such dosages are known in the art.
Furthermore, the dosage can be adjusted according to the typical
dosage for the specific disease or condition to be treated.
Furthermore, culture cells of the target cell type can be used to
optimize the dosage for the target cells in vivo, and transformation from
varying dosages achieved in culture cells of the same type as the target
cell type can be monitored. Often a single dose can be sufficient;
however, the dose can be repeated if desirable. The dosage should not
be so large as to cause adverse side effects. Generally, the dosage will
vary with the age, condition, sex and extent of the disease in the patient
and can be determined by one of skill in the art. The dosage can also
be adjusted by the individual physician in the event of any complication.
Examples of effective doses in non-human animals are provided in the
Examples. Based on art accepted formulas, effective doses in humans
can be routinely calculated from the doses provided and shown to be
effective.
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For administration to a cell in a subject, the compound or
composition, once in the subject, will of course adjust to the subjects
body temperature. For ex vivo administration, the compound or
composition can be administered by any standard methods that would
maintain viability of the cells, such as by adding it to culture medium
(appropriate for the target cells) and adding this medium directly to the
cells. As is known in the art, any medium used in this method can be
aqueous and non-toxic so as not to render the cells non-viable. In
addition, it can contain standard nutrients for maintaining viability of
cells, if desired.
For in vivo administration, the complex can be added to, for
example, a blood sample or a tissue sample from the patient, or to a
pharmaceutically acceptable carrier, e.g., saline and buffered saline,
and administered by any of several means known in the art.
Other examples of administration include inhalation of an aerosol,
subcutaneous or intramuscular injection, direct transfection of a nucleic
acid sequence encoding the compound where the compound is a
nucleic acid or a protein into, e.g., bone marrow cells prepared for
transplantation and subsequent transplantation into the subject, and
direct transfection into an organ that is subsequently transplanted into
the subject.
Further administration methods include oral administration,
particularly when the composition is encapsulated, or rectal
administration, particularly when the composition is in suppository form.
A pharmaceutically acceptable carrier includes any material that is not
biologically or otherwise undesirable, i.e., the material may be
administered to an individual along with the selected complex without
causing any undesirable biological effects or interacting in a deleterious
manner with any of the other components of the pharmaceutical
composition in which it is contained.
Specifically, if a particular cell type in vivo is to be targeted, for
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example, by regional perfusion of an organ or tumor, cells from the
target tissue can be biopsied and optimal dosages for import of the
complex into that tissue can be determined in vitro, as described herein
and as known in the art, to optimize the in vivo dosage, including
concentration and time length.
Alternatively, culture cells of the same cell type can also be used
to optimize the dosage for the target cells in vivo. For example,
intratumoral injection amounts and rates can be controlled using a
controllable pump, such as a computer controlled pump or a micro-
,thermal pump, to control the rate and distribution of the nucleic acid or
vector in the tumor or tissue. Example 4 demonstrates effective
dosages of Ad/FasL-GFPrer used for in vivo treatment of both breast
and brain tumors in mice. One of ordinary skill will readily know how to
extrapolate these figures to determine effective human dosages.
For either ex vivo or in vivo use, the nucleic acid, vector, or
composition can be administered at any effective concentration. An
effective concentration is that amount that results in killing, reduction,
inhibition, or prevention of a transformed phenotype of the cells.
The expression vector of the present invention may be
administered in a composition. For example, the composition may
further comprise other medicinal agents, pharmaceutical agents,
carriers, adjuvants, diluents, etc. Furthermore, the composition can
comprise, in addition to the nucleic acid or vector, lipids such as
liposomes, such as cationic liposomes (e.g., DOTMA, DOPE,
DC-cholesterol) or anionic liposomes. Liposomes may further comprise
proteins to facilitate targeting a particular cell, if desired. Administration
of a composition comprising a nucleic acid or a vector and a cationic
liposome can be administered to the blood afferent to a target organ or
inhaled into the respiratory tract to target cells of the respiratory tract.
Regarding liposomes, see, e.g., Brigham et al. Am. J. Resp. Cell. Mol.
Biol. 1:95-100 (199); Felgner et al. Proc. Natl. Acad. Sci USA
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84:7413-7417 (1987); U.S. Pat. No.4,897,355. Furthermore, the
nucleic acid or a vector can be administered as a component of a
microcapsule that can be targeted to specific cell types, such as
macrophages, or where the diffusion of the compound or delivery of the
compound from the microcapsule is designed for a specific rate or
dosage.
Any cell, specifically a tumor cell, which expresses an apoptosis-
mediating receptor can be treated by the methods of the present
invention. For example, Fas is primarily a surface protein and a cell
expressing Fast can be used to treat the Fas-expressing cell by the
Fas-Fast induction of apoptosis. The cell expressing the Fast can
interact with the Fas-expressing cell via interactions of the Fas and the
Fast on the surface of the cells, and therefore treat Fas-expressing cells
that the Fast-expressing cells can make contact with. Additionally, the
Fast-producing cells may also regulate the Fas-expressing cell by
producing soluble Fast which then interacts with Fas and also induces
apoptosis of the Fas-expressing cells.
The interaction of the Fas and the Fast is typically a ligand-
receptor binding, although the interaction may not have to be binding
per se, but includes any cellular reaction which results from any
interaction of the Fas and the Fast. Therefore any cellular apoptosis via
Fas that results from the expression of a Fast by that same cell or a
second cell which expresses a Fas ligand is hereby contemplated.
Although any cell expressing Fas can be induced to undergo
apoptosis using the methods of the present invention, a preferred
embodiment is inducing Fas+ tumor cells to undergo apoptosis using
these methods. In this embodiment, these tumor cells can selectively
be induced to undergo apoptosis and then die, thereby treating a tumor.
In another preferred embodiment, the tumor is a solid tumor and
the tumor is injected with a recombinant virus which can infect the cells
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of the tumor and thereby cause them to express Fast, and whereby the
interaction of the Fast-expressing cells with the Fas-expressing cells
causes the Fas+ cells to undergo apoptosis.
The Fas-expressing cells which are affected by the FasL-
expressing cells are typically cells adjacent to the Fast-expressing cells
since typically a cell-to-cell contact is necessary for the apoptotic signal
be effectuated. The affected Fas cells can be removed from the
immediate surroundings of the Fast-expressing cell, however, such as
where the Fast-expressing cell has mobilized and/or where the FasL-
expressing cell produces soluble Fast.
The Fast-expressing cells can also cause their own death if
those cells also are Fas+ cells. In this approach, the methods of the
present invention can cause Fas+ cells to die, but the tumor cells that
now express the Fast also will die, thereby eliminating those tumor cells
that might otherwise cause regression of the tumor.
8. Combination therapy
The present invention also provides a method which utilizes a
combination therapy that combines expression of the apoptosis-
signaling ligand with administration of anti-cancer agents. It is believed
that by co-administering anti-cancer drugs, apoptosis of cancer cells can
be enhanced or sensitized, especially in those cancer cells are resistant
to Fast- or TRAIL-mediated apoptosis.
A major hurdle in treating cancer is the development of resistant
tumor cells to drugs and the development of anti-apoptotic machinery
which can spell over Fast or TRAIL sensitivity to apoptosis. It is
desirable to administer subtoxic concentration of chemotherapeutic
drugs and TRAIL (or Fas) on TRAIL (or Fas)-resistant tumor cells, which
should result in maximum tumor suppression and minimum side effects
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associated with administration of high dosage of chemotherapeutics.
