Note: Descriptions are shown in the official language in which they were submitted.
CA 02516652 2005-08-19
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SYSTEM FOR EXTERNAL CONTROL OF ONCOLYTIC VIRUS REPLICATION
Background
Gene therapy forms the basis of innovative and potentially powerFul disease-
fighting
tools, in which an exogenous nucleotide is provided to a cell. This approach
holds great
potential in treating not only cancer, but many other diseases as well,
including cystic
fibrosis, anemia, hemophilia, diabetes, Hungtington's disease, AIDS,
abnormally high serum
cholesterol levels, certain immune deficiencies, and many forms of cancer.
Gene therapy
generally requires a delivery vehicle for the exogenous sequence, such as a
viral or non
viral vector. A variety of viral vectors have shown therapeutic efficacy
against these
diseases. For reviews, see e.g., Sandrin V et al., Curr Top Microbiol Immunol.
2003;281:137-78; Buning H., Gene Ther. 2003 Ju1;10(14):1142-51 and St George
JA, Gene
Ther. 2003 Ju1;10(14):1135-41.
In an alternate approach applicable to cancer treatment, specific attenuated
replication-competent viral vectors have been developed in which selective
replication in
cancer cells preferentially destroys those cells. For example, various cell-
specific
replication-competent adenovirus constructs, which preferentially replicate
(and thus
destroy) certain cell types, are described in, for example, WO 95/19434, WO
98/39465, WO
98/39457, WO 98/394.66, WO 99/06576, WO 98/39464, WO 00/15820.
Factors that make adenovirus a safe therapeutic agent include the facts that:
(a)
infection with adenovirus has minor clinical disease manifestations; (b)
adenovirus has a
stable well-described and characterized genome; (c) adenovirus typically does
not integrate
its viral DNA into host DNA; (d) adenovirus infection results in transient
gene expression; (e)
adenovirus is able to infect both dividing and non-dividing cells; (~
adenovirus can infect a
variety of human cell types; (g) adenovirus is physically stable; and (h)
adenovirus is
amenable to high titer production.
A continuing concern regarding gene therapy in patients is potential toxicity.
It
follows that the ability to regulate virus infection and/or gene expression in
vivo wquld be
advantageous. Regulated gene expression for purposes of gene therapy is
reviewed, for
example, by ~oltick and Wilson (2001 ) Ann NY Acad Sci. 953:53-63; Harrington
et al.
$,(2000) Adv Drug Deliv Rev. 44(2-3):167-84; and Clackson (2000) Gene Ther.
7(2):120-5.
Unlike replication defective vectors which are typically used for gene
therapy, oncolytic
vectors such as those exemplified herein, replicate in vivo producing progeny
which infect
additional target cells. It follows that there is substantial interest in
being able to regulate the
replication of oncolytic vectors in vivo.
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Summary Of The Invention
Replication-competent viral vectors, and methods for their use are provided.
The
invention provides compositions comprising replication pompetent viruses
having at least
one viral gene under transcriptional control of a regulated gene expression
sysfiem, as
exemplified herein by replication competent adenovirus. The regulated gene
expression
system comprises at least two levels of transcriptional regulation. At a first
level of
regulation, a transcriptional transactivator coding sequence is under the
transcriptional
control of a cell type-specific transcriptional response element (CT-TRE). At
a second level
of regulation, a viral gene is under the transcriptional control regulated by
interaction of the
transeriptional transactivator with a transcriptional response element
(referred to herein as a
transcriptional transactivator regulated response element. Transactivators of
interest in
practicing the invention functionally interact with an inducing agent, usually
a non-native
inducing agent which is exogenously provided to the host cells of interest,
e.g. a chemical
entity such as tetracycline, ecdysone, rapamycin, synthetic progesterones,
glucocorticoids,
or an analog thereof, etc. The inducing agent may also be a non-chemical
inducer, i.e.
ultra-sound, heat, external beam radiation; hypoxia, etc.
The transcriptional transactivator is only produced in cells where the cell
type-
specific THE is active. The activity of the transcriptional transactivator is
dependent upon
the presence/concentration of a particular inducing agent or condition. The
inducing agent
or condition therefore provides a means to regulate the function of the
transcriptional
transactivator. In the presence of the appropriate concentration of inducing
agent an/or
condition, an activating transcriptional transactivator will activate
transcription and an
inhibitory transcriptional transactivator will inhibit transcription.
Expression of a gene
essential for viral replication is thus regulated, thereby regulating virus
replication and
spread. Hence, the replication and spread of an oncolytic virus may be
regulated in vitro or
in vivo by providing the inducing agent and/or adjusting the inducing agent
concentration
using the compositions and methods described herein.
Brief Description Of The Drawings
Figure ~ is a schematic illustrating the genome of a prostate specific
adenovirus
vector with tetracycline regulated replication control.
Figure 2 is a schematic illustrating the genome of a prostate specific
adenovirus
vector armed with GM-CSF, with tetracycline regulated replication and
transgene control.
Figure 3 is a schematic illustrating the genome of an adenovirus vector with
dual
tetracycline regulated replication control.
Figure 4 is a schematic illustrating the genome of an adenovirus vector with
rapamycin (ARIAD system) regulated control for use in pan-cancer applications.
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WO 2005/007832 PCT/US2004/005518
Detailed Description of the Embodiments
Regulated replication-competent viral vectors are provided, which comprise a
cell
type-specific transcriptional regulatory element (CT-TRE) and a transactivator
regulated
transcriptional regulatory element, which is inducible. The virus
preferentially replicates in
cells that allow function of the CT-TRE, and based on the presence or absence
of an
inducer that regulates the transactivator regulated transcriptional regulatory
element.
The CT-THE controls transcription of an inducible transcriptional
transactivator (TA)
coding sequence. The transcriptional transactivator (a) requires an inducing
agent to be
functional and (b) controls transcription of a viral gene. The inducer is
preferably an
exogenous compound, not normally present in the host cells of interest, e.g.
tetracycline,
ecdysone, rapamycin, a synthetic progesterone, a glucocorticoid, and the
lilts, including
analogs thereof, or the inducer may be ultra-sound, heat, ultra-sound, heat,
external beam
radiation, hypoxia or some other treatment.
An inhibitory transcriptional transactivator will inhibit transcription in the
presence of
the inducing agent, and an activating transcriptional transactivator will
activate transcription
in the presence of the inducing agent. In this way, expression of a viral gene
essential for
replication is regulated both by the CT-THE and the transactivator regulated
transcriptional
regulatory element, and indirectly by the concentration of the inducing agent
or condition. It
follows that during treatment of a patient with oncolytic virus therapy,
replication and spread
of the virus may be regulated by adjusting the inducing agent concentration.
Depending on the specific uses of the viral vector, the inducing agent may act
to
inhibit transcription, or to enhance transcription. Transcription inhibitors
provide a way to
"shut down" viral replication by delivering the inducing agent to the host
cells. Transcription
activators provide a way to induce viral replicati~n by addition of the
inducing agent.
Preferably the viral gene essential for replication is an early gene. In some
embodiments, the viral vectors of this invention comprise a viral gene under
the
transcriptional control of a transactivator regulated transcriptional
regulatory element, and at
least one other gene, such as an additional viral gene or a transgene, under
control of either
the same transactivator regulated transcriptional regulatory element or a
second
transactivator regulated transcriptional regulatory element that is
substantially identical to
the first transactivator regulated transcriptional regulatory element.
Preferably, the first and
second genes under transcriptional control of the transactivator regulated
transcriptional
regulatory elements) are both viral genes necessary for replication. By
providing for cell-
specific transcription through the use of one or more transactivator regulated
transcriptional
regulatory elements, the invention provides viral vectors that can be used for
cell-specific
replication resulting in selective cytolysis of target cells, where viral
replication is regulated
through exposure of the target cells to an exogenous inducing agent or
condition.
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WO 2005/007832 PCT/US2004/005518
The viral vectors of the invention replicate preferentially in CT-THE
functional cells,
referred to herein as target cells. This replication preference is indicated
by comparing the
level of replication (i.e., titer) in cells in which the CT-THE is active to
the level of replication
in cells in which the CT-THE is not active (i.e., a non-target cell).
Comparison of the viral
titer following infection of a target cell to the titer following infection of
a non-target cell (CT-
TRE inactive) provides a key indication that the overall replication
preference is enhanced
due to the replication in target cells and depressed replication in non-target
cells. This
provides a means for targeted cell killing, such that runaway infection is
prevented. A further
level of replication control is provided by the presence or absence of an
inducing agent or
condition or changes in the concentration thereof. The viral vectors of the
invention are
exemplified herein by replication competent adenovirus which exhibit target
cell specific
replication, the control of which is further regulated by an inducing agent.
General Technidues
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of molecular biol~gy (including recombinant
techniques),
microbiology, cell biology, biochemistry and immunology, which are within the
skill of the art.
Such techniques are explained fully in the literature, such as, "Molecular
Cloning: A
Laboratory Manual", second edition (Sambrook et al., 199); "~ligonucleotide
Synthesis"
(M.J. Gait, ed., 194.); "Animal Cell Culture" (R.I. Freshney, ed., 197);
"Methods in
Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology"
(D.M. Weir
&~ C.C. Blackwell, eds.); "Gene Transfer ~/ectors for Mammalian Cells" (J.M.
Miller ~ M.P.
Calos, eds., 197); "Current Protocols in Molecular Biology" (F.M. Ausubel et
al., eds.,
19137); "PCR: The Polymerase Chain Reaction", (Mullis et al., eds., 1994); and
"Current
Protocols in Immunology" (J.E. Coligan et al., eds., 1991).
Definitions
A "replication competent" or "oncolytic" vector of the present invention (used
interchangeably herein) can be in any of several forms, including, but not
limited to, naked
DNA; a viral vector encapsulated in a virus coat; a viral vector packaged in
another viral or
viral-like form (such as herpes simplex virus and AAV); a viral vector
encapsulated in a
liposome; a viral vector complexed with polylysine or other biocompatible
polymer; a viral
vector complexed with synthetic polycationic molecules; a viral vector
conjugated with
transferrin; a viral vector complexed with compounds such as PEG to
immunologically
"mask" the molecule and/or increase half-life; or a viral vector conjugated to
a non-viral
protein or any delivery vehicle known to those of skill in the art. Such
vectors represent one
v embodiment of the invention. Preferably, the polynucleotide is DNA. As used
herein,
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WO 2005/007832 PCT/US2004/005518
"DNA" includes not only bases A, T, C, and G, but also includes any of their
analogs or
modified forms of these bases, such as methylated nucleotides, internucleotide
modifications such as uncharged linkages and thioates, use of sugar analogs,
and modified
and/or alternative backbone structures, such as polyamides. For purposes of
this invention,
viral vectors are replication-competent in a target cell. Exemplary
replication-competent
viruses included within the scope of the invention include, but are not
limited to,
adenoviruses, vesicular stomatitis viruses (VSV), herpes viruses (e.g., herpes
simplex virus;
HSV), reoviruses, paramyxoviruses, rhinoviruses, Newcastle disease viruses,
polioviruses,
West Nile virus, coxsackie virus, measles viruses and vaccinia viruses, etc.
Any and all
serotypes of replication-competent viruses can be engineered for targeted
expression and
regulated expression using the techniques described herein.
An "adenovirus vector" or "adenoviral vector" (used interchangeably herein) is
a term
well understood in the art and generally comprises a polynucleotide (defined
herein)
including all or a portion of an adenovirus genome. As used herein,
"adenovirus" refers to
the virus itself or derivatives thereof. The term covers all serotypes and
subtypes and both
naturally occurring and recombinant forms, except where otherwise indicated.
For the
purposes of the present invention, regulatable adenovirus vectors are provided
which
contain a cell type-specific transcriptional regulatory element (CT-TRE)
operably linked to
an inducible transactivator gene, and an adenovirus gene operably linked to a
transcriptional transactivator regulated regulatory element regulated by the
inducible
transactivator. The adenovirus vector may optionally contain a second
adenoviral gene or a
transgene operably linked to a transactivator regulated regulatory element or
another type
of transcriptional regulatory element, which is not cell type-specific. For
techniques related
specifically to adenovirus, see, inter alia, Felgner and Ringold (1989) Nafure
337:387-388;
Berkner and Sharp (1983) Nucl. Acids Res. 11:6003-6020; Graham (1984) EMB~ J.
3:2917-2922; Bett et al. (1993) J. Virology 67:5911-5921; Bett et al. (1994)
Proc. Natl.
Acad. Sei. USA 91:8802-8806. Publications describing various aspects of
adenovirus
biology and/or techniques relating to adenovirus include the following. Graham
and Van de
Eb (1973) Virology 52:456-467; Takiff et al. (1981) Lancet ii:832-834; Berkner
and Sharp
(1983) Nucleic Acid Research 6003-6020; Graham (1984) EMB~ J 3:2917-2922; Bett
et al.
(1993) J. Virology67:5911-5921; and Bett et al. (1994) Proc. Natl. Acad. Sci.
USA 91:8802-
8806 describe replication-defective adenoviruses that have been genetically
modified to act
as gene transfer vehicles (i.e., gene therapy). In such vectors, typically the
early adenovirus
gene products E1A and E1 B are deleted and provided in trans by the packaging
cell line
293 developed by Frank Graham (Graham et al. (1987) J. Gen. Birol. 36:59-72
and Graham
(1977) J. Genetic Virology 68:937-940). The gene to be transduced is commonly
inserted
into adenovirus in the deleted E1A and E1 B region of the virus genome Bett et
al. (1994),
5
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WO 2005/007832 PCT/US2004/005518
supra. Adenovirus vectors for gene therapy have been described by Stratford-
Perricaudet
(1990) Human Gene Therapy 1:2-256; Rosenfeld (1991) Science 252:431-434; Wang
et al.
(1991) Adv. Exp. Med. Biol. 309:61-66; Jaffe et al. (1992) Nat Gent. 1:372-
378; Quantin et
al. (1992) Proc Natl. Acad. Sci. USA 89:2581-2584; Rosenfeld et al. (1992)
Cell 68:143-
155; Stratford-Perricaudet et al. (1992) J. Clin. Invest. 90:626-630; Le Gal
La Salle et al.
(1993) Science 259:988-990; Mastrangeli et al. (1993) J. Clin. Invest. 91:225-
234; Ragot et
al. (1993) Nature 361:647-650; Hayaski et al. (1994) J. BioL Chem. 269:23872-
23875.
By "transactivator," "transactivating factor," or "transcriptional activator"
is meant a
polypeptide that facilitates transcription from a promoter. Inducible
transactivators facilitate
transcription in the presence (or absence) or due to a change in concentration
of a specific
inducing agent or condition. For example, a tet regulated inducible
transactivator may
facilitate transcription from the inducible tet~ promoter when the
transactivator is not bound
to the appropriate inducer, e.g., tetracycline or an analog thereof. In
presence of
tetracycline, such a transactivator is prevented from binding to its target
and thus
transcription is blocked. A reverse tet transactivator retains the DNA binding
specificity of a
wild-type tet repressor, but is regulated in a reverse manner, i.e. it binds
to a tet operator
sequence only in the presence of the appropriate inducer, e.g., tetracycline
or an analog
thereof, rather than in the absence of the inducer. Transcriptional activators
generally bind
directly to a transcriptional response element, however in some cases they
bind indirectly to
another protein, which in turn binds to or is bound to the transcriptional
response element.
As used herein, a "transcriptional regulatory element", also referred to as a
"transcriptional response element" or "TRE" is a polynucleotide sequence,
preferably a DNA
sequence, that regulates (i.e., controls) transcription of an operably-linked
polynucleofiide
sequence by an RNA polymerase to form RNA. As used herein, a THE increases
transcription of an operably linked polynucleotide sequence in a host cell
that allows the
THE to function. The THE comprises an enhancer element and/or promoter
element, which
may or may not be derived from the same gene. The promoter and enhancer
components
of a THE may be in any orientation and/or distance from the coding sequence of
interest,
and may comprise multimers of the foregoing, as long as the desired
transcriptional activity
is obtained. As discussed herein, a THE may or may not lack a silencer
element. A THE
may be cell type specific, e.g., specific to cancer cells derived from any of
a variety of tissue
types (cell status specific), prostate cancer cells, bladder cancer cells,
colon cancer cells,
liver cancer cells, kidney cancer cells, breast cancer cells, pancreatic
cancer cells, etc.) or
may be active in a large number of cell types. As used herein, the term cell
type-specific
transcriptional regulatory element (CT-TRE) refers to both cell type specific
and cell status
specific regulatory elements.
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The transcriptional regulatory element upon which the transactivator acts is
referred
to as a "transcriptional transactivator regulated regulatory element". A
transcriptional
transactivator regulated regulatory element regulates transcription of an
operably-linked
polynucleotide sequence, and is regulated by an inducible transactivator, for
example by
binding of an inducible transactivator protein to a specific DNA binding site
within or near
the promoter. As used herein, a transcriptional transactivator regulated
regulatory element
or TA-THE alters transcription levels in the presence of the inducing agent,
either by
downregulating or upregulating transcription.
An "enhancer" is a term well understood in the art and is a nucleotide
sequence
derived from a gene which increases transcription of a gene that is operably-
linked to a
promoter to an extent which is greater than the transcription activation
effected by the
promoter in the absence of the enhancer when operably-linked to the gene, i.e.
it increases
transcription from the promoter. Having "enhancer activity" is a term well
understood in the
art and means that when present the nucleotide sequence which has "enhancer
activity"
increases transcription of a gene which is operably linked to a promoter to a
level which is
greater than the level of transcription effected by the promoter itself when
operably linked to
the gene in the absence of the nucleotide sequence which has "enhancer
activity", i.e., it
increases transcription from the promoter.
"Under transcriptional control" is a term well-understood in the art and
indicates that
transcription of a polynucleotide sequence, usually a DNA sequence, depends on
its being
operably (operatively) linked to an element that contributes to the regulation
of, either
promotes or inhibits, transcription.
The term "operably linked" relates to the orientation of polynucleotide
elements in a
functional relationship. A THE is operably linked to a coding segment if the
THE promotes
transcription of the coding sequence. ~perably linked means that the DNA
sequences
being linked are generally contiguous and, where necessary to join two protein
coding
regions, contiguous and in the same reading frame. However, since enhancers
generally
function when separated from the promoter by several kilobases and intronic
sequences
may be of variable length, some polynucleotide elements may be operably linked
but not
contiguous.
The terms "inducing agent" and "inducing condition" may be used
interchangeably
and refer to a chemical entity or condition which facilitates binding of an
inducible
transactivator transcriptional response element to a specific DNA binding site
within or near
a promoter. As used herein, when the inducing agent/condition acts on a
transactivator
transcriptional response element, transcription levels are altered or
modulated, either by
downregulating or upregulating transcription. Exemplary "inducing agents" and
"inducing
conditions" include, but are not limited to chemical or non-chemical entities
(such as ultra-
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sound, heat, external beam radiation, hypoxia, etc.). It will be understood
that exposure of
an oncolytic vector of the invention to such an "inducing agent" or "inducing
condition may
result in either activation (induction) or suppression (repression) of
transcription, resulting in
an increase or decrease in viral replication, respectively.
A "host cell" includes an individual cell or cell culture which can be or has
been a
recipient of any virus and/or vector of the present invention. Host cells
include progeny of a
single host cell, and the progeny may not necessarily be completely identical
(in
morphology or in terms of total DNA complement) to the original parent cell
due to natural,
accidental, or deliberate mutation and/or change. A host cell includes cells
transfected or
infected in viv~ with a virus and/or vector of the invention. Host cells may
be isolated from a
tissue or animal host for in vitr~ culture.
As used herein, the terms "neoplastic cells", "neoplasia", "tumor", "tumor
cells",
"cancer" and "cancer cells", (used interchangeably herein) refer to cells
which exhibit
relatively autonomous growth, so that they exhibit an aberrant growth
phenotype
characterized by a significant loss of control of cell proliferation.
Neoplastic cells can be
malignant or benign.
In the context of a viral vector, e.g., the viral vector(s), of the invention,
a
"heterologous" promoter or enhancer is one that is not present in wild-type
virus. Examples
of a heterologous promoter or enhancer are the albumin promoter or enhancer
and other
viral promoters and enhancers, such as SV40.
In the context of viruses (viral vectors), an "endogenous" promoter, enhancer,
or
THE is native to, or derived from, the virus.
The term "gene" is well understood in the art and is a polynucleotide encoding
a
polypeptide. In addition to the polypeptide coding regions, a gene includes
non-coding
regions including, but not limited to, introns, transcribed but untranslated
segments, and
regulatory elements upstream and downstream of the coding segments.
In the context of viruses (viral vectors), a "heterologous polynucleotide" or
"transgene" is any gene that is not present in wild-type virus. Preferably,
the transgene will
also not be expressed or present in the target cell prior to introduction by
the virus (viral
vector). Examples of preferred transgenes are provided below.
A sequence, whether polynucleotide or polypeptide, "depicted in" a SEQ ID NO,
means that the sequence is present as an identical contiguous sequence in the
sequence of
the SEQ ID NO.
The terms "polynucleotide" and "nucleic acid", used interchangeably herein,
refer to
a polymeric form of nucleotides of any length, either ribonucleotides or
deoxyribonucleotides. These terms include a single-, double- or triple-
stranded DNA,
genomic DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and
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WO 2005/007832 PCT/US2004/005518
pyrimidine bases, or other natural, chemically, biochemically modified, non-
natural or
derivatized nucleotide bases. The following are non-limiting examples of
polynucleotides: a
gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,
recombinant
polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of
any
sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A
polynucleotide may comprise modified nucleotides, such as methylated
nucleotides and
nucleotide analogs, uracyl, other sugars and linking groups such as
fluororibose and
thioate, and nucleotide branches. The sequence of nucleotides may be
interrupted by non-
nucleotide components. A polynucleotide may be further modified after
polymerization,
such as by conjugation with a labeling component. ~ther types of modifications
included in
this definition are caps, substitution of one or more of the naturally
occurring nucleotides
with an analog, and introduction of means for attaching the polynucleotide to
proteins, metal
ions, labeling components, other polynucleotides, or a solid support.
