Note: Descriptions are shown in the official language in which they were submitted.
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HYPERTHERMIC INDUCIBLE EXPRESSION VECTORS FOR
GENETHERAPY AND METHODS OF USE THEREOF
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of gene therapy. More
particularly, it concerns methods and compositions for increasing transgene
expression.
2. Description of Related Art
Gene therapy now is thought to be widely applicable in the treatment of a
variety of cancers and a number of other diseases. Viral vectors are one
method
employed as a gene delivery system. A great variety of viral expression
systems
have been developed and assessed for their ability to transfer genes into
somatic
cells. In particular, retroviral and adenovirus based vector systems have been
investigated extensively over a decade. Recently, adeno-associated virus (AAV)
has emerged as a potential alternative to the more commonly used retroviral
and
adenoviral vectors. Lipid vectors including cationic lipids and Iiposomes also
are
used to deliver plasmid DNA containing therapeutic genes.
The therapeutic treatment of diseases and disorders by gene therapy involves
the transfer and stable or transient insertion of new genetic information into
cells.
The correction of a genetic defect by re-introduction of the normal allele of
a gene
encoding the desired function has demonstrated that this concept is clinically
feasible (Rosenberg et al., New Eng. J. Med., 323:570 (1990)). Indeed,
preclinical
and clinical studies covering a large range of genetic disorders currently are
underway to solve basic issues dealing with gene transfer efficiency,
regulation of
gene expression, and potential risks of the use of viral vectors. The majority
of
clinical gene transfer trials that employ viral vectors perform ex vivo gene
transfer
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2
into target cells which are then administered in vivo. Viral vectors also may
be
given in vivo but repeated administration may induce neutralizing antibody.
A major issue facing potential clinical application of gene therapy is the
question of how to heterologous genes expressed in clinically significant
quantities
in selected tissues of the subject. Gene regulatory elements provide a
potential
answer to that question. Gene regulatory elements such as promoters and
enhancers possess cell type specific activities and can be activated by
certain
induction factors via responsive elements. The use of such regulatory elements
as
promoters to drive gene expression facilitates controlled and restricted
expression
of heterologous genes in vector constructs. For instance, heat shock promoters
can
be used to drive expression of a heterologous gene following heat shock.
US Patent Nos. 5,614,381, 5,646,410 and WO 89/00603, refer to driving
transgene expression using heat shock at temperatures greater than
42°C. These
temperatures are not practicable in human therapy as they can not be
maintained for
a sustained period of time without harm to the individual.
Gene therapy could be used in combination with a variety of conventional
cancer therapy treatments including cytotoxic drugs and radiation therapies.
It has
been shown that hyperthermia enhances the cell killing effect of radiation in
vitro
(Harisiadis et al., Cancer, 41:2131-2142 (1978)), significantly enhances tumor
response in animal tumors in viva and improves the outcome in randomized
clinical
trials. However, the major problem with the use of hyperthermia treatment is
that
the hyperthermia system can not adequately heat large and deep tumors.
Thus, it would be useful to develop vectors that may be used at temperatures
of 42°C and below, systemically or locally, to treat a patient such
that the
expression of the therapeutic genes) is activated preferentially in regions of
the
body that have been subjected to conditions which induce such expression.
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3
SUMMARY OF THE INVENTION
The present invention provides methods for effecting the inducible
expression of polynucleotides in cells. In particular, the use of heat shock
promoters in methods for effecting the inducible expression of polynucleotides
in
mammalian cells is taught. The present invention overcomes deficiencies in the
prior art by providing heat shock-controlled vectors that may be used at
temperatures of 42°C and below. These methods may be used to treat a
patient via
the inducible expression of a therapeutic gene.
In one embodiment, the present invention provides a method for effecting
l0 transgene expression in a mammalian cell that comprises first providing an
expression construct that comprises both (i) an inducibie promoter operably
linked
to a gene encoding a transactivating factor and (ii) a second promoter
operably
linked to a selected polynucleotide. The second promoter is activated by the
transactivating factor expressed by the same construct. The method then
includes
the step of introducing the expression construct into the cell. Finally, the
cell is
subjected to conditions which activate the inducible promoter and result in
the
expression of the selected polynucleotide.
In a preferred embodiment of the invention, the inducible promoter is a heat
shock promoter and the conditions which activate the heat shock promoter are
hyperthermic conditions. The hyperthermic conditions may comprise a
temperature
between about basal temperature and about 42°C. As used herein the
basal
temperature of the cell is defined as the temperature at which the cell is
normally
found in its natural state, for example, a cell in skin of a mammal may be at
temperatures as low as 33°C whereas a cell in the liver of an organism
may be as
high as 39°C. In specific embodiments, the application of hyperthermia
involves
raising the temperature of the cell from basal temperature, most typically
37°C. to
about 42°C or less. Alternatively, the hyperthermic conditions may
range from
about 38°C to about 41°C, or from about 39°C to about
40°C. The heat shock
promoter is optionally derived from a promoter selected from the group of the
heat
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shock protein (HSP) promoters HSP70, HSP90, HSP60, HSP27, HSP72, HSP73,
HSP25 and HSP28. The ubiquitin promoter may also be used as the heat-shock
inducible promoter in the expression construct. A minimal heat shock promoter
derived from HSP70 and comprising the first approximately 400 by of the HSP70B
promoter may optionally be used in the invention.
In an alternative embodiment, the inducible promoter comprises a hypoxia-
responsive element (HR,E). This hypoxia-responsive element may optionally
contain at least one binding site for hypoxia-inducible factor-1 (HIF-1).
In one embodiment of the invention, the second promoter may be selected
l0 from the group consisting of an human immunodeficiency virus-1 (HIV-1)
promoter and a human immunodeficiency virus-2 (HIV-2) promoter. In preferred
embodiments, the transactivating factor may be a transactivator of
transcription
(TAT).
The selected polynucleotide may code for a protein or a polypeptide. For
instance, the selected polynucleotide may encode any one of the following
proteins:
ornithine decarboxylase antizyme protein, p53, p16, neu, interleukin-1 (IL 1),
interleukin-2 (IL2), interleukin -4 (IL4), interleukin-7 (IL7}, interleukin-12
(IL12),
interleukin-15 (IL 15), FLT-3 ligand, granulocyte-macrophage stimulating
factor
(GM-CSF), granulocyte-colony stimulating factor (G-CSF}, gamma-interferon
(IFNy), alpha-interferon (IFNa), tumor necrosis factor (TNF), herpes simplex
virus
thymidine kinase (HSV-TK), I-CAM1, human leukocyte antigen-B7 (HLA-B7), or
tissue inhibitor of metalloproteinases (TIMP-3). In such an embodiment, the
selected polynucleotide is positioned in a sense orientation with respect to
the
second promoter.
Alternatively, expression of the selected polynucleotide may involve
transcription but not translation and produces a ribozyme. In this embodiment,
the
selected polynucleotide is also positioned in a sense orientation with respect
to the
second promoter.
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In still another alternative embodiment, the expression of the selected
polynucleotide involves transcription but not translation and results in an
RNA
molecule which serves as an antisense nucleic acid. In such an embodiment, the
selected polynucleotide may be the target gene, or a fragment thereof, which
is
5 positioned in the expression construct in an antisense orientation with
respect to
said second promoter.
The expression construct may further comprise a gene encoding a selectable
marker, such as hygromycin resistance, neomycin resistance, puromycin
resistance,
zeocin, gpt, DHFR, green fluorescent protein or histadinol. Alternatively, the
expression construct may further comprise (i) a second selected polynucleotide
which is operably linked to said second promoter, and (ii) an internal
ribosome
entry site positioned between said first and second selected polynucleotides.
The cell may be a tumor cell, a cell located within a tumor, or a cell located
within a mammal. The introduction of the expression construct into the cell
may
occur in vitro or in vivo. In an one embodiment, the introduction of the
expression
construct into the cell is mediated by a delivery vehicle selected from the
group
consisting of liposomes, retroviruses, adenoviruses, adeno-associated viruses,
lentiviruses, herpes simplex viruses, and vaccinia viruses.
In another embodiment of the invention, a method of providing a subject
with a therapeutically effective amount of a product of a selected gene is
provided.
This method involves providing a first expression construct which comprises an
inducible promoter operably linked to a gene encoding a transactivating factor
and
providing a second expression construct which comprises a second promoter
operably linked to a selected polynucleotide, where the second promoter is
activated by the transactivating factor encoded by the first expression
construct.
The first and second expression construct are introduced into the desired cell
of
said subject and that cell is subjected to conditions which activate the
inducible
promoter, so that expression of the selected polynucleotide is induced. In a
preferred embodiment, the first and second expression constructs are present
on the
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same vector. Also, the inducible promoter is preferably a heat shock promoter
and
the activating conditions comprise a temperature below 42°C and above
about basal
temperature.
The introduction of one or both of the expression constructs may be
performed either ih vivo or ex vivo. The expression product of the selected
polynucleotide may optionally be deleterious to a pathogen in the subject,
such as a
virus, bacterium, fungus, or parasite. Alternatively, the expression product
of the
selected polynucleotide may inhibit the growth of the cell of the subject. In
still
another alternative embodiment of the invention, the expression product of the
selected polynucleotide replaces a deficient protein in the subject.
Alternatively,
the expression product of the selected polynucleotide rnay promote nerve
regeneration.
In further embodiments, there is provided a method of treating cancer in a
mammal, such as a human, comprising the steps of (a) providing an expression
construct that comprises (i} an inducible promoter, preferably a heat shock
promoter, which is operably linked to a gene encoding a transactivating
factor; and
(ii) a second promoter operably linked to a selected polynucleotide, wherein
the
second promoter is activated by the transactivating factor; (b) introducing
said
expression construct into a tumor cell; and (c) subjecting the tumor cell to
conditions which activate the inducible promoter so that the selected
polynucleotide
is expressed in high enough quantities to inhibit the growth of the tumor
cell. If the
inducible promoter is a heat shock promoter, the activating conditions
comprise a
temperature below about 42°C and above about basal temperature.
This method further may comprise treating said tumor cell with an
established form of therapy for cancer which is selected from the group
consisting
of external beam radiation therapy, brachytherapy, chemotherapy, and surgery.
The cancer may optionally be selected from the group consisting of cancers of
the
brain, lung, liver, spleen, kidney, lymph node, small intestine, pancreas,
blood cells,
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7
colon, stomach, breast, endometrium, prostate, testicle, ovary, vulva, cervix,
skin,
head and neck, esophagus, bone marrow and blood.
In one particular embodiment of the invention, the selected polynucleotide is
ornithine decarboxylase antizyme protein. .After the cell is subjected to
conditions
which activate the inducible promoter of the expression construct in the tumor
cell,
the tumor cell is treated with the radioprotector WR-33278 or WR-1065. Lastly,
the tumor cell is treated with radiation therapy.
Methods for provoking an immune response in a mammal, such as a human,
are also provided by the present invention. The provoked immune response may
constitute either a humoral immune response or a cellular immune response. In
one
embodiment, the method comprises (a) providing an expression construct that
comprises (i) an inducible promoter, preferably a heat shock promoter, which
is
operably linked to a gene encoding a transactivating factor; and (ii) a second
promoter operably linked to a selected polynucleotide, wherein the second
promoter is activated by the transactivating factor; (b) introducing said
expression
construct into a cell in the mammal; and (c) subjecting the cell to conditions
which
activate the inducible promoter so that the selected polynucleotide is
expressed
highly enough to provoke an immune response in the mammal. If the inducible
promoter is a heat shock promoter, the activating conditions comprise a
temperature
below about 42°C and above about basal temperature.
In one embodiment, the immune response which is provoked is directed
against the cell in the mammal which contains the expression construct. 'The
method may also optionally involve treating the cell with an established form
of
therapy for cancer selected from the group consisting of chemotherapy,
external
beam radiation therapy, brachytherapy, and surgery.
In another embodiment, there is provided an expression construct comprising
(a) a gene encoding a transactivating factor; (b) an inducible promoter
operably
linked to the gene; (c) a selected polynucleotide; and (d) a second promoter
which
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is operably linked to the selected polynucleotide. The second promoter of the
construct is activated by the transactivating factor. In a preferred
embodiment, the
inducible promoter is a heat shock promoter and the expression of the selected
polynucleotide can be induced by hyperthermic conditions comprising a
temperature below about 42°C and above about 37°C. In an
alternative
embodiment, the inducible promoter of the expression construct may comprise a
hypoxia-responsive element. The expression construct may also comprise a
second
selected polynucleotide which is also operably linked to the second promoter
and
separated by the first selected polynucleotide by a IRES.
A cell comprising the expression construct is also provided. The provided
expression construct can also optionally be used in a method of altering the
genetic
material of a mammal.
Other objects, features and advantages of the present invention will become
apparent from the following detailed description. It should be understood,
however, that the detailed description and the specific examples, while
indicating
preferred embodiments of the invention, are given by way of illustration only,
since
various changes and modifications within the spirit and scope of the invention
will
become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are
included to further demonstrate certain aspects of the present invention. The
invention may be better understood by reference to one or more of these
drawings
in combination with the detailed description of specific embodiments presented
herein.
FIG. 1 depicts the basic vector used for quantitating heat shock promoter
activity. The plasmid contains a minimal promoter derived from the HSP70B
promoter (StressGen). A reporter gene, such as Enhanced Green Fluorescence
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9
Protein (EGFP), ~i-gal, or IL-2 is easily inserted into the multiple cloning
site
(MCS) so that it is expressed under control of the minimal HSP70B promoter.
The
plasmid also contains the neomycin and ampicillin resistance genes for
selectability
in mammalian cells as well as the standard elements for growth in a bacterial
system. The S8 plasmid comprises the plasmid shown with EGFP inserted in the
multiple cloning site.
FIG. 2 shows fluorescence activated cell sorting (FACS) histograms for DU-
145 cells stably transfected with the S8 plasmid. Fluorescence increases from
left
to right. The top histogram is from transfected DU-145 cells which have not
been
IO subjected to heat shock. The bottom histogram is from transfected DU-145
cells
which have been subjected to a 42°C heat shock for 1 hour.