The combination therapy of the present invention may overcome
tumor resistance to Fas- or TRAIL-mediated apoptosis by multiple
mechanisms of actions. Anticancer agents such as chemotherapeutic
agents or cytokines may sensitize Fas- or TRAIL-mediated apoptosis by
1 ) suppression of anti-apoptotic molecules, and/or 2) upregulation of
pro-apoptotic molecules. For example, Bcl-x~ and Bcl-2, major inhibitors
of the mitochondria) apoptotic pathway, can be regulated by anti-cancer
drugs. Paclitaxel, a plant-derived anti-cancer drug that at low levels can
reduce the activity of Bcl-2 by inducing phosphorylation of Bcl-2.
In addition, drugs and cytokines can also upregulate the
expression of pro-apoptotic molecules to lower the signaling threshold
required for the induction of TRAIL-mediated apoptosis. For example,
expression of DRS, one of the death-inducing TRAIL receptors, can be
induced by genotoxic drugs and TNF-a. The induction of DR5 appears
to be regulated by both p53-dependent and p53-independent
mechanims. Sheikh et al. (1998) "p53-dependent and independent
regulation of the death receptor KILLER/DR5 gene expression in
response to genotoxic stress and tumor necrosis factor alpha" Cancer
Res. 58:1593-1598. Moreover, the mRNA of caspases (caspases-1, -2,
-6, -8, and -9) can be upregulated by y-interferon. The upregulation of
these caspases should enhance the sensitivity to apoptosis induced by
expression of apoptosis-signaling ligand according to the present
invention.
A wide variety of anti-cancer agents may be co-administered with
the expression vectors of the present invention. Examples of the anti-
cancer agent include, but are not limited to, alkylating agents, antibiotic
agents, antimetabolic agents, hormonal agents, plant-derived agents,
and biologic agents.
Examples of alkylating agents include, but are not limited to,
bischloroethylamines (nitrogen mustards, e.g. chlorambucil,
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cyclophosphamide, ifosfamide, mechlorethamine, melphalan, uracil
mustard), aziridines (e.g. thiotepa), alkyl alkone sulfonates (e.g.
busulfan), nitrosoureas (e.g. carmustine, lomustine, streptozocin),
nonclassic alkylating agents (altretamine, dacarbazine, and
procarbazine), platinum compounds (carboplastin and cisplatin).
Examples of antibiotic agents include, but are not limited to,
anthracyclines (e.g. doxorubicin, daunorubicin, epirubicin, idarubicin and
anthracenedione), mitomycin C, bleomycin, dactinomycin, plicatomycin.
Examples of antimetabolic agents include, but are not limited to,
fluorouracil (5-FU), floxuridine (5-FUdR), methotrexate, leucovorin,
hydroxyurea, thioguanine (6-TG), mercaptopurine (6-MP), cytarabine,
pentostatin, fludarabine phosphate, cladribine (2-CDA), asparaginase,
and gemcitabine.
Examples of such hormonal agents are synthetic estrogens (e.g.
diethylstibestrol), antiestrogens (e.g. tamoxifen, toremifene,
fluoxymesterol and raloxifene), antiandrogens (bicalutamide, nilutamide,
flutamide), aromatase inhibitors (e.g., aminoglutethimide, anastrozole
and tetrazole), ketoconazole, goserelin acetate, leuprolide, megestrol
acetate and mifepristone.
Examples of plant-derived agents include, but are not limited to,
vinca alkaloids (e.g., vincristine, vinblastine, vindesine, vinzolidine and
vinorelbine), podophyllotoxins (e.g., etoposide (VP-16) and teniposide
(VM-26)), camptothecin and its derivatives (e.g., 9-nitro-camptothecin
and 9-amino-camptothecin), and taxanes (e.g., paclitaxel and
docetaxel).
Examples of biologic agents include, but are not limited to,
immuno-modulating proteins such as cytokines, monoclonal antibodies
against tumor antigens, tumor suppressor genes, and cancer vaccines.
Examples of interleukins that may be used in conjunction with the
composition of the present invention include, but are not limited to,
interleukin 2 (IL-2), and interleukin 4 (IL-4), interleukin 12 (IL-12).
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Examples of interferons that may be used in conjunction with CPT
include, but are not limited to, interferon a, interferon ~i (fibroblast
interferon) and interferon y (fibroblast interferon). Examples of such
cytokines include, but are not limited to erythropoietin (epoietin a),
granulocyte-CSF (filgrastin), and granulocyte, macrophage-CSF
(sargramostim). Other immuno-modulating agents other than cytokines
include, but are not limited to bacillus Calmette-Guerin, levamisole, and
octreotide.
Example of monoclonal antibodies against tumor antigens that
can be used in conjunction with CPT include, but are not limited to,
HERCEPTINO (Trastruzumab) and RITUXANO (Rituximab).
Examples of the tumor suppressor genes include, but are not
limited to, DPC-4, NF-1, NF-2, R8, p53, V1/T1, BRCA1 and BRCA2.
Example of cancer vaccines include, but are not limited to
gangliosides (GM2), prostate specific antigen (PSA), a-fetoprotein
(AFP), carcinoembryonic antigen (CEA) (produced by colon cancers and
other adenocarcinomas, e.g. breast, lung, gastric, and pancreas
cancers), melanoma associated antigens (MART-1, gp100, MACE 1,3
tyrosinase), papillomavirus E6 and E7 fragments, whole cells or
portions/lysates of antologous tumor cells and allogeneic tumor cells.
An adjuvant may be used to augment the immune response to
TAAs. Examples of adjuvants include, but are not limited to, bacillus
Calmette-Guerin (BCG), endotoxin lipopolysaccharides, keyhole limpet
hemocyanin (GKLH), interleukin-2 (IL-2), granulocyte-macrophage
colony-stimulating factor (GM-CSF) and cytoxan, a chemotherapeutic
agent which is believed to reduce tumor-induced suppression when
given in low doses.
In an embodiment, actinomycin D, a drug that inhibits RNA
synthesis and decreases expression of Bcl-x~ may be used to sensitize
TRAIL-mediated apoptosis in cancer cells, for example, in cancer cells
of Kaposi's sarcoma (KS) that is associated with AIDS. KS is the most
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common malignancy arising in persons with HIV infection (AIDS-KS).
Although a number of modalities have been used for 15 years, cure or
long-term complete remission from KS is unlikely with the currently
available therapeutic modalities. Lee and Mitsuyasu (1996)
"Chemotherapy of AIDS-related Kaposi's sarcoma" Hematol. Oncol.
Clin. North Am. 10:1051-1068. High levels of Bcl-xand Bcl-x~ are
detected in AIDS-KS lesions, which may be attributed to resistance of
KS cells to killing by chemotherapeutic drugs and NK cells. Thus, co-
administering to KS patients the expression vector encoding TRAIL and
one or more genotoxic drugs should over the resistance by
synergistically sensitizing the cancer cells to TRAIL-mediated apoptosis
through suppression of Bcl-x and Bcl-x~ levels by the genotoxic drugs.