Preferably, the
polynucleotide is DNA. As used herein, "DNA" includes not only bases A, T, C,
and G, but
also includes any of their analogs or modified forms of these bases, such as
methylated
nucleotides, internucleotide modifications such as uncharged linkages and
thioates, use of
sugar analogs, and modified and/or alternative backbone structures, such as
polyamides.
A polynucleotide or polynucleotide region which has a certain percentage (for
example, 30%, 85%, 90%, or 95%) "sequence identity" to another sequence means
that,
when aligned, that percentage of bases are the same in comparing the two
sequences.
This alignment and the percent homology or sequence identity can be determined
using
software programs known in the ark, for example those described in Current
Proloc~Is in
11~~lecular biology (F.NI. Ausubel et al., eds. 1987) Supplement 30, section
7.7.13, Table
7.7.1. A preferred alignment program is ALIGN Plus (Scientific and Educational
Software,
Pennsylvania), preferably using default parameters.
The terms "polypeptide", "peptide" and "protein" are used interchangeably to
refer to
polymers of amino acids of any length. These terms also include proteins that
are post-
translationally modified through reactions that include glycosylation,
acetylation and
phosphorylation.
"Replication" and "propagation" are used interchangeably and refer to the
ability of a
viral vector of the invention to reproduce or proliferate. This term is well
understood in the
art. For purposes of this invention, replication involves production of viral
proteins and is
generally directed to reproduction of virus. Replication can be measured using
assays
standard in the art and described herein, such as a burst assay or plaque
assay.
"Replication" and "propagation" include any activity directly or indirectly
involved in the
process of virus manufacture, including, but not limited to, viral gene
expression, production
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of viral proteins, nucleic acids or other components, packaging of viral
components into
complete viruses, and cell lysis.
A "gene essential for replication" is a gene whose transcription is required
for the
viral vector to replicate in a cell.
As used herein, a "target cell" is a cell that allows (i.e., permits or
induces) a cell
type-specific transcriptional regulatory element (TRE) to function. Typically,
the target cell
is a mammalian cell, preferably a human cell.
As used herein, "cytotoxicity" is a term well understood in the art and refers
to a
state in which one or more of a cell's usual biochemical or biological
functions are perturbed
(i.e., inhibited or elevated). These activities include, but are not limited
to, metabolism,
cellular replication, DNA replication, transcription, translation, and uptake
of molecules.
"Cytotoxicity" includes cell death and/or cytolysis. Assays are known in the
art that indicate
cytotoxicity, such as dye exclusion, 3H-thymidine uptake, and plaque assays.
The term
"selective cytotoxicity", as used herein, refers to the cytotoxicity conferred
by a viral vector
of the present invention on a cell which allows a cell type-specific THE to
function when
compared to the cytotoxicity conferred by a viral vector of the invention on a
cell which does
not allow, or is less permissive for, the same THE to function. Such
cytotoxicity may be
measured, for example, by plaque assays, reduction or stabilization in size of
a tumor
comprising target cells, or the reduction or stabilization of serum levels of
a marker
characteristic of the tumor cells or a tissue-specific marker, e.g., a cancer
marker such as
prostate specific antigen.
As used herein, a "cytotoxic" gene is a gene whose expression in a cell,
either alone
or in conjunction with virus replication, enhances the degree and/or rate of
cytotoxic and/or
cytolytic activity in the cell.
A "therapeutic" gene is a gene whose expression in a cell is associated with a
desirable result. In the cancer context, this desirable result may be, for
example,
cytotoxicity, repression or slowing of cell growth, and/or cell death.
A "biological sample" encompasses a variety of sample types obtained from an
individual and can be used in a diagnostic or monitoring assay. The definition
encompasses blood and other liquid samples of biological origin, solid tissue
samples such
as a biopsy specimen or tissue cultures or cells derived therefrom and the
progeny. thereof.
The definition also includes samples that have been manipulated in any way
after their
procurement, such as by treatment with reagents, solubilization, or enrichment
for certain
components, such as proteins or polynucleotides. The term "biological sample"
encompasses a clinical sample, and also includes cells in culture, cell
supernatants, cell
lysates, serum, plasma, biological fluid, and tissue samples.
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WO 2005/007832 PCT/US2004/005518
An "individual" is a vertebrate, preferably a mammal, more preferably a human.
Mammals include, but are not limited to, rodents, primates, farm animals,
sport animals, and
pets.
An "effective amount" is an amount sufficient to effect beneficial or desired
clinical
results. An effective amount can be administered in one or more
administrations. For
purposes of this invention, an effective amount of a viral vector is an amount
that is
sufficient to palliate, ameliorate, stabilize, reverse, slow or delay the
progression of the
disease state.
As used herein, "treatment" is an approach for obtaining beneficial or desired
clinical
results. For purposes of this invention, beneficial or desired clinical
results include, but are
not limited to, alleviation of symptoms, diminishment of extent of disease,
stabilized (i.e., not
worsening) state of disease, preventing spread (i.e., metastasis) of disease,
delay or
slowing of disease progression, amelioration or palliation of the disease
state, and
remission (whether partial or total), whether detectable or undetectable.
"Treatment" can
also mean prolonging survival as compared to expected survival if not
receiving treatment.
"Palliating" a disease means that the extent and/or undesirable clinical
manifestations of a disease state are lessened and/or time course of the
progression is
slowed or lengthened, as compared to not administering viral vectors of the
present
invention.
Regulatable Transcrit~tional Transactivators and Repressors
The present invention utilizes transactivating proteins that are functional in
mammalian cells capable of serving as host cells for viral infection. Such
transactivating
proteins include synthetic, chimeric and naturally occurring transcriptional
transactivating
proteins or domains of proteins from eukaryotic cells including vertebrate
cells, viral
transactivating proteins or any synthetic amino acid sequence that is able to
stimulate
transcription from a vertebrate promoter. Such a transactivating protein may
be (1 ) natural
(native), (2) chimeric (chimera of a DNA-binding domain of a natural protein
and a
regulatory (activator or repressor) domain of a natural protein, (3)
synthetic, having a novel
DNA-binding domain designed by structural modeling, phage display screening or
other
methods, and (4) may or may not take the form of a fusion protein.
Types of transcriptional activation domains include acidic transcription
activation
domains, proline-rich transcription activation domains, serine/threonine-rich
transcription
activation domains and glutamine-rich transcription activation domains.
Examples of acidic
transcriptional activation domains include the VP16 regions and amino acid
residues 753-
881 of Saccharomyces cerevisiae Gal4 (Braselmann et al., 1993, Proc Natl Acad
Sci USA.
90 (5): 1657-1661 ). Examples of proline-rich activation domains include amino
acid
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CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
residues 399-499 of CTF/NF1 and amino acid residues 31-76 of AP2. Examples of
serine/threonine-rich transcription activation domains include amino acid
residues 1-427 of
ITF1 and amino acid residues 2-451 of ITF2. Examples of glutamine-rich
activation domains
include amino acid residues 175-269 of Oct1 and amino acid residues 132-243 of
Sp1. The
amino acid sequences of each of the above described regions, and of other
useful
transcriptional activation domains, are disclosed in Seipel, K. et al. (EMBO
J. (1992)
13:4961-4968). Examples of transactivating proteins also include the lymphoid
specific
transcription factor identified by Muller et al. (1988, Nature 336:544-551),
the fos protein
(Lucibello et al., 1988, Oncogene 3:43-52); v-jun protein (Bos et al., 1988,
Cell 52:705-712);
factor EF-C (Ostapchuk et al., 1989, Mol. Cell. Biol. 9:2787-2797); HIV-1 tat
protein (Arya et
al., 1985, Science 229:69-73), the papillomavirus E2 protein (Lambert et al.,
1989, J. Virol.
63:3151-3154) and the adenovirus E1A protein (reviewed in Flint and Shenk,
1989, Ann.
Rev. Genet.). In preferred embodiments of the invention, the transactivating
protein is
Herpes simplex virus VP16 (Sadowski et al., 1988, Nature 335:563-564;
Triezenberg et al.,
1988, Genes and Dev. 2:718-729). The human NF-kappaB activation domain (p65)
has
also been used in a number of systems (Vermeulen L et. al., Biochem Pharmacol
64(5-
6):963-70, 2002).
Inducible transactivators include chimeric fusion proteins comprising (i) a
functional
portion of a DNA binding protein and (ii) a functional portion of a
transcriptional activator
protein, as described above. DNA sequences encoding the DNA binding protein
and the
transactivating protein are combined so as to preserve the respective binding
and
transactivating properties of each. Regions not required for function of DNA
binding
proteins or transcriptional transactivating proteins may be identified by any
method known in
the art, including analysis of mapped mutations as well as identification of
regions lacking
mapped mutations, which are presumably less sensitive to mutation than other,
more
functionally relevant portions of the molecule. The appropriate recombinant
constructs may
be produced using standard techniques in molecular biology, including those
set forth in
Maniatis (1982, Molecular Cloning: A Laboratory Manual (Cold Spring Harbor,
N.Y., Cold
Spring Harbor Laboratory)).
The DNA binding protein portion may be derived from any vertebrate,
nonvertebrate,
fungal, plant, or bacterial source and may be natural, engineered or
synthetic. Examples
include Gal 4 (Keegan et al., 1986, Science 231:699-704), ADR1 (Hartshorne et
al., 1986,
Nature 320:283-287), Swl (Stillman et al., 1988, EMBO J. 7:485-495) and as
generally
reviewed in Johnson et al. (1989, Annual Rev. Biochem., 58:799-839). The
transactivator
may be a repressor protein, such as, for example, the IexA repressor. In some
embodiments, a chimeric transactivator protein is derived from a bacterial DNA
binding
protein, which then confers specificity on the transactivator protein by
binding to sites
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CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
engineered into the transcriptional transactivator regulated regulatory
element. DNA
sequences homologous to bacterial DNA binding sites are unlikely to occur
frequently in the
mammalian genome, and therefore selectively control the expression of genes of
interest.
Exemplary activator domains include but are not limited to VP16, NF-kappaB,
TFE3, ITF1,
Oct-1, Spl, Oct-2, NFY-A, ITF2, c-myc, and CTF (Seipel, et al., 1992, EMBO J
13: 4961-
4968). Exempalry repressor domains include but are not limited to Kruppel
(KRAB;
Margolin et al., 1994, Proc Natl Acad Sci USA 91 (10): 4509-13), kox-1
(Deuschle et al.,
1995), even-skipped (Licht et al., 1994), LacR, engrailed (Li et al, 1997, J
Biol Chem., 274
(12): 7803-15), hairy (HES; Fisher et al., 1996, EMBO J 12 (13): 5075-82),
Groucho (TLE;
Fisher et al., 1996), RING1 (Satjin et al., 1997, Mol. Cell. Biol. 17 (7):
4105-4113), SSB16
and SSB24 (Saha et al., 1993), Tupl (Tzamarlas, Struhl, 1994), Nabl (Swirnoff
et al., 1998),
AREB (Ikeda et al., 1998, Mol. Cell. Biol. 18 (1): 10-18), E4BP4 (Cowell ~
Hurst, 1996),
HoxA7 (Schnabell et al, 1996), EBNA3 (Bourillot et al., 1998, J Gen Virol. 79
(Pt 2): 363-
70.), and v-erbA (Busch et al., 1997, Mol Endocrinol. 11 (3): 379-89.). By way
of specific
example, the tTs (TET-silencer) uses the repression domain of the KRAB
protein. See also,
PCT Publication WO0052179 for further examples of activator domains, repressor
domains
and chimeric transcriptional transactivators.
The chimeric transactivator protein may further comprise a nuclear
localisation
sequence, so that the chimeric transactivator protein is selectively
concentrated in the cell
nucleus. For example, nucleic acid sequence encoding the SV40 large T antigen
nuclear
localisation signal, Pro-Lys-Lys-Lys-Arg-Lys-Val (Kalderon et al., 1984, Cell
39:499-509)
may be placed in apposition to the transactivator protein encoding sequences.
A number of methods for control of gene expression by inducing agents or
conditions have been described, for example, those induced by tetracycline,
antiprogestins,
and ecdysone.
Inducible systems applicable to the current invention include the tet (TetT"~)
and
reverse (RevTetT"") systems, which are described in U.S. Pat. Nos. 5,464,758,
5,589,362,
PCT publication WO 94/29442, PCT publication WO 96/01313 and PCT publication
WO
96/40892, each of which is expressly incorporated by reference herein. For
example, one
may use the TetT"" and RevTetT"" systems (BD Biosciences Clontech), which
employ small
molecules, such as tetracycline (Tc) or analogues, e.g. doxycycline, to
regulate (turn on or
off) transcription of the target (Knott A et al., Biotechniques 32(4):796,
798, 800 (2002)).
Both the TetT"" and RevTetT"~ systems have been demonstrated to modulate gene
expression in vivo. Modified vectors based on parvovirus, adenovirus,
retroviruses and
herpes simplex virus have been used to introduce the TET-Technology in vitro.
See, for
example Ho, D.Y. et al. (1996) Mol. Brain Res. 41:200-209; Hwang, J-J. et al.
(1996) J.
Virol. 70:8138-814; Hu, S-X. et al. (1997) Cancer Res. 57:3339-3343; and
Maxwell, I.H. et
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WO 2005/007832 PCT/US2004/005518
al. (1996) Gene Ther.3:28-36. Gossen et al. (1992) Proc Natl Acad Sci U S A.
89(12):5547-
51 describe control of gene expression in mammalian cells by tetracycline-
responsive
promoters. Gossen et al. (1995) Science 268(5218):1766-9, describe
transcriptional
activation by tetracycline in mammalian cells. Urlinger et al. (2000) Proc
Natl Acad Sci U S
A. 97(14):7963-8, discuss mutations in tetracycline-dependent transcriptional
activators.
Freundlieb et al. (1999) J Gene Med. 1 (1 ):4-12 discuss tetracycline
controlled
activationlrepression system with increased potential for gene transfer into
mammalian
cells.
In another exemplary system, transcription is activated by rapamycin (or
analogs
thereof) which bring together two intracellular molecules, each of which is
linked to either a
transactivat~r or a DNA binding protein. When these components come together,
transcription of the gene of interest is activated. Rapamycin mediates the
formation of
heterodimers between the immunophilin FK506-binding protein (FICBP) and the
lipid kinase
homolog FRAP (Standaert, R. F. et al., Nature 346, 671-674 (1990); Brown, E.
J. et al.,
Nature 369, 756-758 (1994); Rivers, V. M. et al. Nat. Med. 2, 1028-1032
(1996); and Ho, S.
N. et al., Nature 382, 822-826(1996)). Rapamycin-based systems (Ariad) have
been shown
to regulate target gene transcription both in cell culture and in animal
models and high dose-
dependent indu~cibility has been demonstrated following addition of rapamycin.
See, e.g., Ye
et al. Science 283 (5398):88-91, 1999; Magari et al. J Clin Invest.
100(11):2865-72,1997;
Rivers et al. Nat Med. 2(9):1028-32,1996. Ariad has two major systems: a
system based on
homodimerization and a system based on heterodimerization (Rivers et x1.,1996,
Nature
Med, 2(9):1028-1032; Ye et al., 2000, Science 283: 88-91; Sawyer TK et al.,
2002, Mini Rev
Med Chem. 2(5):475-88). The heterodimerization system is based on human
FICBP12
(FK506 binding protein) and a 93 as fragment of the large human P13F~ homolog,
FRAP
(RAFT, mT~R). A DNA binding protein called ~.FHD1 (a fusion protein of two
human DNA
binding domains) is joined to FKBP and the human NF-kappa b p65 activation
domain is
fused to FRAP (or if rapamycin analogs are used as inducers, instead a mutant
version of
FRAP). Upon addition of an inducer molecule, which binds to both FKBP12 and
FRAP,
FICBP12 and FRAP come together with the DNA binding protein and the activation
domain.
Gene transcription is therefore initiated.
Other examples of regulated gene expression systems or promoters include the
metallothionein promoter system (Mulherkar et al. Biochem Biophys Res Gommun.
177(1 ):90-6, 1991 ); glucocorticoid promoter systems (Ko et al. (1989) Gene
84(2):383-9),
ecdysone-regulated gene switch (Saez et al. Proc Natl Acad Sci U S A.
97(26):14512-7)
and the macrolide-based transgene control system (Weber et al. (2002) Nat
Biotechnol.
20(9):901-7).
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WO 2005/007832 PCT/US2004/005518
Among ecdysone systems are the Drosophila ecdysone system (Yao and Evans,
1996, Proc. Nat. Acad. Sci., 93:3346), the Bombyx ecdysone system (Suhr et
al., 1998,
Proc. Nat. Acad. Sci., 95:7999). Steroid based systems include a synthetic
progesterone
receptor system which employs RU-486 as the inducer (Wang et al., Biochim
Biophys Acta,
1994, 1218 (3): 308-314; Delort and Capecchi, 1996, Hum Gene Ther 7 (7): 809-
820; and
~sterwalder et al., 2001, Proc Natl Acad Sci 98(22):12596-601).
A "functionally-preserved" variant of a transcriptional transactivator,
repressor or the
response element therefor is a transcriptional transactivator, repressor or
response element
therefor which differs from a reference transcriptional transactivator,
repressor or response
element, but retains the ability to increase transcription of an operably
linked polynucleotide,
in particular, cell type-specific transcriptional activity. The difference can
be due to an
altered linear sequence or conformation, arising from, for example, single or
multiple base
mutation(s), addition(s), deletion(s), and/or modifications) of the bases. The
difference can
also arise from changes in the sugar(s), and/or linkages) between the bases.
The oncolytic viruses of the invention can be used for a wide variety of
purposes.
The purpose will vary with the target cell. Suitable target cells are
characterized by the
transcriptional activation of the cell specific transcriptional response
element in the viral
vector. Regulation of transcriptional activation is the result of interaction
between
transcriptional activators bound to cis-regulatory elements, factors bound to
basal
~transcriptional elements and the activity of transeriptional mediators,
coactivafiors, and the
presence of inducing agents or conditions. The transactivator regulated
transcriptional
regulatory element may be operably linked to a viral gene that is essential
for propagation,
so that replication competence is only achievable in the target cell, and/or
to a transgene.
By transgene it is intended any gene that is not present in wild-type virus,
frequently the
transgene will also not be expressed in the target cell, prior to introduction
by the virus.
One of skill in the art would recognize that some alterations of bases in and
around
transcription factor binding sites are more likely to negatively affect gene
activation and cell-
specificity, while alterations in bases which are not involved in
transcription factor binding
are not as likely to have such effects. Certain mutations are also capable of
increasing
transcriptional transactivator or repressor activity. Testing of the effects
of altering bases
may be performed in vitro or in viv~ by any method known in the art, such as
mobility shift
assays, or transfecting vectors containing these alterations in target non-
target cells.
Additionally, one of skill in the art would recognize that point mutations and
deletions can be
made to a transcriptional transactivator or repressor sequence or the response
element
therefor without altering the ability of the sequence to regulate
transcription.
It will be understood by one of skill in the art that very low basal levels of
transcription may be present in non-targeted cell types. By transcriptional
activation, it is
CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
intended that transcription will be increased above basal levels in the target
cell by at least
about 2-fold; preferably at least about 5-fold; preferably at least about 10-
fold; more
preferably at least about 20-fold, 30-fold or 40-fold; more preferably at
least about 50-fold,
60-fold, 70-fold, i30-fol or 90-fold; more preferably at least about 100-fold;
even more
preferably at least about 200-fold, even more preferably at least about 400-
to about 500-
fold, even more preferably, at least about 1000-fold.
All of the above-described systems for control of gene expression find utility
in
practicing the present invention. An effective dose of inducing agent or
effective inducing
conditions are readily determined from extrapolation of published results,
from empirical
testing, and other methods known and routinely employed by those of skill in
the art. For
example, the inducing agent may be administered to an animal harboring a viral
vector of
the present invention, and effective viral titers determined after
administration of the
inducing agent.
Transcri~tional Regulatory Elements (TREs)
In practicing the current invention, the genetic sequence encoding the
transactivator
protein is desirably placed under the transcriptional control of a suitable
cell type-specific
TRE. A "cell type-specific TRE" is preferentially functional in a specific
type of cell relative
to other types of cells of difFerent functionality. "Cell type" is a
reflection of a differenfiiation
state of a cell which is, under normal physiological conditions, an
irreversible, end-stage
state. For example, a prostate-specific antigen THE is functional in prostate
cells, but is not
substantially functional in other cell types such as hepatocytes, astrocytes,
cardiocytes,
lymphocytes, etc. Generally, a cell type-specific THE is active in only one
cell type. When a
cell type-specific THE is active in more than one cell type, its activity is
restricted to a limited
number of cell types, i.e., it is not active in all cell types. A cell type-
specific THE may or
may not be tumor cell specific. Such cell type specificity refers to a
relative increase in
transcription in a target host cell wherein the THE is active relative to a
cell wherein the THE
is not active.
As used herein, a THE derived from a specific gene is referred to by the gene
from
which it was derived and is a polynucleotide sequence which regulates
transcription of an
operably linked polynucleotide sequence in a host cell that expresses that
gene.
Depending upon the target cell type, various enhancers may be used to provide
for specific
transcription. With lymphocytes, for B cells one may use the Ig enhancer, for
T cells one
may use the T cell antigen receptor promoter. For the different muscle cells,
one may use
the promoters for the different myosins. For endothelial cells, one may use
the different
promoters for the different selectins. For each type of cell, there will be
specific proteins
associated with the cell, which allows for target cell specific transcription.
