Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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STRESS-RESPONSIVE INDUCTION OF A THERAPEUTIC AGENT AND
METHODS OF USE
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority from U.S.
Provisional Application Serial No. 60/141,505, filed June
28, 1999, the disclosure of which is incorporated herein
by reference.
STATEMENT AS TO FEDERALLY-SPONSORED RESEARCH
Pursuant to 35 U.S.C. '202(c), it is acknowledged
that the U.S. Government has certain rights in the
invention described herein, which was made in part with
funds from the National Institutes of Health, Grant Nos.
CA27607 and CA59318.
TECHNICAL FIELD
This invention relates to compositions and methods
for selective expression of a heterologous nucleic acid
sequence in a targeted tissue, and more particularly to
the glucose regulated protein 78 (grp78) stress-
responsive promoter and its use in gene therapy and the
production of transgenic animals.
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BACKGROUND
Targeted gene expression is one of the most
difficult and important goals in the development
effective therapies for a variety of disorders,
including, for example, cell proliferative disorders such
as cancer or biological stress resulting from glucose
starvation in diseases such as diabetes. Two strategies
for specific expression include: 1) targetable entry; and
2) tissue or cell type specific gene expression.
Targetable entry involves vector engineering to change
vector binding tropism thus allowing cell type specific
transduction. Tissue or cell specific expression relies
on restricting expression of the delivered gene
exclusively to a particular type of tissue, such as a
tumor.
Successful application of any method for targeting a
specific tissue or cell for expression of a particular
molecule (e. g., protein or nucleic acid) requires
maximization of expression of the molecule in the
targeted environment. The most common promoter used to
drive expression of a foreign gene has been a
constitutive, general-purpose viral promoter such as the
MuLV LTR. These promoters, while effective in vitro,
often fail to express the sequences under their control
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within a biologically stressed environment (Palmer et
al., Proc. Natl. Acad. Sci. USA, 88:1330, 1991; Gazit et
al., Cancer Res., 55:1660, 1995). These data suggest
that the MuLV promoter and other constitutive or cellular
promoters are not optimal for expressing a nucleic acid
sequence within, for example, a fast growing solid tumor
devoid of nutrients due to insufficient blood supply.
Further, even if a viral promoter escapes genomic
silencing, the expression pattern of the foreign gene
will be constitutive in normal as well as tumor cells.
Such unregulated expression could be highly problematic
in gene therapy methods.
To circumvent these difficulties, stress-responsive
promoters provide an attractive means for tissue-specific
expression of a therapeutic agent. For example, most
fast growing tumors have a heterogeneous distribution of
blood supply; by having a high interstitial and a low
intravascular pressure, a decrease in nutrient supply
results, leading to necrosis in the center of the tumor.
Glucose deprivation, calcium deprivation, chronic anoxia
and low pH known to persist in poorly vascularized solid
tumors induce a class of stress proteins referred to as
the glucose-regulated proteins (GRPs) (Gazit et al.,
Cancer Res., 55:1660, 1995; Koong et al., Int. J. Radiat.
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Oncol. Biol. Phys., 28:661, 1994) including the grp78
gene. A rat grp78 promoter has been used as a potent
internal promoter in a retroviral vector to drive
expression of the neomycin phosphotransfPrase (neo)
reporter gene in a murine fibrosarcoma model system
(Gazit et al., Cancer Res., 55:1660, 1995). Such a
promoter provides an attractive means for specifically
expressing a therapeutic agent in a biologically stressed
tissue using currently available methods in gene therapy.
There are several strategies that have been
developed to accomplish gene therapy for the treatment of
disorders that give rise to a biologically stressed
cellular environment, such as cancer or diabetes, for
example. Within these strategies, there is a need for
controlled, sustained, site-specific expression of a
therapeutic agent such that surrounding healthy tissue
remains unaffected by the effects of the therapeutic
agent.
SUMMARY
The present invention is based, in part, on the
discovery that a stress-responsive promoter specifically
drives the expression of a therapeutic agent in vivo
resulting in the efficient treatment of a biological
stress-related disorder. Accordingly, in one embodiment,
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the invention provides a nucleic acid construct
comprising at least one stress-responsive non-coding
regulatory sequence which comprises at least two
endoplasmic reticulum stress elements (ERSE) as set forth
in SEQ ID NO:1, and a heterologous nucleic acid sequence
operatively linked to the regulatory sequence, wherein
expression of the heterologous sequence is regulated by
the non-coding sequence and wherein the heterologous
sequence encodes a therapeutic agent effective for
treating a cell proliferative disorder.
In another aspect, the invention provides a nucleic
acid construct comprising at least one stress-responsive
non-coding regulatory sequence which comprises at least
two endoplasmic reticulum stress elements (ERSE) as set
forth in SEQ ID NO:1; and a heterologous nucleic acid
sequence operatively linked to the regulatory sequence,
wherein expression of the heterologous sequence is
regulated by the non-coding sequence and wherein the
heterologous sequence encodes a detectable marker.
In one aspect, the present invention provides
vectors comprising the aforementioned nucleic acid
construct.
In another aspect, the present invention provides
compositions useful for gene therapy, such as viral
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vectors comprising a nucleic acid construct of the
invention.
The present invention also relates to the use of the
before described nucleic acid construct and vectors for
the preparation of pharmaceutical compositions for
treating, preventing, and/or delaying a disease in a
subject, such as, for example, a cell proliferative
disease. Furthermore, the recombinant nucleic acid
construct and vectors of the invention can be used for
the preparation of pharmaceutical compositions for
identifying a tumorous disease in a human and non-human
animal.
In a further embodiment, the present invention
provides cells and transgenic non-human animals,
comprising the aforementioned recombinant nucleic acid
sequence or vectors stably integrated into their genome
and their use for the identification of substances
capable of suppressing or activating transcription from a
stress-responsive regulatory sequence.
In a further embodiment, the invention provides a
method of method for producing a transgenic non-human
animal having a phenotype characterized by expression of
a heterologous nucleic acid sequence encoding a
detectable marker otherwise not naturally occurring in
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the animal, wherein the heterologous nucleic acid
sequence is operably associated with at least one stress-
responsive non-coding regulatory sequence comprising at
least two endoplasmic reticulum stress elements (ERSE) as
set forth in SEQ ID NO:l, the method comprising: a)
introducing at least one transgene into a embryo of an
animal, the transgene comprising at least one stress-
responsive non-coding regulatory sequence comprising at
least two endoplasmic reticulum stress elements (ERSE) as
set forth in SEQ ID NO:1 isolated upstream from the
heterologous nucleic acid sequence encoding a detectable
marker; b) transplanting the embryo into a pseudopregnant
animal; c) allowing the embryo to develop to term; and d)
identifying at least one transgenic offspring containing
the transgene.
The details of one or more embodiments of the
invention are set forth in the accompanying drawings and
the description below. Other features, objects, and
advantages of the invention will be apparent from the
description and drawings, and from the claims.
DESCRIPTION OF DRAWINGS
The file of this patent contains at least one
drawing executed in color. Copies of this patent with
color drawings) will be provided by the Patent and
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Trademark Office upon request and payment of the
necessary fee.
Figure 1 shows a schematic drawing of the
recombinant retroviral vectors. In the GlNaGrpTk vector,
the MuLV LTR drives the expression of neomycin
phosphotransferase (neo) gene that is used as a selection
marker. In this same vector, the grp78 promoter,
(spanning nucleotides -520 to +175 of the grp78 gene)
drives the HSVtk gene. The grp78 promoter fragment
contains three copies of the endoplasmic reticulum stress
element (ERSE), the TATA box, and an internal ribosome
entry site (IRES) in the 5' untranslated region
downstream of its transcription initiation site (+1). In
the GlTkSvNa vector, the MuLV LTR drives expression of
the HSVtk gene, while the SV40 promoter drives the neo
gene.
Figure 2 shows induction of HSVTK by the grp78
promoter under glucose starvation conditions. Panel A
shows equal amounts of cell lysates from the parental
B/ClOME cells, independently derived clonal cell lines
transduced with GITKSvNa (LTRtk#5), or transduced with
GlNaGrpTk (grptk#1 and grptk#3) were subjected to Western
blot analysis with antibodies against HSVTK, GRP78 and (3-
actin. The cells were grown under normal culture medium
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(+) or glucose-starved (GS) conditions for 24 h. Panel B
shows a bar graph indicating the intensity of the protein
bands quantitated by densitometry and normalized against
that of actin serving as an internal loading control.
The relative levels of HSVTK under normal culture or
glucose-starved conditions were plotted below the
autoradiograms, with the protein level in control cells
set as 1.
Figure 3 shows the results of an in vitro GCV-
sensitivity assay for B/ClOME cells. Panel A is a line
graph showing about 5 x 103 of GlTkSvNa/clone #3 clones
were seeded in duplicate into 6-well plates and incubated
without (X) or with 0.1 (closed circles, open circles)
~.g/ml GCV starting at day 3 as indicated. The cells were
then incubated in normal medium (-) or pretreated in
glucose-free medium (- - -), and the number of surviving
cells were determined by the trypan blue exclusion
method. Panel B shows data generated by the procedure
used in A except that GlNaGrpTk/clone #3 cells were used.
Panel C, in vitro bystander effect, non-transduced
B/ClOME cells (TK ) were co-cultured with different ratio
of B/ClOME clonal cell lines stably transfected with
GlNaGrpTk. A total of 3,000 cells with various ratios
were plated in quadruplicate in 96 well plate and treated
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with 10 mg/ml GCV for 10 days. The number of remaining
viable cells was measured by cell proliferation assay.
Figure 4 shows tumor growth curves for B/C10ME
fibrosarcoma. Panel A shows B/ClOME cells, B, three
independently derived GlTkSvNa clonal derivatives (#2,
#3, #5) or C, two independently derived GlNaGrpTk clonal
derivatives (#l, #3) were used. Equivalent numbers of 2
x 10' viable cells were subcutaneously injected into
BALB/c mice. Bi-perpendicular measurements were taken
over a period of 29 days. GCV (as indicated by arrows)
was administered daily starting at day 21 at a dosage of
100 mg/kg of body weight.
Figure 5 shows immunohistochemistry staining of
HSVtk protein expression in B/C10ME tumor tissues from
mice. Panel A shows that, after counterstaining the
tissue section with methyl green, no DAB stain can be
detected in tumor from non-transduced B/C10ME cells; B,
isolated patches of HSVtk protein expression can be
observed by cytoplasmic brown DAB staining in tumor from
B/C10ME cells transduced with GlTkSvNa; and C, high level
of HSVtk protein expression as shown by dark cytoplasmic
brown DAB staining in tumor from B/C10ME cells transduced
with GlNaGrpTk. The magnification is 200X.
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Figure 6 shows micropet images of hypoxia inducible
HSVtk expression in a murine mammary adenocarcinoma
model. The mice were bearing tumors derived from a
murine mammary adenocarcinoma cell line, TSA, which has
S been stably transfected with a retroviral vector,
GINaGRP-HSVtk, containing the GRP78 promoter that drives
HSVtk gene expression.
Figure 7 shows the presence of the LacZ transgene in
transgenic mice. Panel A is a diagram of the grp78/LacZ
Transgene construct comprising about 3000 base pairs of
the grp78 regulatory sequence operably linked to the LacZ
gene. Panel B, upper gel, shows a Southern hybridization
resulting in the identification of a LacZ nucleic acid
sequence in transgenic animals (Tg 132-147) containing
the construct shown in Panel A. In the lower gel, a
grp78 cDNA probe which hybridizes to the grp78 gene was
used to demonstrate that similar amounts of total DNA
were loaded in to each lane of the gel. The transgenic
sequences were identified using a suitably labeled LacZ
probe. Non-transgenic (Non-Tg) animals do not contain
the LacZ sequence. Panel C is a bar graph showing the
LacZ activity present in hamster cells tranfected with a
plasmid containing a nucleic acid construct shown in
panel A (grp78/LacZ) or a plasmid expressing LacZ from
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the SV40 large T antigen promoter sequence (SV40/LacZ).
Cells were treated with the calcium ionophore A23187 to
induce biological stress. Untreated and treated activity
is indicated.
Figure 8 shows a diagram of carcinogen treatment of
wild-type (+/+), heterozygous for the grp78/LacZ
transgene (Tg/+) or homozygous for the grp78/LacZ
transgene (Tg/Tg). The carcinogen (7,12-dimethyl bent
[a] anthracene) was applied subcutaneously on a weekly
basis over a period of six months. Subsequently, normal
and tumorous tissue were isolated and stained for
detection of LacZ expression.
Figure 9 shows color photographs of normal (non-
neoplastic) tissue derived from transgenic mice that are
homozygous for the grp78/LacZ transgene (Tg/Tg) or tissue
derived from wild-type (non-transgenic) mice (+/+). The
mice were treated as described in Figure 8.