FIG. 3 shows FACS histograms for three different populations of S8-
transfected MCF7 cells. The MCF7 cells, transfected with the S8 construct,
were
sorted by FACS. The original population came from a polyclonal selected cell
line.
That cell line's activated (i. e., cells expressing EGFP) population was
separated
from the non-activated population. After the sort, the positive population was
grown and then re-sorted to obtain a more purely positive cell line. In this
case, the
polyclonal MCF7-S8-P cells were sorted twice yielding the highly positive
population MCF7-S8-PS2.
FIG. 4 shows expression of EGFP in different cell lines assayed by FACS.
Cell lines were transfected with the plasmid S8. The cells were then cloned or
a
polygonal line was grown. In some cases the cell lines were sorted for EGFP
expression by FACS. The total mean fluorescence was quantified and graphed.
FIG. 5 shows expression of EGFP in stably transfected DU-145 cells which
have been twice sorted (DU-S8-PS2) following heat shock. The DU-S8-PS2 cells
were heated at either 40°C or 42°C and allowed to recover for
various times. The
cells were then analyzed by FACS.
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FIG. 6 shows expression levels of EGFP in stably transfected DU-145 cells
16 hours after exposure to heat stresses. One population of cells (DU-S8-PS2)
was
stably transfected with the S8 plasmid. Another population {DU-V9-PS2) was
stably transfected with the V9 plasnlid, a plasmid identical to S8 except that
the
5 EGFP of the V9 plasmid is operably linked to a CMV promoter, rather than HSP
70B (see FIG. 7). The cells were heated at various temperatures and allowed to
recover for 15 hours. Non-transfected DU-145 cells were included as a control.
FIG. 7 shows a schematic diagram of the plasmid V9 which contains a CMV
promoter that is operably linked to the gene encoding the Enhanced Green
l0 Fluorescence Protein {EGFP)
FIG. 8 shows the basic vector design for a vector containing a second
promoter which allows for amplification of the heat shock response. The
plasmid
contains a multiple cloning site {MCS) operably linked to HSP70B promoter, but
also contains a therapeutic gene operably linked to a second promoter. The
plasmid
also contain the neomycin resistance gene, the ampicillin resistance gene, and
standard elements for growth in bacteria. In the plasmid pCB, the second
promoter
is the HIV-1 long terminal repeat (LTR) and the therapeutic gene is IL2. In
pfl2,
tat is inserted in the MCS, the second promoter is the HIV-1 LTR, and the
therapeutic gene is IL2. Another plasmid, p007, is the same as pfl2, except
that the
HIV-2 LTR is used as the second promoter.
FIG. 9 shows amplifier constructs containing the therapeutic gene IL-2
driven by either the HIV-1 or the HIV-2 promoter. The amplifier part is
controlled
by either the CMV or the HSP 70 promoter driving TAT expression. The plasmids
also contain the neomycin resistance gene and elements for growth in bacteria.
These constructs were used in the amplifier studies of Examples 2 and 3. FIG.
9A
shows a plasmid designated X14 containing a CMV-TAT-HIV-1-IL2 expression
cassette; FIG. 9B shows a plasmid designated Y15 containing a CMV-TAT-HIV-2-
IL2 expression cassette; FIG. 9C shows a plasmid designated pfl2 containing an
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11
HSP-TAT-HIV-1-IL2 expression cassette; and FIG. 9D shows a plasmid designated
p007 containing an HSP-TAT-HIV-2-IL2 expression cassette.
FIG 10. shows the DNA sequence of the BamH 1-HindIII fragment of
p173QR from StressGen Biotechnology Corp. This fragment contains the
approximately 0.4kb minimal HSP70B promoter fragment used in constructs of the
specific examples, Example 1 and 3, below.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
1. The Present Invention
Gene therapy faces two major technical problems: how to both regulate and
enhance the expression of therapeutic genes in vivo. The present invention
addresses both of these questions by combining hyperthermia treatment with
inducible expression constructs. The inventors have demonstrated increases in
the
efficiency of specific, inducible gene expression.
The ability to express therapeutic genes) at very high levels and the ability
to control the levels of expression are important objectives in the
development of
gene therapy. The inventors have created new sets of expression vectors to
address
these objectives. The inventors use an amplifier strategy to drive the
expression of
the genes) of interest. The amplifiers consist of the human HSP70B promoter
driving expression of proteins that are transcriptional activators of other
promoters,
which, in turn, drive reporter genes. These additional promoters and their
operably
linked reporter genes are preferably included in the same vector with the
HSP70B
promoter element and the gene encoding the transactivating protein.
In transfection studies of mammalian cells using human IL-2 as the reporter
gene, the inventors have shown that gene expression was dramatically increased
using their amplifier constructs for all temperature conditions used, compared
to
reporter gene expression produced by the constitutive CMV promoter or by
HSP70B alone (see specific example, Example 3, below). Constructs containing
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12
both the HSF70B promoter, upstream of the human immunodeficiency virus (HIV)
tat gene, and the HIV 1 or HIV2 long terminal repeats, upstream of the
interleukin-2
(IL-2) gene, exhibited promoter activity at 37°C which was further
amplified by
heat shock. Co-transfection experiments indicated that the activities of the
HSP,
HSP/HIV 1 and HSP/HIV2 promoter expression constructs were 0.4, 6.9 and 83.3,
respectively, times that of the CMV promoter expression construct in mammalian
cells. These data indicate that, while less active than the CMV promoter by
itself,
this minimal heat shock promoter can be used in conjunction with a second
promoter to markedly amplify gene expression while still maintaining some
temperature dependence.
Earlier studies have examined the use of the heat shock promoter to drive the
expression of transactivating proteins to conditionally express other
promoters
(Schweinfest et al., Gene, 71(1):207-2I0, 1988; EPO O1 18393; WO 89/00603,
U.S. Patent No. 5,614,381; U.S. Patent No. 5,646,010; EP 0 299 127). The
inventions described herein differ from these earlier approaches, for example,
by
use of 1) different heat shock promoters, (Schweinfest et al., use Drosophila
promoters) 2) different modes of delivery (the present inventors have
incorporated
both promoters into a single construct - whereas others have used co-
transfection)
3) different temperatures for induction (the earlier work used temperatures
greater
than 42°C, whereas the present invention advantageously operates at
temperatures
of 42°C and lower); and 4) use in gene therapy context rather
industrial production.
Furthermore, the present inventors are able to use either HIV-1 or HIV-2
promoters and the present invention shows a clear distinction in the
expression
levels resulting from these two promoters.
In a preferred aspect of the present invention, methods of effecting transgene
expression in a mammalian cell by using a heat shock inducible element are
provided. The heat shock sequence is used to drive the expression of a
transactivating gene. Thus, when the expression construct is subjected to
hyperthermia, the expression of the transactivating element is induced. The
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13
transactivating gene acts upon a second promoter which becomes activated to
drive
the expression of the therapeutic gene of interest. In a particular
embodiment, a
promoter derived from the HSP70 promoter is employed. A particularly useful
aspect of this promoter is that it has a low basal level of expression at
ambient
temperatures and is inducible. The present invention further provides methods
of
providing a subject with a therapeutically effective amount of a gene product
and
for inhibiting the growth of a cell or provoking an immune response.
Compositions and methods employed in order to meet the objectives of the
present invention are discussed in further detail herein below.
2. Heat Shock Response
The heat shock or stress response is a universal response occurring in
organisms ranging from plants to primates. It is a response that can be
elicited as a
result of not only heat shock, but also as a result of a variety of other
stresses
including ischemia, anoxia, glucose deprivation, ionophores glucose and amino
acid
analogues, ethanol, transition series metals, drugs, hormones and bacterial
and viral
infections. Furthermore, there is evidence that overexpression of heat shock
protein
genes may be associated with enhanced proliferation and stress of tumor cells
(Finch et al., Cell Growth and Differentiation 3(5):269-278, 1992). This
response
is characterized by the synthesis of a family of well conserved proteins of
varying
molecular sizes that are differently induced and localized. These proteins are
among the most phylogenetically conserved and are characterized according to
their
weights.
The transcriptional activation of stress protein-encoding genes occurs within
minutes in response to environmental and or physiological trauma. This speedy
response has been attributed to the lack of introns in the vast majority of
heat shock
proteins. This absence of introns allows heat shock proteins to circumvent a
block
in intron processing that occurs at elevated temperature. Thus, the heat shock
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protein is translated with very high efficiency, often at the expense of other
proteins.
The activation of the stress genes is mediated by the conversion of a pre-
existing heat shock transcription factor (HSF) from an inactive to an active
form.
There is a large difference in the molecular weight of this DNA-binding
protein
(e.g., 83 kDa in humans and 150 kDa in yeast). The heat shock element is a
conserved upstream regulatory sequence of HSP70 to which HSF binds. Although
the main function of heat shock proteins is in facilitating protein folding
and
preventing aggregation, it is apparent that these proteins play some role in
providing
1 o an organism with a protective mechanism against environmental insult and
aid
recovery subsequent to trauma.
Like most eukaryotic sequence-specific transcription factors, HSF acts
through a highly conserved response element found in multiple copies upstream
of
the heat shock gene. The heat shock response element is composed of three
contiguous inverted repeats of a 5-base pair sequence whose consensus was
defined
as nGAAn and more recently defined as AGAAn. The regulation of HSF primarily
comprises a change in activity rather than an alteration in synthesis or
stability.
3. Hyperthermia Therapy
Many clinical studies have shown the effectiveness of hyperthermia as an
adjunctive treatment for malignancies, when used in combination with
radiotherapy
or chemotherapy (Hahn, G.M., Hyperthermia and Cancer, 2nd Ed., New York,
Plenum, 1982; Scott, et al., Int. J. Rad. Oc. Biol. Phys. 10{ 11 ) 2219-2123,
1984;
Lindholin, et al., Rec. Res. in Cancer Res. 107:152-156, 1988. The rationale
for
heat application, indication and contraindications, is developed on the basis
of
experimental evidence that desirable physiological responses can be produced
by
the use of heat and on the basis of controlled clinical studies. Lehman
provides a
comprehensive treatise for the therapeutic use of heat in other applications
(Therapeutic Heat and Cold, Rehabilitation Medicine Library, published by
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Williams & Wilkins, 1990, incorporated by reference) the reader is referred in
particular to Chapter 9, which discusses the use of heat in the context of
therapeutic
interventions, both medical and surgical.
"Hyperthermia" is intended to refer to a temperature condition that is greater
5 than the ambient temperature of the subject to which the treatment is being
administered. Hence, a hyperthermic temperature, as used herein, will
typically
range from between about 37°C to about 42°C. In preferred
embodiments, the
temperature will range from about 38°C to about 42°C, in other
embodiments, the
temperature range will be from about 39°C to about 41°C, in
other embodiments,
10 the temperature will be about 40°C. With the devices currently
available for the
application of hyperthermia in adjuvant therapies it is possible to maintain
the
temperature of hyperthermia treatment to within about 0.5°C for
temperatures up to
42°C. Hence, the therapeutic treatments of the present invention may be
carried out
at 37.0°C, 37.2°C, 37.4°C, 37.6°C, 37.8°C,
38.2°C, 38.4°C, 38.6°C, 38.8°C, 39.2°C,
15 39.4°C, 39.6°C, 39.8°C, 40.2°C, 40.4°C,
40.6°C, 40.8°C, 41.2°C, 41.4°C, 41.6°C,
41.8°C, or 42.0°C. Prior to the present invention, efficacy of
hyperthermia required
that temperatures within a tumors) remain above about 43°C for 30 to 60
min,
while safety considerations limit temperatures in normal tissues to below
42°C.
Achieving uniform temperatures above 42°C in tumors is very difficult
and often
not possible.
Tissues in mammals can be heated using a number of technologies including
ultrasound, electromagnetic techniques, including either propagated wave
(e.g.,
microwaves), resistive (e.g., radiofrequency) or inductive (radiofrequency or
magnetic) procedures (Hahn, G.M., Hyperthermia and Cancer, 2nd Ed., New York,
Plenum, 1982; Lehman, L.B., PoStgard Med., 88(3):240-243, 1990; both herein
incorporated by reference). In some simple applications, tissue temperatures
can be
elevated using circulated hot air or water.
U.S. Pat. No. 4,230,129 to Le Veen, herein incorporated by reference, refers
to a method of heating body tissue and monitoring temperature changes in the
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16
tumor in real time with the aid of a scintillation detector. The method
provides for
the coupling of radiofrequency (IZF) energy to the patient's body to avoid any
significant heat absorption in the fatty tissues. This is obtained by focusing
the RF
energy on the tumor with an orbital movement of the applicator so that energy
is
not constantly being applied to the same confined area within the patient's
body.
U.S. Pat. No. 3,991,770 to Le Veen, also herein incorporated by reference,
teaches
a method of treating a tumor in a human by placing the part of the human body
containing the tumor in a radiofrequency electromagnetic field to heat the
tumor
tissue and cause necrosis of the tumor without damaging the adjacent normal
tissue.
In preferred embodiments, of the present invention, hyperthermia is applied
in combination with the gene therapy vectors disclosed herein to achieve
inducible
gene expression at a particular tumor site. Furthermore, the hyperthermia/gene
therapy treatment regimens may be used in combination with other conventional
therapies, such as the chemotherapies and radiotherapies discussed below, to
effectively treat cancer. Other methods for inducing hyperthermia also are
known
in the art. Methods and devices for the regional and/or systemic application
of
hyperthernua are well know to those of skill in the art and are disclosed in
for
example, U.S. Patent Nos. 5,284,144; 4,230,129; 4,186,729; 4,346,716;
4,848,362;
4,815,479; 4,632,128, all incorporated herein by reference.
4. Engineering Expression Constructs
In certain embodiments, the present invention involves the manipulation of
genetic material to produce expression constructs that encode therapeutic
genes.
Such methods involve the use of an expression construct containing, for
example, a
heterologous DNA encoding a gene of interest and a means for its expression,
replicating the vector in an appropriate helper cell, obtaining viral
particles
produced therefrom, and infecting cells with the recombinant virus particles.