In another emobodiment, doxorubicin may be used in
combination of the expression vector expressing TRAIL to treat patients
with prostate cancer. Prostate cancer is one of the most prevalent
cancers in American men and the survival rate of patients with
advanced prostate cancer is currently low. Landis et al. "Cancer
statitics" CA Cancer J. Clin. 49: 8-31. While surgery, hormone therapy,
and chemotherapy can eradicate the majority of prostate cancer,
relapse of advanced cancer metastasis can occur. Since the prostate
cells that are hormone refractory are also insensitive to radiation therapy
and chemotherapy, these cells possibly develop resistance to all
apoptotic programs induced by various stimuli as they progress to
become more malignant. However, co-administrating a genotoxic drug
with the expression vector encoding TRAIL should overcome the
resistance by sensitizing prostate cancer cells to TRAIL-mediated
apoptosis through suppression of apoptosis-inhibiting molecules or
upregulation of pro-apoptotic molecules.
The expression vectors of the present invention, when expressing
the apoptosis-signaling ligand for treating cancer (or other diseases),
may be administered in conjunction with other therapeutic agents
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against the cancer (or the other diseases to be treated) before, during,
or after the administration of the other therapeutic agent. These
therapeutic agents can be administered at doses either known or
determined to be effective and may be administered at reduced doses
due to the presence of the apoptosis-signaling ligand expressed by the
vector of the present invention.
The present invention is more particularly described in the
following examples which is intended as illustrative only since numerous
modifications and variations therein will be apparent to those skilled in
the art.
EXAMPLE
Example 1 Expression vector for Fas Ligand
In an example of the methods described above and depicted in
Figure 1, a recombinant adenovirus containing a nucleic acid encoding a
murine Fas ligand was constructed. Additionally, a recombinant
adenovirus was constructed containing a nucleic acid encoding a murine
Fas ligand and also encoding the jellyfish green fluorescent protein
(GFP) such that a fusion protein was ultimately translated. This fusion
protein was used to monitor the expression and localization of the
protein in cultured cells and in animal tissues following transduction with
the adenovirus vector.
Three different tumor cell lines were isolated from breast cancer
patients, all of which exhibited a high degree of sensitivity to the Fas
ligand treatment via the adenovirus vector. This demonstrates the
tumor cells could be effectively treated, or killed, using these methods.
Parallel experiments also demonstrated several prostate cancer cell
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lines are extremely sensitive to Fas-mediated apoptosis since complete
killing of these cells was obtained using Adenovirus-mediated
introduction of a nucleic acid encoding Fas ligand into these cells.
Example 2: Controlled Delivery of a Fast-GFP Fusion Protein with a
Complex Adenoviral Vector
Fas ligand (Fast) induces apoptosis in cells that express Fas
receptor and plays important roles in immune response, degenerative
and lymphoproliferative diseases and tumorigenesis. It is also involved
in generation of immune privilege sites and is therefore of interest to the
field of gene therapy. We describe the construction and characterization
of replication-deficient adenoviral vectors that express a fusion of murine
Fast and green fluorescent protein (GFP). Fast-GFP retains full
activity of wild-type Fast, at the same time allowing for easy
visualization and quantification in both living and fixed cells. The fusion
protein is under the control of tetracycline-regulated gene expression
system. A tight control is achieved by creating a novel A double
recombinant Ad vector, in which the tet-responsive element and the
transactivator element are built into the opposite ends of the same
vector to avoid enhancer interference. Expression can be conveniently
regulated by tetracycline or its derivatives in a dose-dependent manner.
The vector was able to efficiently deliver Fast-GFP gene to cells in vitro,
and the expression level of the fusion protein was modulated by the
concentration of doxycycline in culture media. This regulation allows us
to produce high titers of the vector by inhibiting Fast expression in a
CrmA-expressing cell line. induction of apoptosis was demonstrated in
all cell lines tested. These results indicate that our vector is a potentially
valuable tool for Fast-based gene therapy of cancer and for the study of
FasL/Fas-mediated apoptosis and immune privilege.
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Materials and Methods
Cells: HeLa and 293 cells were obtained from the American
Type Culture Collection (ATCC CCL-2.1 and ATCC CRL-1573,
respectively) and maintained as monolayers at 37 C under 5% C02 in
Dulbecco's modified Eagle's medium (DMEM; Gibco BRL)
supplemented with 10% bovine calf serum (BCS; HyClone) and 1
penicillin/streptomycin (Cellgro). Cultured rat myoblasts were
maintained in H-21 (Cellgro) media supplemented with 20% Fetal
Bovine Serum (FBS; HyClone) and 1 % each of penicillin/streptomycin
and fungizone.
For DNA transfections, 5x105 cells per well were seeded on 6-
well plates (Greiner) and transfected 24 hours later using
LipofectAMINE (Gibco BRL) according to manufacturer=s instructions.
To produce a cytokine response modifier A (CrmA)-expressing
293 cell line, pCrmA-I-Neo was transfected into HEK293 cells. Neo-
positive clones were selected by adding 6418 to the media at 0.4 g/L for
4 weeks, at the end of which time individual clones were picked up,
propagated and assayed for CrmA expression by their resistance to
Fast-induced apoptosis.
Construction of plasmids and recombinant adenoviral vectors:
Vectors pEGFP-1 and pEGFP1-C1 were obtained from Clontech. They
contain a red-shifted variant of wild type green fluorescent protein (wt
GFP) gene, with brighter fluorescence and "humanized" codon usage.
(Zhang, G., V. Gurtu and S. R. Kain. 1996. "An enhanced green
fluorescent protein allows sensitive detection of gene transfer in
mammalian. cells." (Biochem Biophys Res Commun 227:707-11.) This
protein will be referred to as "GFP" in this Example. The mouse Fast
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cDNA sequence, available in Genbank, was in a Bluescript (Invitrogen)
vector. Vectors pUHDlO-3 and pUHD15-1 (Gossen, M. and H. Bujard,
"Tight control of gene expression in mammalian cells by tetracycline-
responsive promoters" Proc Natl Acad Sci U S A 89:5547-51, 1992) are
available from Clontech. GFP-Fast fusion gene was constructed by
inserting DNA coding for as 11 to as 279 of the murine Fas ligand in-
frame downstream of the GFP sequence in pEGFP-C1, to generate
pC.GFsI. The fusion gene from pC.GFsI was inserted into pUHD10-3 to
produce p10-3.GFsl. Cowpox virus (Chordopoxvirinae) cytokine
response modifier A (crmA; CPV-W2) cDNA in pcDNA3 vector is
available from Genentech. The CrmA gene was excised from pcDNA3
and inserted into pIRES-Neo vector (Clontech) to generate pCrmA-I-
Neo.
GFP, Fast, Fast-GFP and LacZ genes were cloned into the E1
shuttle vector, pLAd-CMV to generate pLAd-C.Gf, pLAd-C.FsI, pLAd-
C.GFsI and pLAd-C.Lz constructs, respectively (Fig. 1A). The Tet-OFF
fusion activator protein expression cassette was extracted from
pUHD15-1 and inserted into pLAd-CMVie to generate pLAd-C.tTA. The
GFP-Fast fusion gene expression cassette was excised from p10-
3.GFsl and inserted into pRAd.mcs, a shuttle vector for transgene
insertion between E4 and right ITR of AdS. The resulting construct was
called pRAd-T.GFsI (Fig. 1 B).