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As used herein, a "cell status-specific transcriptional regulatory element" or
cell
status specific THE is a THE that is induced or becomes active under a
particular
physiological state that permits or induces expression of polynucleotides
under
transcriptional control thereof.
An example of cell status is cell cycle. An exemplary gene whose expression is
associated with the cell cycle is E2F-1, a ubiquitously expressed, growth-
regulated gene,
which exhibits peak transcriptional activity in S phase. Johnson et al. (1994)
Genes Dev.
8:1514-1525. The RB protein, as well as other members of the RB family, form
specific
complexes with E2F-1, thereby inhibiting its ability to activate
transcription. Thus, E2F-1-
responsive promoters are down-regulated by RB. Many tumor cells have disrupted
RB
function, which can lead to de-repression of E2F-1-responsive promoters, and,
in turn, de-
regulated cell division.
Included within the invention are replication competent vectors comprising a
cell
status-specific TRE, e.g., an "E2F-1-THE", a ubiquitously expressed, growth-
regulated
gene, which exhibits peak transcriptional activity in S phase. An "E2F-1-TRE"
is a
polynucleotide sequence, preferably a ~NA sequence, which selectively
increases
transcription (of an operably-linked polynucleotide sequence) in a host cell
that allows an
E2F-1-THE to function, such as a cell (preferably a mammalian cell, even more
preferably a
human cell) that expresses E2F-1. The E2F-1-THE is responsive to transcription
factors
and/or co-factor(s) associated with E2F-1-producing cells and comprises at
least a portion
of the E2F-1 promoter and/or enhancer. See Hernandez-Alcoceba R et al. (Hum
Gene
Ther 2002 Sep 20;13(14):1737-50); Tsukuda et al. (Cancer Res. 62:3238-3447,
2002);
Johnson et al., Cancer Cell. 2002 May;1\(4):325-37. (~nyx); Jakubczak JL et
al., Cancer
Res. 2003 Apr 1;63(7):1490-9; United States Application Serial No. Serial No.
09/392,822,
published as 20010053352 and United States Application Serial No. 10/081969,
published
as 20030104625. The sequence of the E2F promoter is known in the art, and has
been
described, e.g., in Fueyo J et al., Nat Med. 1998 Jun;4(6):685-90.
A further example of cell status is response to hypoxic conditions (such as a
hypoxia
response element or HRE). An important mediator of hypoxic responses is the
transcriptional complex HIF-1, or hypoxia inducible factor-1, which interacts
with a hypoxia-
responsive element (HRE) in the regulatory regions of several genes, including
vascular
endothelial growth factor, and several genes encoding glycolytic enzymes,
including
enolase-1. Murine HRE sequences have been identified and characterized. Firth
et al.
(1994) Proc. Natl. Acad. Sci. USA 91:6496-6500. Hypoxia is an integral
component of the
tumor microenvironment that develops in most solid tumors regardless of their
origin,
location, or genetic alterations and arises from the rapid growth of the tumor
relative to its
vascular supply. Since hypoxia is a major factor in conferring resistance of
cancer cells to
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WO 2005/007832 PCT/US2004/005518
radiotherapy and chemotherapy, selecting tumor clones of high malignancy,
predisposing
tumors to metastasis, the development of novel therapeutic strategies that
target hypoxic
areas of tumors is important. Hypoxia-inducible factor (HIF) is a
heterodimeric transcription
factor that mediates responses to hypoxia by binding to a hypoxia-response
element (HRE)
present within target genes and the HIF/HRE system can therefore be utilized
to specifically
target therapeutic gene expression to tumors. Also included within the
invention are
replication competent vectors comprising a hypoxia responsive element or
"HRE". A
hypoxia responsive element is a polynucleotide sequence, preferably a DNA
sequence,
which selectively increases transcription (of an operably-linked
polynucleotide sequence) in
a host cell under conditions that allow a HRE to function, such as a cell
(preferably a
mammalian cell, even more preferably a human cell) that expresses hypoxia
inducible
factor-1, which interacts with a hypoxia-responsive element (HRE). The
sequence of
hypoxia-response elements are known in the art, e.g., an HRE from a rat
enolase-1
promoter is described in Jiang et al. (1997) Cancer Res. 57:5328-5335.
Another exemplary gene whose expression is associated with the cell cycle is
telomerase. See e.g., United States Application Serial No. 10/081969,
published as
20030104625; PCT Publication WO00/46355; United States Application Serial No.
10/023,969, published as 20030095989 and United States Application Serial N~.
10/206447, published as US20030099616 (Geron); and United States Application
Serial
No. 09/956,335 published as 20020028785 (Saint Louis University), which
describe
regulated expression of adenovirus using the human telomerase promoter. The
invention
provides replication competent vectors comprising a telomerase regulatory
element, e.g., a
telomerase promoter. The sequences of a number of telomerase promoters are
known in
the ark, examples of which may be found in GenBank at Accession Nos.
AF128893.1 and
AF121948.
Other cell status-specific transcriptional response elements include heat-
inducible
(i.e., heat shock) promoters, and promoters responsive to radiation exposure,
including
ionizing radiation and UV radiation. For example, the promoter region of the
early growth
response-1 (Egr-1) gene contains an elements) inducible by ionizing radiation.
Hallahan et
al. (1995) Nat. Med. 1:786-791; and Tsai-Morris et al. (1988) Nucl. Acids.
Res. 16:8835-
8846. Heat-inducible promoters, including heat-inducible elements, have been
described.
See, for example Welsh (1990) in "Stress Proteins in Biology and Medicine",
Morimoto,
Tisseres, and Georgopoulos, eds. Cold Spring Harbor Laboratory Press; and
Perisic et al.
(1989) Cell 59:797-806.
Accordingly, in some embodiments, a cell status-specific transcriptional
response
elements comprises one or more elements responsive to ionizing radiation,
e.g., a 5'
flanking sequence of an Egr-1 gene, a heat shock responsive, or heat-inducible
element.
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WO 2005/007832 PCT/US2004/005518
As used herein, the term "cell type-specific" is intended to mean that the THE
sequences to which a gene, i.e., a gene essential for viral replication, is
operably linked, or
to which a transgene is operably linked, functions specifically in that target
cell so that
transcription (and replication, if the operably linked gene is one essential
for viral replication)
proceeds selectively in target cells, or so that a transgene is expressed in
target cells. This
can occur by virtue of the presence in target cells, and not in non-target
cells, of
transcription factors that activate transcription driven by the operably
linked transcriptional
control sequences. It can also occur by virtue of the absence of transcription
inhibiting
factors that normally occur in non-target cells and prevent transcription
driven by the
operably linked transcriptional control sequences. The term "cell type-
specific", as used
herein, is intended to include cell type specificity, tissue specificity, as
well as specificity for
a cancerous state of a given target cell. In the latter case, specificity for
a cancerous state of
a normal cell is in comparison to a normal, non-cancerous counterpart.
In one embodiment, the invention includes oncolytic viral vectors wherein the
CT-
THE is prostate cell specific. For example, TREs that function preferentially
in prostate cells
and can be used in the present invention to target viral replication to
prostate neoplasia,
include, but are not limited to, TREs derived from the glandular kallikrein-1
gene (from the
human gene, hf~CLdC2-TRE), the prostate-specific antigen gene (PSA-TRE), and
the probasin
gene (PB-TRE). All three of these genes are preferentially expressed in
prostate cells and
2o the expression is androgen-inducible. Generally, expression of genes
responsive to
androgen induction requires the presence of an androgen receptor (AR).
Human glandular kallikrein (hd~CLdC2, encoding the hK2 protein) is expressed
exclusively in the prostate and its expression is up-regulated by androgens
primarily by
transcriptional activation. The levels of hK2 found in various tumors and in
the serum of
patients with prostate cancer differ substantially from those of PSA and
indicate that hK2
antigen may be a significant marker for prostate cancer.
A "human glandular kallikrein transcriptional regulatory element", or "hKLK2-
TRE"
may be included in a replication competent vector of the invention. An "hKLK2-
TRE" is a
polynucleotide sequence, preferably a DNA sequence, which increases
transcription of an
operably linked polynucleotide sequence in a host cell that allows an hKLK2-
THE to
function, such as a cell (preferably a mammalian cell, even more preferably a
human cell)
that expresses androgen receptor. An hKLK2-THE is thus responsive to the
binding of
androgen receptor and comprises at least a portion of an hKLK2 promoter and/or
an hKLK2
enhancer (i.e., the ARE or androgen receptor binding site). hKLK2-TREs are
further
described in US Application Serial No. 09/875,228.
The activity of the hKLK2 5' promoter has been previously described and a
region
up to -2256 relative to the transcription start site was previously disclosed
(SEQ ID N0:3).
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WO 2005/007832 PCT/US2004/005518
Schedlich et al. (1987) DNA 6:429-437. The hKLK2 promoter is androgen
responsive and,
in plasmid constructs wherein the promoter alone controls the expression of a
reporter
gene, expression of the reporter gene is increased approximately 10-fold in
the presence of
androgen. hKLK2 enhancer activity is found within a polynucleotide sequence
approximately nt -12,014 to nt -2257 relative to the start of transcription
(depicted in SEQ ID
N0:3) and, when this sequence is operably linked to an hKLK2 promoter and a
reporter
gene, transcription of operably-linked sequences in prostate cells increases
in the presence
of androgen at levels approximately 30- to approximately 100-fold over the
level of
transcription in the absence of androgen. This induction is generally
orientation
independent and position independent. Enhancer activity has also been
demonstrated in
the following regions (all relative to the transcription start site): about nt
-3993 to about nt -
3643 (nt 8021 to 8371 of SEQ ID N~:3), about nt -4814 to about nt -3643 (nt
7200 to 8371
of SEQ ID N~:3), about nt -5155 to about nt -3387 (nt 6859 to 8627 of SEQ ID
N~:3), about
nt -6038 to about nt -2394 (nt 5976 to 9620 of SE(~ ID N~:3).
Thus, an hKLK2 enhancer can be operably linked to an hKLK2 promoter or a
heterologous promoter to form an hKLK2 transcriptional regulatory element
(hKLK2-TRE).
An hKLK2-THE can then be operably linked to a heterologous polynucleotide to
confer
hKLK2-TRE-specific transcriptional regulation on the linked gene, thus
increasing its
expression.
In another example, a "probasin (PB) transcriptional regulatory element", or
"PB-
TRE" is included in a replication competent vector of the invention. A or "PB-
TRE" is a
polynucleotide sequence, preferably a DNA sequence, which selectively
increases
transcription of an operably-linked polynucleotide sequence in a host cell
that allows q PB-
TRE to function, such as a cell (preferably a mammalian cell, even more
preferably a
human cell) that expresses androgen receptor. A PB-THE is thus responsive to
the binding
of androgen receptor and comprises at least a portion of a PB promoter and/or
a PB
enhancer (i.e., the ARE or androgen receptor binding site). PB-TREs are
further described
in US Pat No. 6,436,394.
For example, the specificity of PB-THE activity for prostate cell that express
the
3o androgen receptor (AR) was demonstrated as follows. The region of the PB 5'-
flanking
DNA (nt -426 to nt +28) (SEQ ID N~:9) including the endogenous promoter
sequences was
inserted upstream of the firefly luciferase gene to generate a chimeric PB-TRE-
luc plasmid.
Cationic-mediated, transient transfection of LNCaP (PSA-producing and AR-
producing
prostate carcinoma cells) and PC-3 (PSA-deficient and AR-deficient prostate
carcinoma
cells) cells was performed. The results showed that LNCaP cells transfected
with PB-TRE-
luc had approximately 400 times more activity than untransfected cells,
indicating that the
PB-THE was intact. Further, the overall luciferase activity recovered in the
cellular extracts
CA 02516652 2005-08-19
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of transfected LNCaP cells was about 30-40-fold higher than that measured in
the cellular
extracts of transfected PC-3 cells. Thus, the results indicate that PB-THE
expression is
preferentially functional in PSA-producing, AR-producing prostate carcinoma
cells as
compared to PSA-deficient, AR-deficient prostate carcinoma cells and that PS-
THE is
capable of mediating specific expression in cells producing the androgen
receptor. The rat
probasin (PB) gene encodes a nuclear and secreted protein, probasin, that is
only
expressed in the dorsolateral prostate. A PB-THE has been shown in an
approximately 0.5
kb fragment of sequence upstream of the probasin coding sequence, from about
nt -426 to
about nt +28 relative to the transcription start site, as depicted in (SEQ ID
NO:4). This
minimal promoter sequence from the PB gene appears to provide sufficient
informafiion to
direct development and hormone -regulated expression of an operably linked
heterologous
gene specifically to the prostate in transgenic mice.
In another example, a "prostate-specific antigen (PSA) transcriptional
regulatory
element", or "PSA-THE", or "PSE-TRE" is included in a replication competent
vector of the
invention. A "PSA-THE", or "PSE-TRE" is a polynucleotide sequence, preferably
a DNA
sequence, which selectively increases transcription of an operably linked
polynucleotide
sequence in a host cell that allows a PSA-THE to function, such as a cell
(preferably a
mammalian cell, even more preferably a human cell) that expresses androgen
receptor. A
PSE-THE is thus responsive to the binding of androgen receptor and comprises
at least a
portion of a PSA promoter and/or a PSA enhancer (i.e., the ARE or androgen
receptor
binding site). PSE-TREs are further described in US Patent Nos. 5,648,478,
6,057,299 and
6,136, 792.
The region of the PSA gene that is used to provide cell specificity dependent
upon
androgens, particular in prostate cells, involves approximately 6.0 kilobases.
Schuur et al.
(1996) J. viol. Chew. 271:7043-7051. An enhancer region of approximately 1.5
kb in
humans is located between nt -5322 and nt -3739, relative to the transcription
start site of
the PSA gene. The PSA promoter consists of the sequence from about nt -540 to
nt +8
relative to the transcription start site. Juxtapositioning of these two
genetic elements yields
a fully functional, minimal prostate-specific enhancer/promoter (PSE) TRE.
Other portions
of the approximately 6.0 kb region of the PSA gene can be used in the present
invention, as
long as requisite functionality is maintained.
The PSE and PSA THE depicted in (SEQ ID NO:1) is the same as that given in
GenBank Accession No. U37672. A variant PSA-THE nucleotide sequence is
depicted in
(SEQ ID N0:2). This is the PSA-THE contained within CN706 clone 35.190.13.
CN706 is
an adenoviral vector in which the E1A gene in Ad5 is under transcriptional
control of a PSA-
TRE. CN706 demonstrates selective cytotoxicity toward PSA-expressing cells in
vitro and
in vivo. Rodriguez et al. (1997).
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The region that is employed to provide cell specificity dependent upon
androgens,
particularly in prostate cells, involves an approximately 1.5kb enhancer
region and a 0.5kb
promoter region. The enhancer region in humans is located between nt -5322 and
nt -3739,
relative to the transcription start site of the prostate specific antigen
(PSA) gene. The
promoter consists of nt -540 to nt +8. Juxtaposition of the two genetic
elements yields a
fully functional, minimal prostate-specific enhancer promoter (PSE). The
enhancer contains
three regions that bind prostate-specific DNA binding proteins, one of which
contains a
putative androgen response element. The promoter region contains typical TATA
and
CHAT boxes as well as a second putative androgen response element.
CEA is a 180,000-Dalton glycoprotein tumor-associated antigen present on
endodermally-derived neoplasia of the gastrointestinal tract, such as
colorectal, gastric
(stomach) and pancreatic cancer, as well as other adenocarcinomas such as
breast and
lung cancers. In yet another example, a "carcinoembryonic antigen (CEA)
transcriptional
regulatory element", or "CEA-TRE" is included in a replication competent
vector of the
invention. A "CEA-THE is a polynucleotide sequence, preferably a DNA sequence,
which
selectively increases transcription of an operably linked polynucleotide
sequence in a host
cell that allows a CEA-THE to function, such as a cell (preferably a mammalian
cell, even
more preferably a human cell) that expresses CEA. The CEA-THE is responsive to
transcription factors and/or co-factor(s) associated with CEA-producing cells
and comprises
at least a portion of the CEA promoter and/or enhancer. The 5' upstream
flanking
sequence of the CEA gene has been shown to confer cell-specific activity. The
CEA
promoter region, approximately the first 424 nucleotides upstream of the
translational start
site in the 5' flanking region of the gene, was shown to confer cell-specific
activity when the
region provided higher promoter activity in CEA-producing cells than in non-
producing HeLa
cells. In addition, cell-specific enhancer regions have been found. The entire
5' CEA
flanking region (containing the promoter, putative silencer, and enhancer
elements) appears
to be contained within approximately 14.5 kb upstream from the transcription
start site. Two
upstream regions, -13.6 to -10.7 kb or -6.1 to -4.0 kb, when linked to the
multimerized
promoter resulted in high-level and selective expression of a reporter
construct in CEA-
producing cells. The promoter region is localized to nt -90 and nt +69
relative to the
transcriptional start site, with region nt -41 to nt -18 as essential for
expression.
W095/14100 describes a series of 5' flanking CEA fragments which confer cell-
specific
activity, such as about nt -299 to about nt +69; about nt -90 to about nt +69;
nt -14,500 to nt
-10,600; nt -13,600 to nt -10,600, nt -6100 to nt -3800. In addition, cell
specific transcription
activity is conferred on an operably linked gene by the CEA fragment from nt -
402 to nt +69,
depicted in (SEQ ID N0:7). CEA-TREs used in the present invention are derived
from
mammalian cells, including but not limited to, human cells. Thus, any of the
CEA-TREs
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WO 2005/007832 PCT/US2004/005518
may be used in the invention as long as requisite desired functionality is
displayed. The
cloning and characterization of CEA sequences have been described in the
literature (e.g.,
in US Application Serial No. 10/045,116, published as 200030026792).
In a further example, an "alpha-fetoprotein (AFP) transcriptional regulatory
element",
or "AFP-TRE" may be included in a replication competent vector of the
invention. AFP is an
oncofetal protein. The serum concentration of AFP is elevated in a majority of
hepatoma
patients, with high levels of AFP found in patients with advanced disease. The
serum AFP
levels in patients appear to be regulated by AFP expression in hepatocellular
carcinoma buff
not in surrounding normal liver. Thus, the AFP gene appears associated with
hepatoma
cell-specific expression. Gell-specific TREs from the AFP gene have been
identified. An
"AFP-TRE" is a polynucleotide sequence, preferably a DNA sequence, which
selectively
increases transcription (of an operably linked polynucleotide sequence) in a
host cell that
allows an AFP-THE to function, such as a cell (preferably a mammalian cell,
even more
preferably a human cell) that expresses AFP. The AFP-THE is responsive to
transcription
factors and/or co-factor(s) associated with AFP-producing cells and comprises
at least a
portion of the AFP promoter and/or enhancer. AFP-TREs are further described in
US
Application Serial No. 09/898,883, expressly incorporated by reference herein.
The entire 5'
AFP flanking region (containing the promoter, putative silencer, and enhancer
elements) is
contained within approximately 5 kb upstream from the transcription start site
(SEQ ID
N~:5).
The AFP enhancer region in humans is located between about nt -3954 and about
nt -3335, relative to the transcription start site of the AFP gene. The human
AFP promoter
encompasses a region from about nt -174 to about nt +29. Juxtapositioning of
these two
genetic elements, yields a fully functional AFP-TRE. Ido et al. (1995)
describe a 259 by
promoter fragment (nt -230 to nt +29) that is specific for HCC. Censer Res.
55:3105-3109.
The AFP enhancer contains two regions, denoted A and B, located between nt -
3954 and nt
-3335 relative to the transcription start site. The promoter region contains
typical TATA and
CART boxes. Preferably, the AFP-THE contains at least one enhancer region.
More
preferably, the AFP THE contains both enhancer regions.
Suitable target cells for oncolytic viral vectors containing AFP-TREs are any
cell type
that allows an AFP-THE to function. Preferred are cells that express, or
produce, AFP,
including, but not limited to, tumor cells expressing AFP. Examples of such
cells are
hepatocellular carcinoma cells, gonadal and other germ cell tumors (especially
endodermal
sinus tumors), brain tumor cells, ovarian tumor cells, acinar cell carcinoma
of the pancreas,
primary gall bladder tumor, uterine endometrial adenocarcinoma cells, and any
metastases
of the foregoing (which can occur in lung, adrenal gland, bone marrow, and/or
spleen). In
some cases, metastatic disease to the liver from certain pancreatic and
stomach cancers
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produces AFP. Especially preferred are hepatocellular carcinoma cells and any
of their
metastases. AFP production can be measured using assays standard in the art,
such as
RIA, ELISA or Western blots (immunoassays) to determine levels of AFP protein
production
or Northern blots to determine levels of AFP mRNA production. Alternatively,
such cells can
be identified and/or characterized by their ability to activate
transcriptionally an AFP-THE
(i.e., allow an AFP-THE to function).
In yet a further example, a urothelial cell-specific transcriptional
regulatory element
is included in a replication competent vector of the invention. A urothelial
cell-specific
transcriptional regulatory element is a polynucleotide sequence, preferably a
DNA
sequence which selectively increases transcription (of an operably-linked
polynucleotide
sequence) in a urothelial host cell. For example, a urothelial cell-specific
THE may be
derived from the 5' flanking region of a uroplakin gene (i.e., a "UP-THE")
such that the THE
selectively increases transcription (of an operably-linked polynucleotide
sequence) in a cell
that allows a UP-THE to function such as a cell (preferably a mammalian cell,
even more
preferably a human cell) that expresses uroplakin. In some of these
embodiments, the
urothelial cell-specific THE is derived from the 5' flanking region of a UPIa
gene. In other
embodiments, the urothelial cell-specific THE is derived from the 5'-flanking
region of a
UPIb gene. In yet other embodiments, the urothelial cell-specific THE is
derived from the 5'-
flanking region of a UPII gene. The UP-THE may comprise a urothelial cell-
specific
promoter and a heterologous enhancer. In other embodiments, a urothelial cell-
specific THE
comprises a urothelial cell-specific promoter. In other embodiments, a
urothelial cell-
specific THE comprises a urothelial cell-specific enhancer and a heterologous
promoter. In
other embodiments, a urothelial sell-specific THE comprises a urothelial cell-
specific
promoter and a urothelial cell-specific enhancer. UP-TREs are further
described in PCT
publication WO 01/72994..