Figure 10 shows color photographs of tumorous
tissues removed from mice treated as described in Figure
8. Tissue from mice heterozygous for the grp78/LacZ
transgene (Tg/+), homozygous for the grp78/LacZ transgene
(Tg/Tg) and wild-type (+/+) are indicated. Note that,
following LacZ-specific histological staining, LacZ
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expression is indicated in tumorous tissue derived from
Tg/+ mice as well as tissue derived from Tg/Tg mice.
Figure 11 shows additional color photographs of
tumorous tissues removed from mice treated as described
in Figure 8. Tissue from mice heterozygous for the
grp78/LacZ transgene (Tg/+) or homozygous for the
grp78/LacZ transgene (Tg/Tg) are indicated. Note that,
following LacZ-specific histological staining, LacZ
expression is indicated in tumorous tissue derived from
Tg/+ mice as well as tissue derived from Tg/Tg mice.
Like reference symbols in the various drawings
indicate like elements.
DETAILED DESCRIPTION
The present invention is directed to compositions
and methods for treating a subject diagnosed as having a
condition that can be treated by gene therapy. The
invention provides a means and method for delivering at
least one stress-responsive non-coding regulatory
sequence comprising at least two endoplasmic reticulum
stress elements (ERSE) as set forth in SEQ ID N0:1; and a
heterologous nucleic acid sequence operatively linked to
the regulatory sequence, wherein expression of the
heterologous sequence is regulated by the non-coding
sequence. The non-coding regulatory sequence comprising
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the ERSE nucleic acid sequences can be derived, for
example, from the transcription regulatory sequence of
the glucose responsive protein 78 (grp78) gene. In
addition, the invention provides transgenic animals the
S cells of which are homozygous or heterozygous for the
expression of a heterologous nucleic acid sequence driven
by a stress-responsive promoter sequence. Such animals
are useful, for example, for identifying glucose starved,
calcium deprived or hypoxic tissue present in the animal
during development or upon exposure to mitogenic
compounds, such as carcinogens. Further, such animals
can be used as models for the development of techniques
for the identification of biologically stressed tissue
associated with, for example, cell proliferative
disorders, such as cancer or disorders associated with
inflammation, such as arthritis.
The identification of endoplasmic reticulum stress
elements (ERSE) allows for the development of a nucleic
acid construct comprising a stress-responsive regulatory
sequence operably associated with a heterologous nucleic
acid sequence. Such a construct can be incorporated in,
for example, a vector suitable for gene therapy. As used
herein, a ERSE nucleic acid sequence derived from a grp78
regulatory sequence means a nucleic acid sequence as set
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forth in SEQ ID NO:1. It is believed that the ERSE
sequence of the invention can be incorporated into any
non-coding regulatory sequence that provides appropriate
transcriptional and translational initiation regions for
expression of a heterologous sequence in an animal cell.
Preferably, a non-coding regulatory sequence comprising
an ERSE nucleic acid sequence of the invention is derived
from the glucose responsive protein 78 (grp78) promoter
sequence comprising a sequence from about 3000 base pairs
5' of the site of initiation of transcription of the
grp78 coding sequence to about 200 base pairs 3' of the
site of initiation of the grp78 coding sequence,
constituting a 3200 base pair regulatory region of the
grp78 gene.
A construct of the invention can be used in
conjunction with a heterologous nucleic acid sequence
encoding a therapeutic agent. A therapeutic agent can
encode a suicide gene for treating a cell proliferative
disorder such as cancer or a therapeutic agent can encode
a protein useful for ameliorating the adverse effects of
glucose starvation in the cell of a diabetic subject, for
example. In addition, the present invention allows for
the production of non-human transgenic animals that
express a heterologous nucleic acid sequence from a grp78
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regulatory sequence. This exemplary animal model
provides a system for identifying, for example, factors
associated with tissue that is biologically stressed,
such as tumorous or inflammatory tissues. As used
herein, the term "biologically stressed" includes any
cellular environment indicative of cellular distress,
damage or trauma resulting in the activation of specific
factors that respond to such an environment. For
example, a biologically stressed tissue can result in a
cellular environment that is glucose starved, calcium
deprived, hypoxic, acidic or in a pathological state.
Biologically stressed further includes tissue generating
free radicals, or tissue that is hot or cold, inflamed or
transformed or any other biological state indicative of
stressed tissue.
The grp78 gene regulatory sequence is located from
about 3000 base pairs 5' of the site of initiation of
transcription of the grp78 coding sequence to about 200
base pairs 3' of the site of initiation of the grp78
coding sequence and exhibits strong expression in
biologically stressed tissue, such as tissue that is
glucose starved or hypoxic. Thus, a nucleic acid
construct of the invention can include a 3200 base pair
regulatory sequence derived from the grp78 gene. The
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genetic code for endoplasmic reticulum stress signaling
leading to grp gene induction consists of two units of a
19 base pair (bp) sequence motif (CCAAT)N9(CCACG) (SEQ ID
N0:1) termed ERSE. This sequence contains a tripartite
S structure, with a high affinity CBF/NF-Y binding site
separated by precisely 9 by of a GC rich sequence motif
to a low affinity YY1 binding site. The transcription
regulatory sequences further include transcriptional
control regions such as TATAA and CAAT box sequences as
well as sequences that regulate the tissue specificity
(i.e., biologically stressed tissue) of the transcribed
product. In the nucleic acid construct of the invention,
the ATG start codon is typically provided by the nucleic
acid sequence expressing the product of interest. As
used herein, a "nucleic acid construct" of the invention
includes at least one, or multiple, stress-responsive
non-coding regulatory sequences and a heterologous
nucleic acid sequence operatively linked to the
regulatory sequence, wherein expression of the
heterologous sequence is regulated by the non-coding
sequence. A nucleic acid construct of the invention can
be included in an expression vector. An "expression
vector" refers to a plasmid, virus or other vehicle known
in the art that has been manipulated by insertion or
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incorporation of the nucleic acid construct of the
invention. The expression vector typically contains an
origin of replication, as well as specific genes that
allow phenotypic selection of the transformed cells.
Vectors suitable for use in the present invention are
well known in the art.
As used herein, the term "regulatory sequence" or
"regulatory element" refers to a nucleic acid sequence
capable of controlling the transcription of an operably
associated gene. A regulatory sequence of the invention
may include a promoter, an enhancer and/or a silencer,
for example. Therefore, placing a gene under the
regulatory control of a promoter or a regulatory element
means positioning the gene such that the expression of
the gene is controlled by the regulatory sequence(s). In
general, promoters are found positioned 5' (upstream) of
the genes that they control. Thus, in the construction
of promoter gene combinations, the promoter is preferably
positioned upstream of the gene and at a distance from
the transcription start site that approximates the
distance between the promoter and the gene it controls in
the natural setting. As is known in the art, some
variation in this distance can be tolerated without loss
of promoter function. Similarly, the preferred
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positioning of a regulatory element, such as an enhancer,
with respect to a heterologous nucleic acid sequence
placed under its control reflects its natural position
relative to the structural gene it naturally regulates.
Enhancers are believed to be relatively position and
orientation independent in contrast to promoter elements.
The noncoding sequences or intron sequences (e. g., which
contain regulatory sequences) that are used in the
invention construct are not more than about 9kbp in
length.
Regulatory sequence function during expression of a
gene under its regulatory control and can be tested at
the transcriptional stage using DNA/RNA and RNA/RNA
hybridization assays (e. g., in situ hybridization,
nucleic acid hybridization in solution or solid support)
and at the translational stage using specific functional
assays for the protein synthesized (e. g., by enzymatic
activity, by immunoassay of the protein, by in vitro
translation of mRNA or expression in microinjected
xenopus oocytes).
As used herein, the term "nucleic acid sequence"
refers to a polymer of deoxyribonucleotides or
ribonucleotides, in the form of a separate fragment or as
a component of a larger construct. Nucleic acids
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expressing the products of interest can be assembled from
cDNA fragments or from oligonucleotides which provide a
synthetic gene which is capable of being expressed in a
recombinant transcriptional unit. Polynucleotide or
nucleic acid sequences of the invention include DNA, RNA
and cDNA sequences.
Nucleic acid sequences utilized in the invention can
be obtained by several methods. For example, the DNA can
be isolated using hybridization procedures that are well
known in the art. These include, but are not limited to:
1) hybridization of probes to genomic or cDNA libraries
to detect shared nucleotide sequences; 2) antibody
screening of expression libraries to detect shared
structural features and 3) synthesis by the polymerase
chain reaction (PCR). Sequences for specific genes can
also be found in GenBank, National Institutes of Health
computer database.
The term "heterologous nucleic acid sequence" as
used herein refers to at least one structural gene that
is operably associated with the regulatory sequence of
the invention. The nucleic acid sequence originates in a
foreign species, or, in the same species if substantially
modified from its original form. For example, the term
"heterologous nucleic acid sequence" includes a nucleic
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acid originating in the same species, where such sequence
is operably linked to a regulatory sequence that differs
from the natural or wild-type regulatory sequence (e. g.,
grp78 regulatory sequence). Thus, a non-coding
regulatory sequence of the invention can be operatively
linked to a heterologous nucleic acid sequence that is
regulated by the non-coding sequence.
The term "operably associated" refers to functional
linkage between the regulatory sequence and the nucleic
acid sequence regulated by the regulatory sequence. The
operably linked regulatory sequence controls the
expression of the product expressed by the nucleic acid
sequence. Alternatively, the functional linkage also
includes an enhancer element.
"Promoter" means the minimal nucleotide sequence
sufficient to direct transcription. Also included in the
invention are those promoter elements that are sufficient
to render promoter-dependent nucleic acid sequence
expression controllable for cell-type specific, tissue
specific, or inducible by external signals or agents;
such elements may be located in the 5' or 3' regions of
the native gene, or in the introns.
"Gene expression" or "nucleic acid sequence
expression" means the process by which a nucleotide
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sequence undergoes successful transcription and
translation such that detectable levels of the delivered
nucleotide sequence are expressed in an amount and over a
time period so that a functional biological effect is
achieved. "Expressible genetic construct" as used herein
means a construct that has the grp78 regulatory sequences
positioned with a heterologous nucleic acid sequence
encoding a desired product, such that the nucleic acid
sequence is expressed.
A heterologous nucleic acid sequence of the
invention can encode a "therapeutic agent" effective for
treating, for example, a cell proliferative disorder or a
disorder associated with glucose starvation, such as
diabetes. As used herein, a "therapeutic agent" can
include a structural gene that encodes a biologically
active protein of interest. The term "structural gene"
excludes the non-coding regulatory sequence that drives
transcription. The structural gene may be derived in
whole or in part from any source known to the art,
including a plant, a fungus, an animal, a bacterial
genome or episome, eukaryotic, nuclear or plasmid DNA,
cDNA, viral DNA or chemically synthesized DNA. A
structural gene may contain one or more modifications in
either the coding or the untranslated regions which could
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affect the biological activity or the chemical structure
of the expression product, the rate of expression or the
manner of expression control. Such modifications
include, but are not limited to, mutations, insertions,
deletions and substitutions of one or more nucleotides.
The structural gene may constitute an uninterrupted
coding sequence or it may include one or more introns,
bound by the appropriate splice junctions. The
structural gene may also encode a fusion protein. It is
contemplated that introduction into animal tissue of
nucleic acid constructs of the invention will include
constructions wherein the structural gene and its
regulatory sequence are each derived from different
animal species.
A structural gene can encode an enzyme, such as a
drug-metabolizing enzyme that confers a dominant,
negatively selectable phenotype to a cell, such as cell
death. Such a gene can encode an enzyme that can convert
a non-therapeutically effective compound in to a
therapeutically effective compound. For example, the
activation of a relatively nontoxic (i.e., non-
therapeutically effective) prodrug to a cytotoxic (i.e.,
therapeutically effective) compound in a specifically
targeted tissue can be used to effectively treat a cell
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proliferative disorder. Enzymes capable of performing
such a function include herpes simplex virus (HSV)
thymidine kinase, vesicular stomatitis virus (VSV)
thymidine kinase, deoxycytidine kinase, cytosine
deaminase or nucleoside phosphorylase. Prodrugs
converted by the aforementioned enzymes include
ganciclovir, acyclovir, 6-methoxypurine arabinoside (Ara-
M), cytosine arabinoside or cytarabine (Ara-C),
fludarabine, 2-chlorodeoxyadenosine,
difluorodeoxycytidine, 5-fluorocytidine and 6-
methylpurine-2'-deoxyriboside (MeP-dr).
Because current gene transfer techniques are unable
to achieve a satisfactorily high level of transfer
efficiency in an in vivo setting, alternative strategies
that do not require 1000 efficiency of gene transfer have
been sought. Two general approaches have evolved that
may be effective when only a minority of the tumor cells
are transduced: (1) cell-targeted suicide, achieved by
directing the synthesis of a toxic metabolite that can
permeate the tumor microenvironment, and (2) engineering
an immune response to the tumor cells by ectopic cytokine
expression or other means for immune recognition or
activation.