The assembly of Ad/FasL-GFPTEr vector is shown in Figure 1 C.
Other rAd genomes used in this study were constructed using a similar
strategy. All vectors were based on Ad5sub360 (0E3) with additional
deletion of all E4 ORFs with the exception of ORF6. (Huang, M. M. and
P. Hearing. 1989) The adenovirus early region 4 open reading frame 6/7
protein regulates the DNA binding activity of the cellular transcription
factor, E2F, through a direct complex. (Genes Dev 3:1699-710).
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Propagation of viral vectors: The 293 cells, which provide Ad5
E1a and E1b functions in trans (Graham, F. L., J. Smiley, W. C. Russell
and R. Nairn; "Characteristics of a human cell line transformed by DNA
from human adenovirus type 5" (J Gen Virol 36:59-74,1977), were
transfected with the ligation mixture containing the rAd vector DNA using
LipofectAMINE method. Transfected cells were maintained until
adenovirus-related cytopathic effects (CPE) were observed (typically
between seven and 14 days), at which point the cells were collected.
Vector propagation and amplification was then achieved by standard
techniques. The stocks were titrated on 293 or 293CrmA cells and
plaques were scored to determine vector yields as PFU/ml. Vectors
were also titrated using GFP fluorescence or X-gal staining, as
appropriate. In both cases, titer estimates were in good agreement with
PFU/ml.
IlIlestern blot analysis: 10 cm plates (Greiner) were seeded with
106 cells of primary rat myoblasts. After 24 hours, plates were infected
with Ad/FasL-GFPTET or control vector at multiplicity of infection (M01) of
2. At 24 hours postinfection, the plates were washed twice with PBS.
The cells were collected and lysed in 200 p,1 of cell lysis buffer containing
50 mM Tris-HCI (pH 7.8), 1 mM EDTA, 2% SDS, 0.1 % Bromophenol
Blue, 1 mM PMSF (Sigma), 50 p,g/ml leupeptin (Sigma), 2 p,g/ml
aprotinin (Sigma) and 1 ng/ml pepstatin (Sigma). The samples were
boiled for 5 minutes and 1/10 of the original amount (106 cells) was
loaded per lane of an 8% SDS-PAGE minigel (BioRad), which was run
at 20 mA for 3 hours. Human recombinant Fast (C-terminal) was
obtained from Santa Cruz Laboratories. The proteins were transferred
to a nitrocellulose membrane (Pharmacia Biotech) using a semi-dry gel
transfer apparatus (BioRad). The membrane was blocked by incubation
(2 hours at 37°C) in a solution containing 10 mM Tris-HCI (pH 7.5), 140
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mM NaCI, 3% (w/v) BSA, 5% (w/v) powdered milk, 0.2% (v/v) Tween-20
(Amresco, Solon, OH) and 0.02% (w/v) sodium azide (Sigma). The
polyclonal rabbit anti-Fast antibody (Santa Cruz) was diluted 1:100 with
blocking solution and incubated with the membrane for 2 hours at
ambient temperature. The blot was washed with 10 mM Tris-HCI (pH
7.5) and 140 mM NaCI solution twice, then incubated with goat anti-
rabbit IgG conjugated with HRPO (Caltag, Burlingame, CA) diluted
1:10000. The blot was developed in ECL reagent (Amersham Life
Science) overnight and visualized with Kodak X-ray film.
Detection of apoptosis: Early detection of apoptosis in cultured
adherent cells was accomplished by utilizing the In Situ Cell Death
Detection Kit, AP (Boehringer Mannheim) according to manufacturers
instructions. This kit utilizes the terminal deoxynucleotidyl transferase-
mediated dUTP nick end-labeling (TUNEL) process to incorporate
fluorescein at free 3'-OH DNA ends and detect it with anti-fluorescein
antibody conjugated to alkaline phosphatase. After substrate reaction,
stained cells can be visualized using light microscopy.
Results:
Functional analysis of Fast and Fast-GFP proteins: In order to
demonstrate that the Fas ligand-GFP (Fast-GFP) fusion protein retains
full Fast activity, we have analyzed and compared the function of the
Fast and Fast-GFP proteins by using transient DNA transfections into
cells susceptible to Fas-mediated apoptosis. Triplicates of wells of
HeLa cells were transfected with vectors expressing Fast, GFP-Fast or
-galactosidase as a control. At 24 hours post-transfection, cells were
fixed and analyzed for apoptosis by using the TUNEL kit. Typically,
transfection efficiencies between 10 and 25% were achieved as
determined by X-Gal staining of cells transfected with pcDNA3-LacZ.
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Large numbers of HeLa cells transfected with vectors expressing either
Fast or Fast-GFP showed typical apoptotic morphology (such as
membrane blebbing and loss of adherence) and stained positive in the
TuNE~ assay. Very few cells transfected with a control plasmid
underwent apoptosis. The numbers of apoptotic cells in wells
transfected with Fast-GFP vector were reproducibly similar to those
transfected with Fast vector, suggesting that the wild-type and fusion
proteins have comparable activity.
Construction and characterization of adenoviral vectors: Our goal
was to produce large amounts of adenoviral vectors in which the Fast
expression could be regulated. This regulation allows control of the
levels of Fast expression in target cells and thus facilitates the study of
its biological effects. In addition, amplification of rAd vectors
constitutively expressing Fast or Fast-GFP in 293 cells would be
expected to produce low titers because Fast expression causes
apoptosis of the virus-producing cells. Muruve, D. A., A. G. Nicolson, R.
C. Manfro, T. B. Strom, V. P. Sukhatme and T. A. Libermann. (1997)
"Adenovirus-mediated expression of Fas ligand induces hepatic
apoptosis after Systemic administration and apoptosis of ex vivo-
infected pancreatic islet allografts and isografts" Hum Gene Ther 8:955-
63. To achieve the controlled Fast-GFP expression, we designed the
Ad/FasL-GFPTETvector in which the Fast-GFP is expressed from a THE
promoter. Gossen, M. and H. Bujard. (1992) "Tight control of gene
expression in mammalian cells by tetracycline- responsive promoters"
Proc Natl Acad Sci U S A 89:5547-51. We inserted CMVie promoter-
driven tTA gene (the "tet-ofP' element) into the Ad5 E1 region and the
TRE-controlled Fast-GFP fusion gene near the right ITR.
This strategy was based on the following considerations. First,
this strategy delivers the entire tet-regulated expression system using a
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single vector, rather than using two Ad vectors as have been described
previously. Harding, T. C., B. J. Geddes, D. Murphy, D. Knight and J. B.
Uney. (1998) "Switching transgene expression in the brain using an
adenoviral tetracycline-regulatable system" see comments, Nat
Biotechnol 16:553-5. Use of a single vector allows a more efficient
delivery to target cells as well as a more uniform regulation of protein
expression. This strategy also achieves maximum possible separation
between the enhancer elements of the CMVie promoter and the THE
promoter, in order to minimize background (unregulated) expression of
Fast-GFP protein (Fig. 1 B and 1 C). By placing the THE promoter at the
right end of the Ad5 genome, a similar result was obtained with respect
to the E1A enhancer elements, which are located within the Ad5
packaging signals Hearing, P. and T. Shenk. 1983. The adenovirus type
5 E1A transcriptional control region contains a duplicated enhancer
element. Cell 33:695-703. These elements have been reported to
interact with some promoters cloned into the E1 region Shi, Q., Y. Wang
and R. Worton. (1997) "Modulation of the specificity and activity of a
cellular promoter in an adenoviral vector" Hum Gene Ther 8:403-10.