The protein product of the MUC1 gene (known as mucin or MUC1 protein;
episialin;
polymorphic epithelial mucin or PEM; EMA; DF3 antigen; NPGP; PAS-O; or CA15.3
antigen) is normally expressed mainly at the apical surface of epithelial
cells lining the
glands or ducts of the stomach, pancreas, lungs, trachea, kidney, uterus,
salivary glands,
and mammary glands. However, mucin is overexpressed in 75-90% of human breast
carcinomas. Mucin protein expression correlates with the degree of breast
tumor
differentiation. This overexpression appears to be controlled at the
transcriptional level.
Overexpression of the MUC1 gene in human breast carcinoma cells MCF-7 and ZR-
75-1
appears to be regulated at the transcriptional level. A "MUC1-TRE" is a
polynucleotide
sequence, preferably a DNA sequence, which selectively increases transcription
(of an
operably-linked polynucleotide sequence) in a host cell that allows an MUC1-
THE to
function, such as a cell (preferably a mammalian cell, even more preferably a
human cell)
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WO 2005/007832 PCT/US2004/005518
that expresses MUC1. The MUC1-THE is responsive to transcription factors
and/or co-
factors) associated with MUC1-producing cells and comprises at least a portion
of the
MUC1 promoter and/or enhancer. MUC1-TREs are further described in US Patent
No.
6,432,700. In an additional example, "a mucin gene (MUC) transcriptional
regulatory
element", or "MUC1-TRE" is included in a replication competent vector of the
invention.
The regulatory sequences of the MUC1 gene have been cloned, including the
approximately 0.9 kb upstream of the transcription start site which contains a
THE that
appears to be involved in cell-specific transcription, depicted in SEQ ID
NO:B.
Any MUC1-TREs used in the present invention are derived from mammalian cells,
including but not limited to, human cells. Preferably, the MUC1-THE is human.
In one
embodiment, the MUC1-THE may contain the entire 0.9 kb 5' flanking sequence of
the
MUC1 gene. In other embodiments, the MUC1-TREs comprise the following
sequences
(relative to the transcription start site of fihe MUC1 gene): about nt -725 to
about nt +31, nt -
743 to about nt +33, nt -750 to about nt +33, and nt -598 to about nt +485
(operably-linked
to a promoter).
The c-erbB2/neu gene (HER-2/neu or HER) is a transforming gene that encodes a
185 kD epidermal growth factor receptor-related transmembrane glycoprotein. In
humans,
the c-erbB2/neu protein is expressed during fetal development, however, in
adults, the
protein is weakly detectable (by immunohistochemistry) in the epithelium of
many normal
tissues. Amplification and/or over-expression of the c-erb~2/neu gene has been
associated
with many human cancers, including breast, ovarian, uterine, prostate, stomach
and lung
cancers.
In yet an additional example, "a HER-2/neu transcriptional regulatory
element", or
"HER-2/neu -TRE" is included in a replication competent vector of the
invention.
Additional tumor- and/or cell type-specific TREs known in the art which may be
included in the oncolytic vectors of the invention include the following:
aromatase,
mammary gland-specific promoter, mammaglobin, plasminogen activator urokinase
(uPA)
and its receptor gene (associated with breast, colon, and liver cancers; TREs
that regulate
uPA and uPAR transcription are provided in SEQ ID N0:6 and further described
in Riccio
et al. Nucleic Acids Res. 13:2759-2771, 1985; Cannio et al., Nucleic Acids
Res. 19:2303-
2308, 1991); human alpha-lactalbumin (associated with breast tissue); BCSG1,
BRCA1,
and BRCA2 (associated with breast cancer); human papilloma virus (HPV) cell
type
dependent regulatory elements (associated with cervical cancer); BLCA4
(associated with
bladder cancer); uroplakins (associated with bladder); NCA (associated with
gastric cancer);
hypoxanthine phosphoribosyltransferase (HPRT; associated with glioma); AVP,
human
pulmonary surfactant protein B gene, and puromycin N-acetyltransferase
associated with
(associated with lung cancer); tyrosinase, gp100; melanoma specific TREs, such
as the
CA 02516652 2005-08-19
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human MART promoter (hMART), the murine tyrosinase gene enhancer and promoter
(mEP), a THE comprising a murine tyrosinase gene enhancer and promoter (mEEP),
a THE
comprising the human tyrosinase promoter (hTYR) and melanocyte specific
factory (MSF);
HER2/neu, urokinase, and CA125 (associated with ovarian cancer); SL3-3 and T
cell
antigen receptor (associated with T cell lymphoma); prostatic acid phosphatase
(associated
with prostate cancer); an EBV-specific THE (associated with EBV-expressing
tumors) such
as an LMP1 Promoter; an LMP2A Promoter, an LMP2B Promoter; or a Cp Promoter
(as
descried e.g., in Sjoblom, A et al, J.Virol. (1998) 72, (2), 1365-1376;
Franken et al, J. Virol
(1995) 69 (12) 8011-8019.; Laux et al, J. Gen. Virol, 1989, 70, 3079-3084);
and Fuentes-
Panana et al, J. Virol., (1999) 73, 826-833 and V01555); a liver specific THE
(associated
with liver cancer) such as a CRGL2 promoter; and a WT1 promoter and enhancer
for
leukemia specific gene expression (Hosen N. Leukemia. 2004 Jan 22). In
addition, tumor
specific promoters may be derived by chimeric construction using different
promoter
elements as exemplified by the artificial hTERT-cHSF1/HSE promoter (described
for
example in Wang J et al. FEBS Lett. Jul 10;546 (2-3):315-20, 2003 or
completely synthetic
in construction (Li X et al Nat Biotechnol. 1999 Mar;17(3):241-5).
Descriptions for these cell-
specific TREs can be found in various scientific publications, and numerous
promoter,
enhancer and regulatory sequences associated with these TREs may be found in
the
GenBank database available on the Internet at
htt~://www.ncbi.nlm.nih.aovlPubMed/. Many
relevant sequences are thus available for practice of this invention and need
not be
described in detail herein.
The TREs listed above are provided as non-limiting examples of TREs that
function
in the instant invention. Additional cell-specific TREs are known in the art,
as are methods
to identify and test cell specificity of candidate TREs. A THE may or may not
lack a silencer.
The presence of a silencer (i.e., a negative regulatory element) may assist in
shutting off
transcription (and thus replication) in non-permissive cells (i.e., cells in a
normal cell state).
Thus, the presence of a silencer may confer enhanced inducible or cell-
specific replication
by more effectively preventing oncolytic viral vector replication in non-
target cells.
Alternatively, the lack of a silencer may assist in effecting replication in
target cells, thus
conferring enhanced cell type-specific replication due to more effective
replication in target
cells. A THE can also comprise multimers. For example, a THE can comprise a
tandem
series of at least two, at least three, at least four, or at least five TREs.
These multimers
may also contain heterologous promoter and/or enhancer sequences.
In one exemplary embodiment, two substantially identical TREs control
transcription
of viral genes, e.g. adenoviral E1A and E1 B genes. It is understood, however,
that any of a
number of combinations of genes may be used with these at least two TREs. In
the
adenoviral example, other preferred embodiments include those which contain
substantially
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WO 2005/007832 PCT/US2004/005518
identical TREs that drive expression of E1A, E1 B, and E4. Such constructs may
or may not
additionally contain a transgene, which may or may not be under control of a
substantially
identical TRE. Preparation of these and other embodiments are provided below
and in the
examples.
Transcriptional activation can be measured in a number of ways known in the
art
(and described in more detail below), but is generally measured by detection
and/or
quantitation of mRNA or the protein product of the coding sequence under
control of (i.e.,
operatively linked to) the TRE. Activity of a THE can be determined as
follows. A THE
polynucleotide sequence or set of such sequences can be generated using
methods known
in the art, such as chemical synthesis, site-directed mutagenesis, PCR, and/or
recombinant
methods. The sequences) to be tested can be inserted into a vector containing
a promoter
(if no promoter element is present in the TRE) and an appropriate reporter
gene encoding a
reporter protein, including, but not limited to, chloramphenicol acetyl
transferase (CAT),
beta-galactosidase (encoded by the lacZ gene), luciferase (encoded by the luc
gene),
alkaline phosphatase, green fluorescent protein, and horseradish peroxidase.
Such vectors
and assays are readily available, from, inter alia, commercial sources.
Plasmids thus
constructed are transfected into a suitable host cell to test for expression
of the reporter
gene as controlled by the putative THE using transfection methods known in the
art, such
as calcium phosphate precipitation, electroporation, liposomes (lipofection),
and DEAE
dextran.
After introduction of the TRE-reporter gene construct into a host cell under
appropriate conditions, THE activity may be measured by detection and/or
quantitation of
reporter gene-derived mRNA or protein product(s), including in the absence of
presence of
a suitable inducing agent or condition. The reporter gene protein can be
detected directly
(e.g., immunochemically) or through its enzymatic activity, if any, with an
appropriate
substrate. Generally, to determine cell specific activity of a TRE, the TRE-
reporter gene
constructs are introduced into a variety of cell types. The amount of THE
activity is
determined in each cell type and compared to that of a reporter gene construct
without the
TRE. A THE is cell specific when it is preferentially functional in a specific
type of cell over
a different type of cell.
As used herein, "a cell that allows a THE to function" or a cell in which the
function
of a THE is "sufficiently preserved" or "functionally preserved", or "a cell
in which a THE is
functional" is a cell in which the TRE, when operably linked to a promoter (if
not included in
the TRE) will increase transcription above basal levels in the target cell by
at least about 2-
fold, preferably at least about 5-fold, preferably at least about 10-fold,
more preferably at
least about 20-fold, more preferably at least about 50-fold, more preferably
at least about
100-fold, even more preferably at least about 200-fold, even more preferably
at least about
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400- to about 500-fold, even more preferably, at least about 1000-fold. Basal
levels are
generally the level of activity, if any, in a non-target cells, or the level
of activity (if any) of a
reporter construct lacking the THE of interest as tested in a target cell
type. Methods for
measuring levels of expression (whether relative or absolute) are known in the
art and are
described herein. Similarly, when employing the regulatable oncolytic
virus.systems of the
invention, an inducible transactivator in the presence of an effective amount
of inducing
agent or under inducing conditions sufficient to induce gene expression, will
increase
expression of a gene at least about 2-fold, preferably at least about 5-fold,
preferably at
least about 10-fold, more preferably at least about 20-fold, more preferably
at least about
50-fold, more preferably at least about 100-fold, more preferably at least
about 200-fold,
even more preferably at least about 400- to about 500 fold, even more
preferably at least
about 1000-fold, when compared to the expression of the same promoter and gene
in the
absence of the inducing agent.
A "functionally-preserved" variant of a THE is a THE which differs from
anofiher TRE,
but still retains the ability to increase transcription of an operably linked
polynucleotide, in
particular, cell type-specific transcription activity. The difference in a THE
can be due to
differences in linear sequence or conformation, arising from, for example,
single or multiple
base mutation(s), addition(s), deletion(s), and/or modification(s). The
difference can also
arise from changes in the sugar(s), and/or linkages) between the bases of a
TRE.
Certain point mutations within sequences of TREs have been shown to decrease
transcription factor binding and gene activation. One of skill in the art
would recognize that
some alterations of bases in and around known fihe transcription factor
binding sites are
more likely to negatively affect gene activation and cell-specifiicity, while
alterations in bases
which are not involved in transcription factor binding are not as likely to
have such effects.
Certain mutations are also capable of increasing THE activity. Testing of the
effects of
altering bases may be performed in vitro or in vivo by any method known in the
art, such as
mobility shift assays, or transfecting vectors containing these alterations in
THE functional
and THE non-functional cells. Additionally, one of skill in the art would
recognize that point
mutations and deletions can be made to a THE sequence without altering the
ability of the
sequence to regulate transcription.
_System For External Control Of Oncolytic Virus Replication
Replication-competent (oncolytic) viral vectors are provided. The vectors
comprise a
cell type-specific transcriptional regulatory element (CT-TRE) and an
inducible
transactivator regulated transcriptional regulatory element. The CT-THE
controls
transcription of an inducible transactivator (TA) coding sequence. The
inducible
transactivator (a) requires an inducing agent or condition to be functional
and (b) controls
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transcription of a viral gene essential for replication. Expression of the
viral gene essential
for replication is regulated both by the CT-THE and by a transactivator
regulated
transcriptional regulatory element, and indirectly by the concentration of the
inducing agent
or condition.
Depending on the specific uses of the virus vector, the inducing agent or
condition
may act to inhibit transcription, or to enhance transcription. Transcription
inhibitors provide
a way to "shut down" viral replication by delivering the inducing agent to the
host cells.
Transcription activators provide a way to induce virus replication by addition
of the inducing
agent, where the virus is otherwise inactive.
Included within the scope of the invention is any oncolytic virus wherein
control of a
viral gene essential for replication may be accomplished using the methods of
the present
invention. Exemplary replication-competent (oncolytic) viruses include, but
are not limited
to, adenoviruses, vesicular stomatitis viruses (VSV), herpes viruses (e.g.,
herpes simplex
virus; HSV), reoviruses, paramyxoviruses, rhinoviruses, Newcastle disease
viruses,
polioviruses, West Nile virus, coxsaclcie virus, measles viruses and vaccinia
viruses, etc.
In embodiments in which two substantially identical TREs are used, the
invention
does not require that the TREs be derived from the same gene. As long as the
THE
sequences are substantially identical, and the requisite functionality is
displayed, the TREs
may be derived from different genes.
In some embodiments, the oncolytic vectors of the invention comprise a first
viral
gene under the transcriptional control of a transactivator regulated
transcriptional regulatory
element and at least one other gene, such as a viral gene or a transgene,
under control of
another heterologous THE which is different from the first TRE, where the
heterologous
TREs are functional in the same cell but do not have the same in
polynucleofiide sequence
(i.e., have different polynucleotide sequences). Preferably, at least two of
the heterologous
TREs in the oncolytic vector are cell specific or inducible for the same cell
or inducing agent
or condition. In one approach, the viral gene is one that enhances cell death,
more
preferably one that is essential for viral replication. Preferably, at least
one of the viral
genes necessary for cell replication is an early gene and the genes under
transcriptional
control of the heterologous THE are necessary for replication. By providing
for cell-specific
transcription through the use of multiple heterologous TREs, the invention
provides
oncolytic vectors that can effect cell-specific cytotoxic effects due to
selective replication.
The novel system of the invention for regulated expression of oncolytic
viruses is
exemplified herein by regulatable adenoviral vectors. In this exemplary
embodiment, the
genes that are regulated by the transactivator regulated transcriptional
regulatory element
may be early or late adenoviral genes and/or transgenes. By providing for
regulated
transcription, one can provide for adenovirus that can be used as a vehicle
for introducing
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WO 2005/007832 PCT/US2004/005518
genetic capability into host target cells, as distinct from other non-target
cell types. The
transgenes serve to modify the genotype or phenotype of the target cell, in
addition to any
modification of the genotype or phenotype resulting from the presence of the
adenovirus.
Typical of the oncolytic vectors of the invention, with replication competent
adenoviruses,
proliferation of the adenovirus may be used for its cytotoxic effect.
It has been demonstrated that adenovirus vectors which include at least two
different heterologous TREs are more stable and provide greater cell
specificity with regard
to replication than previously described adenovirus vectors. See, e.g., U.S.
Patent No.
6,4.32,700. Accordingly, adenovirus vectors have been constructed in which
each of the
E1A and E1 B genes are under transcriptional control of two different
heterologous TREs. It
is understood, however, that any of a number of combinations of genes may be
used with
any combinati~n of at least two TREs.
There are a number of different serotypes of adenovirus, such as Ad2, AdS,
Ad3,
Ad35 and Ad40, which differ to minor or significant degrees. Particularly,
adenoviral
serotypes differ as to host cell tropism. For the purpose of the subject
invention, Ad5 is
exemplified, however any and all serotypes of adenovirus are included within
the scope of
the invention.
The genes of the adenovirus that are of interest for the subject invention may
be
divided into two groups, the early genes and the late genes, the expression of
the latter
being controlled by the major late promoter. Of the early genes, there are
E1A, E1 B, E2, E3
and E4.. The E1A gene is expressed immediately after viral infection (0-2h)
and before any
other viral genes. E1A protein acts as a trans-acting positive-acting
transcriptional
regulatory factor, and is required for the expression of the other early viral
genes and the
promoter proximal major late genes. Despite the nomenclature, the promoter
proximal
genes driven by the major late promoter are expressed during early times after
Ad5
infection. In the absence of a functional E1A gene, viral infection does not
proceed,
because the gene products necessary for viral DNA replication are not
produced.
The E1 B protein functions in trans and is necessary for transport of late
mRNA from
the nucleus to the cytoplasm. Defects in E1 B expression result in poor
expression of late
viral proteins and an inability to shut off host cell protein synthesis.
The E4 gene has a number of transcription products. Open reading frames (ORF)
3
and ORF 6 of the E4 transcription unit increase the accumulation of major late
transcription
unit mRNAs by binding the 55-kDa protein from E1 B and heterodimers of E2F-1
and DP-1.
In the absence of functional protein from ORF3 and ORF6, plaques are produced
with an
efficiency less than 106 of that of wild type virus.
The major late genes relevant to the subject invention are genes such as LI,
L2 and
L3, which encode proteins of the AD5 virus virion.
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Regions of the adenovirus which may be deleted, usually at least 500 nt, more
usually at least about 1000 nt, include in the AD5 genome nucleotides 300 to
3600 in EI,
particularly 342 to 3523; 27000 to 31000, particularly 28133 to 30818 or 27865
to 30995 in
E3. The deletion will be at least sufficient for insertion of the desired
construct and allow for
packaging. In some embodiments E3 sequences are included in the regulatable
replication
competent adenoviruses of the invention, as further described in US
Application Patent No.
6,495,130. In other embodiments, internal ribosome entry sites (IRES) are
included in the
regulatable replication competent adenoviruses of the invention, as further
described in US
Application Serial No. 09/814,351, published as 20030148520.
As exemplified by employing an adenoviral vector with a cell specific response
element comprising a promoter and enhancer construct specific for prostate
cells, various
genetic capabilities may be introduced into prostate cells expressing prostate
specific
antigen. ~f particular interest is the opportunity to introduce cytotoxic
effects that are
controlled by a transcriptional initiation region specifically active in
prostate cells. ~ther cell
types that have specific active transcription factors associated with a state
for which
modulation is desirable include leukocytes, particularly lymphocytes,
epithelial cells,
endothelial cells, hepatic cells, pancreatic cells, neuronal cells, and
keratinocytes. Since
the adenovirus typically results in transient expression (approximately 6 to 8
weeks), one
can provide transient capability to cells, for example in situations where the
desired result
only requires a limited period for response. In other cases, a different
oncolytic vector may
be preferred due the time and level of expression desired.
To further increase specificity of the control mediated by an inducing agent,
it may
also be desirable to control expression of 2 viral genes, i.e. expression of
both E1A and
E1 B adenoviral genes. In this manner, specificity may be increased, i.e.,
replication of the
oncolytic virus in non-target cells in which the CT-THE is not active may be
reduced further.
Both E1A and E1 B may be controlled by the inducer responsive promoter element
as
exemplified in Figure 1, which illustrates a prostate specific oncolytic viral
vector with
tetracycline regulated replication control, wherein a tetracycline responsive
element (TRE)
promoter drives E1A gene expression and a prostate specific Antigen (PSA)
promoter
drives expression of the reverse tet-responsive transactivator (rtTA).
In this approach expression of the E1A and E1B genes may be linked by an IRES
between the E1A and E1 B genes. In the construction of this virus, the
endogenous E1 B
promoter elements are removed and replaced with the IRES element. Therefore
both E1A
and E1 B expression are under the control of the inducer responsive promoter
element. As
an IRES alternative, the 2A peptide sequence derived foot and mouth disease
virus (FMDV)
could be used in place of the IRES sequence (as described in Furler S et al.,
Gene Ther.
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2001 Jun;B(11):864-73) to provide efficient bicistronic expression of both E1A
and a
transgene.
Preferred adenoviral embodiments include those that contain at least two
different
heterologous TREs that drive expression of E1A, E1B, and E4. Such constructs
may or
may not additionally contain a transgene, which may or may not be under
control of a TRE,
wherein the THE may or may not be a CT-TRE. See, e.g., US Patent Nos.
6,436,394 and
6,432,700.
Accordingly, the invention provides an oncolytic virus vector comprising a
viral gene
under transcriptional control of a transactivator regulated transcriptional
regulatory element,
and an inducible transactivator under transcriptional control of a TRE,
preferably a CT-TRE.
In some embodiments, a first transactivator regulated transcriptional
regulatory element
controls expression of a first viral gene, and a second transactivator
regulated
transcriptional regulatory element controls expression of a second viral gene.
The genes to
be controlled under these TREs are preferably viral genes essential for
propagation.
Alternatively, the genes to be controlled under these TREs may be a first
viral gene
essential for propagation wherein the second gene is a transgene. It is
understood that
there may or may not be additional TREs in the viral vectors, and that these
additional
TREs may or may not be substantially identical to the first and/or second
TREs. The
invention includes use of three or more, four or more, TREs.