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Examples of genes encoding therapeutic agents that
can be used in the invention construct include genes
encoding enzymes that convert a prodrug to a toxic
metabolite. As noted above, a variety of enzymes are
capable of performing such a function, and typically kill
cells by activation of a relatively nontoxic prodrug to a
cytotoxic form. Greater selectivity in killing malignant
cells will be obtained if the transferred gene is not
normally found in human beings (e. g., HSV-thymidine
kinase), rather than by overexpressing an endogenous gene
(e. g., deoxycytidine kinase).
The tumoricidal activity of the HSV-TK/ganciclovir
system is due to several factors. In dividing cells, the
phosphorylated ganciclovir inhibits DNA synthesis. This
effect is not confined to cells that are directly
transduced with HSV-TK, as neighboring cells are also
affected. This phenomenon, which likely occurs as a
result of several mechanisms, has been termed the
"bystander effect" and has been observed in several tumor
types, including CNS tumors. Transfer of the
phosphorylated ganciclovir between cells ("metabolic
cooperation") via gap junctions has been proposed as a
possible mechanism. Phagocytosis by neighboring cells of
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-2 6-
ganciclovir phosphate-containing apoptotic vesicles (from
dying transduced cells) also has been proposed.
In addition, a therapeutic agent of the invention
includes nucleic acid sequences encoding tumor suppressor
proteins such as p53 (Takahashi et al. Cancer Res.
62:2340, 1992) and Retinoblastoma (RB); and nucleic acid
sequences encoding apoptosis or cell death promoting
proteins such as Fas (Itoh et al., Cell 66:233, 1991),
GAX (PCT/US95/01882), and FADD (Chinnalyan et al. Cell,
81:505, 1995) which interacts with the death domain of
Fas and initiates apoptosis.
A therapeutic agent of the invention also includes
nucleic acid sequences that encode cell cycle blockers
such as GATA-6 (Suzuki et al, Genomics, 38:283, 1996),
anti-angiogenesis proteins such as endostatin and
angistatin (Folkman J., Nature Med. 1:27, 1995), anti-
sense gene sequences (Wang, Nature Med. 3:887, 1997), and
viral subunit vaccines (Donnelly et al. Nature Med.
1:583, 1995).
A therapeutic agent also encompasses those sequences
encoding proteins, such as asparaginase, that induce cell
death by depriving a cell of a necessary metabolite.
Asparaginase induces apoptosis by catalyzing the
hydrolysis of circulating asparagine to aspartic acid and
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ammonia, thus depriving cells of the asparagine necessary
for protein synthesis, leading to cell death.
A therapeutic agent of the invention also includes
immunomodulators and other biological response modifiers.
The term "biological response modifiers" encompasses
substances that are involved in modifying the immune
response in such manner as to enhance the destruction of
tumor, for example. Examples of immune response
modifiers include such compounds as lymphokines.
Lymphokines include tumor necrosis factor, the
interleukins, lymphotoxin, macrophage-activating factor,
migration inhibition factor, colony stimulating factor,
and interferon. Included in this category are
immunopotentiating agents including nucleic acids
encoding a number of the cytokines classified as
"interleukins". These include, for example, interleukins
1 through 12. Also included in this category, although
not necessarily working according to the same mechanisms,
are interferons, and in particular gamma interferon (Y-
IFN), tumor necrosis factor (TNF) and granulocyte-
macrophage-colony stimulating factor (GM-CSF). Nucleic
acids encoding growth factors, toxic peptides, ligands,
receptors, suicide factors (e. g., TK) or other
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physiologically important proteins can also be introduced
into specific cells of the prostate.
Further, a therapeutic agent includes sense or
antisense nucleic acids encoded by a heterogenous nucleic
acid of the invention. For example, a sense
polynucleotide sequence (the DNA coding strand) encoding
a polypeptide can be introduced into the cell to increase
expression of a "normal" gene. Other cell disorders can
also be treated with nucleic acid sequences that
interfere with expression at the translational level.
This approach utilizes, for example, antisense nucleic
acid, ribozymes, or triplex agents to block transcription
or translation of a specific mRNA, either by masking that
mRNA with an antisense nucleic acid or triplex agent, or
by cleaving it with a ribozyme. Alternatively, the
method includes administration of a reagent that mimics
the action or effect of a gene product or blocks the
action of the gene. Therefore, when a cell proliferative
disorder, such as cancer, is etiologically linked with
over expression of a polynucleotide, it would be
desirable to administer an inhibiting reagent such as an
antisense polynucleotide. For example, overexpression of
the bcl-2 gene that is translocated in nodular non-
Hodgkin's lymphomas, inactivates a key pathway of
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programmed cell death (apoptosis) and leads to continuous
proliferation and survival of highly mutated tumor cells
that have the capacity to survive DNA damage. Similarly,
an increase in expression of the D cyclin (the prad
oncogene) promotes cell entry into DNA synthesis.
Additional oncogenes that promote cell proliferation
include ABL, ERBB-1, ERBB-2 (NEU), GIP, GSP, MYC, L-MYC,
N-MYC, H-RAS, RET, ROS, K-SAM, SIS, SRC, C-FOS, C-JUN AND
TRK. Thus, efforts directed toward restoring apoptosis
in tumor cells, by inhibiting the overexpression of an
apoptosis inhibitor, such as bcl-2, or cell proliferation
promoting oncogene, such as Ras, can be accomplished
using antisense methodology.
The use of antisense methods to inhibit the in vitro
translation of genes is well known in the art (see, e.g.,
Marcus-Sakura, Anal. Biochem., 172:289, 1988). Antisense
nucleic acids are nucleic acid molecules (e. g., molecules
containing DNA nucleotides, RNA nucleotides, or
modifications (e.g., modification that increase the
stability of the molecule, such as 2'-O-alkyl (e. g.,
methyl) substituted nucleotides) or combinations thereof)
that are complementary to, or that hybridize to, at least
a portion of a specific nucleic acid molecule, such as an
RNA molecule (e. g., an mRNA molecule) (see, e.g.,
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Weintraub, Scientific American, 262:40, 1990). The
antisense nucleic acids hybridize to corresponding
nucleic acids, such as mRNAs, to form a double-stranded
molecule, which interferes with translation of the mRNA,
as the cell will not translate a double-stranded mRNA.
Antisense nucleic acids used in the invention are
typically at least 10-12 nucleotides in length, for
example, at least 15, 20, 25, 50, 75, or 100 nucleotides
in length. The antisense nucleic acid can also be as
long as the target nucleic acid with which it is intended
to form an inhibitory duplex. As is described further
below, the antisense nucleic acids can be introduced into
cells as antisense oligonucleotides, or can be produced
in a cell in which a nucleic acid encoding the antisense
nucleic acid has been introduced by, for example, using
gene therapy methods.
Gene Therapy
The present invention also provides gene therapy for
the treatment of a cell proliferative disorder. Such
therapy would achieve its therapeutic effect by
introduction of the nucleic acid construct of the
invention into cells having the disorder such that a
heterologous nucleic acid sequence encoding a therapeutic
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agent or a detectable marker is expressed from a stress-
responsive non-coding regulatory sequence. Preferably,
the regulatory sequence is isolated from a glucose
responsive protein 78 (grp78), however any regulatory
sequence suitable for expression biologically stressed
cells and/or tissue can be used in the present invention.
Delivery of a nucleic acid construct of the
invention can be achieved by introducing the construct
into a cell using a variety of methods known to those of
skill in the art. For example, the construct can be
delivered into a cell using a colloidal dispersion
system. Alternatively, nucleic acid construct of the
invention can be incorporated (i.e., cloned) into an
appropriate vector. For example, a recombinant vector of
the invention can be an expression vector suitable for
expression of the heterologous sequence in a target cell,
such as a cell that is biologically stressed.
Preferably, a recombinant vector comprising nucleic acid
construct of the invention includes a replication
competent or replication incompetent recombinant viral
vector. For example, a recombinant viral vector of the
invention can be derived from an RNA virus (i.e.,
retrovirus) such s lentivirus, or a DNA virus such as
adenovirus. Delivery of a construct of the invention
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into a cell can be performed in vivo or ex vivo.
Further, methods of the invention can be performed alone
or in conjunction with standard medical treatments
currently available for treating a cell proliferative
disorder. For example, when a tumor is being treated, it
may be preferable to remove the majority of a tumor
surgically or by radiation prior to introducing a
construct of the invention in to the cells comprising the
tumor.
Various viral vectors that can be utilized for gene
therapy, as taught herein, include DNA viruses such as
adenovirus, herpes virus, vaccinia, or an RNA virus such
as a retrovirus. The retroviral vector can be a
derivative of a retrovirus capable of infecting a
mammalian host cell. Examples of retroviral vectors in
which a foreign gene can be inserted include, but are not
limited to: Moloney murine leukemia virus (MoMuLV),
Harvey murine sarcoma virus (HaMuSV), murine mammary
tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
Preferably, when the subject is a human, a vector such as
the gibbon ape leukemia virus (GaLV) is utilized. A
number of additional retroviral vectors can incorporate
multiple genes. All of these vectors can transfer or
incorporate a nucleic acid construct of the invention
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into a target cell. By inserting the construct of the
invention into the viral vector along with another gene
that encodes ligand for a receptor on a specific target
cell, for example, the vector is now target cell entry
S specific as well target cell expression specific.
Preferred targeting is accomplished by using an antibody
to target the retroviral vector. Those of skill in the
art will know of, or can readily ascertain without undue
experimentation, specific polynucleotide sequences which
can be inserted into the retroviral genome, for example,
to allow target specific delivery of the retroviral
vector containing the construct of the invention.
Retroviruses are RNA viruses wherein the viral
genome is RNA. When a host cell is infected with a
retrovirus, the genomic RNA is reverse transcribed into a
DNA intermediate which is integrated very efficiently
into the chromosomal DNA of infected cells. The
integrated DNA intermediate is referred to as a provirus.
The family Retroviridae are enveloped single-stranded RNA
viruses that typically infect mammals as well as avian
species. Retroviruses are unique among RNA viruses in
that their multiplication involves the synthesis of a DNA
copy of the RNA that is then integrated into the genome
of the infected cell.
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The Retroviridae family consists of three groups:
the spumaviruses (or foamy viruses) such as the human
foamy virus (HFV); the lentiviruses, as well as visna
virus of sheep; and the oncoviruses (although not all
viruses within this group are oncogenic). The term
"lentivirus" is used in its conventional sense to
describe a genus of viruses containing reverse
transcriptase. The lentiviruses include the
"immunodeficiency viruses" which include human
immunodeficiency virus (HIV) type 1 and type 2 (HIV-1 and
HIV-2) and simian immunodeficiency virus (SIV).
Retroviruses are defined by the way in which they
replicate their genetic material. During replication the
RNA is converted into DNA. Following infection of the
cell a double- stranded molecule of DNA is generated from
the two molecules of RNA that are carried in the viral
particle by the molecular process known as reverse
transcription. The DNA form becomes covalently integrated
in the host cell genome as a provirus, from which viral
RNAs are expressed with the aid of cellular and/or viral
factors. The expressed viral RNAs are packaged into
particles and released as infectious virion.
Retroviruses can be transmitted horizontally and
vertically. Efficient infectious transmission of
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retroviruses requires the expression on the target cell
of receptors that specifically recognize the viral
envelope proteins, although viruses may use receptor-
independent, nonspecific routes of entry at low
efficiency. In addition, the target cell type must be
able to support all stages of the replication cycle after
virus has bound and penetrated. Vertical transmission
occurs when the viral genome becomes integrated in the
germ line of the host. The provirus will then be passed
from generation to generation as though it were a
cellular gene. Hence endogenous proviruses become
established which frequently lie latent, but which can
become activated when the host is exposed to appropriate
agents.
Numerous gene therapy methods, that take advantage
of retroviral vectors, for treating a wide variety of
diseases are known in the art (see, e.g., U.S. Pat. Nos.
4,405,712 and 4,650,764; Friedmann, 1989, Science,
244:1275-1281; Mulligan, 1993, Science, 260:926-932, R.
Crystal, 1995, Science 270:404-410, each of which are
incorporated herein by reference in their entirety). An
increasing number of these methods are currently being
applied in human clinical trials (Morgan, 1993, BioPharm,
6(1):32-35; see also The Development of Human Gene
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Therapy, Theodore Friedmann, Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, NY, 1999. ISBN 0-
87969-528-5, which is incorporated herein by reference in
its entirety). The safety of these currently available
gene therapy protocols can be substantially increased by
using retroviral vectors of the present invention. For
example, where the retroviral vector infects a non-
targeted cell, the retroviral genome will integrate but
the heterologous nucleic acid sequence will not be
transcribed unless the cell or tissue is biologically
stressed. However, when the retroviral vector containing
a nucleic acid construct of the invention infects a
targeted cell (i.e., a cell that is glucose starved,
calcium deprived, hypoxic, etc.) the activation of the
stress-responsive regulatory sequence will result in
transcription and translation of the heterologous nucleic
acid sequence.