The genomes of recombinant adenoviral vectors used in the
present invention were assembled in vitro in large-scale ligation
reactions as schematically diagrammed in Figure 1C. These genomes
were then gel-purified and transfected into 293 cells and the resulting
cultures were propagated until virus-induced CPE was observed. In the
case of vectors expressing ~3-galactosidase or GFP, CPE occurred at
significantly earlier time points than for vectors expressing Fast or FasL-
GFP, indicating that adenoviral vector replication was likely deleteriously
affected by Fast activity. Primary vector stocks were amplified
according to established techniques, and recombinant adenoviral DNA
was extracted and examined for structural integrity by restriction enzyme
digests.
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The titers of Ad/FasL and Ad/FasL-GFPTET in 293 cells were
typically 30 to 100-fold lower then titers of Ad/LacZ or Ad/GFP.
Comparison of titers of Ad vectors with Fast activity demonstrated a
substantial improvement (between 8- and 12-fold) in the yield of these
vectors when they were produced in 293CrmA cells (Figure 2). As
shown in Figure 2, amplification of the control vector Ad/LacZ in either
293 or 293CrmA cells resulted in essentially the same yield.
Subsequently, generation and amplification of all vectors with Fast
activity was carried out in 293CrmA cells.
Induction of apoptosis by adenovirus-mediated Fast expression:
To functionally demonstrate that adenovirus-mediated Fast expression,
we transduced HeLa cells with Ad/FasL-GFPTeTat different MOI. At 24
hours post-transduction, cells were analyzed for apoptosis. Cells
infected with Ad/FasL-GFPTET demonstrated typical apoptotic
morphology. The numbers of apoptotic cells increased with the
increasing vector titers. In contrast, plates transduced with the control
vector Ad/LacZ at the same MOI did not generate apoptotic cells in
excess of untransduced controls. The overall efficiency of transduction
was determined by X-gal staining and shows increasing numbers of ~-
galactosidase-positive cells with increasing MOI. We have observed
that the numbers of apoptotic cells are noticeably higher than those of
the cells with detectable GFP fluorescence, or of the X-gal stained cells
transduced at the same. Thus, apoptosis of cells not infected with the
vector, but adjacent to the cells that are, is caused by the interactions of
Fast on the surface of infected cells with Fas receptors on their
neighbors.
Detection and cellular localization of Fast-GFP fusion protein:
Wild-type Fast is a type II membrane protein. To demonstrate that the
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Fast-GFP fusion protein is also targeted to cellular membrane, we took
advantage of the fluorescence of its GFP component, which can be
detected in living cells using a fluorescent microscope with a FITC filter
set. We have used this technique to observe the expression and
cellular localization of our Fast-GFP fusion protein when expressed
from rAd vector. In HeLa cells, expression of Fast-GFP causes
apoptosis at protein levels close to the detection threshold of GFP.
Therefore, the expression of Fast-GFP was analyzed in primary rat
myoblasts, which we found to be relatively resistant to Fast-induced
IO apoptosis. High levels of Fast-GFP expression can be detected in
myoblasts at 24 hours post-infection with Ad/FasL-GFPTET at MOI of 10.
Membrane-associated expression of Fast-GFP is evident in the
majority of the transduced cells. In contrast, the fluorescence pattern of
GFP itself is evenly distributed in the cytoplasm of the cells, while often
I5 being excluded from the nucleus. These localization differences are
also apparent in transduced 293CrmA cells at higher magnification.
These results indicate that the Fast-GFP fusion protein is directed to
the cell surface, where it can interact with the Fas receptor in a manner
analogous to that of wildtype Fast.
Regulation of Fast-GFP expression from rAd vector. To show
that the present vector has the ability to regulate the amount of Fast
activity produced by our rAd vector in target cells, we have performed
experiments to establish the levels of Fast expression under induced or
uninduced conditions at both the levels of protein synthesis and
function. In Ad/FasL-GFPTET vector, expression of Fast-GFP fusion
protein is designed to be activated by the binding of the tetR-VP16
fusion protein (constituatively expressed from the same vector; see Fig.
1 C) to the heptamer of tet-operators upstream of a minimal CMVie
promoter. Gossen, M. and H. Bujard. (1992) "Tight control of gene
expression in mammalian cells by tetracycline- responsive promoters"
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Proc Natl Acad Sci U S A 89:5547-51. Presence of doxycycline in the
cell should inhibit this binding and therefore the expression of Fast-GFP
in a concentration-dependent manner.
First, we determined the amounts of Fast-GFP produced in
transduced cells by using Western blot analysis. We infected primary
rat myoblasts with Ad/FasL-GFPTE-rat an MOI of 2 and cultured these
cells in the absence or presence of doxycycline, a tetracycline
derivative. Low MOI was chosen to maximize number of cells
transduced with a single copy of the vector. After 48 hours, cells were
lysed and the lysates analyzed by Western blotting using a polyclonal
antibody against the extracellular domain of Fast. A single specific
band larger than the predicted size of wt Fast was detected. The
intensity of the band decreased with the increasing concentration of
doxycycline, and no band could be detected in the cell lysates that have
been cultured in the presence of 0.5 mg/L or higher concentration of
doxycycline. No Fast-specific band was observed in cells transduced
with a control vector. No bands of lesser size, corresponding to the
breakdown or cleavage products, were detected either in the cell lysates
or in the media supernatant. These results indicate that the amount of
GFP-Fast protein produced in the cell from the Ad/FasL-GFPTeTvector
can be regulated by the concentration of doxycycline in culture medium,
and that this protein is stable and does not undergo appreciable
cleavage once on the cell surface.
We have also analyzed the regulation of Fast activity, i.e. the
induction of apoptosis in Fas-positive target cells. Wells of HeLa cells
were transduced with Ad/FasL-GFPTeT at an MOI of 2 and cultured in the
presence of various concentrations of doxycycline. At 24 hours post-
transduction, cells were analyzed for apoptotic phenotype. The results
confirm that the induction of apoptosis in cells transduced with Ad/FasL-
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GFPrEr can be regulated by doxycycline.
In the regulated protein expression system that we chose,
presence of doxycycline inhibits the binding of tTA to THE and turns off
Fast-GFP transcription in a dose-dependent manner. We elected to
insert the constitutively expressed activator into the E1 region and the
Fast-GFP expression cassette into a novel cloning site between the E4
promoter and the right ITR of Ad5, reasoning that this arrangement
would minimize the effect of the E1A enhancer present within the
packaging region of adenovirus and the CMVie enhancer within the tTA
promoter on the TRE, and thus reduce background expression of the
fusion protein in the presence of inhibitor. This system performed
successfully in the context of adenoviral vector, such that the expression
of Fast-GFP could be efficiently regulated by varying the doxycycline
concentrations in cell culture medium.