Transaenes
Use of viral vectors, competent in particular target cells, allows for
proliferation of the
virus in the target cells resulting in the death of the host cells and
proliferation of the virus to
other host cells. To further enhance therapeutic efficacy, the oncolytic
vectors of the
invention may include one or more transgenes that have a therapeutic effect.
Accordingly, the viral vectors of this invention can further include a
heterologous
polynucleotide (transgene) encoding a therapeutic gene product under the
control of a
transactivator regulated transcriptional regulatory element. Alternatively,
the viral vector
may comprise a heterologous transgene encoding a therapeutic gene product
under the
control of a constitutive or inducible promoter. Numerous examples of
constitutive and
inducible promoters are known in the art and routinely employed in transgene
expression in
the context of viral vectors. In this way, various genetic capabilities may be
introduced into
target cells. For example, in certain instances, it may be desirable to
enhance the degree
therapeutic efficacy by enhancing the rate of cytotoxic activity. This could
be accomplished
by coupling the cell-specific replicative cytotoxic activity with expression
of, one or more
metabolic enzymes such as HSV-tk, nitroreductase, cytochrome P450 or cytosine
deaminase (cd) which render cells capable of metabolizing 5-fluorocytosine (5-
FC) to the
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chemotherapeutic agent 5-fluorouracil (5-FU) and carboxypeptidase G2 (CPG2).
This type
of transgene may also be used to confer a bystander effect.
Additional transgenes that may be introduced into a viral vector of the
invention
include a factor capable of initiating apoptosis, antisense or ribozymes,
which among other
capabilities may be directed to mRNAs encoding proteins essential for
proliferation, such as
structural proteins, transcription factors, polymerases, etc., viral or other
pathogenic
proteins, where the pathogen proliferates intracellularly, cytotoxic proteins,
e.g., the chains
of diphtheria, ricin, abrin, etc., genes that encode an engineered cytoplasmic
variant of a
nuclease (e.g., RNase A) or protease (e.g., trypsin, pepsin, proteinase K,
carboxypeptidase,
1o etc.), chemokines, such as MCP3 alpha or MIP-1, pore-forming proteins
derived from
viruses, bacteria, or mammalian cells, fusgenic genes, chemotherapy
sensitizing genes and
radiation sensitizing genes. ~ther genes of interest include cytokines,
antigens,
transmembrane proteins, and the like, such as IL-1, -2, -6, -12, GM-CSF, G-
CSF, M-CSF,
IFN-a, -~3, -y, TNF-a, -Vii, TGF-a, -Vii, NGF, MDA-7 (Melanoma differentiation
associated
gene-7, mda-7/interleukin-24), and the like. Further examples include,
proapoptotic genes
such as Fas, Bax, Caspase, TRAIL, Fas ligands, and the like; fusion genes
which can lead
to cell fusion or facilitate cell fusion such as V22, VSV and the like; tumor
suppressor gene
such as p53, RB, p16, p17, W9 and the like; genes associated with the cell
cycle and genes
which encode anti-angiogenic proteins such as endostatin, angiostatin and the
like.
~ther opportunities for specific genetic modification include T cells, such as
tumor
infiltrating lymphocytes (TILs), where the TILs may be modified to enhance
expansion,
enhance cytotoxicity, reduce response to proliferation inhibitors, enhance
expression of
lymphokines, etc. ~ne may also wish to enhance target cell vulnerability by
providing for
expression of specific surface membrane proteins, e.g., B7, SV4.0 T antigen
mutants, etc.
In some embodiments, the adenovirus death protein (ADP), encoded within the E3
region, is maintained (i.e., contained) in the adenovirus vector. The ADP
gene, under
control of the major late promoter (MLP), appears to code for a protein (ADP)
that is
important in expediting host cell lysis. Tollefson et al. (1996) J. Virol.
70(4):2296; Tollefson
et al. (1992) J. Virol. 66(6):3633. Thus, adenoviral vectors containing the
ADP gene may
render the adenoviral vector more potent, making possible more effective
treatment and/or
a lower dosage requirement.
Accordingly, in one embodiment the invention provides adenovirus vectors in
which
an adenovirus gene is under transcriptional control of a first transactivator
regulated
transcriptional regulatory element and a polynucleotide sequence encoding an
ADP under
control of a second transactivator regulated transcriptional regulatory
element, and wherein
preferably the adenovirus gene is essential for replication. A DNA sequence
encoding an
ADP and the amino acid sequence of an ADP are depicted in SEQ ID N0:10 and SEQ
ID
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WO 2005/007832 PCT/US2004/005518
N0:11, respectively. Briefly, an ADP coding sequence is obtained preferably
from Ad2
(since this is the strain in which ADP has been more fully characterized)
using techniques
known in the art, such as PCR. Preferably, the Y leader (which is an important
sequence
for correct expression of late genes) is also obtained and ligated to the ADP
coding
sequence. The ADP coding sequence (with or without the Y leader) can then be
introduced
into the adenoviral genome, for example, in the E3 region (where the ADP
coding sequence
will be driven by the MLP). The ADP coding sequence could also be inserted in
other
locations of the adenovirus genome, such as the E4 region. Alternatively, the
ADP coding
sequence could be operably linked to a different type of TRE, including, but
not limited to,
another viral TRE.
It is understood that the present invention does not exclude oncolytic vectors
containing additional genes under control of transactivator regulated
transcriptional
regulatory elements. Accordingly, the invention provides viral vectors
comprising a third
gene under transcriptional control of a third TRE. The third THE may or may
not be
substantially identical to the first and/or second TA- TREs, with all three
TREs functional in
the same cell. Preferably, the third gene is one that contributes to
cytotoxicity (whether
direct and/or indirect), more preferably one that contributes to and/or
enhances cell death.
Delivery ~f ~ncolytic ~/ectors To Cells
The oncolytic vectors can be used in a variety of forms, including, but not
limited to,
naked polynucleotide (usually DNA) constructs; polynucleotide constructs
complexed with
agents to facilitate entry into cells, such as cationic liposomes or other
compounds such as
polylysine; packaged into infectious adenovirus particles (which may render
the adenoviral
vectors) more immunogenic); packaged into other particulate viral forms such
as HSV or
AAV; complexed with agents to enhance or dampen an immune response; complexed
with
agents that facilitate in vivo transfection, such as DOTMATM, D~TAPT"', and
polyamines.
If an oncolytic vector is packaged into a virus, the virus itself may be
selected to
further enhance targeting. For example for an adenoviral vector, adenovirus
fibers mediate
primary contact with cellular receptors) aiding in tropism. See, e.g., Arnberg
et al. (1997)
Virol. 227:239-244. If a particular subgenus of an adenovirus serotype
displayed tropism for
a target cell type and/or reduced affinity for non-target cell types, such a
subgenus (or
subgenera) could be used to further increase cell-specificity of cytotoxicity
and/or cytolysis.
Adenovirus fiber, hexon or other surface proteins may be modified to enhance
the
specificity of uptake by target cells.
The modified oncolytic vectors may be delivered to the target cell in a
variety of
ways, depending upon whether the cells are in culture, ex vivo or in vivo. In
situations
where in vivo delivery is desired, delivery can be achieved in a variety of
ways, employing
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liposomes, direct injection, subcutaneous injection, intramuscular injection,
catheters,
intravenous inhalation, topical applications, intravenous infusion, etc. Due
to the high
efficiency of transfection of various oncolytic vectors, one can achieve a
large number of
modified cells. In the case of neoplasia, where toxins are produced, the
toxins may be
released locally, so as to affect cells that may not have been transfected. In
this manner,
one can specifically eliminate neoplastic cells, without a significant effect
on the normal
cells. In addition, expression of viral proteins will serve to activate the
immune system
against the target cells. Finally, proliferation of the replication competent
viral vector in a
host cell will lead to cell death. The means of delivery will depend in large
part on the
particular vector (including its form) as well as the type and location of the
target cells (i.e.,
whether the cells are in vifir~ or in viv~).
In the example of a packaged virus, e.g., an adenovirus, the adenovirus may be
administered in an appropriate physiologically acceptable carrier at a dose of
about 10~ to
1 O". The multiplicity of infection will generally be in the range of about
0.001 to 100. The
virus may be administered one or more times, depending upon the immune
response
potential of the host. If necessary, the immune response may be diminished by
employing
a variety of immunosuppressants or treatments to decrease the level of
circulating antibody,
so as to permit repetitive administration, without a strong immune response.
If administered as a polynucleotide construct (i.e., not packaged as a virus)
about
0.01 micrograms to 1000 micrograms of viral vector can be administered. The
oncolytic
vector may be administered one or more times, or may be administered as
multiple
simultaneous injections. Dependent upon the type of replication competent
virus employed,
such as herpes simplex virus (HS!/), reovirus, vesicular stomatitis virus
(!!SV), Newcastle
Disease virus, vacinia virus, Vilest Nile virus, coxsackie virus, poliovirus
and measles virus,
the amount of virus to be administered is based on standard knowledge about
that
particular virus (which is readily obtainable from, for example, published
literature) and can
be determined empirically.
In some embodiments, a packaged viral vectors) is complexed to a hydrophilic
polymer to create a masked virus. The hydrophilic polymer is attached
(covalently or non-
covalently) to the capsid proteins of the virus, in the case of adenovirus,
particularly the
hexon and fiber proteins. In preferred embodiments, the viral vectors of the
instant
invention are complexed with masking agents to create masked viral vectors.
(See, e.g.,
US Application Serial No. 10/139,089, published as 20030152553. In the
adenoviral vector
embodiments of the invention, masked viruses are advantageous due to (a) the
masking of
the adenovirus surface to adenovirus neutralizing antibodies or opsonins which
are in
circulation and (b) increasing the systemic circulation time of adenovirus
particles by
reduction of non-specific clearance mechanisms in the body (i.e. macrophages,
etc.). In the
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in vivo context, the systemic delivery of a masked virus results in a longer
circulation of
virus particles, less immunogenicity, and increased biodistribution with a
decrease in
clearance by the liver and spleen.
Host Cells and Target Cells
The present invention also provides host cells and target cells comprising
(i.e.,
transformed with) the viral vectors described herein. Host cells include both
prokaryotic and
eukaryotic host cells as long as sequence requisite for maintenance in that
host, such as
appropriate replication origin(s), are present. For convenience, selectable
markers are also
provided. Prokaryotic host include bacterial cells, for example, E. coli and
mycobacteria.
Among eukaryotic host cells are yeast, insect, avian, amphibian, plant and
mammalian host
cells. Numerous host cells are known in the art and need not be described in
detail herein.
Suitable target cells for the viral vectors of the invention include any
eukaryotic cell
type that allows function of the TREs and transactivator regulated
transcriptional regulatory
elements, preferably mammalian, more preferably human, even more preferably
neoplastic
cells. Suitable target cells also include any cells that produce proteins and
other factors
necessary for expression of the gene under control of the TREs, such factors
necessary for
said expression are produced naturally or recombinantly. For example, if the
TRE(s) used
is prostafie cell-specific, the cells are preferably prostate cells. The
prostate cells used may
2.0 or may not be producing an androgen receptor, depending on whether the
promoter used is
androgen-inducible. If an androgen-inducible promoter is used, non-androgen
receptor
producing cells, such as HLF, HLE, and 3T3 and the non-AR-producing prostate
cancer
cells PC3 and ~1J14.5 can be used, provided an androgen receptor-encoding
expression
vector is introduced into the cells along with the adenovirus. For example,
ifi the oncolytic
vector comprises a THE derived from the AFP gene, suitable host cells include
any cell type
that produces AFP, including but not limited to, Hep3B, HepG2, HuH7, HuH1/C12.
Activity
of a given THE in a given cell can be assessed by measuring the level of
expression of an
operably-linked reporter gene using standard assays. The comparison of
expression
between cells in which the THE is suspected of being functional and the
control cell
indicates the presence or absence of transcriptional enhancement.
Comparisons between or among various TREs can be assessed by measuring and
comparing levels of expression within a single target cell line. It is
understood that absolute
transcriptional activity of a THE will depend on several factors, such as the
nature of the
target cell, delivery mode and form of a TRE, and the coding sequence that is
to be
selectively transcriptionally activated. To compensate for various plasmid
sizes used,
activities can be expressed as relative activity per mole of transfected
plasmid.
Alternatively, the level of transcription (i.e., mRNA) can be measured using
standard
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Northern analysis and hybridization techniques. Levels of transfection (i.e.,
transfection
efficiencies) are measured by co-transfecting a plasmid encoding a different
reporter gene
under control of a different TRE, such as the CMV immediate early promoter.
This analysis
can also indicate negative regulatory regions, i.e., silencers.
Compositions
The present invention also includes compositions, including pharmaceutical
compositions, containing the viral vectors described herein. Such compositions
are useful
for administration in vivo, for example, when measuring the degree of
transduction and/or
effectiveness of cell killing in an individual. Preferably, these compositions
further comprise
a pharmaceutically acceptable excipient. These compositions, which comprise an
effective
amount of a viral vector of the invention in a pharmaceutically acceptable
excipient, are
suitable for systemic administration to individuals in unit dosage forms,
sterile parenteral
solutions or suspensions, sterile non-parenteral solutions or oral solutions
or suspensions,
oil in water or water in oil emulsions and the like. Formulations for
parenteral and
nonparenteral drug delivery are known in the art and are set forth in
Remington's
Pharmaceutical Sciences, 18t" Edition, Mack Publishing (1990). Compositions
also include
lyophilized and/or reconstituted forms of the viral vectors (including those
packaged as a
virus, such as adenovirus) of the invention.
~ther compositions are used, and are useful for, detection methods described
herein. For these compositions, the viral vector usually is suspended in an
appropriate
solvent or solution, such as a bufFer system. Such solvent systems are well
known in the
art.
Kits
The present invention also encompasses kits containing an oncolytic vector of
the
invention. These kits can be used for diagnostic and/or monitoring purposes,
preferably
monitoring. Procedures using these kits can be performed by clinical
laboratories,
experimental laboratories, medical practitioners, or private individuals. Kits
embodied by
this invention allow one to detect the presence of target cells in a suitable
biological sample,
such as biopsy specimens.
The kits of the invention comprise an oncolytic vector as described herein in
suitable
packaging. The kit may optionally provide additional components that are
useful in the
procedure, including, but not limited to, buffers, developing reagents,
labels, reacting
surfaces, means for detection, control samples, instructions, and interpretive
information.
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Preparation Of The Viral Vectors Of The Invention
The viral vectors of this invention can be prepared using recombinant
techniques
that are standard in the art. Generally, TREs are inserted 5' to the
adenoviral and
transactivator genes of interest, preferably one or more early genes (although
late genes)
may be used). TREs can be prepared using oligonucleotide synthesis (if the
sequence is
known) or recombinant methods (such as PCR and/or restriction enzymes).
Convenient
restriction sites, either in the natural DNA sequence or introduced by methods
such as PCR
or site-directed mutagenesis, provide an insertion site for the TREs.
Accordingly,
convenient restriction sites for annealing (i.e., inserting) TREs can be
engineered onto the
5' and 3' ends of the THE using standard recombinant methods, such as PCR.
Polynucleotides used for making the oncolytic viral vectors of the invention
may be
obtained using standard methods in the art such as chemical synthesis
recombinant
methods andlor obtained from biological sources.
In the case of adenovirus, the vectors are typically prepared by employing two
plasmids, one plasmid providing for the left-hand region of adenovirus and the
other
plasmid providing for the right hand region, where the two plasmids share at
least about
500nt of middle region for homologous recombination. In this way, each
plasmid, as
desired, may be independently manipulated, followed by cotransfection in a
competent host,
providing complementing genes as appropriate, or the appropriate transcription
factors for
initiation of transcription from the PSE for propagation of the adenovirus.
For convenience, plasmids are available that provide the necessary portions of
the
adenovirus. Plasmid pXC.1 (fVlcKinnon (1982) Gene 19:33-4.2) contains the wild-
type left-
hand end of AdS, pBHG10 provides the right-hand end of AdS, with a deletion in
E3. The
deletion in E3 provides room in the virus to insert the 2kb minimal PSE
without deleting the
wild-type enhancer-promoter. The gene for E3 is located on the opposite strand
from E4 (r-
strand).
For manipulation of the early genes, the transcription start site of Ad5 E1A
is at nt
560 and the ATG start site of the E1A protein is at nt 610 in the virus
genome. This region
can be used for insertion of the cell specific element, e.g., PSE.
Conveniently, a restriction
site may be introduced by employing the polymerase chain reaction (PCR), where
the
primer that is employed may be limited to the Ad5 genome, or may involve a
portion of the
plasmid carrying the Ad5 genomic DNA. For example, where pBR322 is the
backbone, the
primers may use the EcoRl site in the pBR322 backbone and the Xpal site at nt
1339 of
AdS. By carrying out the PCR in two steps, where overlapping primers at the
center: of the
region introduce a sequence change resulting in a unique restriction site, one
can provide
for insertion of the cell specific response element at that site.
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A similar strategy may also be used for insertion of the cell specific
response
element to regulate E1 B. The E1 B promoter of Ad5 consists of a single high-
affinity
recognition site for Spl and a TATA box. This region extends from 1636 to 1701
nt. By
insertion of the cell specific response element in this region, one can
provide for cell specific
transcription of the E1 B gene. By employing the left-hand region modified
with the cell
specific response element regulating E1A, as the template for introducing the
cell specific
response element to regulate E1 B, the resulting adenovirus will be dependent
upon the cell
specific transcription factors for expression of both E1A and E1 B.
For example, we have introduced an Agel site 12 by 5' to the E1A initiation
colon
(Ad5 nucleotide 547) by oligo-directed mutagenesis and linked PCR. In
addition, an Eagl
site was created upstream of the E1 B start site by inserting a G residue at
Ad5 nt 1682 by
oligonucleotide directed mutagenesis. To simplify insertion of a THE in the
Eagl site, the
endogenous Eagl site in CN95 was removed by digestion with Eagl, treatment
with mung
bean nuclease, and religation to construct CN114. In this way, we generated an
adenovirus
vector containing unique Agel and Eagl sites in the proximal upstream region
to E1A and
E1 B, respectively. Using these unique sites, one can insert a THE which has
engineered
Agel or Eagl sites, thus simplifying construction of recombinant adenovirus
vectors.
Accordingly, the invention includes an adenoviral vector comprising a unique
Agel site 5' of
the E1A initiation colon and a unique Eagl site 5' of E1B.
Similarly, a THE may be inserted upsfiream of the E2 gene. The E2 early
promoter,
mapping in Ad5 from 27050-27150, consists of a major and a minor transcription
initiation
site, the latter accounting for about 5°/~ of the E2 transcripts, two
non-canonical TATA
boxes, two E2F transcription factor binding sites and an ATF transcription
factor binding site
(for a detailed review of the E2 promoter architecture see Swaminathan et al.,
Curr. Topics
in ntlicrobiol. and Immunol. (1995) 199 part 3:177-194).
The E2 late promoter overlaps with the coding sequences of a gene encoded by
the
counterstrand and is therefore not amenable to genetic manipulation. However,
the E2
early promoter overlaps only for a few base pairs with sequences coding for a
33-kD protein
on the counterstrand. Notably, the Spel restriction site (Ad5 position 27082)
is part of the
stop colon for the above mentioned 33 kD protein and conveniently separates
the major E2
early transcription initiation site and TATA-binding protein site from the
upstream
transcription factor biding sites E2F and ATF. Therefore, insertion of a THE
having Spel
ends into the Spel site in the plus-strand would disrupt the endogenous E2
early promoter
of Ad5 and should allow THE regulated expression of E2 transcripts.
For E4, one must use the right hand portion of the adenovirus genome. The E4
transcription start site is predominantly at nt 35609, the TATA box at nt
35638 and the first
AUG/GUG of ORF1 is at nt 35532. Virtanen et al. (1984) J. Virol. 51: 822-831.
Using any of
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the above strategies for the other genes, a heterologous THE may be introduced
upstream
from the transcription start site. For the construction of mutants in the E4
region, the co-
transfection and homologous recombination are performed in W162 cells
(Vlleinberg et al.
(1983) Proc. Natl. Acad. Sci. USA 80:5383-5386) which provide E4 proteins in
trans to
complement defects in synthesis of these proteins.
Methods of packaging adenovirus polynucleotides into adenovirus particles are
known in the art and are described in the Examples.
Methods Using The Oncolytic Vectors Of The Invention
The subject oncolytic vectors can be used for a wide variety of purposes,
which will
vary with the desired or intended result. Accordingly, the present invention
includes
methods using the oncolytic viral vectors described above. In one embodiment,
methods
for using oncolytic vectors comprise introducing the vector into a cell,
preferably a
eukaryotic cell, more preferably a mammalian cell, in vitro or in vivo. In one
preferred
embodiment, an oncolytic vector of the invention is administered in vivo for
treatment of
cancer.
Purposes for introducing transient expression include indications that may be
treated
involving undesired proliferation other than tumors, such as psoriatic
lesions, restenosis,
wound healing, tissue repair, enhanced immune response, resistance to
infection,
production of factors, enhanced proliferation, investigation of metabolic or
other
physiological pathways, comparison of activity of cells in fihe presence and
absence of the
virus introduced transgene, by comparing the activity of the cell before,
during and after the
modification with the virus, etc. The subject vectors can be used to free a
mixture of cells of
a particular group of cells, where the group of cells is the target cells. By
having the
oncolytic virus be selectively competent for propagation in the target cells,
only those cells
will be killed on proliferation of the oncolytic virus. By combining the virus
with the mixture
of cells, for example, in culture or in vivo, the oncolytic virus will only be
capable of
proliferation in the target cells, and will be regulated by the presence of
the inducing agent.
In this way cells other than the target cells will not be affected by the
oncolytic virus, while
the target cells will be killed. The expansion of the oncolytic virus due to
propagation in the
target cells will ensure that the mixture is substantially freed of the target
cells. Once the
target cells are destroyed, the oncolytic virus will no longer be capable of
propagation, but in
culture may be retained so as to continually monitor the mixture for
recurrence of the target
cell, e.g., a mutated cell or neoplastic cell. The presence or concentration
of an inducing
agent and/or condition provides further control for viral replication.