Recombinant retroviruses defective for replication
require assistance in order to produce infectious vector
particles. This assistance can be provided, for example,
by using helper cell lines that contain plasmids encoding
all of the structural genes of the retro virus under the
control of regulatory sequences within the LTR. These
plasmids are missing a nucleotide sequence that enables
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the packaging mechanism to recognize an RNA transcript
for encapsidation. Helper cell lines which have
deletions of the packaging signal include but are not
limited to iY2, PA317 and PA12, for example. These cell
lines produce empty virions, since no genome is packaged.
If a retroviral vector is introduced into such cells in
which the packaging signal is intact, but the structural
genes are replaced by other genes of interest, the vector
can be packaged and vector virion produced.
Another targeted delivery system useful for
introducing a nucleic construct of the invention into a
target cell is a colloidal dispersion system. Colloidal
dispersion systems include macromolecule complexes,
nanocapsules, microspheres, beads, and lipid-based
systems including oil-in-water emulsions, micelles, mixed
micelles, and liposomes. The preferred colloidal system
of this invention is a liposome. Liposomes are
artificial membrane vesicles that are useful as delivery
vehicles in vitro and in vivo. It has been shown that
large unilamellar vesicles (LUV), which range in size
from 0.2-4.0 um can encapsulate a substantial percentage
of an aqueous buffer containing large macromolecules.
RNA, DNA and intact virions can be encapsulated within
the aqueous interior and be delivered to cells in a
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biologically active form (Fraley, et al., Trends Biochem.
Sci., 6:77, 1981). In order for a liposome to be an
efficient gene transfer vehicle, the following
characteristics should be present: (1) encapsulation of
the nucleic acid of interest (i.e., a nucleic acid
construct of the invention or a vector comprising the
construct) at high efficiency while not compromising
their biological activity; (2) preferential and
substantial binding to a target cell in comparison to
non-target cells; (3) delivery of the aqueous contents of
the vesicle to the target cell cytoplasm at high
efficiency; and (4) accurate and effective expression of
genetic information (Mannino, et al., Biotechniques,
6:682, 1988).
In preferred embodiments, the grp78 regulatory
sequence comprises at least one stress-responsive nucleic
acid sequence regulatable by factors present in
biologically stressed cells and tissues such as glucose
starved or hypoxic cells or tissue. In one aspect of the
invention, the expression of a heterologous nucleic acid
sequence encoding a therapeutic agent or detectable
marker is regulated by fusion of the heterologous nucleic
acid, or a fragment thereof, to at least one stress-
responsive regulatory sequence, such as, for example, a
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grp78 regulatory sequence. A grp78 regulatory sequence
is one that is not normally associated with, and does not
normally regulate, the expression of a heterologous
nucleic acid that it regulates in the practice of the
invention. Grp78 regulatory elements can comprise
transcriptional, post-transcriptional, translational, and
post-translational elements; as well as regulatory
elements related to replication. By way of example,
grp78 transcriptional regulatory elements can include
promoters, enhancers, operators, and elements that
modulate the rate of transcription initiation, elongation
and/or termination; post-transcriptional regulatory
elements can include those influencing messenger
stability, processing and transport; translational
regulatory elements can include those which modulate the
frequency of translation initiation and the rate of
translational elongation; post-translational regulatory
elements can include those which influence protein
processing, stability and transport; and replication-
associated regulatory elements can include those related
to gene dosage.
In one embodiment, the invention provides
recombinant vectors comprising a nucleic acid construct
of the invention. The recombinant vectors are made using
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standard methods of molecular biology and biotechnology
to incorporate a nucleic acid construct of the invention
containing a heterologous nucleic acid sequence in
operative linkage with a stress-responsive regulatory
S sequence, such as a grp-78 regulatory sequence. In
preferred embodiments, the grp-78 regulatory sequence
will be upstream of the heterologous sequence when they
are placed in operative linkage. Locations of
restriction enzyme recognition sequences can be easily
determined by one of skill in the art. Alternatively,
various in vitro techniques can be used for insertion of
a restriction enzyme recognition sequence at a particular
site, or for insertion of nucleic acid construct at a
site that does not contain a restriction enzyme
recognition sequence. Such methods include, but are not
limited to, oligonucleotide-mediated heteroduplex
formation for insertion of one or more restriction enzyme
recognition sequences (see, for example, Zoller et al.
(1982) Nucleic Acids Res. 10:6487-6500; Brennan et al.
(1990) Roux's Arch. Dev. Biol. 199:89-96; and Kunkel et
al. (1987) Meth. Enzymology 154:367-382) and PCR-mediated
methods for insertion of longer sequences. See, for
example, Zheng et al. (1994) Virus Research 31:163- 186.
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Operative linkage refers to an arrangement of one or
more regulatory sequences with one or more coding
sequences, such that the regulatory sequences) is
capable of exerting its regulatory effect on the coding
sequence.
By way of illustration, a stress responsive-
transcriptional regulatory sequence or a promoter is
operably linked to a heterologous sequence if the
transcriptional regulatory sequence or promoter promotes
transcription of the heterologous sequence. Similarly,
an operator is considered operatively linked to a
promoter or to a heterologous sequence if binding of a
repressor to the operator inhibits initiation at the
promoter so as to prevent or diminish expression of the
heterologous sequence. An operably linked
transcriptional regulatory sequence is generally joined
in cis with the coding sequence, but it is not
necessarily directly adjacent to it.
Recombinant vectors comprising a nucleic acid
construct of the invention can also comprise other types
of sequence including, but not limited to, replication
origins, detectable markers (including, but not limited
to, those encoding antibiotic resistance), transcription
termination sites, sequences specifying translation
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initiation and termination, sequences mediating mRNA
processing and/or stability and multiple cloning sites.
Recombinant vectors can exist as freely-replicating
extrachromosomal elements, such as plasmids or episomes,
S or can exist as chromosomal recombinants, such as would
be achieved either by integration of a nucleic acid
construct into the chromosome of a cell. Methods for
obtaining chromosomal integration of recombinant vectors
have been described, for example, by Gerhardt et al.,
METHODS FOR GENERAL AND MOLECULAR MICROBIOLOGY, American
Society for Microbiology, Washington, D.C., 1994; Link et
al. (1997) J Bacteriol. 179:6228-6237; and Metcalf et al.
(1996) Plasmid 35:1-13.
A coding sequence, as present in a recombinant
construct, can encode a full-length nucleic product
(i.e., the length normally found in the wild- type cell)
or any fragment of a gene product. A gene product can be
RNA or a polypeptide; untranslated RNA gene products can
include structural, catalytic and regulatory RNA
molecules. Examples of untranslated RNA gene products
include, but are not limited to, tRNA, rRNA, antisense
RNAs and ribozymes. In one embodiment, a coding sequence
comprises a gene, which can encode a therapeutic agent,
or a gene product whose function is to act as a
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detectable marker under a particular set of environmental
conditions. It is understood that any gene of interest
can be placed in operative linkage with grp-78 regulatory
region sequences, so that its expression is regulated by
the grp78 regulatory region sequences.
The phrase "non-dividing" cell refers to a cell that
does not go through mitosis. Non-dividing cells may be
blocked at any point in the cell cycle, (e. g., GO/G1,
G1/S, G2/M), as long as the cell is not actively
dividing. For ex vivo infection, a dividing cell can be
treated to block cell division by standard techniques
used by those of skill in the art, including,
irradiation, aphidocolin treatment, serum starvation, and
contact inhibition. However, it should be understood
that ex vivo infection is often performed without
blocking the cells since many cells are already arrested
(e. g., stem cells). For example, a recombinant
lentivirus vector of the invention is capable of
infecting any non-dividing cell, regardless of the
mechanism used to block cell division or the point in the
cell cycle at which the cell is blocked. Examples of
pre-existing non-dividing cells in the body include
neuronal, muscle, liver, skin, heart, lung, and bone
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marrow cells, and their derivatives. For dividing cells
onco-retroviral vectors can be used.
By "dividing" cell is meant a cell that undergoes
active mitosis, or meiosis. Such dividing cells include
stem cells, skin cells (e.g., fibroblasts and
keratinocytes), gametes, and other dividing cells known
in the art. Of particular interest and encompassed by
the term dividing cell are cells having cell
proliferative disorders, such as neoplastic cells. The
term "cell proliferative disorder" refers to a condition
characterized by an abnormal number of cells. The
condition can include both hypertrophic (the continual
multiplication of cells resulting in an overgrowth of a
cell population within a tissue) and hypotrophic (a lack
or deficiency of cells within a tissue) cell growth or an
excessive influx or migration of cells into an area of a
body. The cell populations are not necessarily
transformed, tumorigenic or malignant cells, but can
include normal cells as well.
The present invention provides gene therapy for the
treatment of cell proliferative disorders or disorders
associated with glucose starvation such as diabetes.
Such therapy would achieve its therapeutic effect by
introduction of a nucleic acid construct encoding an
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appropriate therapeutic agent (e. g., suicide gene, tumor
suppressor genes, antisense, ribozymes), into cells of
subject having the disorder. Delivery of such a nucleic
acid constructs can be achieved using a viral vector of
the present invention.
Cell proliferative disorders include disorders
associated with an overgrowth of connective tissues, such
as various fibrotic conditions, including scleroderma,
arthritis and liver cirrhosis. Cell proliferative
disorders include neoplastic disorders such as head and
neck carcinomas. Head and neck carcinomas would include,
for example, carcinoma of the mouth, esophagus, throat,
larynx, thyroid gland, tongue, lips, salivary glands,
nose, paranasal sinuses, nasopharynx, superior nasal
vault and sinus tumors, esthesioneuroblastoma, squamous
call cancer, malignant melanoma, sinonasal
undifferentiated carcinoma (SNUG) or blood neoplasia.
Also included are carcinoma's of the regional lymph nodes
including cervical lymph nodes, prelaryngeal lymph nodes,
pulmonary juxtaesophageal lymph nodes and submandibular
lymph nodes (Harrison's Principles of Internal Medicine
(eds., Isselbacher, et al., McGraw-Hill, Inc., 13th
Edition, pp1850-1853, 1994). Other cancer types,
include, but are not limited to, lung cancer, colon-
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rectum cancer, breast cancer, prostate cancer, urinary
tract cancer, uterine cancer lymphoma, oral cancer,
pancreatic cancer, leukemia, melanoma, stomach cancer and
ovarian cancer.
Disorders associated with glucose starvation include
diabetes or any other disorder wherein tissue is
constantly or periodically subjected to low glucose
availability such that the cells of the tissue are
biologically stressed.
In addition, the therapeutic methods (e. g., the gene
therapy or gene delivery methods) as described herein can
be performed in vivo or ex vivo. It may be preferable to
remove the majority of a tumor prior to gene therapy, for
example surgically or by radiation.
The invention also provides a method of nucleic acid
transfer to a target cell to provide expression of a
particular nucleic acid sequence (e. g., a heterologous
sequence). Therefore, in another embodiment, the
invention provides a method for introduction and
expression of a heterologous nucleic acid sequence in a
target cell comprising infecting the target cell with a
recombinant virus of the invention containing a nucleic
acid construct of the invention and expressing the
heterologous nucleic acid sequence in the target cell.
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As mentioned above, the target cell can be any cell type
including dividing, non-dividing, neoplastic,
immortalized, modified and other cell types recognized by
those of skill in the art, so long as they are capable of
infection by a retrovirus.
In another embodiment, the invention provides a
method of treating a subject having a cell proliferative
disorder. The subject can be any mammal, and is
preferably a human. The subject is contacted with a
recombinant vector of the present invention. The
recombinant vector is preferably a recombinant viral
vector and more preferably a recombinant retroviral
vector. The contacting can be in vivo or ex vivo.
Methods of administering the vector of the invention are
known in the art and include, for example, systemic
administration, topical administration, intraperitoneal
administration, intra-muscular administration, as well as
administration directly at the site of a tumor or cell-
proliferative disorder and other routes of administration
known in the art.
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Pharmaceutical Compositions
The invention further includes various
pharmaceutical compositions useful for treating a cell
proliferative disorder or a disorder associated with
glucose starvation such as, for example, diabetes. The
present invention provides a nucleic acid construct
capable of driving the expression of a therapeutic agent
in a cell associated with biologically stressed tissue.
A biologically stressed tissue of the invention
includes those tissues where the cellular environment is
"naturally" glucose starved, calcium deprived, hypoxic,
acidic or in a pathological state. Biologically stressed
further includes tissue generating free radicals, or
tissue that is hot or cold, inflamed or transformed or
any other biological state indicative of stressed tissue.