In the course of our experiments, we have observed that 293
cells are susceptible to Fast-induced apoptosis. This effect acts to
significantly limit the titers of rAd vectors expressing Fast. This is true
even if regulated or tissue-specific promoters are used to express Fast
protein, since high levels of protein expression are unavoidable in the
course of vector replication in 293 cells. In order to overcome this
problem, we have generated a 293 cell line which constitutively
expresses CrmA. This protein acts specifically to inhibit the activity of
regulatory caspases, which are integral to the Fas apoptosis pathway.
By producing our Fast-containing vectors in these cells, we have
obtained significant improvements in the vector titers.
In summary, we have developed and tested a rAd vector that
expresses a novel Fast-GFP fusion protein under the control of
tetracycline-regulated gene expression system. This vector combines
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high titers and efficient transgene delivery to multiple types of dividing
and non-dividing cells with convenient regulation of protein expression
and easy detection of the fusion protein in both living and fixed cells.
This vector is a valuable tool for treating disease through immunology,
transplantation and cancer therapy.
Example 3: Bystander Gene Therapy Using Adenoviral Delivery of a
Fas Ligand Fusion Gene
This example describes a type of bystander gene therapy utilizing
a Fas Ligand-fusion gene approach that induces prostatic
adenocarcinoma to undergo apoptosis (programmed cell death) through
a paracrine/autocrine mechanism. This work provides a novel and
potent therapy for treatment of prostate cancer (PCa). Furthermore,
specificity for the prostate or any other tissue may be achieved using
tissue-specific promoters to allow parenteral delivery of virus for
treatment of metastatic disease.
Our therapeutic approach is to deliver and express a Fas Ligand
(CD95L-fusion gene) with a second generation adenovirus deleted for
E1A, E3 and E4. CD95L expression is controlled by a Tet operator
allowing for doxycycline regulation in vitro and in vivo. The CD95L
used in this proposal is the mouse CD95L cDNA truncated by 10 amino
acids at its N terminus and fused in frame with a four-amino acid linker
to the C terminus of an enhanced GFP.
Table 1 presents our data using five PCa cell lines and generally
confirms literature reports (Hedlund et al. The Prostate 36:92-101,
1998; and Rokhlin et al. Can. Res. 57:1758-1768, 1997) that
demonstrate PCa cell lines are resistant to CH-11 agonist activity. In
contrast, we now demonstrate sensitivity to AdGFP-Fast and C2-
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ceramide in all five PCa cell lines tested to date.
Percent cytotoxicity was determined using the MTS assay. In
brief, cells were seeded in a 12-well plate with 1 ml of media. Prior to
treatments, cells were grown to 75% confluency and treated with either
500ng/ml CH-11 anti-Fas antibody, 500ng/ml Normal Mouse Serum or
30~,M C2-ceramide. For adenoviral transduction, approximately 1x105
cells/well were treated with either Ad/CMVGFP or Ad/GFP-FasLTer at an
MOI between 10-1000. For each cell line, positive controls were left
untreated, and 1 ml of media was used as a negative control. The cells
were incubated for 48 hours at 37 °C for maximal cell killing. Media
was
aspirated and replaced with 0.5m1 fresh media + 100 ~,I of Cell Titer 967
Aqueous One Solution Reagent per well. Cells were incubated for an
additional 1-3 hours at 37°C. After incubation, 120 ~,I of media was
placed into a 96-well plate and absorbance readings were taken using a
Vmax kinetic microplate reader at 490nm. Percent cytotoxicity was
calculated as follows: % cytotoxicity = [1-(OD of experimental well/ OD
of positive control well)] x 100. For ceramide assays, 1x104 cells/well
were seeded in a 96-well plate. The following morning cells were
washed and incubated with 100p1 of 30~,M Dihydro- or C2-ceramide
(diluted from a 10mM stock in ethanol) in serum-free RPMI 1640. After
24 hours, 20 ~,I Celltiter 967 Aqueous One Solution Reagent was added
to each well and plates were incubated an additional 1-4 hours.
Absorbance and % cytotoxicity were determined as above. In each
experiment, data points were run in triplicate.
Results:
Clearly, the five PCa cell lines analyzed in Table 1 are largely
insensitive to CH-11. Sensitivity to C2-ceramide is relatively uniform at
the 30 ~,M dose suggesting that the apoptosis pathway is intact. Most
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importantly, all the cell lines are responsive to AdGFP-Fast
administration with DU145 being the least sensitive.
Several important points are made by these experiments. First,
S we show using FACS analysis that CD95 (Fas receptor) was expressed
on all candidate PCa cell lines, for all lines we used. Second, we show
that the fas receptor blocking antibody (ZB4) does not prevent induction
of apoptosis by AdGFP-Fast. We have performed this experiment
several times with different doses of ZB4, always with the result that the
virus induced the same extent of apoptosis in the presence or absence
of the antibody. This suggests that newly synthesized CD95-CD95L may
interact perhaps in the golgi (Bennett et al. Science 282:290-293,
1998), on the way to the plasma membrane, or on arrival at the cell
surface as a preformed and functional apoptotic signaling complex.
Third, our results show that there is no intrinsic property of the
adenovirus that facilitated induction of apoptosis in PCa. This was
demonstrated by infecting PCa with control virus (AdCMVGFP) plus CH-
11 at 500ng/ml. The result was that CH-11 still failed to induce
apoptosis. These results show that apoptosis only occurs in CD95+-CH-
11 resistant PCa cell lines when viral directed intracellular expression of
CD95L occurs and this was not virus-dependent.
The final and most relevant piece of information pertains to
whether we can administer AdGFP-FasLTEr without lethality to the
subject. This is critically important because a dose as low as 2x10$ pfu
of virus kills the mouse when administered parenterally. To address this
issue, xenografts of PPC1 were developed in Balbc nu/nu mice and
treated with various doses of AdCMVGFP control or AdGFP-Fast virus.
From these single dose studies, we have evidence that tumor cell
growth is retarded or stopped. Further, out of 14 animals treated with
virus, none have died from the virus. In summary, we conclude that the
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GFP-Fast fusion protein in our Ad5 delivery system has strong
therapeutic potential for treating PCa.
Development of a version of AdGFP-Fast that is up-regulated by
doxycycline.
Our present virus is designed to be administered orthotopically to
PCa. If the virus escapes the tumor and enters the body it could be
lethal if sufficient virus reaches~the reticuloendothelial system (mostly
the liver). By administration of doxycycline (dox), expression of CD95L
from AdGFP-Fast can be down-regulated, and this danger avoided. A
viral vector induced by doxycycline that exhibits "very low" basal activity
is constructed by using the Tet regulatable elements set forth in
Example 1. This vector is completely repressed relative to GFP-Fast
expression in the absence of dox and induced starting at 10ng/ml with
maximal induction between 100-500ng/ml. These are easily achievable
doses in humans (1-3 p,g/ml at typical dosage levels). Should adverse
effects be observed, dox administration is terminated. However,
doxycycline has a serum half-life of 16 hours which we believe argues
that the addition of dox to down-regulate expression of Fas Ligand may
be better for treating adverse effects in patients since we can rapidly
achieve effective doxycycline doses within minutes by parenteral
administration. If necessary, addition of a PEST signal can speed
degradation (see Clontech catalogue).