By identifying genes that are expressed specifically by the target host cells,
based
on the nature of the cells, their level of maturity or their condition, the
target cell specific
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response element can be used to provide genetic capability to such cells,
where the genetic
capability will be absent in other cells, even when transfected with the
oncolytic virus.
In one embodiment, methods for using oncolytic virus vectors comprise
introducing
an oncolytic virus vector into a target cell, preferably a neoplastic cell. In
another
embodiment, methods for using oncolytic virus vectors comprise introducing an
oncolytic
virus vector into a prostate cell. In another embodiment, methods for using
oncolytic virus
vectors comprise introducing an oncolytic virus vector into a liver cell. In
another
embodiment, methods for using oncolytic virus vectors comprise introducing an
oncolytic
virus vector into a breast cancer cell. In another embodiment, methods for
using oncolytic
virus vectors comprise introducing an oncolytic virus vector into a colon
cancer cell.
In one embodiment, methods are provided for conferring selective cytotoxicity
in
cells which allow function of the TRE, comprising contacting cells with an
oncolytic virus
vector described herein, such that the oncolytic virus vectors) enters, i.e.,
transduces the
cell(s). Cytotoxicity can be measured using standard assays in the art, such
as dye
exclusion, 3H-thymidine incorporation, and/or lysis.
In another embodiment, methods are provided for propagating an oncolytic virus
specific for cells which allow function of the cell type-specific and
transactivator regulated
transcriptional regulatory element(s), preferably eukaryotic cells, more
preferably
mammalian cells. These methods entail combining an oncolytic virus vector with
mammalian cells that allow function of the TREs, whereby said oncolytic virus
is
propagated.
Another embodiment provides methods of killing cells that allow a THE to
function
(i.e., target cells) comprising combining the mixture of cells with an
oncolytic virus vector of
the present invention. The mixture of cells is generally a mixture of
cancerous cells in which
the TREs are functional and normal cells, and can be an in vivo mixture or in
vitro mixture.
The invention also includes methods for detecting cells in which a CT-THE
andlor
transactivator regulated transcriptional regulatory element is functional in a
biological
sample. These methods are particularly useful for monitoring the clinical
and/or
physiological condition of an individual (i.e., mammal), whether in an
experimental or clinical
setting. For these methods, cells of a biological sample are contacted with an
oncolytic
virus vector, and replication of the oncolytic viral vector is detected. A
suitable biological
sample is one in which target cells may be or are suspected to be present.
Generally, in
mammals, a suitable clinical sample is one in which target cancerous cells are
suspected to
be present. Such cells can be obtained, for example, by needle biopsy or other
surgical
procedure. Cells to be contacted may be treated to promote assay conditions
such as
selective enrichment and/or solubilization. In these methods, target cells can
be detected
using in vitro assays that detect proliferation, which are standard in the
art. Examples of
41
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such standard assays include, but are not limited to, burst assays (which
measure virus
yields) and plaque assays (which measure infectious particles per cell). Also,
propagation
can be detected by measuring specific oncolytic viral DNA replication, which
are also
standard assays.
The invention also provides methods of modifying the genotype of a target
cell,
comprising contacting the target cell with an oncolytic virus vector described
herein, wherein
the oncolytic viral vector enters the cell. ,
The invention further provides methods of suppressing tumor cell growth,
comprising
contacting a tumor cell with an oncolytic viral vector of the invention such
that the oncolytic
viral vector enters the tumor cell and exhibits selective cytotoxicity for the
tumor cell. Tumor
cell growth can be assessed by any means known in the art, including, but not
limited to,
measuring tumor size, determining whether tumor cells are proliferating using
a 3H-
thymidine incorporation assay, or counting tumor cells. "Suppressing" tumor
cell growth
means any or all of the following states: slowing, delaying, and stopping
tumor growth, as
well as tumor shrinkage. "Suppressing" tumor growth indicates a growth state
that is
curtailed when compared to growth without contact with, i.e., transfection by,
an oncolytic
viral vector described herein.
The invention also provides methods of lowering the levels of a tumor cell
marker in
an individual, comprising administering to the individual an oncolytic viral
vector of the
present invention, wherein the oncolytic viral vector is selectively cytotoxic
in cells producing
the tumor cell marker. Tumor cell markers include, but are not limited to,
PSA, GEA and
hl<2. Methods of measuring the levels of a tumor cell marker are known to
those of ~rdinary
skill in the art and include, but are not limited to, immunological assays,
such as enzyme-
linked immunosorbent assay (ELISA), using antibodies specific for the tumor
cell marleer. In
general, a biological sample is obtained from the individual to be tested, and
a suitable
assay, such as an ELISA, is performed on the biological sample.
The invention also provides methods of treatment, in which an effective amount
of
an oncolytic viral vector described herein is administered to an individual.
For example,
treatment using an oncolytic viral vector in which at least one cell type-
specific THE is
specific for prostate cells (e.g., PSE TRE, PB-TRE, and/or hKLK2-TRE) is
indicated in
individuals with prostate-associated diseases as described above, such as
hyperplasia and
cancer. In this example, also indicated are individuals who are considered to
be at risk for
developing prostate-associated diseases, such as those who have had disease
which has
been resected and those who have had a family history of prostate-associated
diseases.
Determination of suitability of administering oncolytic viral vectors) of the
invention will
depend, inter alia, on assessable clinical parameters such as serological
indications and
histological examination of tissue biopsies. Generally, a pharmaceutical
composition
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comprising an oncolytic viral vector is administered. Pharmaceutical
compositions are
described above.
The following examples are offered by way of illustration and not by way of
limitation.
EXAMPLE 1
T_ etracycline Regulated Oncolytic Virus Replication Specific for Prostate
Tissue
A prostate-specific oncolytic adenovirus is provided in which replication is
regulated
using the Tet-On system to control expression of at least one adenoviral gene.
In the case
of the Tet-On regulated gene expression system to control oncolytic viral
replication, the
expression of the immediate early adenoviral E1A gene region is placed under
the control of
the chimeric promoter element - the tetracycline responsive element (Fig. 1 )
by removal of
the endogenous adenoviral promoter elements and insertion of the tetracycline
responsive
element. An expression cassette for production of the modified reverse
tetracycline
transactivator (rtTA(2)s-M2), which binds to the tetracycline responsive
element to induce
expression in the presence of tetracycline and its derivatives (such as
Doxycycline), is
placed in the E3 region of the virus under the control of a tissue/tumor
specific promoter (a
CT-TRE), exemplified herein by the human prostate specific antigen (PSA)
promoter. In this
manner, expression of the rtTA transactivator (a TA-TRE) is limited to the
tissue/or tumor in
which the PSA promoter is active e.g. prostate-derived tissue. lJpon the
addition of
tetracycline (or a derivative thereof) the rtTA transactivator binds to the
tetracycline
responsive element and switches on E1A transcription and thus expression. From
expression of the E1A profiein, the adenovirus replication process continues,
resulting in the
eventual death of the host cell and release of further adenoviral progeny. In
comparison, in
the absence of the inducer or the virus entering a non-target cell, rtTA
cannot bind to the
tetracycline responsive element, E1A proteins are not expressed and viral
replication does
not continue. The E1A gene region is described in this example, however, any
essential
ORF of adenovirus can be regulated in this manner, i.e. the tetracycline
responsive element
can control the E1 a, E1 b, E2 or E4 region. Furthermore multiple ORFs may be
placed
under tetracycline regulated control i.e. E1 and E4, E1 and E2, to increase
the control of the
system.
_1A Ad5 with PSE Driving Expression of E1A
The cloning and characterization of a minimal prostate-specific enhancer (PSE)
is
described in Schuur et al., 1996. Plasmid CN71 contains a minimal PSE (from-
5322 by to -
3875bp relative to the transcription start site of the PSA gene) and -532 to
+11 of the PSA
promoter. CN71 was cut with Xhol/Hindlll which removes the PSA promoter. A
shorter
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promoter, from -230 to +7 was amplified by PCR. The PCR product was cut with
Xhol/Hindlll and ligated back into Xhol/Hindlll cut CN71 creating CN105. The
plasmid,
pUHrt62-1 (2) (a generous gift from W. Hillen) was then cut with EcoRl, the
site blunted by
klenow fragment, and then cut with Xho I to remove the CMV promoter from the
construct.
The PSA promoter fragment from above was then placed 5' of the rtTA2(s)-M2
transgene
(in pUHrt62-1 (2)) in place of CMV to create pUH-PSA-rt62-1 (2). The PSA-
rtTA2(s)-M2
fragment (incorporating the SV40 Late polyA) was then liberated from pUH-PSA-
rt62-1 (2)
using Xho I / Hind III digestion and then ligated /inserted via Xba I
restriction sites into of
pABS4 (Microbix, Toronto), a shuttle plasmid containing the kanamycin-
resistance, gene to
create pABS4-PSA-rtTA. By digesting pABS4-PSA-rtTA with Pacl, a fragment
containing
the FCan~ gene and the PSA-rtTA2(s)-M2 expression cassette was isolated and
ligated into
similarly cut BHG11 (Microbix), which contains a unique Pacl site engineered
in the E3
region of Ad5 to create BHG11-PSA-rtTA-kan~. The kanR gene was removed by
digesting
BHG11-PSA-rtTA-kanR with Swal and religating the vector (BHG11-PSA-rfiTA).
1 B Attenuated Ad5 with Tetracycline responsive promoter Driving E1A and
Retaining the
Endoctenous Ad5 E1A Promoter and Enhance
In the absence of a functional E1A gene, viral infection does not proceed for
the
gene products necessary for viral DNA replication are not produced (Nevins
(1939) Adv.
Virus Res. 31:35-31). The transcription start site of Ad5 E1A is at nt 560 and
the ATG start
site of the E1A protein is at nt 610 in the virus genome.
pXC.1 was purchased from Microbix Biosystems Inc. (Toronto). pXC.1 contains
Adenovirus 5 sequences from bp22 to 5790. An Agel site was introduced 12 by 5'
to the
E1A initiation codon (Ad5 nucleotide 547) by oligo-directed mutagenesis and
linked PCR.
The plasmid pXC.1 was PCR amplified using primers confiaining an extra A to
introduce an
Agel site. This created a segment from the EcoRl site in the pBR322 backbone
to Ad5 nt
560. A second segment of pXC.1 from Ad nucleotide 541 to the Xbal site at Ad
nucleotide
1339 was amplified using primers containing an extra T to introduce an Agel
site. A mixture
of these two PCR amplified DNA segments was mixed and amplified with primers 3
and 4 to
create a DNA segment from the EcoRl site to the Xbal site of pXC.1. This DNA
segment
encompasses the leftmost 1317 bases of Adenovirus sequence and contained an
Agel site
at Ad nucleotide 547. This DNA segment was used to replace the corresponding
segment
of pXC.1 to create CN95. Similarly, a Tetracycline responsive element with
Agel ends was
PCR amplified from the plasmid pTRE2 (purchased from BD Biosciences Clontech)
to
create pXC-TRE-E1 a .
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1 C. The virus created by homologous recombination - CG1974
An Ad5 recombinant virus containing the PSA promoter driving rtTA(2)s-M2 gene
in
the E3 region and the tetracycline responsive element driving E1a in the E1
region was thus
constructed. Virus was generated by homologous recombination in low passage
293 cells,
a human kidney cell line that expresses Ad E1A and E1B proteins. This was
accomplished
by co-transfection of pXC-TRE-E1a and BHG11-PSA-rtTA. Genomic integrity of the
resulting recombinant virus construct was verified using Hind III digestion
and was
designated CG1974 (Figure 1).
EXAMPLE 2
In an further example, an oncolytic vector is "armed" with a therapeutic
transgene to
increase efficacy, e.g., granulocyte macrophage colony stimulating factor
(GMCSF) or
thymidine kinase (TK). The expression of these therapeutic transgenes may be
placed
under the control of the regulated gene expression system. In this manner, the
inducer
used within the regulated gene expression system switches on both virus
replication and
therapeutic gene expression at the same time. An example of such a vector is
given in
Figure 2 which illustrates a prostate specific oncolytic viral vector armed
with GMCSF with
tetracycline regulated replication and expression control. In this example, a
tetracycline
responsive element (TRE) drives E1 and GMGSF expression. An IRES allows 2
coding
sequences to be expressed from a single promoter and a prostate specific
antigen (PSA)
promoter drives expression of the reverse tet-responsive transactivator
(rtTA). Here, the
vector functions just as set forth above, with both E1 and GMCSF expression
switched on
by the addition of Tet to the system. In this example, an internal ribosome
entry site (IRES)
or 2A sequence allows 2 coding regions to be expression using a single
promoter.
EXAMPLE 3
A number of variant tetracycline regulated gene control systems for use in the
manner described above. For example, the tetracycline controlled
transcriptional silencer
(tTS) system (Freundlieb et al 1999) in conjunction with the Tet-On system
described above
may be used to more tightly control gene expression (see Figure 3 which
illustrates an
oncolytic viral vector with dual tetracycline regulated replication control
for use in bladder
cancer). In this example a tetracycline responsive element (TRE) drives E1
gene
expression and a human uroplakin II (hUPll) promoter drives expression of the
reverse tet-
responsive transactivator (rtTA) and tet-controlled transcriptional silencer
(tTS). The use of
the tTS repressor molecule, in addition to the rtTA transactivator, decreases
basal or "leaky"
gene expression in the "ofP' state to increase the degree of gene regulation.
For example
the system in Figure 3 expresses both the Tet-on transactivator and tTs
repressor from the
CA 02516652 2005-08-19
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human uroplakin II promoter using an internal ribosome entry site (IRES). In
this manner,
expression of the rtTA and tTS are specific to the bladder tissue cell type.
The expression of
the essential E1 gene is again under the control of the TRE. In the absence of
Tet the tTS
molecule binds to the THE element and actively represses E1 gene expression.
This active
repression by the tTS molecule may be required since adenovirus has a number
of
transcription enhancer elements present within its genome which could cause
"leaky"
expression of the E1 promoter, even in the absence of its endogenous promoter,
leading to
a limited amount of virus formation in the absence of the inducer. Upon the
addition of
tetracycline, a conformational change in the tTS molecule will prevent binding
to and active
repression of expression from the TRE. The rtTA, Tet-~n transactivator, would
then be able
to induce expression of E1 by the resulting ability to bind to the TRE, such
that replication
proceeds.
An alternative system is presented in Figure 4 which represents an example of
an
oncolytic viral vector with rapamycin (ARIAD system) regulated replication
control for use in
pan-cancer applications. This system relies on a chimeric promoter
encompassing 8 copies
of the recognition site for ZFHD1 upstream of a minimal IL-2 promoter driving
E1 gene
expression and a human E2F promoter (E2F) driving expression of the
"activation domain"
(the rapamycin binding domain of FRAP (FRB) fused to the
activation domain of p65 sub-unit of NF-I<B) and "DNA binding domain" (which
encodes a
chimeric DNA-binding molecule with ZFHD1 fused to 3 copies of FhCBP)
It is evident from the above description that regulatable replication-
competent
viruses can be provided as vehicles specific for parfiicular host cells, where
the viruses
selectively replicate in particular target cells and viral replication may be
regulated.
All publications and patent applications mentioned in this specification are
herein
incorporated by reference to the same extent as if each individual publication
or patent
application was specifically and individually indicated to be incorporated by
reference.
The invention now being fully described, it will be apparent to one of
ordinary skill in
the art that many changes and modifications can be made thereto without
departing from
the spirit or scope of the appended claims.