A naturally biologically stressed tissue is a tissue
wherein normal cellular metabolism in conjunction with a
pathological state has induced the biological stress.
For example, a fast growing solid tumor devoid of
nutrients due to insufficient blood supply exposes the
neoplastic cells contained in such an environment to
glucose deprivation, calcium deprivation, chronic anoxia
and low pH. Thus, the cells in such an environment are
subjected to biological stress that is induced by a
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pathological state resulting from tumor growth. The
nucleic acid construct of the invention can be used to
express a therapeutic agent, such as, for example, a
suicide gene, or an apoptosis-inducing gene, such that
the targeted cell is killed. The surrounding healthy
tissue remains unaffected by the treatment because they
do not provide a biologically stressed necessary for
expression of the therapeutic agent.
In addition, diabetes is a disease that results in
glucose starvation in a wide range of tissues. Cells
subjected to glucose deprivation must utilize other
sources of energy in order to survive. The consequences
of this type metabolism is a cellular environment that
is, for example, acidic. Again, a nucleic acid construct
of the invention can be used to express a therapeutic
agent such that the acidic environment of a targeted cell
can be ameliorated by expression of the agent.
A biologically stressed tissue of the invention also
includes those tissues where a biologically stressed
cellular environment has been "artificially" induced.
For example, photodynamic therapy involves the combined
use of photosensitizing drugs and light for the treatment
of malignant or benign disease. The photosensitized
chemical reaction requires oxygen. Light, delivered to
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the tissue, activates porphyrin molecules. These
molecules transfer their energy to form cytotoxic singlet
oxygen, which results in lethal alteration of cellular
membranes and subsequent tissue destruction. Artificial
means for inducing biological stress also include
compounds such as combretastatin A4-phosphate (CA4DP).
CA4DP has been used as an antiangiogenesis agent to
prevent or reduce the blood supply to, for example,
tumorous tissue. Reduced blood supply facilitated by
CA4DP, or any other antiangiogenic agent, promotes
biological stress in the affected tissue and provides the
appropriate environment for expression of a therapeutic
agent of the invention.
Thus, a nucleic acid construct of the invention can
be used in conjunction with a method for "artificially"
inducing a biologically stressed cellular environment.
For example, the construct can be introduced into a cell
as part of a pharmaceutical composition comprising, for
example, a liposomal delivery vehicle or a viral delivery
vehicle, prior to, during, or subsequent to artificial
induction of biological stress. Potential uses in
dermatology include the treatment of malignant cutaneous
lesions and nononcologic conditions, including psoriasis,
alopecia, viral infections, and vascular malformations.
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Photodynamic therapy also has been employed for bladder,
endobronchial, and esophageal carcinoma.
The pharmaceutical compositions according to the
invention are prepared by placing a nucleic acid
construct of the invention into a form suitable for
administration to a subject using carriers, excipients
and additives or auxiliaries. The nucleic acid construct
can be contained in a recombinant vector, preferably a
recombinant viral vector and most preferably a
recombinant retroviral vector. A pharmaceutical
composition can include a nucleic acid construct of the
invention comprising at least one stress-responsive non-
coding regulatory sequence comprising at least two
endoplasmic reticulum stress elements (ERSE).
Preferably, the stress-responsive non-coding regulatory
sequence is derived from a glucose responsive protein 78
(grp78) gene. A heterologous nucleic acid sequence
operatively linked to the regulatory sequence. The
expression of the heterologous sequence is regulated by
the non-coding sequence and the heterologous sequence can
encode a therapeutic agent effective for treating, for
example, a cell proliferative disorder.
Generally, the terms "treating", "treatment", and
the like are used herein to mean obtaining a desired
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pharmacologic and/or physiologic effect. The effect may
be therapeutic in terms of a partial or complete cure for
a cell proliferative disorder. "Treating" as used herein
covers any treatment of (e.g., complete or partial), or
prevention of, a cell proliferation disorder or for
ameliorating the pathogenic effect of biological stress,
such as biological stress induced by glucose deprivation,
in a mammal, particularly a human, and includes:
(a) preventing the disease from occurring in a
subject that may be predisposed to the disease,
but has not yet been diagnosed as having it;
(b) inhibiting the disorder, i.e., arresting the
development of, for example, a tumor; or
(c) relieving or ameliorating the disorder or
disease, i.e., cause regression of the disorder
or disease.
Thus, the invention includes various pharmaceutical
compositions useful for ameliorating symptoms
attributable to a cell proliferative disorder or,
alternatively, for inducing a protective immune response
to treat a cell proliferative disorder or for
ameliorating the pathogenic effect of biological stress.
For example, a pharmaceutical composition according to
the invention can be prepared to include a nucleic acid
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construct according to the invention into a form suitable
for administration to a subject using carriers,
excipients and additives or auxiliaries. Frequently used
carriers or auxiliaries include magnesium carbonate,
titanium dioxide, lactose, mannitol and other sugars,
talc, milk protein, gelatin, starch, vitamins, cellulose
and its derivatives, animal and vegetable oils,
polyethylene glycols and solvents, such as sterile water,
alcohols, glycerol and polyhydric alcohols. Intravenous
vehicles include fluid and nutrient replenishers.
Preservatives include antimicrobial, anti-oxidants,
chelating agents and inert gases. Other pharmaceutically
acceptable carriers include aqueous solutions, non-toxic
excipients, including salts, preservatives, buffers and
the like, as described, for instance, in Remington's
Pharmaceutical Sciences, 15th ed. Easton: Mack
Publishing Co., 1405-1412, 1461-1487 (1975) and The
National Formulary XIV., 14th ed. Washington: American
Pharmaceutical Association (1975), the contents of which
are hereby incorporated by reference. The pH and exact
concentration of the various components of the
pharmaceutical composition are adjusted according to
routine skills in the art. See Goodman and Gilman's The
Pharmacological Basis for Therapeutics (7th ed.).
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The pharmaceutical compositions according to the
invention may be administered locally or systemically.
By "therapeutically effective dose" is meant the quantity
of a compound according to the invention necessary to
prevent, to cure or at least partially arrest the
symptoms of the disease and its complications. Amounts
effective for this use will, of course, depend on the
severity of the disease and the weight and general state
of the patient. Typically, dosages used in vitro may
provide useful guidance in the amounts useful for in situ
administration of the pharmaceutical composition, and
animal models may be used to determine effective dosages
for treatment of particular disorders. Various
considerations are described, e.g., in Langer, Science,
249: 1527, (1990); Gilman et al. (eds.) (1990), each of
which is herein incorporated by reference.
As used herein, "administering a therapeutically
effective amount" is intended to include methods of
giving or applying a pharmaceutical composition of the
invention to a subject that allow the composition to
perform its intended therapeutic function. The
therapeutically effective amounts will vary according to
factors such as the degree of infection in a subject, the
age, sex, and weight of the individual. Dosage regima
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can be adjusted to provide the optimum therapeutic
response. For example, several divided doses can be
administered daily or the dose can be proportionally
reduced as indicated by the exigencies of the therapeutic
situation.
The pharmaceutical composition can be administered
in a convenient manner such as by injection
(subcutaneous, intravenous, etc.), oral administration,
inhalation, transdermal application, or rectal
administration. Depending on the route of
administration, the pharmaceutical composition can be
coated with a material to protect the pharmaceutical
composition from the action of enzymes, acids and other
natural conditions that may inactivate the pharmaceutical
composition. The pharmaceutical composition can also be
administered parenterally or intraperitoneally.
Dispersions can also be prepared in glycerol, liquid
polyethylene glycols, and mixtures thereof and in oils.
Under ordinary conditions of storage and use, these
preparations may contain a preservative to prevent the
growth of microorganisms.
Pharmaceutical compositions suitable for injectable
use include sterile aqueous solutions (where water
soluble) or dispersions and sterile powders for the
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extemporaneous preparation of sterile injectable
solutions or dispersions. In all cases, the composition
must be sterile and must be fluid to the extent that easy
syringability exists. It must be stable under the
conditions of manufacture and storage and must be
preserved against the contaminating action of
microorganisms such as bacteria and fungi. The carrier
can be a solvent or dispersion medium containing, for
example, water, ethanol, polyol (for example, glycerol,
propylene glycol, and liquid polyetheylene glycol, and
the like), suitable mixtures thereof, and vegetable oils.
The proper fluidity can be maintained, for example, by
the use of a coating such as lecithin, by the maintenance
of the required particle size in the case of dispersion
and by the use of surfactants. Prevention of the action
of microorganisms can be achieved by various
antibacterial and antifungal agents, for example,
parabens, chlorobutanol, phenol, ascorbic acid,
thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example,
sugars, polyalcohols such as mannitol, sorbitol, sodium
chloride in the composition. Prolonged absorption of the
injectable compositions can be brought about by including
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in the composition an agent which delays absorption, for
example, aluminum monostearate and gelatin.
Sterile injectable solutions can be prepared by
incorporating the pharmaceutical composition in the
required amount in an appropriate solvent with one or a
combination of ingredients enumerated above, as required,
followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the
pharmaceutical composition into a sterile vehicle which
contains a basic dispersion medium and the required other
ingredients from those enumerated above.
The pharmaceutical composition can be orally
administered, for example, with an inert diluent or an
assimilable edible carrier. The pharmaceutical
composition and other ingredients can also be enclosed in
a hard or soft shell gelatin capsule, compressed into
tablets, or incorporated directly into the individual's
diet. For oral therapeutic administration, the
pharmaceutical composition can be incorporated with
excipients and used in the form of ingestible tablets,
buccal tablets, troches, capsules, elixirs, suspensions,
syrups, wafers, and the like. Such compositions and
preparations should contain at least to by weight of
active compound. The percentage of the compositions and
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preparations can, of course, be varied and can
conveniently be between about 5 to about 800 of the
weight of the unit.
The tablets, troches, pills, capsules and the like
can also contain the following: a binder such as gum
gragacanth, acacia, corn starch or gelatin; excipients
such as dicalcium phosphate; a disintegrating agent such
as corn starch, potato starch, alginic acid and the like;
a lubricant such as magnesium stearate; and a sweetening
agent such as sucrose, lactose or saccharin or a
flavoring agent such as peppermint, oil of wintergreen,
or cherry flavoring. When the dosage unit form is a
capsule, it can contain, in addition to materials of the
above type, a liquid carrier. Various other materials
can be present as coatings or to otherwise modify the
physical form of the dosage unit. For instance, tablets,
pills, or capsules can be coated with shellac, sugar or
both. A syrup or elixir can contain the agent, sucrose as
a sweetening agent, methyl and propylparabens as
preservatives, a dye and flavoring such as cherry or
orange flavor. Of course, any material used in preparing
any dosage unit form should be pharmaceutically pure and
substantially non-toxic in the amounts employed. In
addition, the pharmaceutical composition can be
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incorporated into sustained-release preparations and
formulations.
As used herein, a "pharmaceutically acceptable
carrier" is intended to include solvents, dispersion
media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like.
The use of such media and agents for pharmaceutically
active substances is well known in the art. Except
insofar as any conventional media or agent is
incompatible with the pharmaceutical composition, use
thereof in the therapeutic compositions and methods of
treatment is contemplated. Supplementary active
compounds can also be incorporated into the compositions.
It is especially advantageous to formulate
parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit
form as used herein refers to physically discrete units
suited as unitary dosages for the individual to be
treated; each unit containing a predetermined quantity of
pharmaceutical composition is calculated to produce the
desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for
the novel dosage unit forms of the invention are dictated
by and directly dependent on (a) the unique
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characteristics of the pharmaceutical composition and the
particular therapeutic effect to be achieve, and (b) the
limitations inherent in the art of compounding such an
pharmaceutical composition for the treatment of a
pathogenic infection in a subject.
The principal pharmaceutical composition is
compounded for convenient and effective administration in
effective amounts with a suitable pharmaceutically
acceptable carrier in an acceptable dosage unit. In the
case of compositions containing supplementary active
ingredients, the dosages are determined by reference to
the usual dose and manner of administration of the said
ingredients.
Transgenic Animal Production
Transgenesis is a term used to describe the
artificial introduction of new genetic material into the
germ line of an organism. As such, it is a form of
genetic manipulation that includes not only the
introduction of foreign DNA into the germ line but also
designer gene modifications which to date usually involve
the insertion of new extraneous DNA. Transgenic animals
are useful as models for diseases for the testing of
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pharmacological agents prior to clinical trials or the
testing of therapeutic modalities.