The
gene will be a therapeutic gene, for example to treat cancer cells, to express
immunomodulatory genes to fight viral infections, or to replace a gene's
function as
a result of a genetic defect. In the context of the gene therapy vector, the
gene will
CA 02308575 2000-OS-03
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17
be a heterologous DNA, meant to include DNA derived from a source other than
the viral genorne which provides the backbone of the vector. Finally, the
virus may
act as a live viral vaccine and express an antigen of interest for the
production of
antibodies thereagainst. The gene may be derived from a prokaryotic or
eukaryotic
source such as a bacterium, a virus, a yeast, a parasite, a plant, or an
animal. The
heterologous DNA also may be derived from more than one source, i.e., a
multigene construct or a fusion protein. 'The heterologous DNA also may
include a
regulatory sequence which may be derived from one source and the gene from a
different source.
a) Therapeutic Genes
The selected polynucleotide of the present invention may optionally be a
therapeutic gene. Any of a wide variety of therapeutic genes are suitable for
use in
the vectors and methods described herein. Therapeutic genes which are suitable
for
application of the present invention to a particular disorder, medical
condition, or
disease will be discernible to one skilled in the art.
In one embodiment of the invention, the selected polynucleotide is the gene
encoding for ornithine decarboxylase antizyme protein. The ornithine
decarboxylase (ODC} antizyme protein is an important component of feedback
regulation of intracellular polyamine pool sizes (Hayashi et al., Trends in
Biochemical Sciences 21(1}:27-30, 1996, herein incorporated by reference). The
levels of this protein are directly related to levels of intracellular
polyarnines, which
stimulate translation of antizyme message. Antizyme protein targets ornithine
decarboxylase, the first and often rate-limiting enzyme in polyamine
synthesis, for
degradation. This protein also suppresses polyamine uptake. Thus, low levels
of
endogenous polyamines lead to low levels of antizyme which in turn maximizes
polyamine synthesis via ODC and polyamine uptake. Conversely, high levels of
endogenous polyamines cause high levels of antizyme protein, which in turn
minimize polyamine synthesis via ODC and suppress polyamine uptake.
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The radioprotector WR-33278 (N,N"-(dithiodi-2,1-ethanediyl)bis-1,3-
propanediamine) is a disulfide-containing polyamine analog, which is taken up
by
cells using the polyamine transporter (Mitchell et al., Carcinogenesis,
16:3063-
3068, 1995, herein incorporated by reference). This transporter is inhibited
by
antizyme. Evidence from animal models indicates that this radioprotector is
taken
up by at least some normal tissues to a greater extent than some tumors {Ito
et al.,
International Journal o, f'Radiation Oncolo~r, Biology, Physics 28:899-903,
1994).
Agents like WR-33278 have been used in clinical radiotherapy in attempts to
protect dose-limiting normal tissues from toxicity, without reducing the tumor
I0 control effectiveness of radiotherapy (Spencer and Goa, Drugs, 50{6):1001-
31,
1995, herein incorporated by reference). Rationale for the difference in
uptake of
WR-33278 may be that proliferating tumor cells often contain higher levels of
polyamines than do non-proliferating cells in normal tissues. Thus, tumors
would
express higher levels of antizyme than would normal tissues.
The inventors have placed an antizyme cDNA lacking the sequences
necessary for polyamine-dependent regulation under the control of the human
heat
shock 70B promoter. The inventors have stably transfected human prostate
cancer
derived DU-145 cells with this construct and have selected clones which
display
heat-inducible suppression of polyamine uptake (indicating heat-inducible
antizyme
activity). The therapeutic application of this gene therapy (HSP70B promoter
regulation of antizyme expression) will be put to use in future clinical
trials in men
with localized prostate cancer. Patients are treated with this gene therapy,
administered intratumorally, combined with systemic WR-33278 and localized
radiotherapy. Expression of antizyme intratumorally is then activated by
localized
hyperthermia. Dose-limiting normal tissues adjacent to these prostate tumors
will
not express antizyme in response to hyperthermia and will take up the
radioprotector WR-33278, while the tumor tissue will not take up the
radioprotector
because they will express antizyrne in response to hyperthermia. This strategy
will
allow higher doses of radiotherapy to be given to the prostate, with the
intent to
improve local control of prostate cancers.
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19
In an alternative embodiment, othex metabolic products of the cytoprotective
drug ethyol (also known as amifostine, WR-2721, or S-2-(3-
aminopropylamino)ethylphosphororthioic acid) other than WR33278 may be used
in conjunction with the expression constructs described herein. For instance,
WR-
1065 (2-(3-aminopropylamino)ethanethiol) may be instead used as the
radioprotector.
There are many other genes that may be delivered using the vectors of the
present invention. For instance, it is contemplated that the vectors of the
present
invention may be used to transfer tumor suppressors, antisense oncogenes and
1 o prodrug activators, such as the HSV-TK gene {Rosenfeld et al., Annals of
Surgery,
225:609-618, 1997; Esandi et al., Gene Therapy, 4:280-287, 1997), for the
treatment of cancer. Other genes which could optionally be used in the
expression
constructs of the present invention include p53, p16, p21, p27, C-CAM, HLA-B7
(Gleich, et al., Arch Otolaryngol Head Neck Surg, 124:1097-104, 1998; Heo et
al.,
Hum. Gene Ther. 9:2031-8, 1998; Nabel et al., Proc. Nat. Acad. Sciences USA,
90: 15388-15393, 1996; Stopeck et al., Journal of Clinical Oncology, 15: 341-
349,
1997), IL2 (O'Malley et al., Molecular Endocrinology, 11:667-673, 1997; Otova
et al., Folia Biologica, 43:25-32, 1997), IL4 (Kling, Nature Biotechnology,
15:316-
317, 1997), IL7 (Toloza et al., Annals of Surgical Oncology, 4:70-79, 1997;
Sharma et al., Cancer Gene Therapy, 3:303-313, 1996), IL12 (Hiscox and Jiang,
In
Vivo, 11:125-137, 1997; Chen et al., Journal of Immunology, 159:351-359,
1997),
GM-CSF (Kreitman and Pastan, Blood, 90:252-259, 1997; Homick et al., Blood,
89:4437-4447, 1997; Lama et al., Haematologica, 82:239-245, 1997), IFNy
(Noguchi et al., Clinical Infectious Diseases, 24:992-994, 1997; Kanemaru et
al.,
European Archives of Oto-Rhino-Laryngology, 254:158-162, 1997; Tanaka et al.,
Journal of Gastroenterolo~ and Hepatology, 11:1155-1160, 1996; Imai et al.,
Liver, 17:88-92, 1997), I-CAM 1, and TNF {Corcione et al., Annals of the New
York
Academy of Sciences, 815:364-366, 1997). (All articles cited in this paragraph
are
herein incorporated by reference.)
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p53 currently is recognized as a tumor suppressor gene (Montenarh, Crit.
Rev. Oncogen, 3:233-256, 1992). High levels of mutant p53 have been found in
many cells transformed by chemical carcinogenesis, ultraviolet radiation, and
several viruses, including SV40. The p53 gene is a frequent target of
mutational
5 inactivation in a wide variety of human tumors and already is documented to
be the
most frequently-mutated gene in common human cancers. It is mutated in over
50% of human NSCLC and in a wide spectrum of other tumors.
P 16"''K4 belongs to a newly described class of CDK-inhibitory proteins that
also includes pl6B, p21"''~~° CIP1, SD11' ~d p27KIP1. .Lhe pl6i''tK4
gene maps to 9p21, a
1 o chromosome region frequently deleted in many tumor types. Homozygous
deletions and mutations of the p16~''~K4 gene are frequent in human tumor cell
lines.
This evidence suggests that the pl6a''KQ gene is a tumor suppressor gene. This
interpretation has been challenged, however, by the observation that the
frequency
of the p 16n''K4 gene alterations is much lower in primary uncultured tumors
than in
15 cultured cell lines. Restoration of wild-type p16~K4 function by
transfection with a
plasmid expression vector reduced colony formation by some human cancer cell
lines (Okamoto, et al., Proc. Natl. Acad. Sci. USA, 91:11045-11049, 1994;
Arap, et
al., Cancer Res., 55:1351-1354, 1995, both herein incorporated by reference).
C-CAM is expressed in virtually all epithelial cells. C-CAM, with an
20 apparent molecular weight of 105 kD, originally was isolated from the
plasma
membrane of the rat hepatocyte by its reaction with specific antibodies that
neutralize cell aggregation. Recent studies indicate that, structurally, C-CAM
belongs to the immunoglobulin (Ig) superfamily and its sequence is highly
homologous to carcinoembryoruc antigen (CEA). The first Ig domain of C-CAM
has been shown to be critical for cell adhesion activity.
Cell adhesion molecules, or CAMs are known to be involved in a complex
network of molecular interactions that regulate organ development and cell
differentiation (Edelman, Annu. Rev. Biochem., 54:135-169, 1985). Recent data
indicate that aberrant expression of CAMS may be involved in the tumorigenesis
of
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21
several neoplasms; for example, decreased expression of E-cadherin, which
predominantly is expressed in epithelial cells, is associated with the
progression of
several kinds of neoplasms. Also, Giancotti and Ruoslahti, Cell, 60:849-859,
1990,
incorporated herein by reference, demonstrated that increasing expression of
asl~1
integrin by gene transfer can reduce tumorigenicity of Chinese hamster ovary
cells
in vivo. C-CAM now has been shown to suppress tumor growth in vitro and in
VIVO.
Other tumor suppressors that may be employed according to the present
invention. For example, the selected polynucleotide may be any one of the
l0 following genes: retinoblastoma (Rb); adenomatous polyposis coli gene
(APC);
deleted in colorectal carcinomas (DCC); neurofibromatosis 1 (NF-1);
neurofibromatosis 2 (NF-2); Wilin's tumor suppressor gene (WT-1); multiple
endocrine neoplasia type 1 (MEN-1); multiple endocrine neoplasia type 2 (MEN-
2); BRCA1; von Hippel-Lindau syndrome (VHL); mutated in colorectal cancer
(MCC); p 16; p21; p57; p27; and BRCA2.
In an alternative embodiment of the invention, the methods and vectors of
the present invention may be used to promote regeneration processes, such as
nerve
regeneration, by stimulating the production of growth factors or cytokines. In
such
an embodiment the selected polynucleotide may be a neurotrophic factor. For
instance, the selected polynucleotide may encode ciliary neurotrophic factor
(CNTF), brain-derived neurotrophic factor (BDNF), or glial cell line-derived
neurotrophic factor (GDNF) (Mitsumoto et al., Science, 265:1107-1110, 1994 and
Gash et al., Ann. Neurol., 44(3 Suppl 1):5121-125, 1998, both herein
incorporated
by reference). Alternatively, the selected polynucleotide of the expression
construct may optionally encode tyrosine hydroxylase, GTP cyclohydrolase 1, or
aromatic L-amino acid decarboxylase (Kang, Mov. Disord., 13 Suppl 1:59-72,
1998, herein incorporated by reference). In still another embodiment, the
therapeutic expression construct may express; a growth factor such as insulin-
like
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22
growth factor-I (IGF-I) (Webster, Mult. Scler., 3:113-I20, 1997, incorporated
herein by reference).
Examples of other diseases for which the present vectors are useful include
but are not limited to hyperproliferative diseases and disorders, such as
rheumatoid
arthritis or restenosis by transfer of therapeutic genes, e.g., gene encoding
angiogenesis inhibitors or cell cycle inhibitors.
b) Antisense constructs
Oncogenes such as ras, myc, neu, raf, erb, src, fms, jun, trk, ret, gsp, hst,
bcl
and abl also are suitable targets. However, for therapeutic benefit, these
oncogenes
would be expressed as an antisense nucleic acid, so as to inhibit the
expression of
the oncogene. The term "antisense nucleic acid" is intended to refer to the
oligonucleotides complementary to the base sequences of oncogene-encoding DNA
and RNA. Antisense nucleic acid, when expressed in a target cell, specifically
bind
to their target nucleic acid and interfere with transcription, RNA processing,
transport and/or translation. Targeting double-stranded (ds) DNA with
polynucleotides leads to triple-helix formation; targeting RNA will lead to
double-
helix formation.
Antisense constructs may be designed to bind to the promoter and other
control regions, exons, introns or even exon-intron boundaries of a gene.
Antisense
RNA constructs, or DNA encoding such antisense RNAS, may be employed to
inhibit gene transcription or translation or both within a host cell, either
in vitro or
in vivo, such as within a host animal, including a human subject. Nucleic acid
sequences comprising "complementary nucleotides" are those which are capable
of
base-pairing according to the standard Watson-Crick complementary rules. That
is,
that the larger purines will base pair with the smaller pyrimidines to form
only
combinations of guanine paired with cytosine ~(G:C) and adenine paired with
either
thyrnine (A:T), in the case of DNA, or adenine paired with uracil (A:U) in the
case
of RNA.
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23
As used herein, the terms "complementary" or "antisense sequences" mean
nucleic acid sequences that are substantially complementary over their entire
length
and have very few base mismatches. For example, nucleic acid sequences of
fifteen bases in length may be termed complementary when they have a
complementary nucleotide at thirteen or fourteen positions with only single or
double mismatches. Naturally, nucleic acid sequences which are "completely
complementary" will be nucleic acid sequences which are entirely complementary
throughout their entire length and have no base mismatches.
While all or part of the gene sequence may be employed in the context of
antisense construction, statistically, any sequence 17 bases long should occur
only
once in the human genome and, therefore, suffice to specify a unique target
sequence. Although shorter oligomers are easier to make and increase in vivo
accessibility, numerous other factors are involved in determining the
specificity of
hybridization. Both binding affinity and sequence specificity of an
oligonucleotide
to its complementary target increases with increasing length. It is
contemplated that
oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more
base
pairs will be used. One can readily determW a whether a given antisense
nucleic
acid is effective at targeting of the corresponding host cell gene simply by
testing
the constructs in vitro to determine whether the endogenous gene's function is
affected or whether the expression of related genes having complementary
sequences is affected.
In certain embodiments, one may wish to employ antisense constructs which
include other elements, for example, those which include C-5 propyne
pyrimidines.