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SEQUENCE LISTING
<110> Harding, Thomas
Yu, De Chao
<120> SYSTEM FOR EXTERNAL CONTROL OF ONCOLYTIC VIRUS REPLICATION
<210> 1
<211> 5836
<212> DNA
<213> Homo Sapiens
<400> 1
aagcttctag ttttcttttc ccggtgacat cgtggaaagc actagcatct ctaagcaatg 60
atctgtgaca atattcacag tgtaatgcca tccagggaac tcaactgagc cttgatgtcc 120
agagattttt gtgttttttt ctgagactga gtctcgctct gtgccaggct ggagtgcagt 180
ggtgcaacct tggctcactg caagctccgc ctcctgggtt cacgccattc tcctgcctca 240
gcctcctgag tagctgggac tacaggcacc cgccaccacg cctggctaat ttttttgtat 300
ttttagtaga gatggggttt cactgtgtta gccaggatgg tctcagtctc ctgacctcgt 360
gatctgccca ccttggcctc ccaaagtgct gggatgacag gcgtgagcca ccgcgcctgg 420
ccgatatcca gagatttttt ggggggctcc atcacacaga catgttgact gtcttcatgg 480
ttgactttta gtatccagcc cctctagaaa tctagctgat atagtgtggc tcaaaacctt 540
cagcacaaat cacaccgtta gactatctgg tgtggcccaa accttcaggt gaacaaaggg 600
actctaatct ggcaggatat tccaaagcat tagagatgac ctcttgcaaa gaaaaagaaa 660
tggaaaagaa aaagaaagaa aggaaaaaaa aaaaaaaaaa gagatgacct ctcaggctct 720
gaggggaaac gcctgaggtc tttgagcaag gtcagtcctc tgttgcacag tctccctcac 780
agggtcattg tgacgatcaa atgtggtcac gtgtatgagg caccagcaca tgcctggctc 840
tggggagtgc cgtgtaagtg tatgcttgca ctgctgaatg cttgggatgt gtcagggatt 900
atcttcagca cttacagatg ctcatctcat cctcacagca tcactatggg atgggtatta 960
ctggcctcat ttgatggaga aagtggctgt ggctcagaaa ggggggacca ctagaccagg 1020
gacactctgg atgctgggga ctccagagac catgaccact caccaactgc agagaaatta 1080
attgtggcct gatgtccctg tcctggagag ggtggaggtg gaccttcact aacctcctac 1140
cttgaccctc tcttttaggg ctctttctga cctccaccat ggtactagga ccccattgta 1200
ttctgtaccc tcttgactct atgaccccca ctgcccactg catccagctg ggtcccctcc 1260
tatctctatt cccagctggc cagtgcagtc tcagtgccca cctgtttgtc agtaactctg 1320
aaggggctga cattttactg acttgcaaac aaataagcta actttccaga gttttgtgaa 1380
tgctggcaga gtccatgaga ctcctgagtc agaggcaaag gcttttactg ctcacagctt 1440
agcagacagc atgaggttca tgttcacatt agtacacctt gcccccccca aatcttgtag 1500
ggtgaccaga gcagtctagg tggatgctgt gcagaagggg tttgtgccac tggtgagaaa 1560
cctgagatta ggaatcctca atcttatact gggacaactt gcaaacctgc tcagcctttg 1620
tctctgatga agatattatc ttcatgatct tggattgaaa acagacctac tctggaggaa 1680
catattgtat cgattgtcct tgacagtaaa caaatctgtt gtaagagaca ttatctttat 1740
tatctaggac agtaagcaag cctggatctg agagagatat catcttgcaa ggatgcctgc 1800
tttacaaaca tccttgaaac aacaatccag aaaaaaaaag gtgttgctgt ctttgctcag 1860
aagacacaca gatacgtgac agaaccatgg agaattgcct cccaacgctg ttcagccaga 1920
gccttccacc cttgtctgca ggacagtctc aacgttccac cattaaatac ttcttctatc 1980
acatcctgct tctttatgcc taaccaaggt tctaggtccc gatcgactgt gtctggcagc 2040
actccactgc caaacccaga ataaggcagc gctcaggatc ccgaaggggc atggctgggg 2100
atcagaactt ctgggtttga gtgaggagtg ggtccaccct cttgaatttc aaaggaggaa 2160
gaggctggat gtgaaggtac tgggggaggg aaagtgtcag ttccgaactc ttaggtcaat 2220
gagggaggag actggtaagg tcccagctcc cgaggtactg atgtgggaat ggcctaagaa 2280
tctcatatcc tcaggaagaa ggtgctggaa tcctgagggg tagagttctg ggtatatttg 2340
tggcttaagg ctctttggcc cctgaaggca gaggctggaa ccattaggtc cagggtttgg 2400
ggtgatagta atgggatctc ttgattcctc aagagtctga ggatcgaggg ttgcccattc 2460
ttccatcttg ccacctaatc cttactccac ttgagggtat caccagccct tctagctcca 2520
tgaaggtccc ctgggcaagc acaatctgag catgaaagat gccccagagg ccttgggtgt 2580
catccactca tcatccagca tcacactctg agggtgtggc cagcaccatg acgtcatgtt 2640
gctgtgacta tccctgcagc gtgcctctcc agccacctgc caaccgtaga gctgcccatc 2700
ctcctctggt gggagtggcc tgcatggtgc caggctgagg cctagtgtca gacagggagc 2760
ctggaatcat agggatccag gactcaaaag tgctagagaa tggccatatg tcaccatcca 2820
tgaaatctca agggcttctg ggtggagggc acagggacct gaacttatgg tttcccaagt 2880
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ctattgctct cccaagtgag tctcccagat acgaggcact gtgccagcat cagccttatc 2940
tccaccacat cttgtaaaag gactacccag ggccctgatg aacaccatgg tgtgtacagg 3000
agtagggggt ggaggcacgg actcctgtga ggtcacagcc aagggagcat catcatgggt 3060
ggggaggagg caatggacag gcttgagaac ggggatgtgg ttgtatttgg ttttctttgg 3120
ttagataaag tgctgggtat aggattgaga gtggagtatg aagaccagtt aggatggagg 3180
atcagattgg agttgggtta gataaagtgc tgggtatagg attgagagtg gagtatgaag 3240
accagttagg atggaggatc agattggagt tgggttagag atggggtaaa attgtgctcc 3300
ggatgagttt gggattgaca ctgtggaggt ggtttgggat ggcatggctt tgggatggaa 3360
atagatttgt tttgatgttg gctcagacat ccttggggat tgaactgggg atgaagctgg 3420
gtttgatttt ggaggtagaa gacgtggaag tagctgtcag atttgacagt ggccatgagt 3480
tttgtttgat ggggaatcaa acaatggggg aagacataag ggttggcttg ttaggttaag 3540
ttgcgttggg ttgatggggt cggggctgtg tataatgcag ttggattggt ttgtattaaa 3600
ttgggttggg tcaggttttg gttgaggatg agttgaggat atgcttgggg acaccggatc 3660
catgaggttc tcactggagt ggagacaaac ttcctttcca ggatgaatcc agggaagcct 3720
taattcacgt gtaggggagg tcaggccact ggctaagtat atccttccac tccagctcta 3780
agatggtctt aaattgtgat tatctatatc cacttctgtc tccctcactg tgcttggagt 3840
ttacctgatc actcaactag aaacagggga agattttatc aaattctttt tttttttttt 3900
ttttttttga gacagagtct cactctgttg cccaggctgg agtgcagtgg cgcagtctcg 3960
gctcactgca acctctgcct cccaggttca agtgattctc ctgcctcagc ctcctgagtt 4020
gctgggatta caggcatgca gcaccatgcc cagctaattt ttgtattttt agtagagatg 4080
gggtttcacc aatgtttgcc aggctggcct cgaactcctg acctggtgat ccacctgcct 4140
cagcctccca aagtgctggg attacaggcg tcagccaccg cgcccagcca cttttgtcaa 4200
attcttgaga cacagctcgg gctggatcaa gtgagctact ctggttttat tgaacagctg 4260
aaataaccaa ctttttggaa attgatgaaa tcttacggag ttaacagtgg aggtaccagg 4320
gctcttaaga gttcccgatt ctcttctgag actacaaatt gtgattttgc atgccacctt 4380
aatctttttt tttttttttt taaatcgagg tttcagtctc attctatttc ccaggctgga 4440
gttcaatagc gtgatcacag ctcactgtag ccttgaactc ctggccttaa gagattctcc 4500
tgcttcggtc tcccaatagc taagactaca gtagtccacc accatatcca gataattttt 4560
aaattttttg gggggccggg cacagtggct cacgcctgta atcccaacac catgggaggc 4620
tgagatgggt ggatcacgag gtcaggagtt tgagaccagc ctgaccaaca tggtgaaact 4680
ctgtctctac taaaaaaaaa aaaaatagaa aaattagccg ggcgtggtgg cacacggcac 4740
ctgtaatccc agctactgag gaggctgagg caggagaatc acttgaaccc agaaggcaga 4800
ggttgcaatg agccgagatt gcgccactgc actccagcct gggtgacaga gtgagactct 4860
gtctcaaaaa aaaaaaattt tttttttttt tttgtagaga tggatcttgc tttgtttctc 4920
tggttggcct tgaactcctg gcttcaagtg atcctcctac cttggcctcg gaaagtgttg 4980
ggattacagg cgtgagccac catgactgac ctgtcgttaa tcttgaggta cataaacctg 5040
gctcctaaag gctaaaggct aaatatttgt tggagaaggg gcattggatt ttgcatgagg 5100
atgattctga cctgggaggg caggtcagca ggcatctctg ttgcacagat agagtgtaca 5160
ggtctggaga acaaggagtg gggggttatt ggaattccac attgtttgct gcacgttgga 5220
ttttgaaatg ctagggaact ttgggagact catatttctg ggctagagga tctgtggacc 5280
acaagatctt tttatgatga cagtagcaat gtatctgtgg agctggattc tgggttggga 5340
gtgcaaggaa aagaatgtac taaatgccaa gacatctatt tcaggagcat gaggaataaa 5400
agttctagtt tctggtctca gagtggtgca gggatcaggg agtctcacaa tctcctgagt 5460
gctggtgtct tagggcacac tgggtcttgg agtgcaaagg atctaggcac gtgaggcttt 5520
gtatgaagaa tcggggatcg tacccacccc ctgtttctgt ttcatcctgg gcatgtctcc 5580
tctgcctttg tcccctagat gaagtctcca tgagctacaa gggcctggtg catccagggt 5640
gatctagtaa ttgcagaaca gcaagtgcta gctctccctc cccttccaca gctctgggtg 5700
tgggaggggg ttgtccagcc tccagcagca tggggagggc cttggtcagc ctctgggtgc 5760
cagcagggca ggggcggagt cctggggaat gaaggtttta tagggctcct gggggaggct 5820
ccccagcccc aagctt 5836
<210> 2
<211> 5835
<212> DNA
<213> Homo Sapiens
<400> 2
aagcttctag ttttcttttc ccggtgacat cgtggaaagc actagcatct ctaagcaatg 60
atctgtgaca atattcacag tgtaatgcca tccagggaac tcaactgagc cttgatgtcc 120
agagattttt gtgttttttt ctgagactga gtctcgctct gtgccaggct ggagtgcagt 180
ggtgcaacct tggctcactg caagctccgc ctcctgggtt cacgccattc tcctgcctca 240
gcctcctgag tagctgggac tacaggcacc cgccaccacg cctggctaat ttttttgtat 300
CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
ttttagtaga gatggggttt cactgtgtta gccaggatgg tctcagtctc ctgacctcgt 360
gatctgccca ccttggcctc ccaaagtgct gggatgacag gcgtgagcca ccgcgcctgg 420
ccgatatcca gagatttttt ggggggctcc atcacacaga catgttgact gtcttcatgg 480
ttgactttta gtatccagcc cctctagaaa tctagctgat atagtgtggc tcaaaacctt 540
cagcacaaat cacaccgtta gactatctgg tgtggcccaa accttcaggt gaacaaaggg 600
actctaatct ggcaggatac tccaaagcat tagagatgac ctcttgcaaa gaaaaagaaa 660
tggaaaagaa aaagaaagaa aggaaaaaaa aaaaaaaaaa gagatgacct ctcaggctct 720
gaggggaaac gcctgaggtc tttgagcaag gtcagtcctc tgttgcacag tctccctcac 780
agggtcattg tgacgatcaa atgtggtcac gtgtatgagg caccagcaca tgcctggctc 840
tggggagtgc cgtgtaagtg tatgcttgca ctgctgaatg gctgggatgt gtcagggatt 900
atcttcagca cttacagatg ctcatctcat cctcacagca tcactatggg atgggtatta 960
ctggcctcat ttgatggaga aagtggctgt ggctcagaaa ggggggacca ctagaccagg 1020
gacactctgg atgctgggga ctccagagac catgaccact caccaactgc agagaaatta 1080
attgtggcct gatgtccctg tcctggagag ggtggaggtg gaccttcact aacctcctac 1140
cttgaccctc tcttttaggg ctctttctga cctccaccat ggtactagga ccccattgta 1200
ttctgtaccc tcttgactct atgaccccca ccgcccactg catccagctg ggtcccctcc 1260
tatctctatt cccagctggc cagtgcagtc tcagtgccca cctgtttgtc agtaactctg 1320
aaggggctga cattttactg acttgcaaac aaataagcta actttccaga gttttgtgaa 1380
tgctggcaga gtccatgaga ctcctgagtc agaggcaaag gcttttactg ctcacagctt 1440
agcagacagc atgaggttca tgttcacatt agtacacctt gcccccccca aatcttgtag 1500
ggtgaccaga gcagtctagg tggatgctgt gcagaagggg tttgtgccac tggtgagaaa 1560
cctgagatta ggaatcctca atcttatact gggacaactt gcaaacctgc tcagcctttg 1620
tctctgatga agatattatc ttcatgatct tggattgaaa acagacctac tctggaggaa 1680
catattgtat cgattgtcct tgacagtaaa caaatctgtt gtaagagaca ttatctttat 1740
tatctaggac agtaagcaag cctggatctg agagagatat catcttgcaa ggatgcctgc 1800
tttacaaaca tccttgaaac aacaatccag aaaaaaaaag gtgttactgt ctttgctcag 1860
aagacacaca gatacgtgac agaaccatgg agaattgcct cccaacgctg ttcagccaga 1920
gccttccacc ctttctgcag gacagtctca acgttccacc attaaatact tcttctatca 1980
catcccgctt ctttatgcct aaccaaggtt ctaggtcccg atcgactgtg tctggcagca 2040
ctccactgcc aaacccagaa taaggcagcg ctcaggatcc cgaaggggca tggctgggga 2100
tcagaacttc tgggtttgag tgaggagtgg gtccaccctc ttgaatttca aaggaggaag 2160
aggctggatg tgaaggtact gggggaggga aagtgtcagt tccgaactct taggtcaatg 2220
agggaggaga ctggtaaggt cccagctccc gaggtactga tgtgggaatg gcctaagaat 2280
ctcatatcct caggaagaag gtgctggaat cctgaggggt agagttctgg gtatatttgt 2340
ggcttaaggc tctttggccc ctgaaggcag aggctggaac cattaggtcc agggtttggg 2400
gtgatagtaa tgggatctct tgattcctca agagtctgag gatcgagggt tgcccattct 2460
tccatcttgc cacctaatcc ttactccact tgagggtatc accagccctt ctagctccat 2520
gaaggtcccc tgggcaagca caatctgagc atgaaagatg ccccagaggc cttgggtgtc 2580
atccactcat catccagcat cacactctga gggtgtggcc agcaccatga cgtcatgttg 2640
ctgtgactat ccctgcagcg tgcctctcca gccacctgcc aaccgtagag ctgcccatcc 2700
tcctctggtg ggagtggcct gcatggtgcc aggctgaggc ctagtgtcag acagggagcc 2760
tggaatcata gggatccagg actcaaaagt gctagagaat ggccatatgt caccatccat 2820
gaaatctcaa gggcttctgg gtggagggca cagggacctg aacttatggt ttcccaagtc 2880
tattgctctc ccaagtgagt ctcccagata cgaggcactg tgccagcatc agccttatct 2940
ccaccacatc ttgtaaaagg actacccagg gccctgatga acaccatggt gtgtacagga 3000
gtagggggtg gaggcacgga ctcctgtgag gtcacagcca agggagcatc atcatgggtg 3060
gggaggaggc aatggacagg cttgagaacg gggatgtggt tgtatttggt tttctttggt 3120
tagataaagt gctgggtata ggattgagag tggagtatga agaccagtta ggatggagga 3180
tcagattgga gttgggttag ataaagtgct gggtatagga ttgagagtgg agtatgaaga 3240
ccagttagga tggaggatca gattggagtt gggttagaga tggggtaaaa ttgtgctccg 3300
gatgagtttg ggattgacac tgtggaggtg gtttgggatg gcatggcttt gggatggaaa 3360
tagatttgtt ttgatgttgg ctcagacatc cttggggatt gaactgggga tgaagctggg 3420
tttgattttg gaggtagaag acgtggaagt agctgtcaga tttgacagtg gccatgagtt 3480
ttgtttgatg gggaatcaaa caatggggga agacataagg gttggcttgt taggttaagt 3540
tgcgttgggt tgatggggtc ggggctgtgt ataatgcagt tggattggtt tgtattaaat 3600
tgggttgggt caggttttgg ttgaggatga gttgaggata tgcttgggga caccggatcc 3660
atgaggttct cactggagtg gagacaaact tcctttccag gatgaatcca gggaagcctt 3720
aattcacgtg taggggaggt caggccactg gctaagtata tccttccact ccagctctaa 3780
gatggtctta aattgtgatt atctatatcc acttctgtct ccctcactgt gcttggagtt 3840
tacctgatca ctcaactaga aacaggggaa gattttatca aattcttttt tttttttttt 3900
tttttttgag acagagtctc actctgttgc ccaggctgga gtgcagtggc gcagtctcgg 3960
3
CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
ctcactgcaa cctctgcctc ccaggttcaa gtgattctcc tgcctcagcc tcctgagttg 4020
ctgggattac aggcatgcag caccatgccc agctaatttt tgtattttta gtagagatgg 4080
ggtttcacca atgtttgcca ggctggcctc gaactcctga cctggtgatc cacctgcctc 4140
agcctcccaa agtgctggga ttacaggcgt cagccaccgc gcccagccac ttttgtcaaa 4200
ttcttgagac acagctcggg ctggatcaag tgagctactc tggttttatt gaacagctga 4260
aataaccaac tttttggaaa ttgatgaaat cttacggagt taacagtgga ggtaccaggg 4320
ctcttaagag ttcccgattc tcttctgaga ctacaaattg tgattttgca tgccacctta 4380
atcttttttt tttttttttt aaatcgaggt ttcagtctca ttctatttcc caggctggag 4440
ttcaatagcg tgatcacagc tcactgtagc cttgaactcc tggccttaag agattctcct 4500
gcttcggtct cccaatagct aagactacag tagtccacca ccatatccag ataattttta 4560
aattttttgg ggggccgggc acagtggctc acgcctgtaa tcccaacacc atgggaggct 4620
gagatgggtg gatcacgagg tcaggagttt gagaccagcc tgaccaacat ggtgaaactc 4680
tgtctctact aaaaaaaaaa aaaatagaaa aattagccgg gcgtggtggc acacggcacc 4740
tgtaatccca gctactgagg aggctgaggc aggagaatca cttgaaccca gaaggcagag 4800
gttgcaatga gccgagattg cgccactgca ctccagcctg ggtgacagag tgagactctg 4860
tctcaaaaaa aaaaaatttt tttttttttt ttgtagagat ggatcttgct ttgtttctct 4920
ggttggcctt gaactcctgg cttcaagtga tcctcctacc ttggcctcgg aaagtgttgg 4980
gattacaggc gtgagccacc atgactgacc tgtcgttaat cttgaggtac ataaacctgg 5040
ctcctaaagg ctaaaggcta aatatttgtt ggagaagggg cattggattt tgcatgagga 5100
tgattctgac ctgggagggc aggtcagcag gcatctctgt tgcacagata gagtgtacag 5160
gtctggagaa caaggagtgg ggggttattg gaattccaca ttgtttgctg cacgttggat 5220
tttgaaatgc tagggaactt tgggagactc atatttctgg gctagaggat ctgtggacca 5280
caagatcttt ttatgatgac agtagcaatg tatctgtgga gctggattct gggttgggag 5340
tgcaaggaaa agaatgtact aaatgccaag acatctattt caggagcatg aggaataaaa 5400
gttctagttt ctggtctcag agtggtgcat ggatcaggga gtctcacaat ctcctgagtg 5460
ctggtgtctt agggcacact gggtcttgga gtgcaaagga tctaggcacg tgaggctttg 5520
tatgaagaat cggggatcgt acccaccccc tgtttctgtt tcatcctggg catgtctcct 5580
ctgcctttgt cccctagatg aagtctccat gagctacaag ggcctggtgc atccagggtg 5640
atctagtaat tgcagaacag caagtgctag ctctccctcc ccttccacag ctctgggtgt 5700
gggagggggt tgtccagcct ccagcagcat ggggagggcc ttggtcagcc tctgggtgcc 5760
agcagggcag gggcggagtc ctggggaatg aaggttttat agggctcctg ggggaggctc 5820
cccagcccca agctt 5835
<210> 3
<211> 12047
<212> DNA
<213> Homo Sapiens
<400> 3
gaattcagaa ataggggaag gttgaggaag gacactgaac tcaaagggga tacagtgatt 60
ggtttatttg tcttctcttc acaacattgg tgctggagga attcccaccc tgaggttatg 120
aagatgtctg aacacccaac acatagcact ggagatatga gctcgacaag agtttctcag 180
ccacagagat tcacagccta gggcaggagg acactgtacg ccaggcagaa tgacatggga 240
attgcgctca cgattggctt gaagaagcaa ggactgtggg aggtgggctt tgt,agtaaca 300
agagggcagg gtgaactctg attcccatgg gggaatgtga tggtcctgtt acaaattttt 360
caagctggca gggaataaaa cccattacgg tgaggacctg tggagggcgg ctgccccaac 420
tgataaagga aatagccagg tgggggcctt tcccattgta ggggggacat atctggcaat 480
agaagccttt gagacccttt agggtacaag tactgaggca gcaaataaaa tgaaatctta 540
tttttcaact ttatactgca tgggtgtgaa gatatatttg tttctgtaca gggggtgagg 600
gaaaggaggg gaggaggaaa gttcctgcag gtctggtttg gtcttgtgat ccagggggtc 660
ttggaactat ttaaattaaa ttaaattaaa acaagcgact gttttaaatt aaattaaatt 720
aaattaaatt ttactttatt ttatcttaag ttctgggcta catgtgcagg acgtgcagct 780
ttgttacata ggtaaacgtg tgccatggtg gtttgctgta cctatcaacc catcacctag 840
gtattaagcc cagcatgcat tagctgtttt tcctgacgct ctccctctcc ctgactccca 900
caacaggccc cagtgtgtgt tgttcccctc cctgtgtcca tgtgttctca ttgttcagct 960
cccacttata agtgagaaca tgtggtgttt ggttttctgt ttctgtgtta gtttgctgag 1020
gataatggct tccacctcca tccatgttcc tgcaaaggac gtgatcttat tcttttttat 1080
ggttgcatag aaattgtttt tacaaatcca attgatattg tatttaatta caagttaatc 1140
taattagcat actagaagag attacagaag atattaggta cattgaatga ggaaatatat 1200
aaaataggac gaaggtgaaa tattaggtag gaaaagtata atagttgaaa gaagtaaaaa 1260
aaaatatgca tgagtagcag aatgtaaaag aggtgaagaa cgtaatagtg actttttaga 1320
ccagattgaa ggacagagac agaaaaattt taaggaattg ctaaaccatg tgagtgttag 1380
CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
aagtacagtc aataacatta aagcctcagg aggagaaaag aataggaaag gaggaaatat 1440
gtgaataaat agtagagaca tgtttgatgg attttaaaat atttgaaaga cctcacatca 1500
aaggattcat accgtgccat tgaagaggaa gatggaaaag ccaagaagcc agatgaaagt 1560
tagaaatatt attggcaaag cttaaatgtt aaaagtccta gagagaaagg atggcagaaa 1620
tattggcggg aaagaatgca gaacctagaa tataaattca tcccaacagt ttggtagtgt 1680
gcagctgtag ccttttctag ataatacact attgtcatac atcgcttaag cgagtgtaaa 1740
atggtctcct cactttattt atttatatat ttatttagtt ttgagatgga gcctcgctct 1800
gtctcctagg ctggagtgca atagtgcgat accactcact gcaacctctg cctcctctgt 1860
tcaagtgatt ttcttacctc agcctcccga gtagctggga ttacaggtgc gtgccaccac 1920
acccggctaa tttttgtatt ttttgtagag acggggtttt gccatgttgg ccaggctggt 1980