Thus, in another embodiment, the present invention
provides a transgenic non-human animal containing a
nucleic acid construct of the invention. As previously
noted, a "nucleic acid construct" of the invention
includes at least one, or multiple, stress-responsive
non-coding regulatory sequences and a heterologous
nucleic acid sequence operatively linked to the
regulatory sequence, wherein expression of the
heterologous sequence is regulated by the non-coding
sequence. Thus, a "transgene", as used herein, refers to
a nucleic acid construct of the invention that is
inserted by artifice into a cell, and becomes part of the
genome of the organism that develops from that cell. For
example, a transgene of the present invention can contain
multiple grp78 regulatory elements driving expression of
a heterologous nucleic acid sequence in biologically
stressed tissue, such as glucose starved or hypoxic
tissue.
Phenotypically, a transgenic animal of the present
invention can appear normal because of the unique stress-
responsive regulatory sequence used to develop the
animal. Such a promoter is fully active only in a
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cellular environment that exhibits the biochemical
manifestations of biological stress. As previously
noted, such a cellular environment can include, but is
not limited to, glucose starvation, calcium deprivation
or hypoxia. Thus, a transgene of the present invention
may not be active under normal cellular conditions.
However, when an animal having such a transgene
incorporated in to its genome is exposed to conditions
that induce biological stress in the whole animal or in
specific tissues, the transgene can become activated in
the whole animal or only in specific tissues. For
example, exposure of a transgenic animal of the invention
to a mitogenic agent can induce a cell proliferative
disorder such that a tumor develops as a result of the
exposure. As previously noted, a fast growing solid
tumor devoid of nutrients due to insufficient blood
supply exposes the neoplastic cells contained in such an
environment to glucose deprivation, calcium deprivation,
chronic anoxia and low pH. Thus, a transgene containing
a stress-responsive regulatory sequence, such as grp78,
can become active in this environment.
The present invention provides transgenic animals
that are heterozygous for the transgene and animals that
are homozygous for the transgene of the invention. As
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shown in Figures 10 and 11, both heterozygous and
homozygous animals display activity of the stress
responsive regulatory sequence in tissues that have
developed tumors, i.e. are biologically stressed. Thus,
it is understood that both heterozygous and homozygous
transgenic animals of the invention are useful, for
example, for identifying compounds that induce biological
stress in such an animal.
A transgene of the invention includes a nucleic acid
construct comprising at least one stress-responsive
regulatory sequence operably associated with a
heterologous nucleic acid sequence. A heterologous
nucleic acid sequence can encode a detectable marker
expressed under the control of a stress-responsive
regulatory sequence that is active in a targeted,
biologically stressed, tissue. For example, grp78
regulatory sequences can be used in conjunction with a
heterologous nucleic acid sequence encoding a visually
detectable marker, such as green fluorescent protein
(GFP), or a biologically active protein detectable by
antibodies or enzymatic assay, to provide a means for
identifying biologically stressed tissue in a transgenic
animal. A heterogenous nucleic acid can further include
antisense polynucleotides and dominant negative encoding
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polynucleotides, which may be expressed in a transgenic
non-human animal.
The term "transgenic" as used herein additionally
includes any organism whose genome has been altered by in
vitro manipulation of the early embryo or fertilized egg
or by any transgenic technology to induce a specific gene
knockout. The term "gene knockout" as used herein,
refers to the targeted disruption of a gene in vivo with
complete loss of function that has been achieved by any
transgenic technology familiar to those in the art. In
one embodiment, transgenic animals having gene knockouts
are those in which the target gene has been rendered
nonfunctional by expression of antisense nucleic acid.
As used herein, the term "transgenic" includes any
transgenic technology familiar to those in the art which
can produce an organism carrying an introduced transgene
or one in which an endogenous gene has been rendered non-
functional or "knocked out".
The "non-human animals" of the invention include
vertebrates such as rodents, non-human primates, sheep,
dog, cow, pig, amphibians, and reptiles. Preferred non-
human animals are selected from the rodent family
including rat and mouse, most preferably mouse. The
"transgenic non-human animals" of the invention are
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produced by introducing "transgenes" into the germline of
the non-human animal. Embryonal target cells at various
developmental stages can be used to introduce transgenes.
As previously noted, different methods are used depending
on the stage of development of the embryonal target cell.
A "transgenic" animal can be produced by cross-
breeding two chimeric animals which include exogenous
genetic material within cells used in reproduction.
Various methods to make the transgenic animals of the
subject invention can be employed. Generally speaking,
three such methods may be employed. In one such method,
an embryo at the pronuclear stage (a "one cell embryo")
is harvested from a female and the transgene is
microinjected into the embryo, in which case the
transgene will be chromosomally integrated into both the
germ cells and somatic cells of the resulting mature
animal. The use of a one cell embryo as a target for
gene transfer has a major advantage in that in most cases
the injected DNA will be incorporated into the host gene
before the first cleavage (Brinster et al., Proc. Natl.
Acad. Sci. USA 82:4438-4442, 1985). As a consequence,
all cells of the transgenic non-human animal will carry
the incorporated transgene. This will in general also be
reflected in the efficient transmission of the transgene
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to offspring of the founder since 50% of the germ cells
will harbor the transgene. Microinjection of such an
embryo is the preferred method for incorporating
transgenes in practicing the invention.
Retroviral infection can also be used to introduce
transgene into a non-human animal. The developing non-
human embryo can be cultured in vitro to the blastocyst
stage. During this time, the blastomeres can be targets
for retro viral infection (Jaenich, R., Proc. Natl. Acad.
Sci USA 73:1260-1264, 1976). Efficient infection of the
blastomeres is obtained by enzymatic treatment to remove
the zona pellucida (Hogan, et al. (1986) in Manipulating
the Mouse Embryo, Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.). The viral vector system used
to introduce the transgene is typically a replication-
defective retro virus carrying the transgene (Jahner, et
al., Proc. Natl. Acad. Sci. USA 82:6927-6931, 1985; Van
der Putten, et al., Proc. Natl. Acad. Sci USA 82:6148-
6152, 1985). Transfection is easily and efficiently
obtained by culturing the blastomeres on a monolayer of
virus-producing cells (Van der Putten, supra; Stewart, et
al., EMBO J. 6:383-388, 1987). Alternatively, infection
can be performed at a later stage. Virus or virus-
producing cells can be injected into the blastocoele (D.
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Jahner et al., Nature 298:623-628, 1982). Most of the
founders will be mosaic for the transgene since
incorporation occurs only in a subset of the cells that
formed the transgenic nonhuman animal. Further, the
founder may contain various retroviral insertions of the
transgene at different positions in the genome that
generally will segregate in the offspring. In addition,
it is also possible to introduce transgenes into the germ
line, albeit with low efficiency, by intrauterine retro
viral infection of the midgestation embryo (D. Jahner et
al., supra). Methods to make transgenic animals
described generally above are described in U.S.
5,162,215, incorporated herein by reference.
In another such method, embryonic stem cells are
isolated and the transgene incorporated therein by
electroporation, plasmid transfection or microinjection,
followed by reintroduction of the stem cells into the
embryo where they colonize and contribute to the germ
line. Methods for microinjection of mammalian species is
described in United States Patent No. 4,873,191,
incorporated herein by reference. ES cells are obtained
from pre-implantation embryos cultured in vitro and fused
with embryos (M. J. Evans et al. Nature 292:154-156,
1981; M.O. Bradley et al., Nature 309: 255-258, 1984;
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Gossler, et al., Proc. Natl. Acad. Sci USA 83: 9065-9069,
1986; and Robertson et al., Nature 322:445-448, 1986).
Transgenes can be efficiently introduced into the ES
cells by as described above. Such transformed ES cells
can thereafter be combined with blastocysts from a
nonhuman animal.
The analysis of expression of a transgene is
essential in determining the utility of the transgenic
animal produced. As with integration analysis, the
presence or absence of similar or identical endogenous
counterparts will determine, to a degree, the strategies
that may be most useful. For transgenes that are unique
(no endogenous counterpart) or contain some unique
sequences, the strategies that can be used are more
straightforward. The presence of a novel RNA transcript
or a unique protein (or enzyme activity) is more easily
determined than it is when the transcript or protein
products are very similar to endogeneous transcripts or
proteins. As with integration analysis, molecular "tags"
are also sometimes useful in that the transcripts will
contain some unique identifying sequence that can be
readily and unequivocally determined.
A nucleic acid construct of the invention can
comprise a suitable detectable marker expressed under the
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control of a stress-responsive regulatory sequence that
is active in a targeted, biologically stressed, tissue.
"Detectable marker", as used herein, refers to any
identifiable composition useful for distinguishing cells
containing a nucleic acid construct of the present
invention from those cells that do not contain such a
construct.
It is also envisioned that biologically stressed
tissue of a transgenic animal of the invention can be
identified by the presence of a biologically active
protein product encoded by the construct of the
invention. Thus, a detectable marker of the invention
also includes biologically active protein products. The
term "biologically active protein product", as used
herein, refers to products produced or synthesized by a
host cell as a result of the insertion of a transgene
into the cell. In the present example, the cell can be
part of a transgenic animal. In addition, the term
encompasses those biological products that are secondary
products of the activity encoded by a transgene.
In those cases where it is desirable for the
detectable marker to encode a biologically active protein
product, it is envisioned that antibodies can be used to
detect the presence of an antigenic determinant resulting
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from expression of the protein encoded by the
heterologous DNA sequence. Such antibodies may, for
example, recognize a specific epitope unique to the
expressed protein. The term "epitope", as used herein,
S refers to an antigenic determinant on an antigen, such as
a protein encoded by the heterologous nucleic acid, to
which the paratope of an antibody, such as a protein
encoded by the heterologous nucleic acid, binds.
Antigenic determinants usually consist of chemically
active surface groupings of molecules, such as amino
acids or sugar side chains, and can have specific three-
dimensional structural characteristics, as well as
specific charge characteristics.
An antibody suitable for binding to a protein
encoded by the heterologous nucleic acid is specific for
at least one portion of an extracellular region of the
protein encoded by the heterologous nucleic acid
polypeptide. For example, one of skill in the art can
use the peptides to generate appropriate antibodies of
the invention. Antibodies of the invention include
polyclonal antibodies, monoclonal antibodies, and
fragments of polyclonal and monoclonal antibodies.
The preparation of polyclonal antibodies is well-
known to those skilled in the art. See, for example,
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Green et al., Production of Polyclonal Antisera, in
Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana
Press 1992); Coligan et al., Production of Polyclonal
Antisera in Rabbits, Rats, Mice and Hamsters, in Current
Protocols in Immunology, section 2.4.1 (1992), which are
hereby incorporated by reference. The preparation of
monoclonal antibodies likewise is conventional. See, for
example, Kohler & Milstein, Nature 256:495 (1975);
Coligan et al., sections 2.5.1-2.6.7; and Harlow et al.,
Antibodies: A Laboratory Manual, page 726 (Cold Spring
Harbor Pub. 1988), which are hereby incorporated by
reference.
In addition, a detectable marker of the invention
can be, for example, a visually detectable marker. In
one embodiment, the invention utilizes a visually
detectable marker protein that fluoresces directly upon
illumination with light of an appropriate wavelength.
Any fluorescent protein can be used in the invention,
including proteins that fluoresce due to intramolecular
rearrangements or the addition of cofactors that promote
fluorescence. For example, green fluorescent proteins of
cnidarians, which act as their energy-transfer acceptors
in bioluminescence, are suitable fluorescent proteins for
use in the fluorescent indicators. A green fluorescent
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protein ("GFP") is a protein that emits green light, and
a blue fluorescent protein ("BFP") is a protein that
emits blue light. GFPs have been isolated from the
Pacific Northwest jellyfish, Aequorea victoria, the sea
pansy, Renilla reniformis, and Phialidium gregarium.
See, Ward, W.W., et al., Photochem. Photobiol., 35:803,
1982); and Levine, L.D., et al., Comp. Biochem. Physiol.,
72B:771982.
A variety of Aequorea-related GFPs having useful
excitation and emission spectra have been engineered by
modifying the amino acid sequence of a naturally
occurring GFP from Aequorea victoria. (See, Prasher,
D.C., et al., Gene, 111:229, 1992); Heim, R., et al.,
Proc. Natl. Acad. Sci., USA, 91:12501, 1994); U.S. Patent
No. 5491084; 5,625,048, incorporated herein by
reference). The cDNA of GFP can be concatenated with
those encoding many other proteins; the resulting
chimerics often are fluorescent and retain the
biochemical features of the partner proteins. (See,
Cubitt, A.B., et al., Trends Biochem. Sci. 20:448, 1995).
Mutagenesis studies have produced may GFP mutants, some
having shifted wavelengths of excitation or emission.
Such proteins are included in the invention sensor. A
fluorescent protein is an "Aequorea-related fluorescent
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protein" if any contiguous sequence of 150 amino acids of
the fluorescent protein has at least 85o sequence
identity with an amino acid sequence, either contiguous
or non-contiguous, from the wild type Aequorea green
fluorescent protein. More preferably, a fluorescent
protein is an Aequorea-related fluorescent protein if any
contiguous sequence of 200 amino acids of the fluorescent
protein has at least 95o sequence identity with an amino
acid sequence, either contiguous or non-contiguous, from
the wild type Aequorea green fluorescent protein.