Oligonucleotides which contain C-5 propyne analogues of uridine and cytidine
have been shown to bind RNA with high affinity and to be potent antisense
inhibitors of gene expression (Wagner et al., Science, 260:1510-1513, 1993,
herein
incorporated by reference).
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24
c) Ribozyme constructs
As an alternative to targeted antisense delivery, targeted ribozymes may be
used. The term "ribozyme" refers to an RNA-based enzyme capable of targeting
and cleaving particular base sequences in DNA or, more typically, RNA. In the
present invention, ribozymes are introduced into the cell as an expression
construct
encoding the desired ribozymal RNA. The targets of the ribozymes are much the
same as described for antisense nucleic acids.
Many ribozymes are known to catalyze the hydrolysis of phosphodiester
bonds under physiological conditions. The ribozymes of the present invention
catalyze the sequence specific cleavage of a second nucleic acid molecule,
preferably an mRNA transcript, and optionally an mRNA transcript of an
oncogene.
In general, ribozymes bind to a target RNA through the target binding portion
of
the ribozyme which flanks the enzymatic portion of the ribozyme. The enzymatic
portion of the ribozyme cleaves the target RNA. Strategic cleavage of a target
RNA
destroys its ability to directly or indirectly encode protein. After enzymatic
cleavage of the target has occurred, the ribozyme is released from the target
and
searches for another target where the process is repeated.
In a preferred embodiment of the invention, the ribozyme is a hammerhead
ribozyme, a small RNA molecule derived from plant viroids (Symons, Ann. Rev.
Biochem. 61: 641-671, 1992; Clouet-D'Orval and Uhlenbeck, RNA, 2:483-491,
1996; Haseloff and Gerlach, Nature 334:585-591, 1988; Jeffries and Syrnons,
Nucleic Acids Res. 17: 1371-1377, 1989; Uhlenbeck, Nature 328:596-600, 1987;
all
herein incorporated by reference).
In other embodiments, the ribozyme may be a group I intron, a hairpin
ribozyme, VS RNA, a hepatitis Delta virus ribozyme or an Rnase P-RNA ribozyme
(in association with an RNA guide sequence). Examples of hairpin motifs are
described by Hampel et al., Nucleic Acids Res. 18:299, 1990 and Hampel and
Tritz,
Biochemistry 28:4929, 1989; an example of the hepatitis delta viius motif is
described by Perrotta and Been, Biochemistry 31:16, 1992; an example of the
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RNAseP motif (associated with an external guide sequence) is described by Yuan
et
al., Patent No. 5,624,824; a Neurospora VS RNA ribozyme motif is described in
Saville and Collins, Cell 61: 685-696, 1990, Saville and Coliins, Proc. Natl.
Acad.
Sci. USA 88: 8826-8830, 1991, Collins and Olive, Biochemistry 32: 2795-2799,
5 1993; the group I intron is described in Cech et al., U.S. Patent No.
5,354,855. The
above-mentioned motifs should not be considered limiting with respect to the
present invention and those skilled in the art will recognize that ribozymes
that may
be utilized herein comprise a specific substrate binding site which is
complementary to a target mRNA. Such ribozymes also comprise an enzymatic
l0 portion which imparts RNA cleaving activity to the molecule. The enzymatic
portion resides within or surrounds the substrate binding site.
d) Selectable Markers
In certain embodiments of the invention, the therapeutic vectors of the
present invention contain nucleic acid constructs whose expression may be
15 identified in vitro or in vivo by including a marker in the expression
construct.
Such markers would confer an identifiable change to the cell permitting easy
identification of cells containing the expression construct. Usually the
inclusion of
a drug selection marker aids in cloning and in the selection of transformants.
For
example, genes that confer resistance to neomycin, purornycin, hygromycin,
20 DHFR, GPT, zeocin and histidinol are useful selectable markers.
Alternatively,
enzymes such as herpes simplex virus thymidine kinase (tk) may be employed.
Immunologic markers also can be employed. The selectable marker employed is
not believed to be important, so long as it is capable of being expressed
simultaneously with the nucleic acid encoding a gene product. Further examples
of
25 selectable markers are well known to one of skill in the art and include
reporters
such as EGFP, ~3-gal and chloramphenicol acetyltransferase (CAT).
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26
e) Multigene Constructs and IRES
In certain embodiments of the invention, the use of internal ribosome
binding sites (IRES) elements are used to create multigene, or polycistronic,
messages. IRES elements are able to bypass the ribosome scanning model of 5'
methylated Cap dependent translation and begin translation at internal sites.
IRES
elements from two members of the picanovirus family (polio and
encephalomyocarditis) have been described (Pelletier and Sonenberg, Nature,
334:320-325, 1988), as well as an IRES from a mammalian message (Macejak and
Sarnow, Nature, 353:90-94, 1991). IRES elements can be linked to heterologous
l0 open reading frames. Multiple open reading frames can be transcribed
together,
each separated by an IRES, creating polycistronic messages. By virtue of the
IR.ES
element, each open reading frame is accessible to ribosomes for e~cient
translation. Multiple genes can thus be efficiently expressed using a single
promoter/enhancer to transcribe a single message.
Any heterologous open reading frame can be linked to IRES elements. This
includes genes for secreted proteins, multi-subunit proteins encoded by
independent
genes, intracellular or membrane-bound proteins and selectable markers.
Control Regions
In order for the expression construct to affect expression of a transcript
encoding a therapeutic gene, the polynucieotide encoding the therapeutic gene
will
be under the transcriptional control of a promoter and a polyadenylation
signal. A
"promoter" refers to a DNA sequence recognized by the synthetic machinery of
the
host cell, or introduced synthetic machinery, that is required to initiate the
specific
transcription of a gene. A polyadenylation signal refers to a DNA sequence
recognized by the synthetic machinery of the host cell, or introduced
synthetic
machinery, that is required to direct the addition of a series of nucleotides
on the
end of the MRNA transcript for proper processing and trafficking of the
transcript
out of the nucleus into the cytoplasm for translation. The phrase "under
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27
transcriptional control" means that the promoter is in the correct location in
relation
to the polynucleotide to control RNA polymerase initiation and expression of
the
polynucleotide.
The term promoter will be used here to refer to a group of transcriptional
control modules that are clustered around the initiation site for RNA
polymerase II.
Much of the thinking about how promoters are organized derives from analyses
of
several viral promoters, including those for the HSV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more recent work,
have shown that promoters are composed of discrete functional modules, each
consisting of approximately 7-20 by of DNA, and containing one or more
recognition sites for transcriptional activator or repressor proteins.
At least one module in each promoter functions to position the start site for
RNA synthesis. The best known example of this is the TATA box, but in some
promoters lacking a TATA box, such as the promoter for the mammalian terminal
deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a
discrete element overlying the start site itself helps to fix the place of
initiation.
Additional promoter elements regulate the frequency of transcriptional
initiation. Typically, these are located in the region 30-110 by upstream of
the start
site, although a number of promoters have recently been .shown to contain
2o functional elements downstream of the start site as well. The spacing
between
promoter elements frequently is flexible, so that promoter function is
preserved
when elements are inverted or moved relative to one another. In the tk
promoter,
the spacing between promoter elements can be increased to 50 by apart before
activity begins to decline. Depending on the promoter, it appears that
individual
elements can function either cooperatively or independently to activate
transcription.
Where a human cell is targeted, it is preferable to position the
polynucleotide
coding region adjacent to and under the control of a promoter that is capable
of
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28
being expressed in a human cell. Generally speaking, such a promoter might
include either a human or viral promoter. A list of promoters is provided in
Table
1.
TABLE 1
PROMOTER
Immunoglobulin Heavy Chain
Immunoglobulin Light Chain
T-Cell Receptor
HLA DQ a and DQ (3
', ~i-Interferon
Interleukin-2
Interleukin-2 Receptor
MHC Class II 5
MHC Class II HLA-DRa
(3-Actin
Muscle Creatine Kinase
Prealbumin (Transthyretin)
Elastase~ I
Metallothionein
Collagenase
Albumin Gene
a-Fetaprotein
i-Globin
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29
(3-Globin
c-fos
c-HA-ras
Insulin
Neural Cell Adhesion Molecule (NCAM)
aL.Antitrypsin
H2B (TH2B) Histone
Mouse or Type I Collagen
Glucose-Regulated Proteins (GRP94 and
GRP78)
Rat Growth Hormone
Human Serum Amyloid A (SAA)
Troponin I (TN I)
Platelet-Derived Growth Factor
Duchenne Muscular Dystrophy
SV40
Polyoma
Retroviruses
Papilloma Virus
Hepatitis B Virus
Human Immunodeficiency Virus
Cytomegalovirus
Gibbon Ape Leukemia Virus
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The particular promoter that is employed to control the expression of the
therapeutic gene is not believed to be critical, so long as it is capable of
being
activated by the gene product linked to the inducible promoter. In a preferred
embodiment of the invention, the transactivating protein is tat, and the
promoter
5 which is operably linked to the therapeutic gene is the HIV-1 or HIV-2 LTRs.
For
example, a promoter element containing an AP-1 site would respond to the
inducible expression of the c-jun or c-fos proteins. Other suitable
transactivating
factor/promoter combination would be known by one skilled in the art.
The promoter which controls expression of the gene encoding the
1 o transactivating factor must be an inducible promoter. An inducible
promoter is a
promoter which is inactive or exhibits relatively low activity except in the
presence
of an inducer substance. Some examples of promoters that may be included as a
part of the present invention include, but are not limited to, MT II, MMTV,
collagenase, stromelysin, SV40, marine MX gene, a-2-macroglobulin, MHC class I
15 gene h-2kb, proliferin, tumor necrosis factor, or thyroid stimulating
hormone a
gene. The associated inducers of these promoter elements are shown in Table 2.
The Egr-1 promoter and the multidrug resistance gene (MDR 1 ) promoter are
also
options for inducible promoters. In preferred embodiments the inducible
promoter
is heat shock inducible and is derived from one of the following promoters:
20 HSP70, HSP90, HSP60, HSP27, HSP72, HSP73, HSP25, ubiquitin, and HSP28. In
another preferred embodiment, the inducible promoter comprises a hypoxia-
responsive element, such as those responsive to HIF-1. It is understood that
any
inducible promoter may be used in the practice of the present invention and
that all
such promoters would fall within the spirit and scope of the claimed
invention.
TABLE 2
Element ~ Inducer
MT II Phorbol Ester (TPA)
Heavy metals
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31
MMTV (mouse Glucocorticoids
mammary tumor virus)
(3-Interferon poly(rI)X
poly(rc)
Adenovirus 5 E2 Ela
c-jun Phorbol Ester (TPA), H202
Collagenase Phorbol Ester (TPA)
Stromelysin Phorbol Ester (TPA), IL-1
SV40 Phorbol Ester (TPA)
Murine MX Gene Interferon, Newcastle Disease
Virus
GRP78 Gene A23187
a-2 Macroglobulin IL-6
Vimentin Serum
MHC Class I Gene H- Interferon
2kB
HSP70 Ela, SV40 Large T Antigen
Proliferin Phorbol Ester-TPa
Tumor Necrosis FactorFMA
Thyroid Stimulating Thyroid Hormone
Hormone a Gene
In particularly preferred embodiments, the tat protein is used as the
transactivating factor. The genome of HIV-1 and HIV-2 share a great deal of
similarities with the Simian immunodeficiency viruses (SIVS) and they have
been
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32
extensively studied. It was discovered that in addition to the gag, env, pal
genes
that are common to all retroviruses, there are a number of regulatory genes
that are
important in HIV transcription. The viral tat protein is one such regulatory
factor
and it is characterized by its ability to greatly increase the activity of the
HIV-1 and
HIV-2 promoter (Sodroski et al., J. Virol, 55(3):831-835,1985a; Sodroski, et
al.,
Science, 229(4708):74-77, 1985b; Sodroski, et al., Science, 228(4706):1430-
1434,
Sodroski, et al., Science, 228(4706):1430-1434, 1985c; Sodroski et al.,
Science,
227(4683):171-173, 1985d; which are all incorporated by reference herein). Tat
is
thought to bind with the transactivation response element (TAR) in the HIV LTR
and increase the steady state levels of the HIV specific RNA. There is also
evidence suggesting that tat can act more like a traditional transcription
factor in
that it can interact with several transactivator proteins. Tat and adenovirus
transactivator EIA can act synergistically in increasing the levels of steady
state
RNA (Laspia et al., Genes Dev., 4(12B):2397-2408, 1990, herein incorporated by
reference). Thus, a way to increase further the activity of the HIV-LTR/TAT
constructs is to incorporate ElA into the same construct.
Where a cDNA insert is employed, one will typically desire to include a
polyadenylation signal to effect proper polyadenylation of the gene
transcript. The
nature of the polyadenylation signal is not believed to be crucial to the
successful
practice of the invention, and any such sequence may be employed. Such
polyadenylation signals as that from SV40, bovine growth hormone, and the
herpes
simplex virus thymidine kinase gene have been found to function well in a
number
of target cells.
5. Methods of Gene Transfer
In order to effect transgene expression in a cell, it is necessary to first
introduce or transfer the therapeutic expression constructs of the present
invention
into a cell. Such transfer may employ viral or non-viral methods of
gene.transfer.
This section provides a discussion of methods and compositions of gene
transfer.
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A. Non-viral Transfer
In a preferred embodiment, the therapeutic constructs of the present
invention e.g., various genetic (i.e., DNA) constructs must be delivered into
a cell.
In certain preferred situations, the introduction of the expression construct
into a
cell is mediated by non-viral means.
Several non-viral methods for the transfer of expression constructs into
cultured mammalian cells are contemplated by the present invention. These
include
calcium phosphate precipitation {Graham and Van Der Eb, virology, 52:456-467,
1973; Chen and Okayama, Mol. Cell. Biol., 7:2745-2752, 1987; lZippe et al.,
Mol.