Methods:
We replace our current Tet repressor and operator system with
the rTSk'd B/C and rtTA system (Freundlieb et al. J. Gene Med. 1:4-12,
1999). It has already been pointed out that we can place our prostate
specific promoters (PSA, PSADBam, PB and ARRPB2, Appendix) into
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the virus (replacing CMVie) to achieve tissue specificity where only
prostate epithelial cells will be able to regulate rtTA. All viruses are
grown by standard techniques from 3X plaque-purified samples
assessed to be negative for wild-type adenovirus by PCR. All viruses
are grown in the presence of l~,g/ml doxycycline in the HEK 293
packaging cell line that constitutively expresses the cowpox virus
cytokine response modifier, crmA Rubinchik et al. This is necessary to
prevent GFP-Fast induced apoptosis in the packaging cell line. Virus is
always purified by isopycnic centrifugation on CsCI, desalted by
chromatography, concentrated by filtration and stored frozen in PBS
10% glycerol in small aliquots at -80°C. Virus is thawed only once and
administered to the animals under anesthesia, by infusion as described
above, at 15 ~.I/min or via the tail vein with a tuberculin syringe. Tumor
and animal tissues are collected for frozen sections or, fixed and
embedded, where appropriate, and analyzed by H & E, by tunel assays
for apoptosis, and by immunostaining to determine neutrophil infiltration
and GFP expression where relevant.
Testing the original AdGFP-FasLTeta (dox dov~n-regulated) on
prostate cancerxenografts in Balbc nulnu mice. These experiments are
carried out to establish both toxicological and efficacy parameters.
Specifically, we infuse increasing doses 1x109 - 5x10'° pfu AdGFP-
FasLTeta into 75 to 100 mm3 tumors to determine: A) lowest successful
dose required to decrease tumor volume by 75% or more following
orthotopic administration of virus with one dose and with three doses
administered every four days. Tumors are developed from CD95L
sensitive PPC1, intermediately sensitive LnCAP C2-4, and more
resistant Du145 cell lines. Other parameters of administration are
developed based on results with the endpoint always being tumor
remission. B) Highest tolerated viral dose following orthotopic
administration (up to 5 x 10'° pfu). C) Determine if tumor will reoccur
at
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a later time (6-12 months) in the same or distant site (C4-2). D) Highest
dose administered i.v. (tail vein) that 50% of mice survive. E) Using
data from D, test the effect of doxycycline administration on the animal
survival curve and duration of doxycycline protection (Balbc nu/nu mice
have no CTL response so adenovirus may survive for a long time).
Statistical analysis using a one sided t-test is employed. F) Determine
the half-life of GFP-Fast in K562 cells (CD95L resistant, see Tabfe 1) by
monitoring GFP (as the GFP-Fast fusion) over time in the presence of
1 ~,g/ml dox using FACS analysis.
The same set of experiments as in B1 above is carried out with
the Tet inducible virus constructed as described above.
Toxicology testing Of AdGFP-FaSL,.etu (upregulated) and AdGFP-
FasL,-era (down-regulated) administered to normal laboratory beagles.
Although there are a number of animal models for PCa, none but the
dog model well-represent human disease in pathology and anatomy. It
has recently been shown that human AdRSVbgal (serotype 5)
adenovirus will infect dog epithelial cells, including prostate tumor cells,
both in vitro and in vivo Andrawiss et al. Prostatic Can. Prostatic Dis.
2:25-35, 1999. Comparison of the present Ad/GFP-FasTET in dogs
(immunocompetent) verses immunocompromised mice (Balbc nu/nu)
provides additional support for a human phase I trial of this gene therapy
approach.
In the following section, experiments are carried out on sexually
mature normal dogs to see if orthotopic delivery Ad/GFP-FasLTer to
hormonal prostate is safe with minimal or no collateral damage.
Purified concentrated adenovirus (Ad/GFP-FasLTer both up- and
down-regulated and a reporter virus Ad/CMV-LacZ all serotype 5) is
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injected via an abdominal surgical approach into one lobe of the dog
prostate. This approach is preferable to transrectal introduction
because it is believed that direct visualization of the prostate provide for
a more accurate introduction of virus in these first series of experiments.
Second, because of the highly vascular nature of the dog prostate
direct visualization allows us to seal the needle track with topical tissue
glue and digital pressure to prevent viral leakage from the injection site.
Based on these results, a 3D ultrasound guided transrectal introduction
is used to mimic one of the proposed human approaches.
Virus dosages of 5x109, 1x10'°, and 5x10'° in a constant
400u1
volume are used: one set of 2 dogs receives AdICMV-LacZ at 5x10'° pfu
to allow histochemical monitoring of viral spread. Dogs are monitored
closely the first 72 hours for any signs of distress. Feces is collected
and analyzed for viral shedding by PCR. Urine is also collected by foley
catheter and assayed on 293 cells for shed virus and by PCR. At day 7
(2 dogs per viral dose) are euthanized with sodium pentobarbital and
processed as described. (Andrawiss et al. Prostatic Can. Prostatic Dis.
2:25-35, 1999). Samples of all tissues are frozen in OCT while the
remainder are either fixed and processed for histology (tunel,
immunohistochemistry), or stored frozen at -80°C for DNA extraction
and PCR using viral-specific primers. Expression of LacZ is examined
in the Ad/CMV-LacZ group to monitor systemic viral spread.
Example 4: Intratumoral Introduction of Ad/GFP-FasLTEr
Suppresses Breast Tumor and Brain Tumor Growth in Mice
In this experiment, we implanted 106 MCF-7 cells bilaterally into
Balbc nu/nu mice (Figure 6). When tumor sizes reached 5 mm in
diameter, we infused at 15 ~,I per minute, 2 X 109 pfu Ad/GFP-FasLTeT
into the tumors on the right side of the mouse or 2 X 109 pfu Ad/LacZ
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into the left side, over a period of 10 minutes using a Harvard infusion
pump. At three weeks post-injection, all tumors injected with Ad/FasL-
GFPTEr exhibited about 80-100% regression of the tumor in comparison
with the control-treated tumor. In particular, in four of the six mice
treated, most of the tumor masses disappeared after one injection
(indicated by yellow arrows). In the other two of the six mice,
suppression of tumor growth wad greater than 80% (indicated by black
arrows) in comparison to tumors on the control side of the same mice.
In contrast, all tumors injected with Ad/LacZ grew to about 2 cm in
diameter at three weeks after implantation. Histological analysis of the
residual tumors in some of the mice showed only infiltrating immune
cells and fibroblasts with no apparent cancer cells remaining. This
demonstrates that Fast-induced apoptosis may be used as a novel
treatment for breast cancer.
Similarly, we implanted 106 SF767 cells bilaterally into Balbc
nu/nu mice. When tumor sizes reached 5 mm in diameter, we infused at
15 ~,I per minute, 2 X 109 pfu Ad/GFP-FasLTer into the tumors on the
right side of the mouse or 2 X 109 pfu Ad/LacZ into the left side, over a
period of 10 minutes using a Harvard infusion pump. Tumor
suppression was about 80-100% in treated tumors as compared to
untreated tumors. In contrast, all tumors injected with Ad/LacZ grew to
about 2 cm in diameter at three weeks after implantation. This
demonstrates that Fast-induced apoptosis may be used as a novel
treatment for brain cancer.