Similarly, the fluorescent protein can be related to
Renilla or Phialidium wild-type fluorescent proteins
using the same standards.
Other selectable markers include DNA sequences
encoding membrane bound polypeptides. Such polypeptides
are well known to those skilled in the art and contain a
secretory sequence, an extracellular domain, a
transmembrane domain and an intracellular domain. When
expressed as a positive selection marker, such
polypeptides associate with the target cell membrane.
Fluorescently labeled antibodies specific for the
extracellular domain may then be used in a fluorescence
activated cell sorter (FAGS) to select for cells
expressing the membrane bound polypeptide.
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It may also be useful in some circumstances to use a
version of a detectable marker that is targeted to a
specific subcellular compartment. "Targeting signal
sequence", as used herein, refers to any nucleic acid or
amino acid sequence useful for predetermining the
intracellular or extracellular location of a molecule
containing such a sequence. Subcellular targeting of the
detectable marker would be achieved by fusing the marker
gene to a targeting sequence. For example, the nuclear
localization signal from SV40 T antigen could be fused
to, for example, a visually detectable marker such as
GFP, which would lead to an accumulation of GFP in the
nucleus. Numerous subcellular targeting sequences are
known in the art. Using this well-known method, GFP has
been targeted to subcellular locations including the
nucleus, the mitochondria, the cell membrane, nuclear
pores, the actin cytoskeleton, the golgi apparatus,
transport vescicles and other locations.
The use of a targeting signal sequence is
advantageous for three reasons. First, concentration of
the detectable marker in a smaller area within the cell
gives a brighter, more easily visualized fluorescent
signal. Second, some cells tend to exhibit a basal level
of background autofluorescence, which is generally
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distributed throughout the cytoplasm. Targeting GFP, for
example, to a specific subcellular location permits the
fluorescent signal generated by the transgene to be more
easily distinguished from background autofluorescence. A
third advantage of using subcellular targeting is that it
allows sequential integration of different vector DNAs.
This might be desirable in situations when it is
desirable to have more than one gene expressed in a
transgenic animal. For example, it might be desirable to
express a specific monoclonal antibody in a transgenic
animal. In this case, a first vector DNA expressing the
heavy chain of a desirable antibody could be integrated
using GFP fused to an actin cytoskeletal targeting
sequence. A second vector DNA carrying a GFP fusion to a
nuclear localization signal and a light chain expression
cassette could then be integrated. Another example of
when this technique might be useful is in the case of
proteins that must be processed by a particular protease
in order to attain their mature, active forms.
Materials and Methods
Cell Culture Conditions. The tumor cell line B/ClOME
was cultured in high glucose Dulbecco's modified Eagle's
medium (DMEM) containing 4.5 mg/ml glucose supplemented
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with loo fetal calf serum, 2 mM glutamine, and 1%
penicillin-streptomycin-neomycin antibiotics. Transduced
B/C10ME cells were maintained in 2 mg/ml of 6418
respectively. To generate individual transduced clones,
transduced cells were plated into 96 well plates by
serial dilution, to a final concentration of 0.3
cell/well. Individual clones were then isolated and
expanded.
Retroviral Vector Construction. The GlTkSvNa
retroviral construct (Lyons et al., Cancer Gene Ther.,
2:273, 1995) was obtained from Genetic Therapy
Inc./Novartis (Summit, NJ). GlNaGrpTk (Figure 1) was
constructed by removing the 356 by SV40 promoter region
of a retroviral vector GlNaSvTk (Hung et al., Int. J.
Pediatr. Oncol., 4:317, 1997) by Sall and BglII and
replaced with a 695 by rat grp78 promoter spanning -520
to +175 (Chang et al., Proc. Natl. Acad. Sci. USA,
84:684, 1987). Retroviral vector plasmid DNA was prepared
by Qiagen Maxi Kit and transfected into ecotropic
retroviral producer cell line PE501. The viral
supernatant was harvested and an amphotropic retroviral
producer cell line PA317 was transduced and drug (G418)
resistant clones were selected. Retroviral vectors were
collected and titered by NIH3T3 cells.
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Western Blot. For the detection of HSVTK, GRP78 and
(3-actin, 20 mg of cell lysate were prepared as previously
described (Zhou et al., J. Natl. Cancer Inst., 90:381,
1998) and resolved on a denaturing sodium dodecyl
sulphate-8% polyacrylamide gel and transferred onto
Hybond nitrocellulose membrane (Amersham Life Science
Inc., Arlington Heights, IL). The membrane was blocked
with 5o non-fat milk (Bio-Rad Laboratories, Hercules, CA)
in TBS buffer (20 mM Tris-HC1, pH 7.5, 14 mM NaCl) for 1
h at room temperature prior to the incubation with
polyclonal rabbit anti-HSVtk antibody or monoclonal mouse
anti-GRP78 antibody (StressGen, British Columbia,
Canada), or monoclonal mouse anti-b-actin antibody (Sigma
Chemical Co.) 1 h at room temperature. For all the
primary antibodies, 1:1000 dilutions were used. The
secondary antibodies used were: goat anti-rabbit IgG
conjugated with horseradish peroxidase (Promega, WI) and
diluted 1:3000 in TBS buffer for detecting HSVTK; and
goat anti-mouse IgG conjugated with horseradish
peroxidase (Promega, WI) and diluted 1:5000 in TBS buffer
for detecting GRP78 and (3-actin. The immunocomplexes were
detected with the Enhanced Chemiluminescence (ECL) kit
(Amersham Life Science Inc.).
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In Vitro GCV-sensitivity Assay. Individual clones of
B/C10ME cells transduced with either the GlTkSvNa or the
GlNaGrpTk retroviral vector were seeded in duplicate at 5
x 103 cells/well in a 6-well plate. On day three after
seeding, the cells were incubated with either control
medium or 0.1 mg/ml GCV. Fresh GCV was added daily to the
cells, which were counted every 3 days using the trypan
blue dye exclusion method. For glucose starvation
treatment, on the second day after seeding, the cells
were maintained on glucose-free DMEM supplemented with
dialyzed fetal calf serum for a period of 30 h. After the
30 h, the cells were incubated with 0.1 mg/ml GCV. GCV
was added daily while the culture medium was changed
every third day for all cell cultures. When the cells
reached approximately 700 confluency, the cultures were
transferred to 10-cm diameter dishes.
Assay for In Vitro Bystander Effect. To measure the
GCV killing effect, non-transduced B/C10ME cells were c0-
cultured with different ratio of B/ClOME clonal cell
lines stably transfected with GlNaGrpTk. Typically, a
total of 3000 cells with various ratios (900:100;
750:250; 500:500) were plated in quadruplicate in 96 well
plate and treated with 10 mg/ml GCV for 10 days. The
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number of remaining viable cells was measured by cell
proliferation assay (Promega, WI).
Tumor Formation. Confluent cultures of B/ClOME
clones were harvested with trypsin-EDTA (Gibco/BRL) and
washed three times in PBS. Approximately 2 x 10' viable
cells were resuspended in 200 ml of PBS. Six- to eight-
week-old BALB/c mice obtained from Jackson Laboratory
were subcutaneously injected with an 18-gauge needle in
their right flank. Tumors were palpable within 12 days of
inoculation and bi-perpendicular measurements were taken
of the progressively growing tumor daily. Tumor growth
was monitored by measurement of the larger and smaller
diameters. At the indicated times post-injection, mice
were injected with GCV daily at a dosage of 100 mg/kg of
body weight for about 10 days. Tumors were judged to have
regressed after losing both measurability and
palpability. For each retroviral construct, multiple
injections of 2 to 3 independently derived transduced
clonal cell lines were performed.
Immunohistochemistry. Tumor tissues were removed,
stored at -80°C and cut by cryostat to 4 mm sections. The
frozen sections were fixed by loo formalin solution for
15 min and treated with 3o hydrogen peroxide. A rabbit
polyclonal antibody against HSVtk was added to the
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sections for 1 h at room temperature. After washing three
times with PBS, a HRP labeled polymer conjugated to goat
anti-rabbit antibody (Dako, Carpenteria, CA) was added
and incubated for 30 min. After three washes with PBS,
the slides were stained with 3, 3-diaminobenzidine (DAB)
and counterstained with methylgreen and covered with
regular permount and viewed under a Zeiss microscope.
MicroPET Imaging of HSVtk Expression. The hypoxia-
inducibility of the GRP78 promoter was demonstrated in
vivo by microPET scanning to detect GRP78-driven HSV-tk
gene expression, using the isotope-labeled substrate [18F]
FHBG. Tumors were established by subcutaneous
inoculation of TSA murine breast cancer cells that had
been stably pre-transduced with a standard replication-
defective retrovirus containing the GRP78-driven HSV-tk
cassette. These tumors were then examined by microPET
scanning in the absence or presence of further hypoxia
induction by photodynamic treatment. Figure 6 shows
microPET images of hypoxia inducible HSVtk expression in
a murine mammary adenocarcinoma model. The mcroPET scan
was performed as described (Gambhir et a1. PNAS, 96:2333,
1999). The isotope-labeled substrate was [1gF]FHBG
(Alauddin, Nuclear Medicine & Biology. 25:175, 1998). In
Panel A, the grp78 promoter is able to drive high level
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HSVtk expression in sizable solid tumors. A tumor was
formed in the left (L) shoulder area in a BALB/C mouse by
injecting s.c. 2 X 10~ of GINaGRP-HSVtk transfected TSA
cells. The microPET scan was performed when the tumor
was about l.5cm in diameter. The red color denotes high
HSVtk activity. In Panel B, the grp78 promoter is
inducible by hypoxia activated by photodynamic treatment
(PDT). Two tumors were formed simultaneously on the left
(L) and right (R) shoulder areas of a BALB/C mouse the
same as described in A. The microPET scan was performed
when tumor sizes reached about 0.6 cm in diameter and
about 12 hours after the tumor on the right had received
PDT which induces hypoxia in vivo. The two tumors were
of approximately the same size before PDT treatment and
the relatively larger contour seen on the image on the R
tumor is due to hemorrhage and edema after PDT treatment.
Production of Transgenic Animals. The present
invention further provides a transgenic mouse line
expressing the (3-galactosidase (lacZ) gene driven by the
GRP78 promoter. Significantly, lacZ staining was
observed in a variety of tumor tissues that developed in
the transgenic mice after exposure to chemical
carcinogens, but was not observed in any normal organs.
Furthermore, using a plasmid containing the grp78
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endoplasmic reticulum stress response element (ERSE)
linked to the minimal MMTV promoter, extremely low basal
levels and a 25-fold induction by glucose starvation was
observed upon transient transfection in the human
prostate cancer cell line PC3.
The transgene of the invention was injected into
fertilized eggs from superovaluated 4 to 5 week old F1
(C57BL/6J xCBA/J) females impregnated by F1 (C57BL/6J x
CBA/J) adult males. Psuedopregant females for embryo
transfer were produced by coatings between CDl adult
females and vasectomized CD1 adult males.
Mice of about 1.5 to 2 years of age were treated
with the chemical carcinogen every week for 6 months.
Tumors developed in both transgenic mice as well as non-
transgenic controls. Tumors and normal organs were
excised and stained for (3-galactosidase expression. Blue
color indicates that the grp78 promoter was active and
driving the expression of the(3-gal gene. The results
showed no expression in normal organs indicating low
grp78 promoter activity in normal tissues but elevated
expression in tumorous and/or inflammatory tissues.
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Results
Features of Stress-inducible grp78/BiP Promoter.
Under glucose starvation and anaerobic conditions, the
grp78 promoter is highly induced. The mammalian grp78
S promoter is functionally redundant and contains multiple
stress-inducible elements interacting with the CBF and
YY1 transcription factors (Li et al., J. Biol. Chem.,
268:12003, 1993; Roy et al., J. Biol. Chem., 271:28995,
1996; Li et al., Mol. Cell. Biol., 17:54, 1997). The
genetic code for endoplasmic reticulum stress signaling
leading to grp gene induction consists of two units of a
19 base pair (bp) sequence motif (CCAAT)N9(CCACG) (SEQ ID
NO:1) termed ERSE. This sequence contains a tripartite
structure, with a high affinity CBF/NF-Y binding site
separated by precisely 9 by of a GC rich sequence motif
to a low affinity YYl binding site.