Cell Biol., 10:689-695, 1990) DEAE-dextran (Gopal, Mol. Cell Biol., 5:1188-
1190,
1985), electroporation (Tur-Kaspa et al., Mol. Cell Biol., 6:716-718, 1986;
Potter
et al., Proc. Nat. Acad. Sci. USA, 81:7161:7165, 1984), direct microinjection
(Harland and Weintraub, J. Cell Biol., 1 D 1:1094-1099, 1985), DNA-loaded
liposomes (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, 1982; Fraley
et al., Proc. Natl. Acad. Sci. USA, 76:3348-3352, 1979), cell sonication
(Fechheimer et al., Proc. Natl. Acad. Sci. USA, 84:8463-8467, 1987), gene
bombardment using high velocity microprojectiles (Yang et al., Proc. Natl.
Acad.
Sci. USA, 87:9568-9572, 1990}, and receptor-mediated transfection (Wu and Wu,
J.
Biol. Chem., 262:4429-4432, 1987; Wu and Wu, Biochemistry, 27:887-892, 1988).
(Articles cited in this paragraph are herein incorporated by reference.}
Once the construct has been delivered into the cell the nucleic acid encoding
the therapeutic gene may be positioned and expressed at different sites. In
certain
embodiments, the nucleic acid encoding the therapeutic gene may be stably
integrated into the genome of the cell. This integration may be in the cognate
location and orientation via homologous recombination {gene replacement) or it
may be integrated in a random, non-specific location (gene augmentation). In
yet
further embodiments, the nucleic acid may be stably maintained in the cell as
a
separate, episomal segment of DNA. Such nucleic acid segments or "episomes"
encode sequences sufficient to permit maintenance and replication independent
of
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34
or in synchronization with the host cell cycle. How the expression construct
is
delivered to a cell and where in the cell the nucleic acid remains is
dependent on
the type of expression construct employed.
In a particular embodiment of the invention, the expression construct may be
entrapped in a liposome. Liposomes are vesicular structures characterized by a
phospholipid bilayer membrane and an inner aqueous medium. Multilamellar
liposomes have multiple lipid layers separated by aqueous medium. They form
spontaneously when phospholipids are suspended in an excess of aqueous
solution.
The lipid components undergo self rearrangement before the formation of closed
structures and entrap water and dissolved solutes between the lipid bilayers.
The
addition of DNA to cationic liposomes causes a topological transition from
liposomes to optically birefringent liquid-crystalline condensed globules.
These
DNA-lipid complexes are potential non-viral vectors for use in gene therapy.
Liposome-mediated nucleic acid delivery and expression of foreign DNA in
vitro has been very successful. Using the (3-lactamase gene, Wong et al.,
Gene,
10:87-94, 1980, demonstrated the feasibility of liposome-mediated delivery and
expression of foreign DNA in cultured chick embryo, HeLa, and hepatoma cells.
Nicolau et al., (Methods Enzymol., 149:157-176, 1987, herein incorporated by
reference) accomplished successful liposome-mediated gene transfer in rats
after
intravenous injection. Also included are various commercial approaches
involving
"lipofection" technology.
In certain embodiments of the invention, the liposome may be complexed
with a hemagglutinating virus (HVJ}. This has been shown to facilitate fusion
with
the cell membrane and promote cell entry of liposome-encapsulated DNA. In
other
embodiments, the liposome may be complexed or employed in conjunction with
nuclear nonhistone chromosomal proteins (HMG-1). In yet further embodiments,
the liposome may be complexed or employed in conjunction with both HVJ and
HMG-1. In that such expression constructs have been successfully employed in
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transfer and expression of nucleic acid in vitro and in vivo, then they are
applicable
for the present invention.
Other vector delivery systems which can be employed to deliver a nucleic
acid encoding a therapeutic gene into cells are receptor-mediated delivery
vehicles.
5 These take advantage of the selective uptake of macromolecules by receptor-
mediated endocytosis in almost all eukaryotic cells. Because of the cell type-
specific distribution of various receptors, the delivery can be highly
specific (Wu
and Wu, Adv. DrugDelivety Rev., 12:159-167, 1993).
Receptor-mediated gene targeting vehicles generally consist of two
10 components: a cell receptor-specific ligand and a DNA-binding agent.
Several
ligands have been used for receptor-mediated gene transfer. The most
extensively
characterized ligands are asialoorosomucoid (ASOR) (Wu and Wu, J. Biol. Chem.,
262:4429-4432, 1987) and transferrin (Wagner et al., Proc. Natl. Acad. Sci.
87(9):3410-3414, 1990). Recently, a synthetic neoglycoprotein, which
recognizes
15 the same receptor as ASOR, has been used as a gene delivery vehicle (Ferkol
et al.,
FASEB J., 7:1081-1091, 1993; Perales et al., Proc. Natl. Acad. Sci USA,
91:4086-
4090, 1994) and epidermal growth factor (EGF) has also been used to deliver
genes
to squamous carcinoma cells (Myers, EPO 0273085).
In other embodiments, the delivery vehicle may comprise a ligand and a
20 liposome. For example, Nicolau et al. Methods Enzymol., 149:157-176, 1987,
employed lactosyl-ceramide, a galactose-terminal asialganglioside,
incorporated
into Iiposomes and observed an increase in the uptake of the insulin gene by
hepatocytes. Thus, it is feasible that a nucleic acid encoding a therapeutic
gene also
may be specifically delivered into a cell type such as prostate, epithelial or
tumor
25 cells, by any number of receptor-ligand systems with or without liposomes.
For
example, the human prostate-specific antigen (Watt et al., Proc. Natl. Acad.
Sci.,
83(2):3166-3170, 1986) may be used as the receptor for mediated delivery of a
nucleic acid in prostate tissue.
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3fi
In another embodiment of the invention, the expression construct may
simply consist of naked recombinant DNA or plasmids. Transfer of the construct
may be performed by any of the methods mentioned above which physically or
chemically permeabilize the cell membrane. This is applicable particularly for
transfer in vitro, however, it may be applied for in vivo use as well.
Dubensky
et al. {Proc. Nat. Acad. Sci. USA, 81:7529-7533, 1984), successfully. injected
polyomavirus DNA in the form of CaP04 precipitates into liver and spleen of
adult
and newborn mice demonstrating active viral replication and acute infection.
Benvenisty and Neshif, Proc. Nat. Acad. Sci. USA, 83:9551-9555, 1986, also
demonstrated that direct intraperitoneal injection of CaP04 precipitated
plasmids
results in expression of the transfected genes. It is envisioned that DNA
encoding a
CAM may also be transferred in a similar manner in vivo and express CAM.
Another embodiment of the invention for transferring a naked DNA
expression construct into cells may involve particle bombardment. This method
depends on the ability to accelerate DNA coated microprojectiles to a high
velocity
allowing them to pierce cell membranes and enter cells without killing them
(Klein
et al., Nature, 327:70-73, 1987, herein incorporated by reference). Several
devices
for accelerating small particles have been developed. One such device relies
on a
high voltage discharge to generate an electrical current, which in turn
provides the
motive force (Yang et al., Proc. Natl. Acad. Sci. USA, 87:9568-9572, 1990).
The
microprojecriles used have consisted of biologically inert substances such as
tungsten or gold beads.
B. Viral Vector-Mediated Transfer
Another method of achieving gene transfer is via viral transduction using
infectious viral particles as a delivery vehicle, for example, by
transformation with
an adenovirus vector of the present invention as described herein below.
Alternatively, retroviral or bovine papilloma virus may be employed, both of
which
permit permanent transformation of a host cell with a genes) of interest.
Thus, in
one example, viral infection of cells is used in order to deliver
therapeutically
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37
significant genes to a cell. Typically, the virus simply will be exposed to
the
appropriate host cell under physiologic conditions, permitting uptake of the
virus.
Though adenovirus is exemplified, the present methods may be advantageously
employed with other viral vectors, as discussed below. Such methods will be
familiar to those of ordinary skill in the art.
a) Adenovirus
Adenovirus is particularly suitable for use as a gene transfer vector because
of its mid-sized DNA genome, ease of manipulation, high titer, wide, target-
cell
range, and high infectivity. The roughly 36 kB viral genome is bounded by 100-
200 base pair (bp) inverted terminal repeats (ITR), in which are contained cis-
acting elements necessary for viral DNA replication and packaging. The early
(E)
and late (L) regions of the genome that contain different transcription units
are
divided by the onset of viral DNA replication.
The E1 region (ElA and E1B) encodes proteins responsible for the
regulation of transcription of the viral genome and a few cellular genes. The
expression of the E2 region (E2A and E2B) results in the synthesis of the
proteins
for viral DNA replication. These proteins are involved in DNA replication,
late
gene expression, and host cell shut off (Renan, 1990). The products of the
late
genes (L1, L2, L3, L4 and L5), including the majority of the viral capsid
proteins,
are expressed only after significant processing of a single primary transcript
issued
by the major late promoter (MLP). The MLP (located at 16.8 map units) is
particularly efficient during the late phase of infection, and all the mRNAs
issued
from this promoter possess a 5' tripartite leader (TL) sequence which makes
them
preferred mRNAs for translation.
In order for adenovirus to be optimized for gene therapy, it is necessary to
maximize the carrying capacity so that large segments of DNA can be included.
It
also is very desirable to reduce the toxicity and immunologic reaction
associated
with certain adenoviral products. The two goals are, to an extent, coterminous
in
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38
that elimination of adenoviral genes serves both ends. By practice of the
present
invention, it is possible to achieve both these goals while retaining the
ability to
manipulate the therapeutic constructs with relative ease.
The large displacement of DNA is possible because the cis elements required
for viral DNA replication all are localized in the inverted terminal repeats
(ITR)
( 100-200 bp) at either end of the linear viral genome. Plasmids containing
ITR's
can replicate in the presence of a non-defective adenovirus. Therefore,
inclusion of
these elements in an adenoviral vector should permit replication.
In addition, the packaging signal for viral encapsulation is localized between
194-385 by (0.5-1.1 map units) at the left end of the viral genome. This
signal
mimics the protein recognition site in bacteriophage ~, DNA where a specific
sequence close to the left end, but outside the cohesive end sequence,
mediates the
binding to proteins that are required for insertion of the DNA into the head
structure. E1 substitution vectors of Ad have demonstrated that a 450 by (0-
1.25
map units) fragment at the left end of the viral genome could direct packaging
in
293 cells.
Previously, it has been shown that certain regions of the adenoviral genome
can be incorporated into the genome of mammalian cells and the genes encoded
thereby expressed. These cell lines are capable of supporting the replication
of an
adenoviral vector that is deficient in the adenoviral function encoded by the
cell
line. There also have been reports of complementation of replication deficient
adenoviral vectors by "helping" vectors, e.g., wild-type virus or
conditionally
defective mutants.
Replication-deficient adenoviral vectors can be complemented, in traps, by
helper virus. This observation alone does not permit isolation of the
replication-
deficient vectors, however, since the presence of helper virus, needed to
provide
replicative functions, would contaminate any preparation. Thus, an additional
element was needed that would add specificity to the replication and/or
packaging
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39
of the replication-deficient vector. That element, as provided for in the
present
invention, derives from the packaging function of adenovirus.
It has been shown that a packaging signal for adenovirus exists in the left
end of the conventional adenovirus map. Later studies showed that a mutant
with a
deletion in the ElA (194-358 bp) region of the genome grew poorly even in a
cell
line that complemented the early (ElA) function. When a compensating
adenoviral
DNA (0-353 bp) was recombined into the right end of the mutant, the virus was
packaged normally. Further mutational analysis identified a short, repeated,
position-dependent element in the left end of the Ad5 genome. One copy of the
l0 repeat was found to be su~cient for efficient packaging if present at
either end of
the genome, but not when moved towards the interior of the Ad5 DNA molecule.
By using mutated versions of the packaging signal, it is possible to create
helper viruses that are packaged with varying efficiencies. Typically, the
mutations
are point mutations or deletions. When helper viruses with low efficiency
packaging are grown in helper cells, the virus is packaged, albeit at reduced
rates
compared to wild-type virus, thereby permitting propagation of the helper.
When
these helper viruses are grown in cells along with virus that contains wild-
type
packaging signals, however, the wild-type packaging signals are recognized
preferentially over the mutated versions. Given a limiting amount of packaging
factor, the virus containing the wild-type signals are packaged selectively
when
compared to the helpers. If the preference is great enough, stocks approaching
homogeneity should be achieved.
b) Retrovirus
The retroviruses are a group of single-stranded RNA viruses characterized
by an ability to convert their RNA to double-stranded DNA in infected cells by
a
process of reverse-transcription. The resulting DNA then stably integrates
into
cellular chromosomes as a provirus and directs synthesis of viral proteins.
The
integration results in the retention of the viral gene sequences in the
recipient cell
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and its descendants. The retroviral genome contains three genes - gag, pol and
env
- that code for capsid proteins, polymerase enzyme, and envelope components,
respectively. A sequence found upstream from the gag gene, termed ~f,
functions
as a signal for packaging of the genome into virions. Two long terminal repeat
5 (LTR) sequences are present at the 5' and 3' ends of the viral genome. These
contain strong promoter and enhancer sequences and also are required for
integration in the host cell genome.
In order to construct a retroviral vector, a nucleic acid encoding a promoter
is inserted into the viral genome in the place of certain viral sequences to
produce a
10 virus that is replication-defective. In order to produce virions, a
packaging cell line
containing the gag, pol and env genes but without the LTR and 'IJ components
is
t
constructed. When a recombinant plasmid containing a human cDNA, together
with the retroviral LTR and ~l' sequences is introduced into this cell, line
(by
calcium phosphate precipitation for example), the ~ sequence allows the RNA
15 transcript of the recombinant plasmid to be packaged into viral particles,
which are
then secreted into the culture media. The media containing the recombinant
retroviruses is collected, optionally concentrated, and used for gene
transfer.
Retroviral vectors are able to infect a broad variety of cell types. However,
integration and stable expression of many types of retroviruses require the
division
20 of host cells.
An approach designed to allow specific targeting of retrovirus vectors
recently was developed based on the chemical modification of a retrovirus by
the
chemical addition of galactose residues to the viral envelope. This
modification
could permit the specific infection of cells such as hepatocytes via
25 asialoglycoprotein receptors, should this be desired.