Example 5: Comparison of sensitivities of cancer cells to Fast- and
TRAIL-induced apoptosis in vitro
In this example, sensitivities of different cancer cell lines (derived
from prostate, cervical and liver cancers) to Fast- and TRAIL-mediated
apoptosis were compared in vitro.
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An expression vector for TRAIL, Ad.TRAIL/GFPTET, was used to
express TRAIL in these cancer cells. The construction of
Ad.TRAIL/GFPTeT is illustrated in Figure 3. Similar to Ad/FasL-GFP'~ET,
this vector contains a transactivator driven by the CMV promoter in the
E1 region, and the TRAIL-IRES-GFP expression cassette under the
control of the THE promoter in the E4 region, so that the expression of
both TRAIL and GFP can be regulated by the addition of doxicycline to
the culture media. The internal ribosome entry site (IRES) of the
encephalomyocarditis virus allows expression of two genes from the
same mRNA transcript. Although the GFP is not fused to the apoptotic
protein TRAIL, its expression is correlated with that of TRAIL. Since
TRAIL is in front of the GFP and the IRES sequence, the level of its
expression should be several folds higher than GFP. Liu et al. (2000)
"Generation of mammalian cells stably expressing multiple genes at
predetermined levels" Anal. Biochem. 280:20-28. This will assure high
levels of TRAIL expression in cells that GFP expression can be
observed with UV microscope.
To determine if TRAIL expression can induce apoptosis of cancer
cells, we transduced TRAIL into four different cancer cell lines: LNCaP
(prostate), HeLa (cervical), A549 (lung), and C3A (liver). We infected
these cells with Ad.TRAIL/GFPTer at the same MOI of 10. All these cells
demonstrated sensitivity to TRAIL induced apoptosis at different levels
of sensitivities, and the sensitivities appeared lower than those of the
cancer cells to Fast. To confirm this observation, we have compared
the efficacy of the Fast-GFP and TRAIL in inducing apoptosis in parallel
experiments. We infected the cancer cells with Ad/GFP, Ad/FasL-
GFPTET and Ad.TRAIL/GFPTET at comparable MOI. Similar to the FasL-
sensitivity studies (described earlier), the susceptibility of the these cells
was analyzed by the number of GFP-expressing cells and the
sensitivities to Fast and TRAIL induced apoptosis are determined
based on cell morphology in these initial experiments. As shown in
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Figure 4, panels "TRAIL", cells showed different levels of apoptosis. In
all the cells tested, less cells underwent apoptosis in Ad.TRAIL/GFPTEr
infected wells than those infected with Ad/FasL-GFPTer (panels labeled
with "Fast") suggesting that LNCaP, HeLa, A549 and C3A cells are
more sensitive to Fast than TRAIL-induced apoptosis.
Example 6. Adenovirus-mediated TRAIL expression induces
apoptosis in cancer cells, but not in normal fibroblasts.
It is believed that one of the major advantages of TRAIL tumor
therapy is that TRAIL expression is supposed to be much less toxic to
normal cells than that of Fast, while still inducing apoptosis in tumor
cell. To test this "tumor specificity", we transduced normal human
fibroblasts with Ad.TRAIUGFPTET at MOI about 10. We obtained
primary early passage human fibroblasts from foreskin samples and
tested them for their sensitivity to apoptosis induced by our Ad/FasL-
GFPTer and Ad.TRAIL/GFPTEr vectors. We have found that primary
human fibroblasts were quite sensitive to Fast-GFP induced apoptosis,
so that even at a low transduction efficiency, most of them displayed
standard apoptotic morphology (Figure 5, panel FasL). In contrast,
primary fibroblasts transduced with adenovirus vector expressing TRAIL
were essentially unaffected, even at MOI five-fold higher than those
used to deliver Fast-GFP (Figure 5, panel TRAILXS). We therefore
confirm the findings that TRAIL does not induce significant apoptosis in
normal cells, even at high expression levels from our adenovirus vector.
These results suggest that vector-mediated intratumoral delivery of
TRAIL can be even safer than Fast.
Although the present invention has been described with reference
to specific details of certain embodiments thereof, it is not intended that
such details should be regarded as limitations upon the scope of the
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invention except as and to the extent that they are included in the
accompanying claims.
Throughout this application, various publications are referenced.
The disclosures of these publications in their entireties are hereby
incorporated by reference into this application in order to more fully
describe the state of the art to which this invention pertains.
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TABLE 1: Fas-Mediated Cytotoxicity in Prostate Cancer Cell Lines Treated
for 48 hours with either Anti-Fas Antibody, C2-Ceramide (22 hours) or
AdIGFP-FasLTeT (expressed as % cytotoxicity _ SD)
Cell Normal Mouse C2-Ceramide Anti-Fas IgM Ad/CMV-GFP Ad~GFP
FasLrer
Line Serum (30~M (CH-11)(500 ng/ml) (M01 100) (M01
100)
(500 nglml) for 22 hrs)
DU145 0.87.1 61 5 6.010.4 1.92.8 69.6
4.5
PC-3 1.35.8 769 1.32.2 0.95.0 84.8
1S 1.1
PPC-1 2.30.3 587 29.22.3 2.36.1* 98.0
7.1
LNCaP 7.514.2 ND 11.613.7 1.63.2 96.4
4.3
TSU-Pr1 -3.52.3 729 -1.92.8 11.67.0 81.3
5.0
Jurkat(+ctrl) 98 2 72.3 0.9 -19.5 22.5"93.0
2.1 5.3
3.4"
K-562(-ctrl) - - -1.3 5.5" -114
2S . 8.1"
*MOI 10, "M01 1000. In all experiments N=3 (except N=2 for ceramide
experiments using TSU and PC-3). Percent cytotoxicity was determined
using the MTS assay. In brief, cells were seeded in a 12-well plate with
one ml of media. Prior to treatments, cells were grown to 75%
confluency and treated with either 500 mg/ml CH-11 anti-Fas antibody,
500 ng/ml Normal Mouse Serum or 30 p,M C2-ceramide. For adenoviral
3S transduction, approximately 1x 105 cells/well were treated with either
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AdCMVGFP or AdGFPFasLTEZ at an MOI between 10-1000. For each
cell line, positive controls were left untreated, and 1 ml of media was
used as a negative control. The cells incubated for 48 hours at 37°C
for
maximal cell killing. Media was aspirated and replaced with 0.5m1 fresh
media + 100,1 of CeIITiter 96 AQueous One Solution Reagent per well.
Cells were incubated for an additional 1-3 hours at 37°C. After
incubation 120~,i of media was placed into a 96 wet( plate and
absorbance readings were taken using a Vmax kinetic microplate reader
at 490nm. Percent cytotoxicity was calculated as follows: % cytotoxicity
= [1-(OD of experimental well/ OD of positive control well)] x 100. For
ceramide assays, 1x 104 cells/well were seeded in a 96-well plate. The
following morning cells were washed and incubated with 100.1 of 30~.M
Dihydro- or C2-ceramide (diluted from a 10mM stock in ethanol) in
serum-free RPMI 1640. After 24 hours 20,1 Celltiter 96 AQueous One
Solution Reagent was added each well and plates were incubated for an
additional 1-4 hours. Absorbance and % cytotoxicity were determined
as above. In each experiment, data points were run in triplicate.