In the construction of the retroviral vector
GlNaGrpTk, the rat grp78 promoter, spanning 520 by
upstream and 175 by downstream of the site of initiation
of transcription, serves as an internal promoter driving
the expression of the HSVtk gene (Figure 1). This 695 by
grp78 promoter subfragment contains three ERSEs, a TATA
element and an internal ribosome entry site, a unique and
useful feature of the 5' untranslated region of grp78
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that allows internal initiation of translation (Macejak
et al., Nature, 353:90, 1991). In the GlNaGrpTk vector,
the MuLV LTR directs the expression of the neo gene that
is used as a selection marker. For comparison, instead
of using a retroviral vector with another internal
promoter such as SV40 that has previously been shown to
be ineffective to drive a reporter gene in a tumor
environment (Gazit et al., Cancer Res., 55:1660, 1995),
the GlTkSvNa retroviral vector was used. In this vector,
the viral LTR drives the expression of the HSVtk gene,
while the Simian virus SV40 promoter drives neo
expression (Figure 1). The rationale for choosing
GlTkSvNa is that it represents an improved retroviral
vector for suicide gene therapy and is the vector of
choice in current clinical protocols (Anderson, Nature,
392(Suppl):25, 1998). Both vectors were transduced into
B/ClOME, a murine fibrosarcoma cell line that is
syngeneic with the Balb/c mice. The advantage of the
B/C10ME as a model system is that it has been previously
established that kinetics of tumor growth and subsequent
regression can be readily monitored in the recipient
mice.
Glucose Deprivation Induces grp78-driven HSVtk
Expression in vitro. To create clonal B/ClOME cell lines
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with stably integrated retroviral vectors, the cells
infected with the retroviruses were selected with 6418.
Serial dilution plating was performed after selection to
isolate individual clones. The individual clones were
expanded and analyzed. Under standard culture conditions,
B/ClOME cells transduced with either retroviral construct
exhibited equivalent plating efficiencies and growth
rates (see below). Thus, the basic growth properties of
the transduced cells in vitro were similar.
To test for the efficacy of the LTR and the grp78
promoter to drive expression of the HSVTK protein, total
cell lysates were prepared from individual clonal lines
under normal culture and glucose starved conditions. The
proteins were separated by SDS-PAGE and subjected to
Western blot analysis. The levels of HSVTK, GRP78 and (3-
actin in each sample was measured. As expected, there was
no detectable HSVTK in the non-transduced B/C10ME cells
(Figure 2). In the clonal line with the HSVtk gene
driven by the LTR, there was HSVTK expression under
normal culture conditions. However, when the cells were
subjected to glucose starvation for 24 h, while the level
of GRP78 was induced and the level of HSVTK was reduced.
In contrast, in the clonal cell lines with the HSVtk gene
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driven by the grp78 promoter, the level of HSVTK was
upregulated in glucose-starved cells (Figure 2).
To analyze HSVtk activity under normal and glucose-
starved conditions, clonal cell lines derived from
B/C10ME transduced cells with each respective retroviral
construct were analyzed using an in vitro GCV-sensitivity
assay (Figure 3). For this purpose, about 5,000 cells
were seeded in duplicates in 6-well plates, and on the
third day of seeding, either remained untreated, or
incubated with 0.1 ~tg/ml of GCV. One set of cells was
cultured in normal culture medium containing 4.5 mg/ml of
glucose, and an identical set of cells was maintained in
glucose-free medium supplemented with dialyzed fetal calf
serum for 30 h prior to the addition of GCV. Example of
the GCV survival test for a typical B/C10ME derived clone
transduced with GlTkSvNa (GlTkSvNa/clone #3) is shown in
Figure 3, Panel A. Without the addition of GCV the cells
continued to grow exponentially, and by the end of the
12th day, the cell number had reached 4 x 106. Addition of
GCV resulted in loss of live cells at a similar rate for
both sets of cells. By the end of the 12th day, about
1,000 cells survived (Figure 3, Panel A). Thus, for the
LTR-driven HSVtk, the sensitivity to GCV was similar in
cells cultured in normal or glucose-free medium.
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The results of the GCV survival assay for a typical
clonal line (GlNaGrpTk/clone #3) derived from B/C10ME
cells transduced with GlNaGrpTk is shown in Figure 3,
Panel B. Under normal culture conditions, the growth rate
as well as sensitivity to GCV was similar to that driven
by the LTR. However, in contrast to the LTR-driven HSVtk
cells, when GlNaGrpTk transduced cells were pretreated
with the glucose-free medium, decrease in viable cells
was much more pronounced. Thus by day 9 there were no
more surviving cells. Further, to demonstrate these cells
exhibit a bystander effect, HSVTK-positive cells were co-
cultured with various ratios of non-transduced HSVTK-
negative cells. Over 90o killing was observed when only
l00 of GlNaGrpTk cells are present in the culture (Figure
3, Panel C). Collectively, these in vitro studies show
that the retroviral construct containing an internal
grp78 promoter produces higher levels of HSVtk inducible
by glucose deprivation, thereby enhancing the sensitivity
of tumor cells to GCV.
Complete Eradication of Tumors in GlNaGrpTk
Transduced Cells. To directly compare the therapeutic
efficacy of the GlNaGrpTk vector with GlTkSvNa, B/C10ME
clones transduced with the respective retroviral
constructs were injected subcutaneously at a dose of 2 x
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10' cells per BALB/c mouse. As controls, the parental,
non-transduced cells were also injected. Tumors were
palpable after 12 days of injection. At day 21, when the
average tumor diameter reached about 2 cm, GCV was
administered. The rationale for starting the GCV
treatment when the tumor had reached a sizable mass
instead of just being palpable is that this will offer a
more vigorous test for the potency of the retroviral
vectors. For the parental B/C10ME cells, upon addition of
GCV, the tumors continued to grow at various rates and
growth was arrested as tumors reached substantial mass
(Figure 4, Panel A). In the nine mice injected with three
different GlTkSvNa clonal cell lines, the majority of
tumor growth was arrested upon GCV treatment for two to
three days but subsequently, tumor growth continued
(Figure 4, Panel B). Thus, at this stage of tumor growth,
the LTR-driven HSVtk was insufficient to mediate
efficient GCV toxicity. In contrast, in mice injected
with the GlNaGrpTk clonal cell lines containing the
internal grp78 promoter driving HSVtk expression, tumor
regression was observed in all four mice injected with
two independently derived clonal lines following GCV
treatment. By day 29, there were no visible tumors in
any of the animals (Figure 4, Panel C). Complete tumor
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eradication was also observed in mouse mammary tumor
clonal cell lines transduced with GlNaGrpTk. All mice
remained healthy and developed no tumors after withdrawal
of the GCV treatment.
To confirm that higher efficacy of GlNaGrpTk is due
to higher expression of HSVTK within the tumor,
immunohistochemistry-staining for the HSVTK protein was
performed with the tumor tissues. Examples of the
immunohistochemistry-staining using antibody against
HSVTK in B/C10ME tumors are shown in Figure 5. The
parental cells showed the absence of HSVTK protein
staining (Figure 5, Panel A). A much higher level of
staining was detected in tumors derived from GlNaGrpTk
transduced cells (Figure 5, Panel C) as compared to that
derived from GlTkSvNa (Figure 5, Panel B). Notably, the
HSVTK staining for the GlTkSvNa was in isolated patches,
suggesting there were areas within the tumor unfavorable
for LTR-driven gene expression. In contrast, the
staining for GlNaGrpTk was much more enhanced across the
tumor section as previously observed with the endogenous
grp78 transcript and the neo mRNA driven by the grp78
promoter. Thus, within the tumor environment, GlNaGrpTk
containing an internal stress-inducible grp78 promoter is
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more effective in directing high level HSVTK expression
than the retroviral LTR.
In cancer gene therapy, a major technical difficulty
is the lack of specificity in targeting suicide gene
expression in the anatomic site of tumors. The present
invention provides a novel approach to this problem by
using a stress-inducible promoter from the grp78 gene to
direct the expression of the HSVtk gene in solid tumors.
Increased grp78 protein expression is detected in
chemical- and radiation-transformed cells, as well as in
tumor cells that become drug-resistant. Within the tumor
environment, glucose deprivation, chronic anoxia, and
acidic pH induce the GRPs, in particular grp78. Thus,
grp78 mRNA levels are elevated in a variety of tumors,
correlating with tumor size. These results indicate that
in regions of the tumors deprived of glucose and oxygen,
the cells experience a stress response resulting in the
specific activation of the grp78 promoter.
The present invention provides a truncated rat grp78
promoter with most of the distal basal elements removed
while retaining its array of stress-inducible elements
(Figure 1). When used as an internal promoter in a
retroviral construct, the truncated rat grp78 promoter
can drive increased expression of HSVTK in vitro under
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glucose-starved conditions (Figures 2 and 3). These in
vitro studies confirm that the internal grp78 promoter is
capable of inducing a high level of marker gene
transcript in glucose-deprived cells, in contrast to the
HaMSV LTR that was repressed. In vivo, the GlNaGrpTk
retroviral vector was highly effective in directing HSVTK
expression within the tumor environment (Figure 5),
leading to complete eradication of sizable tumors in
their syngeneic host after GCV treatment. The potency of
GlNaGrpTk, coupled with the known bystander effects of
suicide gene approach, suggests that this type of vector
could offer distinct advantages in solid tumor cancer
therapy.
In addition, the present study shows that mice with
regressed tumors remained tumor free after withdrawal of
GCV treatment. These data indicate that protective
immunity might have been induced in such mice, preventing
regrowth of tumors. In support, there are several
examples of long-lasting antitumor immunity in various
tumor models in response to HSVtk transduction and GCV
treatments. For example, the immune response elicited by
mammary adenocarcinoma cells transduced with interferon-y
and suicide genes may induce regression of lung
metastases (Nanni et al., Hum. Gene Ther., 9:217, 1998).
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These data indicate that tumors transduced with suicide
genes can be used as live anti-tumor vaccines
(Santodonato et al., Gene Ther., 4:1246-, 1997). In
support of the this, it has recently been discovered that
induction of apoptosis in tumor cells leads to a dramatic
change in antigen presentation which could lead to
enhancement of the cell mediated immune response to the
tumor.
Transgenic animals containing a nucleic acid
construct of the invention were produced and used to
identify biologically stressed tissue in the animal.
Figure 7 shows the presence of the LacZ transgene in
transgenic mice. Panel A is a diagram of the grp78/LacZ
Transgene construct comprising about 3000 base pairs of
the grp78 regulatory sequence operably linked to the LacZ
gene. Panel B, upper gel, shows a Southern hybridization
resulting in the identification of a LacZ nucleic acid
sequence in transgenic animals (Tg 132-147) containing
the construct shown in Panel A. In the lower gel, a
grp78 cDNA probe that hybridizes to the grp78 gene was
used to demonstrate that similar amounts of total DNA
were loaded in to each lane of the gel. The transgenic
sequences were identified using a suitably labeled LacZ
probe. Non-transgenic (Non-Tg) animals do not contain
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the LacZ sequence. Panel C is a bar graph showing the
LacZ activity present in hamster cells tranfected with a
plasmid containing a nucleic acid construct shown in
panel A (grp78/LacZ) or a plasmid expressing LacZ from
the SV40 large T antigen promoter sequence (SV40/LacZ).
Cells were treated with the calcium ionophore A23187 to
induce biological stress. Untreated and treated activity
is indicated.
Carcinogen treatment of wild-type (+/+),
heterozygous for the grp78/LacZ transgene (Tg/+) or
homozygous for the grp78/LacZ transgene (Tg/Tg). The
carcinogen (7,12-dimethyl bent [a] anthracene) was
applied subcutaneously on a weekly basis over a period of
six months. Subsequently, normal and tumorous tissue
were isolated and stained for detection of LacZ
expression (Figure 8).
Color photographs of normal (non-neoplastic) tissue
derived from transgenic mice that are homozygous for the
grp78/LacZ transgene (Tg/Tg) or tissue derived from wild-
type (non-transgenic) mice (+/+) are sown in Figure 9.
The mice, and tissue derived therefrom, were treated as
described in Figure 8.
Color photographs of tumorous tissues removed from
mice treated as described in Figure 8 are shown in Figure
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10. Tissue from mice heterozygous for the grp78/LacZ
transgene (Tg/+), homozygous for the grp78/LacZ transgene
(Tg/Tg) and wild-type (+/+) are indicated. Note that,
following LacZ-specific histological staining, LacZ
expression is indicated in tumorous tissue derived from
Tg/+ mice as well as tissue derived from Tg/Tg mice.
Additionally, photographs of tumorous tissues
removed from mice treated as described in Figure 8 are
shown in Figure 11. Tissue from mice heterozygous for
the grp78/LacZ transgene (Tg/+) or homozygous for the
grp78/LacZ transgene (Tg/Tg) are indicated. Note that,
following LacZ-specific histological staining, LacZ
expression is indicated in tumorous tissue derived from
Tg/+ mice as well as tissue derived from Tg/Tg mice.
A number of embodiments of the invention have been
described. Nevertheless, it will be understood that
various modifications may be made without departing from
the spirit and scope of the invention. Accordingly,
other embodiments are within the scope of the following
claims .