A different approach to targeting of recombinant retroviruses was designed
in which biotinylated antibodies against a retroviral envelope protein and
against a
specific cell receptor were used. The antibodies were coupled via the biotin
components by using streptavidin (Roux et al., Proc. Natl Acad. Sci. USA,
86:9079-
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41
90$3, 19$9). Using antibodies against major histocompatibility complex class I
and
class II antigens, the infection of a variety of human cells that bore those
surface
antigens was demonstrated with an ecotropic virus in vitro.
c) Adeno-associated Irrus
AAV utilizes a linear, single-stranded DNA of about 4700 base pairs.
Inverted terminal repeats flank the genome. Two genes are present within the
genome, giving rise to a number of distinct gene products. The first, the cap
gene,
produces three different virion proteins (VP), designated VP-1, VP-2 and VP-3.
The second, the rep gene, encodes four non-structural proteins (NS). One or
more
of these rep gene products is responsible for transactivating AAV
transcription.
The three promoters in AAV are designated by their location, in map units,
in the genome. These are, from left to right, p5, p19 and p40. Transcription
gives
rise to six transcripts, two initiated at each of three promoters, with one of
each pair
being spliced. The splice site, derived from map units 42-46, is the same for
each
transcript. The four non-structural proteins apparently are derived from the
longer
of the transcripts, and three virion proteins all arise from the smallest
transcript.
AAV is not associated with any pathologic state in humans. Interestingly,
for efficient replication, AAV requires "helping" functions from viruses such
as
herpes simplex virus I and II, cytomegalovirus, pseudorabies virus and, of
course,
adenovirus. The best characterized of the helpers is adenovirus, and many
"early"
functions for this virus have been shown to assist with AAV replication. Low
level
expression of AAV rep proteins is believed to hold AAV structural expression
in
check, and helper virus infection is thought to remove this block.
The terminal repeats of the AAV vector can be obtained by restriction
endonuclease digestion of AAV or a plasmid such as p201, which contains a
modified AAV genome {Sarnulski et al. J. hirol., 61(10):3096-3101, 19$7,
herein
incorporated by reference), or by other methods known to the skilled artisan,
including but not limited to chemical or enzymatic synthesis of the terminal
repeats
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42 -
based upon the published sequence of AAV. The ordinarily skilled artisan can
determine, by well-known methods such as deletion analysis, the minimum
sequence or part of the AAV ITRs which is required to allow function, i.e.,
stable
and site-specific integration. The ordinarily skilled artisan also can
determine
which minor modifications of the sequence can be tolerated while maintaining
the
ability of the terminal repeats to direct stable, site-specific integration.
AAV-based vectors have proven to be safe and effective vehicles for gene
delivery in vitro, and these vectors are being developed and tested in pre-
clinical
and clinical stages for a wide range of applications in potential gene
therapy, both
ex vivo and in vivo.
AAV-mediated efficient gene transfer and expression in the lung has led to
clinical trials for the treatment of cystic fibrosis (Carter and Flotte, Ann.
N. Y. Acad.
Sci., 770:79-90, 1995; Flotte et al., Proc.. Natl. Acad. Sci. USA, 90:10613-
10617,
1993). Similarly, the prospects for treatment of muscular dystrophy by A.AV-
mediated gene delivery of the dystrophin gene to skeletal muscle, of
Parkinson's
disease by tyrosine hydroxylase gene delivery to the brain, of hemophilia B by
Factor IX gene delivery to the liver, and potentially of myocardial infarction
by
vascular endothelial growth factor gene to the heart, appear promising since
AAV-
mediated transgene expression in these organs has recently been shown to be
highly
efficient (Fisher et al., J. Yirol, 70:520-532, 1996; Flotte et al., Proc.
Natl. Acad.
Sci. USA, 90:10613-10617, 1993; Kaplitt et al., Nat. Genet., 8:148-153, 1994;
Kaplitt et al., Arm Thord. Surg., 62:1669-1676, 1996; Koeberl et al., Proc.
Natl.
Acad. Sci. USA, 94:1426-1431, 1997; McCown et al., Brain Res., 713:99-107,
1996; Ping et al., Microcirculation, 3:225-228, 1996; Xiao et al., J. virol.,
70:8098-8108, 1996).
d) Oth er oral Vectors
Other viral vectors may be employed as expression constructs in the present
invention. Vectors derived from viruses such as vaccinia viruses (Ridgeway,
In:
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43
Vectors: A survey of molecular cloning vectors and their uses, Rodriguez RL,
Denhardt DT. ed., Stoneham: Butterworth, pp. 467-492, 1988; Baichwal and
Sugden, In: Gene Transfer, Kucherlapati R, ed., New York, Plenum Press, pp.
117
148, 1986; Coupar et al., Gene, 68:1-10, 1988) canary pox viruses,
lentiviruses and
herpes viruses may be employed.
6. Cell Targets
The methods and vectors of the present invention may be used to target a
wide variety of cells, organs, and tissues within a mammal.
In some embodiments, the expression constructs described herein are used to
l0 treat cancer. The cell which is targeted may be either a tumor cell, a cell
within a
tumor, or a cell near a tumor. The tumor may optionally be in the brain, lung,
liver,
spleen, kidney, bladder, lymph node, small intestine, pancreas, colon,
stomach,
breast, endometrium, prostate, testicle, vulva, cervix, ovary, skin, head and
neck,
esophagus, bone marrow, or blood. One of ordinary skill in the art will be
able to
readily discern an appropriate therapeutic gene to be expressed in a given
tumor
type.
In alternative embodiments, a medical condition other than cancer is being
treated. For instance, the present invention provides for highly effective
protein
replacement therapy. In such a case, a specific type of cell, tissue, or organ
may be
targeted for expression of a protein which is underexpressed in the subject,
especially if the activity of the protein is limited to that specific cell
type, tissue, or
organ. Again, one of ordinary skill in the art will be able to discern which
cells are
most appropriately targeted.
The expression construct may be introduced into the cell of interest through
an in vitro, ex vivo, or in vivo method. Much gene therapy is currently
performed
ex vivo, since the transfection or transduction of an isolated cell is often
more
efficient. The choice of method of introduction will be dependent upon the
cell
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44
type, tissue, or organ being targeted, as well as the particular delivery
vehicle
chosen. One of ordinary skill in the art can readily navigate such a choice.
Since the expression constructs of the present invention require induction to
be active, in many cases the expression construct may be delivered to a larger
part
of the subject's body than just the cell, tissue, or organ in which expression
is
desired. Exposure of the subject to the activating conditions which induce
expression of the transferred expression constructs can then be limited to
achieve
specificity of expression. In many cases, this is preferred. For instance,
exposure
of a subject to radiation therapy is preferably limited to only those areas
necessary.
Application of hyperthermia will generally also be limited in its range. In
other
embodiments, the activating conditions may be conditions inherent to the
targeted
cell itself. For instance, the hypoxic environment of a tumor will trigger
expression
when the expression construct has an inducible promoter containing a hypoxia-
responsive element. In such cases, the resulting expression, will be by its
very
nature, very localized, even if delivery of the expression construct was not
7. Combination Therapy
The expression constructs of the present invention may advantageously be
combined with one or more traditional clinical therapies. One goal of current
cancer
research is to fmd ways to improve the efficacy of chemo- and radiotherapy.
One
way is by combining such traditional therapies with gene therapy. For example,
the
herpes simplex-thymidine kinase (HS-tk) gene, when delivered to brain tumors
by a
retroviral vector system, successfully induces susceptibility to the antiviral
agent
ganciclovir. However, the effective use of gene therapy in combination with
traditional cancer therapies has been hindered by the need for clinically
significant
expression of the genes once they have been transferred to the target cell.
To kill cells, inhibit cell growth, inhibit metastasis, decrease tumor size
and
otherwise reverse or reduce the malignant phenotype of tumor cells, using the
methods and compositions of the present invention, one would generally
introduce
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an expression construct of the present invention into the "target" cell and
induce
expression by the application of hyperthermia or other conditions which
activate
the inducible promoter. This gene therapy may be combined with compositions
comprising other agents effective in the treatment of cancer. These
compositions
5 would be provided in a combined amount effective to kill or inhibit
proliferation of
the cell. This process may involve introducing the expression construct and
the
agents} or factors) into the cell at the same time. This may be achieved by
contacting the cell with a single composition or pharmacological formulation
that
includes both agents, or by exposing the cell to two distinct compositions or
10 formulations, at the same time, wherein one composition includes the
expression
construct and the other includes the agent.
Alternatively, the gene therapy/hyperthermia treatment may precede or
follow the other agent treatment by intervals ranging from minutes to weeks.
In
embodiments where the other agent and expression construct are applied
separately
15 to the cell, one would generally ensure that a significant period of time
did not
expire between the time of each delivery, such that the agent and expression
construct would still be able to exert an advantageously combined effect on
the cell.
In such instances, it is contemplated that one would contact the cell with
both
modalities within about 12-24 hours of each other and, more preferably, within
20 about 6-12 hours of each other, with a delay time of only about 12 hours
being
most preferred. In some situations, it may be desirable to extend the time
period for
treatment significantly, however, where several days (2, 3, 4, 5, 6 or 7) to
several
weeks (1, 2, 3, 4, 5, 6, 7 or 8) lapse between the respective administrations.
It also is conceivable that more than one administration of either expression
25 construct or the other agent will be desired. Various combinations may be
employed, where the expression construct is "A" and the other agent is "B", as
exemplified below:
A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
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AIA/B/B A/B/A/B ABIBIA BB/A/A BIAIBIA BIA/A/B BIBIBIA
A/A/AIB B/A/A/A A/B/A/A AIAIBIA AIBBB B/A/B/B B/B/AIB
Other combinations are contemplated. Again, to achieve cell killing, both
agents
are delivered to a cell in a combined amount effective to kill the cell.
Agents or factors suitable for use in a combined therapy are any chemical
compound or treatment method that induces DNA damage when applied to a cell.
Such agents and factors include radiation and waves that induce DNA damage
such
as, y-irradiation, X-rays, UV-irradiation, microwaves, electronic emissions,
and the
like.
In one embodiment of the invention, the radiation therapy which is
combined with the gene therapy constitutes external beam radiation. The
external
beam radiation treatment typically delivers high-energy radiation, such as
high-
energy x-ray beams.
Alternatively, internal radiation, or brachythcrapy, may be used in
combination with the gene therapy. Methods of delivering brachytherapy include
intracavitary or interstitial placement of radiation sources, instillation of
colloidal
solutions, and parenteral or oral administration. Sealed sources are
encapsulated in
a metal, wire, tube, needle, or the like. Unsealed radioactive sources are
prepared
in a suspension or solution.
Encapsulated radioactive elements are placed in body cavities or inserted
directly into tissues with suitable applicators. The applicator is usually
placed into
the body cavity or tissue surgically or using fluoroscopy. The applicators,
usually
plastic or metal tubes, may be sutured into or near the tumor to hold them in
place.
The radioactive isotope is later placed into the applicator ("afterloading").
Radiative implants are used in the treatment of cancers of the tongue, lip,
breast,
vagina, cervix, endometrium, rectum, bladder, and brain. Encapsulated sources
may also be left within a patient as permanent implants. "Seeding" with small
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beads of radioactive material is an approach used for the treatment of
localized
prostate cancers, and localized, but inoperable, lung cancers.
A variety of chemical compounds, also described as "chemotherapeutic
agents," function to induce DNA damage, all of which are intended to be of use
in
the combined treatment methods disclosed herein. Chemotherapeutic agents
contemplated to be of use, include, e.g., adriamycin, 5-fluorouracil (SFU),
etoposide (VP-16), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP)
and even hydrogen peroxide. The invention also encompasses the use of a
combination of one or more DNA damaging agents, whether radiation-based or
l0 actual compounds, such as the use of X-rays with cisplatin or the use of
cisplatin
with etoposide.
For example, in treating cancer according to the invention, one would
contact the tumor cells with an agent in addition to the expression construct
and
induce the expression of the gene by application of hyperthermia. This may be
achieved by applying hyperthermia locally at the tumor site or systemically to
the
individual. This treatment may be in combination with irradiation of the tumor
with radiation such as X-rays, UV-light, gamma-rays or even microwaves.
Alternatively, the tumor cells may be contacted with the agent by
administering to
the subject a therapeutically effective amount of a pharmaceutical composition
comprising a compound such as, adriamycin, 5-fluorouracil, etoposide,
camptothecin, actinomycin-D, mitomycin C, or more preferably, cisplatin. The
agent may be prepared and used as a combined therapeutic composition, or kit,
by
combining it with a therapeutic expression construct, as described above.
Agents that directly cross-link nucleic acids, specifically DNA, are
envisaged to facilitate DNA damage leading to a synergistic, antineoplastic
combination with the expression constructs of the present invention. Agents
such
as cisplatin, and other DNA alkylating agents may be used. Cisplatin is not
absorbed orally and must therefore be delivered via injection intravenously,
subcutaneously, intratumorally or intraperitoneally.
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Agents that damage DNA also include compounds that interfere with DNA
replication, mitosis and chromosomal segregation. Such chemotherapeutic
compounds include adriamycin, also known as doxorubicin, etoposide,
veraparnil,
podophyllotoxin, and the like. Widely used in a clinical setting for the
treatment of
neoplasms, these compounds are administered through bolus injections
intravenously at doses ranging from 25-75 mg/m2 at 21 day intervals for
adriamycin, to 100 mg/m2 for etoposide intravenously or double the intravenous
dose orally.
Agents that disrupt the synthesis and fidelity of nucleic acid precursors and
subunits also lead to DNA damage. As such a number of nucleic acid precursors
have been developed. Particularly useful are agents that have undergone
extensive
testing and are readily available. As such, agents such as 5-fluorouracil (5-
FU), are
preferentially used by neoplastic tissue, making this agent particularly
useful for
targeting to neoplastic cells. Although quite toxic, 5-FU, is applicable in a
wide
range of carriers, including topical, however intravenous administration with
doses
ranging from 450-1000 mg/m2/day being commonly used.
Other factors that cause DNA damage and have been used extensively
include what are commonly known as y-rays, X-rays, and/or the directed
delivery
of radioisotopes to tumor cells. Other forms of DNA damaging factors are also
contemplated such as microwaves and UV-irradiation. It is most likely that all
of
these factors effect a broad range of damage on DNA, on the precursors of DNA,
on the replication and repair of DNA, and on the assembly and maintenance of
chromosomes. Dosage ranges for X-rays range from daily doses of 50 to 200
roentgens for prolonged periods of time (3 to 4 weeks), to single doses of
2000 to
6000 roentgens. Dosage ranges for radioisotopes vary widely, and depend on the
half life of the isotope, the strength and type of radiation emitted, and the
uptake by
the neoplastic cells.
The skilled artisan is directed to "Remington's Pharmaceutical Sciences"
15th Edition, chapter 33, in particular pages 624-652. Some variation in
dosage
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will necessarily occur depending on the condition of the subject being
treated. The
person responsible for administration will, in any event, determine the
appropriate
dose for the individual subject. Moreover, for human administration,
preparations
should meet sterility, pyrogenicity, general safety and purity standards as
required
by FDA Office of Biologics standards.
The regional delivery of the therapeutic expression constructs of the present
invention to patients with cancers is a preferred method for delivering a
therapeutically effective gene to counteract the clinical disease being
treated.
Similarly, the chemo- or radiotherapy may be directed to a particular,
affected
region of the subject's body. Alternatively, systemic delivery of expression
construct and/or the agent may be appropriate in certain circumstances, for
example, where extensive metastasis has occurred.
In addition to combinations of gene therapies with chemo- and
radiotherapies, it also is contemplated that combination of multiple gene
therapies
will be advantageous. For example, targeting of p53 and p16 mutations at the
same
time may produce an improved anti-cancer treatment. Any other tumor-related
gene conceivably can be targeted in this manner, for example, p21, Rb, APC,
DCC,
NF-1, NF-2, BRCA2, p16, FHIT, WT-1, MEN-I, MEN-II, BRCA1, VHL, FCC,
MCC, ras, myc, neu, raf, erb, src, jyrts, jun, trk, ret, gsp, hst, bcl and
abl.
8. Pharmaceutical Compositions and Routes of Administration
It is contemplated that the therapeutic compositions of the present invention
may be administered, in vitro, ex vivo or in vivo. Thus, it will be desirable
to
prepare the complex as a pharmaceutical composition appropriate for the
intended
application. Generally this will entail preparing a pharmaceutical composition
that
is essentially free of pyrogens, as well as any other impurities that could be
harmful
to humans or animals. One also will generally desire to employ appropriate
salts
and buffers to render the complex stable and allow for complex uptake by
target
cells.
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The compositions of the present invention comprise an effective amount of
the expression construct or a viral vector or .liposome carrying the
expression
construct, dissolved or dispersed in a pharmaceutically acceptable carrier or
aqueous medium. Such compositions also can be referred to as inocula. The
5 phrases "pharmaceutically or pharmacologically acceptable" refer to
molecular
entities and compositions that do not produce an adverse, allergic or other
untoward
reaction when administered to an animal, or a human, as appropriate. As used
herein, "pharmaceutically acceptable carrier" includes any and all solvents,
dispersion media, coatings, antibacterial and antifungal agents, isotonic and
1 o 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 active ingredient, its
use in
the therapeutic compositions is contemplated. Supplementary active ingredients
also can be incorporated into the compositions.
15 Solutions of the active compounds as free base or pharmacologically
acceptable salts can be prepared in water suitably mixed with a surfactant,
such as
hydroxypropylcellulose. Dispersions also can be prepared in glycerol, liquid
polyethylene glycols, and mixtures thereof and in oils. Under ordinary
conditions
of storage and use, these preparations contain a preservative to prevent the
growth
20 of microorganisms.
The therapeutic compositions of the present invention may include classic
pharmaceutical preparations for use in therapeutic regimens, including their
administration to humans. Administration of therapeutic compositions according
to
the present invention will be via any common route so long as the target
tissue or
25 cell is available via that route. This includes oral, nasal, buccal,
rectal, vaginal or
topical. Alternatively, administration will be by orthotopic, intradermal
subcutaneous, intramuscular, intraperitoneal, or intravenous injection. Such
compositions would normally be administered as pharmaceutically acceptable
compositions that include physiologically acceptable carners, buffers or other
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excipients. For application against tumors, direct intratumoral injection,
injection
of a resected tumor bed, regional (e.g., lymphatic) or systemic administration
is
contemplated. It also may be desired to perform continuous perfusion over
hours or
days via a catheter to a disease site, e.g., a tumor or tumor site.
The therapeutic compositions of the present invention are administered
advantageously in the form of injectable compositions either as liquid
solutions or
suspensions; solid forms suitable for solution in, or suspension in, liquid
prior to
injection may also be prepared. These preparations also may be emulsified. A
typical composition for such purpose comprises a pharmaceutically acceptable
carrier. For instance, the composition may contain about 100 mg of human serum
albumin per milliliter of phosphate buffered saline. Other pharmaceutically
acceptable carriers include aqueous solutions, non-toxic excipients, including
salts,
preservatives, buffers and the like may be used. Examples of non-aqueous
solvents
are propylene glycol, polyethylene glycol, vegetable oil and injectable
organic .
esters such as ethyloleate. Aqueous carriers include water, alcoholiclaqueous
solutions, saline solutions, parenteral vehicles such as sodium chloride,
R.inger's
dextrose, etc. Intravenous vehicles include fluid and nutrient replenishers.
Preservatives include antimicrobial agents, anti-oxidants, chelating agents
and inert
gases. The pH and exact concentration of the various components of the
pharmaceutical composition are adjusted according to well known parameters.
Additional formulations which are suitable for oral administration also are
contemplated. Oral formulations include such typical excipients as, for
example,
pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium
saccharine, cellulose, magnesium carbonate and the like. The compositions take
the form of solutions, suspensions, tablets, pills, capsules, sustained
release
formulations or powders. When the route is topical, the form may be a cream,
ointment, salve or spray.
An effective amount of the therapeutic agent is determined based on the
intended goal, for example (i) inhibition of tumor cell proliferation, (ii)
elimination
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or killing of tumor cells, or (iii) gene transfer for short- or long-term
expression of a
therapeutic gene. The term "unit dose" refers to physically discrete units
suitable
for use in a subject, each unit containing a predetermined quantity of the
therapeutic composition calculated to produce the desired responses, discussed
above, in association with its administration, i. e., the appropriate route
and
treatment regimen. The quantity to be administered, both according to number
of
treatments and unit dose, depends on the subject to be treated, the state of
the
subject and the result desired. Multiple gene therapeutic regimens are
contemplated
by the present inventors
In one embodiment, a vector encoding a therapeutic gene is used to treat
cancer patients. Typical amounts of a viral vector used in gene therapy of
cancer is
106-lOls PFU/dose e. 106 107 10g 109 10~° 1011 1012 1013 1014 and 101s)
( &> > > > > > > > >
wherein the dose is divided into several injections at different sites within
a solid
tumor. The treatment regimen also involves several cycles of administration of
the
gene transfer vector over a period of 3-10 wk. Administration of the vector
for
longer periods of time from months to years may be necessary for continual
therapeutic benefit.
In another embodiment of the present invention, a viral vector encoding a
therapeutic gene may be used to vaccinate humans or other mammals. Typically,
an amount of virus effective to produce the desired effect, in this case
vaccination,
would be administered to a human or other mammal so that long term expression
of
the transgene is achieved and a host immune response develops. It is
contemplated
that a series of injections, for example, a primary injection followed by two
booster
injections, would be sufficient to induce an long term immune response. A
typical
dose would be from 106 to 1015 PFU/injection depending on the desired result.
Low doses of antigen generally induce a strong cell-mediated response, whereas
high doses of antigen generally induce an antibody-mediated immune response.
Precise amounts of the therapeutic composition also depend on the judgment of
the
practitioner and are peculiar to each individual.
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9. Examples
The following specific examples are intended to illustrate the invention and
should not be construed as limiting the scope of the claims.
EXAMPLE 1
The Heat Shock Promoter Can Induce Expression of a Reporter Gene
Vector Constructs. To study the ability of the HSP70 promoter to induce
gene expression, either a minimal heat shock (HS) promoter or a minimal CMV
promoter was inserted upstream of a reporter gene in a plasmid containing
l0 neomycin and ampicillin selectable markers. The basic design of a plasmid
(MS)
containing one multiple cloning site in operable position to a promoter
derived from
HSP70 is shown in Figure 1. MS was constructed by replacing the CMV promoter
in pcDNA3.0 (Invitrogen, Inc.) with a minimal HSP70B promoter (SEQ ID NO:1,
Figure 10), a 0.4 kb fragment (HindIII-BarnHl) of the human heat shock protein
70B {HSP70B) promoter, obtained from StressGen, Inc.
Activity of the minimal HS and CMV promoters were determined by
transfecting human cancer cells, MCF7 human breast carcinoma cells and DU 145
human prostate carcinoma cells, with the plasmid S8. The S8 plasmid, derived
from the MS vector of Figure 1, contains the minimal HSP70B promoter operably
linked to the gene encoding Enhanced Green Fluorescence Protein (EGFP). S8 was
constructed by inserting the EGFP gene from pEGFP-1 (Clonetech, Inc.) into the
multiple cloning site (MCS) of M5.
Cell Culture and Transfection. Human DU-145 prostate cancer derived
cells and MCF-7 human breast cancer derived cells were transfected with the S8
vector described above. To isolate cells stably transfected with S8, cultures
were
transfected using standard calcium phosphate precipitation methods. Cells
containing the integrated plasmids were selected for their ability to
proliferate in the
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presence of neomycin. Heat shock was administered by completely submersing
culture flasks in a temperature controlled (t0.1 °C) waterbath.
One positive clone, clone 4, and a polyclonal line were selected with
geneticin from the MCF7 cells' transfection. One polyclonal line was selected
with
geneticin from the DU-145 cell's transfection. (In each case, the cells were
selected with geneticin for 2 weeks.) The selected lines were then analyzed
and
sorted by FACS.
Isolation of Positive Cell Lines. Cells expressing high levels of EGFP in
l0 response to heat shock were selected both using conventional serial
dilution
methods and by fluorescence activated cell sorting {FACS) methods. Expression
of
EGFP was quantitated using flow cytometry. The Enhanced Green Fluorescence
Protein (EGFP) excites at 490 run allowing it to be viewed under a
fluorescence
microscope or analyzed by FACS. Cells expressing EGFP were sorted from cells
not expressing EGFP by using the FACS method. This was done with Geneticin-
selected cell lines. The reason that this is required is that in a polyclonal
cell line
there are populations that have the S8 plasmid integrated in a way that
interferes
with the expression of the reporter gene. By sorting the cells these
populations can
be removed leaving the purely positive population for further analysis.
As seen in Figure 2, mean fluorescence from EGFP in DU-145 cells stably
transfected with the minimal heat shock promoter driving EGFP (S8) and growing
at 37°C was approximately 10 relative fluorescence units. Four hours
after
exposure to 42°C heat shock for one hour, the mean relative
fluorescence was 7-9
times greater. Relative gene expression was subsequently quantitated by
measuring
changes in relative fluorescence in stably transfected cells. The sorting by
FACS of
MCF7 cells transfected with the S8 plasmid is illustrated in Figure 3.
Kinetic Studies. Heat exposure survival studies were conducted to evaluate
the optimal times/temperatures at which MCF7 cells could be heated without
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causing massive cell death. For 40°C and 42°C up to 1 hour cell
death was found to
be negligible with less than a 3% cell death. At 44°C for a time of
only 30 minutes
almost 50% of the cells had died.
Using the optimal survival times above, initial kinetic studies were
5 performed. Heating transfected MCF7 cells for 1 hour at 40°C and
42°C produced
more EGFP than heating for only 30 minutes when assayed by FACS. The optimal
recovery time for the cells after heating was 4 hour. Any additional recovery
time
did not increase the levels of EGFP. For heat treatments done at 44°C
for 30 min,
the recovery time took longer with 8 hours being maximal.
10 EGFP Expression at 37°C-44°C in Various Cell Lines. The
following
heating/recovering times were used as identified in the kinetic studies for
testing the
inducibility of EGFP driven by the HSP70-derived promoter in all of the
inventors'
transfected cell lines:
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40°C - 1 h of heat treatment, 4 h of recovery
42°C - 1 h of heat treatment, 4 h of recovery
44°C - 30 min of heat treatment, 8 h of recovery
Using these temperatures/times the following cell lines were tested for EGFP
expression:
MCF7: breast carcinoma parental cell line.
Du145: prostate carcinoma parental cell line.
MCF7-S8-P: MCF7 cells transfected with the S8 plasmid, polyclonal
line.
MCF7-S8-PS1: MCF7-S8-P cells that were sorted for EGFP
expression by FACS once.
MCF7-S8-PS2: MCF7-S8-PS 1 cells that were resorted for EGFP
expression by FACS.
MCF7-S8-4: Clone 4 of the MCF7 S8 transfection.
MCF7-S8-451: MCF7-S8-4 cells sorted once for EGFP expression.
Du145-S8-P: Du145 cells transfected with the S8 plasmid, polyclonal
line.
The expression seen from the transfected lines of EGFP driven by the
HSP70-derived promoter is shown in Figure 4. As the temperature increases the
relative amount of EGFP also increases. These data show that the inventors'
heat
shock promoter does indeed respond to heat. However, at 37°C there was
still
expression of EGFP.
EGFP Expression in Stably Transfected DU-145 Cells After Heat Shock.
The induction of endogenous heat shock promoters is transient and temperature
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dependent. When DU-145 cells, stably transfected with the minimal HS promoter
driving EGFP expression (S8) and selected twice by FACS {DU-S8-PS2 cells),
were heat shocked for various times and at various temperatures, reporter gene
expression was temperature-dependent and expression was transient with maximal
values at 15-24 hours after the inducing stress (Figure 5). These results
indicate
that the promoter is transiently activated under the conditions used here and
that
EGFP is unstable, since fluorescence decreases after 15-24 hours in these
cells.
The minimal heat shock promoter activity increases transiently by
approximately 3
fold after a 40°C heat shock for either 1 or 2 hours. Promoter activity
increases 13
l0 and 25 fold after 42°C heat shock for either 1 or 2 hours,
respectively.
Comparison of Expression of EGFP Under Control of Heat Shock and
CMV Promoters. The data presented in Figure 6 show that minimal heat shock
promoter activity in DU-S8-PS2 cells is temperature-dependent over the range
from
37-43°C. In contrast, DU-145 cells stably transfected with V9, a vector
in which
the CMV promoter drives EGFP expression (Figure 7), express nearly 50% higher
levels of promoter activity than do these same cells transfected with the
minimal
heat shock promoter and induced with 43°C heat shock. The CMV promoter
activity is essentially unaffected by temperature in these cells. The
temperature-
dependence of the minimal HS promoter is not specific to the DU-145 cells.
EXAMPLE 2
Expression of IL-2 Can Be Amplifeed by the Use of a
HIV Promoter and tat in a Construct.
Initial Amplifier Studies. Studies involving new constructs capable of
amplifying a therapeutic gene's expression were performed. To demonstrate the
principle of the amplifier idea, several constructs were produced. The
constructs
contain a constitutive promoter, the CMV promoter, rather than a heat-shock
induible promoter. These constructs are the plasmids L-27, X 14; RR I 3, Y 15,
and
SS 10. Table 3, below, shows the promoters/genes present in each plasmid and
the
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~8
amount of iL-2 produced. Four of the plasmids were obtained from a plasmid
containing two multiple cloning sites. In these four plasmids, the CMV
promoter
was inserted upstream of either the tat gene or a multiple cloning site (MCS)
and
either the HIV 1 or HIV2 long terminal repeats (LTRs) was inserted upstream of
the
mouse interleukin-2 (IL-2) gene. The plasmids X 14 and Y 15 are shown
schematically in Figure 9A and 9B. The L-27 plasmid served as a reference. IL-
2
was measured from tissue culture supernatants by ELISA using the IL-2 EASIA
kit
(Medgenix Diagnostics, Fleurus, Belgium). The sensitivity of the kit is
estimated at
O.lIU IL-2/ml. In this study, SW480 cells were transfected with the lipid
Dosper
l0 (see the transfection protocol of Example 3, below.).
TABLE 3
Plasmid Name Promoter/gene Amount of IL-2 in LU.
Lipid alone Dosper .48
L-27 CMV/IL-2 15.63
RR13 HIV 1/IL-2 17.56
CMV/multiple cloning
site
X 14 HI V 1 /IL-2 173 . 7
CMV/TAT
SS 10 HIV2/IL-2 12.83
CMV/multiple cloning
site
Y 15 HIV2/IL-2 440.55
CMV/TAT
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It can be seen from this study that the complete amplifier constructs are
capable of increased expression over the CMV promoter. Also, the production of
the transactivating factor, TAT, is required for this increased production.
EXAMPLE 3
Heat Inducible Amplifiers.
Vector Construction. To determine if a second promoter could be used to
increase the activity of the minimal HS promoter, MCF-7 cells were transiently
transfected with a series of vectors, including pCB, pfl2, and p007 (Figures 8
and
9). Using a plasmid containing two multiple cloning sites, the minimal heat
shock
promoter was inserted upstream of either the tat gene or a multiple cloning
site
(MCS) and either the HIV 1 or HIV2 long terminal repeats (LTRs) was inserted
upstream of the mouse interleukin-2 (IL-2) gene. The plasmids also each
carried
neomycin and ampicillin selectable markers.
The plasmid fll was first created by inserting a 0.5 kb EcoRI fragment,
containing the interleukin-2 (IL-2) coding region (see GenBank accession no.
577834), from plasmid CS into the EcoRI site of the vector MS (see specific
example, Example 1, above). The plasmid C8 was constructed by inserting a 1 kb
BarnHI fragment, containing the 0.4 kb HSP70B fragment upstream of a MCS from
plasmid B4527 (see Tsang, et al., Biotechniques 20:51-52, 1996 and Tsang et
al.,
2o Biotechniques 22:68, 1997, both herein incorporated by reference}, into the
BamHI
site of plasmid DNP-1 (Tsang et al., 1996, and Tsang et al., 1997), which
contains
the HIV1 LTR upstream of the IL-2 coding region. The vector f12 (Figure 9) was
then constructed by inserting a 0.4 kb NotI fragment, containing the coding
region
for the HIV tat gene, into the NotI site of C8. An intermediate vector D10 was
constructed by inserting the 1 kb BamH I fragment containing the minimal
HSP70B
promoter into plasmid MNP-7 (Tsang et al., 1996, and Tsang et al., 1997),
which
contains the HIV2 LTR upstream of the IL-2 coding region. Plasmid 007 (Figure
9)
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was created by inserting the 0.4 kb Not I fragment, encoding the tat gene,
into the
Not I site of D 10.
Transfection Protocol. Transfection were performed according to the
published procedure (Stopeck, et al., Cancer Gene Therapy, 5:119-126, 1998.)
5 MCF-7 cells were plated in either a 6 well or 12 well plates. The next day,
cells
were washed with Hanks Buffered Saline Solution and replaced with a 1 rnl
transfection solution. The transfection solution was a 4:1 lipid to DNA mass
ratio
of either Dosper (1,3-Di-Oleoyloxy-2-(6-Carboxy-spermyl)-propylamid, from
Boehringer Mannheim) or Dmrie C ( 1,2-dimyristyloxypropyl-3dimethyl-hydroxy
10 ethyl ammonium bromide, from Gibco BRL) with either 1.25 ~.g or 2.5 p,g of
plasmid DNA in serum-free OptiMEM (from Gibco BRL). Fetal bovine serum
(FBS) was immediately added to each well to a final concentration of 10%
(vol/vol). Dmrie C was determined to be a better lipid then Dosper. Cells were
incubated for 24 hours before heating and 24 hours after heating prior to IL-2
15 quantitation.
Heat-Induced Amplification Studies. In one set of experiments, cells
transfected with the pCB, pfl2, or p007 plasmids were assayed for IL-2
expression
activity. IL-2 was measured from tissue culture supernatants by ELISA using
the
IL-2 EASIA kit (Medgenix Diagnostics, Fleurus, Belgium). The sensitivity of
the
20 kit is estimated at 0. lIU IL-2/ml. The data from this set of experiments
are shown
in Table 4, below. This table shows IL-2 expression levels in MCF7 cells which
were transfected with Dosper, heated 24 hours later, and were then assayed by
ELISA 49 hours after the transfection. The plasmids L-27 (a plasmid used for
reference that expresses IL-2 driven by the CMV promoter), 007, f12, and C8
were
25 all tested.
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TABLE 4
__ _. -LU. of II,-2
. .
temperature:37C 39C 41C 42C 44C
heat shock continuous continuous1 hr lhr 0.5 hr
.
duration:
Lipid alone2.03 0.50 0.41 0.53 0.53
L-27 14.2 9.88 5.95 9.88 7.80
007 336.76 318.49 334.02 373.74 389.27
F12 8.40 6.88 49.93 60.02 88.13
C8 9.19 8.03 11.74 8.73 16.37
From this study it can be seen that pfl2 is responsive to heating and
produces larger quantities of the heat shock amplifier construct IL-2 than
either pC8
or pL-27 does. At 37°C, pfl2 produced 5-fold more IL-2 than its CMV
driven
control, L-27. When cells were heat shocked at 39°C overnight, pfl2
produced 7-
fold more IL-2 than the CMV driven controls at 37°C. A 1 hour heat
shock
treatment at 41°C or 42°C increased expression from the
amplifier constructs by as
much as 26-fold, compared to the CMV-driven control vector at 37°C.
(However,
the p007 plasmid at 37°C is already near its maximal activity and does
not increase
expression levels greatly with heat.) The activity of pfl2 is also at a high
level at
37°C. These results showed that the amplifier strategy can augment the
levels of
gene expression at temperatures between about 37°C and about
42°C.
In a different set of experiments, variations in transfection efficiencies
were
accounted for by co-transfection with a control plasmid in which the CMV
promoter was upstream of ~i-galactosidase. The general protocol for these
experiments was to transfect cells 24 hours after subculture, heat shock
cultures an
additional 24 hours later, change culture medium and then collect medium for
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measurement of IL-2 levels 24 hours later. As seen in Table 5, below, the
activity
of the CMV promoter was only minimally affected by heat shock. The minimal
heat shock promoter activity was very low in cells maintained at 37°C
and was
induced over 20 fold by heat shock at 42°C. As seen in the stably
transfected cells,
the minimal heat shock promoter activity was only about one half that of the
CMV
promoter.
TABLE 5
Interleukin-2 (IL2) Expression*
Vector Promoter 37°C 42°C** Fold (42/3?) Relative***
L27 CMV-IL2 82.6 93.4 1.1 1.0
C8 HSP-MCS 84.7 70.6 0.8 1.9
HIV 1-IL2
fll HSP-IL2 2.3 54.0 23.7 0.4 (1)
f12 HSP-TAT 107.6 347.4 3.2 , 6.9 (17)
HIV 1-IL2
007 HSP-TAT 747.5 1642.9 2.2 83.3 (208)
HIV2-IL2
* values in IU IL2 produced per mg cell protein in 24 hours
** heat shock was for 1 hour
*** based on 42°C values and co-transfection with CMV-~-gal
The HIV1 promoter, in the absence of tat expression, was similar to that of
the CMV promoter and was nearly independent of heat shock. However, when the
minimal heat shock promoter was used to express tat, reporter gene expression
was
dramatically increased after 42°C heat shock. In cells transiently
transfected with
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63
heat shock promoter/tat and HIV1/IL-2, IL-2 production was similar to that for
heat
shock promoter/MCS and HIV 1/IL-2 in cells maintained at 37°C. This
activity was
increased over 3 fold, and to levels nearly 7 fold greater than CMV promoter
activity by itself, after 42°C HS.
The HS promoter/tat and HIV2/IL-2 transfected cells showed substantial
reporter gene expression in cells maintained at both 37 and after 42°C
heat shock.
Relative promoter activity, measured by IL-2 production was over 80 fold
higher
than that for the CMV promoter. alone. Temperature regulation was reduced,
with
reporter gene expression approximately 2 times higher after 42°C heat
shock
1 o compared to the same activity in cells maintained at 37°C.
The temperature dependence of reporter gene expression was not influenced
by the presence of a second promoter. As shown in Table b, below, reporter
gene
expression, in cells transiently transfected with the minimal heat shock
promoter/tat
and HIV2/IL-2 containing plasmid, increased in a temperature-dependent manner
between 37 and 44°C. These results are qualitatively similar to those
seen in
Figures 4 and 6 for cells stably transfected with only the minimal heat shock
promoter.
TABLE 6
Ii.-2 Expression ~IU/ml)*
Vector Promoter 37°C 39°C 40°C 41°C
42°C 44°C
C8 HSP-MCS 7.2 9.3 6.0 4.8 5.3 7.0
HIV 1-IL2
fl2 HSP-TAT 40.6 -- -- -- 133.1 -
HIV 1-IL2
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64
007 HSP-TAT 224 222 230 250 375 470
HIV2-IL2
MCF7 breast cancer cells were transiently transfected with vectors as shown;
heat shocked for 1 hr
24 hrs later; media were collected and IL2 measured 24 hrs after heat shock
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EXAMPLE 4
Animal Studies
Mouse models of human cancer, with the histologic features and metastatic
5 potential resembling tumors seen in humans, can be treated with the
therapeutic
compositions of the present invention. In one embodiment of the present
invention,
SCID mice are injected with human tumor cells stably transfected with reporter
constructs in which the HSP70B promoter is driving the expression of TAT and
in
which the HIV-1 or HIV-2 promoter is driving either EGFP or IL-2 expression.
10 After growing the tumors to an appropriate measurable size of for example,
1 cm in
diameter, the tumors are heated using ultrasound to temperatures up to about
42°C.
Gene expression is quantitated at various times after heating by either
removing the
tumor, making tissue slices and measuring fluorescence from EGFP or measuring
tumor tissue levels of IL2 using ELISA. Using another embodiment of the
present
15 invention, human tumor cells are injected into SCID mice. The tumors grown
to an
appropriate measurable size and injected with DNA-lipid complexes. Tumors are
heated using ultrasound and gene expression measured at times after heating.
The
efficacy of these treatments is indicated by a decrease in the size of the
tumor, a
decrease in metastatic activity, a decrease in cell proliferation or a halt in
the tumor
20 growth as a result of the administration of the therapeutic compositions of
the
present invention.
Various modifications and variations of the present invention will be
apparent to those skilled in the art without departing from the scope and
spirit of the
invention. Although the invention has been described in connection with
specific
25 preferred embodiments, it should be understood that the invention as
claimed
should not be unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention which are
obvious to those skilled in the art are intended to be within the scope of the
following claims.
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SEQUENCE LINING
<110> Tsang, Tom
Gerner, Eugene vl.
Harris, David T.
Hersh, Evan
<120> Hyperthermic Inducible Erpression Vectors for Gene
Therapy and Methods of Use Thereof
<130> 15907-0016
<140>
<141>
<150> US 60/064,088
<151> 1997-11-03
<160> 1
<170> PatentIn Ver. 2.0
<210> 1
<211> 469
<212> DNA
<213> Homo sapiens
<400> 1
ggatcctcca cagccccggg gagaccttg~ ~tctaaagtt gctgcttt~g cagctctgcc 60
acaaccgcgc gtcctcagag ccagccggc~ ~gagctaaaa ccttccccgc gtttctttca 120
gcagccctga gtcagaggcg ggctggcct= gcaagtagcc ccccagcc~t cttcggtctc 180
acggaccgat ccgcccgaac cttctcccg: ggtcagcgcc gcgctgcgcc gcccggctga 240
ctcagcccgg gcgggcgggc gggaggctct cgactgggcg ggaaggtgcg ggaaggttcg 300
cggcggcggg gtcggggagg tgcaaaagg~ tgaaaagccc gtggacggag ctgagcagat 360
ccggccgggc tggcggcaga gaaaccgcag ggagagcctc actgctgagc gcccctcgac 420
gcgggcggca gcagcctccg tggcctccac catccgacaa gaagcttac 469
SUBSTITUTE SHEET (RULE 28)