cttgaactcc tgacatcagg tgatccacct gccttggcct cctaaagtgc tgggattaca 2040
ggcatgagcc accgtgccca accactttat ttatttttta tttttatttt taaatttcag 2100
cttctatttg aaatacaggg ggcacatata taggattgtt acatgggtat attgaactca 2160
ggtagtgatc atactaccca acaggtaggt tttcaaccca ctccccctct tttcctcccc 2220
attctagtag tgtgcagtgt ctattgttct catgtttatg tctatgtgtg ctccaggttt 2280
agctcccacc tgtaagtgag aacgtgtggt atttgatttt ctgtccctgt gttaattcac 2340
ttaggattat ggcttccagc tccattcata ttgctgtaaa ggatatgatt catttttcat 2400
ggccatgcag tattccatat tgcgtataga tcacattttc tttctttttt ttttttgaga 2460
cggagtcttg ctttgctgcc taggctggag tgcagtagca cgatctcggc tcactgcaag 2520
cttcacctcc ggggttcacg tcattcttct gtctcagctt cccaagtagc tgggactaca 2580
ggcgcccgcc accacgtccg gctaattttt ttgtgtgttt ttagtagaga tgggggtttc 2640
actgtgttag ccaggatggt cttgatctcc tgaccttgtg gtccacctgc ctcggtctcc 2700
caaagtgctg ggattacagg ggtgagccac tgcgcccggc ccatatatac cacattttct 2760
ttaaccaatc caccattgat gggcaactag gtagattcca tggattccac agttttgcta 2820
ttgtgtgcag tgtggcagta gacatatgaa tgaatgtgtc tttttggtat aatgatttgc 2880
attcctttgg gtatacagtc attaatagga gtgctgggtt gaacggtggc tctgtttaaa 2940
attctttgag aattttccaa actgtttgcc atagagagca aactaattta catttccacg 3000
aacagtatat aagcattccc ttttctccac agctttgtca tcatggtttt tttttttctt 3060
tattttaaaa aagaatatgt tgttgttttc ccagggtaca tgtgcaggat gtgcaggttt 3120
gttacatagg tagtaaacgt gagccatggt ggtttgctgc acctgtcaac ccattacctg 3180
ggtatgaagc cctgcctgca ttagctcttt tccctaatgc tctcactact gccccaccct 3240
caccctgaca gggcaaacag acaacctaca gaatgggagg aaatttttgc aatctattca 3300
tctgacaaag gtcaagaata tccagaatct acaaggaact taagcaaatt tttacttttt 3360
aataatagcc actctgactg gcgtgaaatg gtatctcatt gtggttttca tttgaatttc 3420
tctgatgatc agtgacgatg agcatttttt catatttgtt ggctgcttgt acgtcttttg 3480
agaagtgtct cttcatgcct tttggccact ttaatgggat tattttttgc tttttagttt 3540
aagttcctta tagattctgg atattagact tcttattgga tgcatagttt gtgaatactc 3600
tcttccattc tgtaggttgt ctgtttactc tattgatggc ttcttttgct gtgccgaagc 3660
atcttagttt aattagaaac cacctgccaa tttttgtttt tgttgcaatt gcttttgggg 3720
acttagtcat aaactctttg ccaaggtctg ggtcaagaag agtatttcct aggttttctt 3780
ctagaatttt gaaagtctga atgtaaacat ttgcattttt aatgcatctt gagttagttt 3840
ttgtatatgt gaaaggtcta ctctcatttt ctttccctct ttctttcttt ctttcttttc 3900
tttctttctt tctttctttc tttctttctt tctttctttc tttctttttg tccttctttc 3960
tttctttctt tctctttctt tctctctttc tttttttttt ttgatggagt attgctctgt 4020
tgcccaggct gcagtgcagc ggcacgatct cggctcactg caacctctgc ctcctgggtt 4080
caactgattc tcctgcatca gccttccaag tagctgggat tataggcgcc cgccaccacg 4140
cccgactaat ttttgtattt ttagtagaga cggggttgtg ccatgttggc caggctggtt 4200
tgaaactcct gacctcaaac gatctgcctg ccttggcctc ccaaagtgct gggattacag 4260
gtgtgagcca ctgtgcccag ccaagaatgt cattttctaa gaggtccaag aacctcaaga 4320
tattttggga ccttgagaag agaggaattc atacaggtat tacaagcaca gcctaatggc 4380
aaatctttgg catggcttgg cttcaagact ttaggctctt aaaagtcgaa tccaaaaatt 4440
tttataaaag ctccagctaa gctaccttaa aaggggcctg tatggctgat cactcttctt 4500
gctatacttt acacaaataa acaggccaaa tataatgagg ccaaaattta ttttgcaaat 4560
aaattggtcc tgctatgatt tactcttggt aagaacaggg aaaatagaga aaaatttaga 4620
ttgcatctga cctttttttc tgaattttta tatgtgccta caatttgagc taaatcctga 4680
attattttct ggttgcaaaa actctctaaa gaagaacttg gttttcattg tcttcgtgac 4740
acatttatct ggctctttac tagaacagct ttcttgtttt tggtgttcta gcttgtgtgc 4800
cttacagttc tactcttcaa attattgtta tgtgtatctc atagttttcc ttcttttgag 4860
aaaactgaag ccatggtatt ctgaggacta gagatgactc aacagagctg gtgaatctcc 4920
tcatatgcaa tccactgggc tcgatctgct tcaaattgct gatgcactgc tgctaaagct 4980
atacatttaa aaccctcact aaaggatcag ggaccatcat ggaagaggag gaaacatgaa 5040
CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
attgtaagag ccagattcgg ggggtagagt gtggaggtca gagcaactcc accttgaata 5100
agaaggtaaa gcaacctatc ctgaaagcta acctgccatg gtggcttctg attaacctct 5160
gttctaggaa gactgacagt ttgggtctgt gtcattgccc aaatctcatg ttaaattgta 5220
atccccagtg ttcggaggtg ggacttggtg gtaggtgatt cggtcatggg agtagatttt 5280
cttctttgtg gtgttacagt gatagtgagt gagttctcgt gagatctggt catttaaaag 5340
tgtgtggccc ctcccctccc tctcttggtc ctcctactgc catgtaagat acctgctcct 5400
gctttgcctt ctaccataag taaaagcccc ctgaggcctc cccagaagca gatgccacca 5460
tgcttcctgt acagcctgca gaaccatcag ccaattaaac ctcttttctg tataaattac 5520
cagtcttgag tatctcttta cagcagtgtg agaacggact aatacaaggg tctccaaaat 5580
tccaagttta tgtattcttt cttgccaaat agcaggtatt taccataaat cctgtcctta 5640
ggtcaaacaa ccttgatggc atcgtacttc aattgtctta cacattcctt ctgaatgact 5700
cctcccctat ggcatataag ccctgggtct tgggggataa tggcagaggg gtccaccatc 5760
ttgtctggct gccacctgag acacggacat ggcttctgtt ggtaagtctc tattaaatgt 5820
ttctttctaa gaaactggat ttgtcagctt gtttctttgg cctctcagct tcctcagact 5880
ttggggtagg ttgcacaacc ctgcccacca cgaaacaaat gtttaatatg ataaatatgg 5940
atagatataa tccacataaa taaaagctct tggagggccc tcaataattg ttaagagtgt 6000
aaatgtgtcc aaagatggaa aatgtttgag aactactgtc ccagagattt tcctgagttc 6060
tagagtgtgg gaatatagaa cctggagctt ggcttcttca gcctagaatc aggagtatgg 6120
ggctgaagtc tgaagcttgg cttcagcagt ttggggttgg cttccggagc acatatttga 6180
catgttgcga ctgtgatttg gggtttggta tttgctctga atcctaatgt ctgtccttga 6240
ggcatctaga atctgaaatc tgtggtcaga attctattat cttgagtagg acatctccag 6300
tcctggttct gccttctagg gctggagtct gtagtcagtg acccggtctg gcatttcaac 6360
ttcatataca gtgggctatc ttttggtcca tgtttcaacc aaacaaccga ataaaccatt 6420
agaacctttc cccacttccc tagctgcaat gttaaaccta ggatttctgt ttaataggtt 6480
catatgaata atttcagcct gatccaactt tacattcctt ctaccgttat tctacaccca 6540
ccttaaaaat gcattcccaa tatattccct ggattctacc tatatatggt aatcctggct 6600
ttgccagttt ctagtgcatt aacatacctg atttacattc ttttacttta aagtggaaat 6660
aagagtccct ctgcagagtt caggagttct caagatggcc cttacttctg acatcaattg 6720
agatttcaag ggagtcgcca agatcatcct caggttcagt gattgctggt agccctcata 6780
taactcaatg aaagctgtta tgctcatggc tatggtttat tacagcaaaa gaatagagat 6840
gaaaatctag caagggaaga gttgcatggg gcaaagacaa ggagagctcc aagtgcagag 6900
attcctgttg ttttctccca gtggtgtcat ggaaagcagt atcttctcca tacaatgatg 6960
tgtgataata ttcagtgtat tgccaatcag ggaactcaac tgagccttga ttatattgga 7020
gcttggttgc acagacatgt cgaccacctt catggctgaa ctttagtact tagcccctcc 7080
agacgtctac agctgatagg ctgtaaccca acattgtcac cataaatcac attgttagac 7140
tatccagtgt ggcccaagct cccgtgtaaa cacaggcact ctaaacaggc aggatatttc 7200
aaaagcttag agatgacctc ccaggagctg aatgcaaaga cctggcctct ttgggcaagg 7260
agaatccttt accgcacact ctccttcaca gggttattgt gaggatcaaa tgtggtcatg 7320
tgtgtgagac accagcacat gtctggctgt ggagagtgac ttctatgtgt gctaacattg 7380
ctgagtgcta agaaagtatt aggcatggct ttcagcactc acagatgctc atctaatcct 7440
cacaacatgg ctacagggtg ggcactacta gcctcatttg acagaggaaa ggactgtgga 7500
taagaagggg gtgaccaata ggtcagagtc attctggatg caaggggctc cagaggacca 7560
tgattagaca ttgtctgcag agaaattatg gctggatgtc tctgccccgg aaagggggat 7620
gcactttcct tgacccccta tctcagatct tgactttgag gttatctcag acttcctcta 7680
tgataccagg agcccatcat aatctctctg tgtcctctcc ccttcctcag tcttactgcc 7740
cactcttccc agctccatct ccagctggc,c aggtgtagcc acagtaccta actctttgca 7800
gagaactata aatgtgtatc ctacagggga gaaaaaaaaa aagaactctg aaagagctga 7860
cattttaccg acttgcaaac acataagcta acctgccagt tttgtgctgg tagaactcat 7920
gagactcctg ggtcagaggc aaaagatttt attacccaca gctaaggagg cagcatgaac 7980
tttgtgttca catttgttca ctttgccccc caattcatat gggatgatca gagcagttca 8040
ggtggatgga cacaggggtt tgtggcaaag gtgagcaacc taggcttaga aatcctcaat 8100
cttataagaa ggtactagca aacttgtcca gtctttgtat ctgacggaga tattatcttt 8160
ataattgggt tgaaagcaga cctactctgg aggaacatat tgtatttatt gtcctgaaca 8220
gtaaacaaat ctgctgtaaa atagacgtta actttattat ctaaggcagt aagcaaacct 8280
agatctgaag gcgataccat cttgcaaggc tatctgctgt acaaatatgc ttgaaaagat 8340
ggtccagaaa agaaaacggt attattgcct ttgctcagaa gacacacaga aacataagag 8400
aaccatggaa aattgtctcc caacactgtt cacccagagc cttccactct tgtctgcagg 8460
acagtcttaa catcccatca ttagtgtgtc taccacatct ggcttcaccg tgcctaacca 8520
agatttctag gtccagttcc ccaccatgtt tggcagtgcc ccactgccaa ccccagaata 8580
agggagtgct cagaattccg aggggacatg ggtggggatc agaacttctg ggcttgagtg 8640
cagagggggc ccatactcct tggttccgaa ggaggaagag gctggaggtg aatgtccttg 8700
6
CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
gaggggagga atgtgggttc tgaactctta aatccccaag ggaggagact ggtaaggtcc 8760
cagcttccga ggtactgacg tgggaatggc ctgagaggtc taagaatccc gtatcctcgg 8820
gaaggagggg ctgaaattgt gaggggttga gttgcagggg tttgttagct tgagactcct 8880
tggtgggtcc ctgggaagca aggactggaa ccattggctc cagggtttgg tgtgaaggta 8940
atgggatctc ctgattctca aagggtcaga ggactgagag ttgcccatgc tttgatcttt 9000
ccatctactc cttactccac ttgagggtaa tcacctactc ttctagttcc acaagagtgc 9060
gcctgcgcga gtataatctg cacatgtgcc atgtcccgag gcctggggca tcatccactc 9120
atcattcagc atctgcgcta tgcgggcgag gccggcgcca tgacgtcatg tagctgcgac 9180
tatccctgca gcgcgcctct cccgtcacgt cccaaccatg gagctgtgga cgtgcgtccc 9240
ctggtggatg tggcctgcgt ggtgccaggc cggggcctgg tgtccgataa agatcctaga 9300
accacaggaa accaggactg aaaggtgcta gagaatggcc atatgtcgct gtccatgaaa 9360
tctcaaggac ttctgggtgg agggcacagg agcctgaact tacgggtttg ccccagtcca 9420
ctgtcctccc aagtgagtct cccagatacg aggcactgtg ccagcatcag cttcatctgt 9480
accacatctt gtaacaggga ctacccagga ccctgatgaa caccatggtg tgtgcaggaa 9540
gagggggtga aggcatggac tcctgtgtgg tcagagccca gagggggcca tgacgggtgg 9600
ggaggaggct gtggactggc tcgagaagtg ggatgtggtt gtgtttgatt tcctttggcc 9660
agataaagtg ctggatatag cattgaaaac ggagtatgaa gaccagttag aatggagggt 9720
caggttggag ttgagttaca gatggggtaa aattctgctt cggatgagtt tggggattgg 9780
caatctaaag gtggtttggg atggcatggc tttgggatgg aaataggttt gtttttatgt 9840
tggctgggaa gggtgtgggg attgaattgg ggatgaagta ggtttagttt tggagataga 9900
atacatggag ctggctattg catgcgagga tgtgcattag tttggtttga tctttaaata 9960
aaggaggcta ttagggttgt cttgaattag attaagttgt gttgggttga tgggttgggc 10020
ttgtgggtga tgtggttgga ttgggctgtg ttaaattggt ttgggtcagg ttttggttga 10080
ggttatcatg gggatgagga tatgcttggg acatggattc aggtggttct cattcaagct 10140
gaggcaaatt tcctttcaga cggtcattcc agggaacgag tggttgtgtg ggggaaatca 10200
ggccactggc tgtgaatatc cctctatcct ggtcttgaat tgtgattatc tatgtccatt 10260
ctgtctcctt cactgtactt ggaattgatc tggtcattca gctggaaatg ggggaagatt 10320
ttgtcaaatt cttgagacac agctgggtct ggatcagcgt aagccttcct tctggtttta 10380
ttgaacagat gaaatcacat tttttttttc aaaatcacag aaatcttata gagttaacag 10440
tggactctta taataagagt taacaccagg actcttattc ttgattcttt tctgagacac 10500
caaaatgaga tttctcaatg ccaccctaat tctttttttt tttttttttt tttttgagac 10560
acagtctggg tcttttgctc tgtcactcag gctggagcgc agtggtgtga tcatagctca 10620
ctgaaccctt gacctcctgg acttaaggga tcctcctgct tcagcctcct gagtagatgg 10680
ggctacaggt gcttgccacc acacctggct aattaaattt tttttttttt tttgtagaga 10740
aagggtctca ctttgttgcc ctggctgatc ttgaacttct gacttcaagt gattcttcag 10800
ccttggactc ccaaagcact gggattgctg gcatgagcca ctcaccgtgc ctggcttgca 10860
gcttaatctt ggagtgtata aacctggctc ctgatagcta gacatttcag tgagaaggag 10920
gcattggatt ttgcatgagg acaattctga cctaggaggg caggtcaaca ggaatccccg 10980
ctgtacctgt acgttgtaca ggcatggaga atgaggagtg aggaggccgt accggaaccc 11040
catattgttt agtggacatt ggattttgaa ataataggga acttggtctg ggagagtcat 11100
atttctggat tggacaatat gtggtatcac aaggttttat gatgagggag aaatgtatgt 11160
ggggaaccat tttctgagtg tggaagtgca agaatcagag agtagctgaa tgccaacgct 11220
tctatttcag gaacatggta agttggaggt ccagctctcg ggctcagacg ggtataggga 11280
ccaggaagtc tcacaatccg atcattctga tatttcaggg catattaggt ttggggtgca 11340
aaggaagtac ttgggactta ggcacatgag actttgtatt gaaaatcaat gattggggct 11400
ggccgtggtg ctcacgcctg taatctcatc actttgggag accgaagtgg gaggatggct 11460
tgatctcaag agttggacac cagcctaggc aacatggcca gaccctctct ctacaaaaaa 11520
attaaaaatt agctggatgt ggtggtgcat gcttgtggtc tcagctatcc tggaggctga 11580
gacaggagaa tcggttgagt ctgggagttc aaggctacag ggagctgcga tcacgccgct 11640
gcactccagc ctgggaaaca gagtgagact gtctcagaat ttttttaaaa aagaatcagt 11700
gatcatccca acccctgttg ctgttcatcc tgagcctgcc ttctctggct ttgttcccta 11760
gatcacatct ccatgatcca taggccctgc ccaatctgac ctcacaccgt gggaatgcct 11820
ccagactgat ctagtatgtg tggaacagca agtgctggct ctccctcccc ttccacagct 11880
ctgggtgtgg gagggggttg tccagcctcc agcagcatgg ggagggcctt ggtcagcatc 11940
taggtgccaa cagggcaagg gcggggtcct ggagaatgaa ggctttatag ggctcctcag 12000
ggaggccccc cagccccaaa ctgcaccacc tggccgtgga caccggt 12047
<210> 4
<211> 454
<212> DNA
<213> Homo Sapiens
CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
<400> 4
aagcttccac aagtgcattt agcctctcca gtattgctga tgaatccaca gttcaggttc 60
aatggcgttc aaaacttgat caaaaatgac cagactttat attcttacac caacatctat 120
ctgattggag gaatggataa tagtcatcat gtttaaacat ctaccattcc agttaagaaa 180
atatgatagc atcttgttct tagtcttttt cttaataggg acataaagcc cacaaataaa 240
aatatgcctg aagaatggga caggcattgg gcattgtcca tgcctagtaa agtactccaa 300
gaacctattt gtatactaga tgacacaatg tcaatgtctg tgtacaactg ccaactggga 360
tgcaagacac tgcccatgcc aatcatcctg aaaagcagct ataaaaagca ggaagctact 420
ctgcaccttg tcagtgaggt ccagatacct acag 454
<210> 5
<211> 5224
<212> DNA
<213> Homo Sapiens
<400> 5
gaattcttag aaatatgggg gtaggggtgg tggtggtaat tctgttttca ccccataggt 60
gagataagca ttgggttaaa tgtgctttca cacacacatc acatttcata agaattaagg 120
aacagactat gggctggagg actttgagga tgtctgtctc ataacacttg ggttgtatct 180
gttctatggg gcttgtttta agcttggcaa cttgcaacag ggttcactga ctttctcccc 240
aagcccaagg tactgtcctc ttttcatatc tgttttgggg cctctggggc ttgaatatct 300
gagaaaatat aaacatttca ataatgttct gtggtgagat gagtatgaga gatgtgtcat 360
tcatttgtat caatgaatga atgaggacaa ttagtgtata aatccttagt acaacaatct 420
gagggtaggg gtggtactat tcaatttcta tttataaaga tacttatttc tatttattta 480
tgcttgtgac aaatgttttg ttcgggacca caggaatcac aaagatgagt ctttgaattt 540
aagaagttaa tggtccagga ataattacat agcttacaaa tgactatgat ataccatcaa 600
acaagaggtt ccatgagaaa ataatctgaa aggtttaata agttgtcaaa ggtgagaggg 660
ctcttctcta gctagagact aatcagaaat acattcaggg ataattattt gaatagacct 720
taagggttgg gtacattttg ttcaagcatt gatggagaag gagagtgaat atttgaaaac 780
attttcaact aaccaaccac ccaatccaac aaacaaaaaa tgaaaagaat ctcagaaaca 840
gtgagataag agaaggaatt ttctcacaac ccacacgtat agctcaactg ctctgaagaa 900
gtatatatct aatatttaac actaacatca tgctaataat gataataatt actgtcattt 960
tttaatgtct ataagtacca ggcatttaga agatattatt ccatttatat atcaaaataa 1020
acttgagggg atagatcatt ttcatgatat atgagaaaaa ttaaaaacag attgaattat 1080
ttgcctgtca tacagctaat aattgaccat aagacaatta gatttaaatt agttttgaat 1140
ctttctaata ccaaagttca gtttactgtt ccatgttgct tctgagtggc ttcacagact 1200
tatgaaaaag taaacggaat cagaattaca tcaatgcaaa agcattgctg tgaactctgt 1260
acttaggact aaactttgag caataacaca catagattga ggattgtttg ctgttagcat 1320
acaaactctg gttcaaagct cctctttatt gcttgtcttg gaaaatttgc tgttcttcat 1380
ggtttctctt ttcactgcta tctatttttc tcaaccactc acatggctac aataactgtc 1440
tgcaagctta tgattcccaa atatctatct ctagcctcaa tcttgttcca gaagataaaa 1500
agtagtattc aaatgcacat caacgtctcc acttggaggg cttaaagacg tttcaacata 1560
caaaccgggg agttttgcct ggaatgtttc ctaaaatgtg tcctgtagca catagggtcc 1620
tcttgttcct taaaatctaa ttacttttag cccagtgctc atcccaccta tggggagatg 1680
agagtgaaaa gggagcctga ttaataatta cactaagtca ataggcatag agccaggact 1740
gtttgggtaa actggtcact ttatcttaaa ctaaatatat ccaaaactga acatgtactt 1800
agttactaag tctttgactt tatctcattc ataccactca gctttatcca ggccacttat 1860
ttgacagtat tattgcgaaa acttcctaac tggtctcctt atcatagtct tatccccttt 1920
tgaaacaaaa gagacagttt caaaatacaa atatgatttt tattagctcc cttttgttgt 1980
ctataatagt cccagaagga gttataaact ccatttaaaa agtctttgag atgtggccct 2040
tgccaacttt gccaggaatt cccaatatct agtattttct actattaaac tttgtgcctc 2100
ttcaaaactg cattttctct cattccctaa gtgtgcattg ttttccctta ccggttggtt 2160
tttccaccac cttttacatt ttcctggaac actataccct ccctcttcat ttggcccacc 2220
tctaattttc tttcagatct ccatgaagat gttacttcct ccaggaagcc ttatctgacc 2280
cctccaaaga tgtcatgagt tcctcttttc attctactaa tcacagcatc catcacacca 2340
tgttgtgatt actgatacta ttgtctgttt ctctgattag gcagtaagct caacaagagc 2400
tacatggtgc ctgtctcttg ttgctgatta ttcccatcca aaaacagtgc ctggaatgca 2460
gacttaacat tttattgaat gaataaataa aaccccatct atcgagtgct actttgtgca 2520
agacccggtt ctgaggcatt tatatttatt gatttattta attctcattt aaccatgaag 2580
gaggtactat cactatcctt attttatagt tgataaagat aaagcccaga gaaatgaatt 2640
aactcaccca aagtcatgta gctaagtgac agggcaaaaa ttcaaaccag ttccccaact 2700
ttacgtgatt aatactgtgc tatactgcct ctctgatcat atggcatgga atgcagacat 2760
CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
ctgctccgta aggcagaata tggaaggaga ttggaggatg acacaaaacc agcataatat 2820
cagaggaaaa gtccaaacag gacctgaact gatagaaaag ttgttactcc tggtgtagtc 2880
gcatcgacat cttgatgaac tggtggctga cacaacatac attggcttga tgtgtacata 2940
ttatttgtag ttgtgtgtgt atttttatat atatatttgt aatattgaaa tagtcataat 3000
ttactaaagg cctaccattt gccaggcatt tttacatttg tcccctctaa tcttttgatg 3060
agatgatcag attggattac ttggccttga agatgatata tctacatcta tatctatatc 3120
tatatctata tctatatcta tatctatatc tatatctata tatgtatatc agaaaagctg 3180
aaatatgttt tgtaaagtta taaagattt~ agactttata gaatctggga tttgccaaat 3240
gtaacccctt tctctacatt aaacccatgt tggaacaaat acatttatta ttcattcatc 3300
aaatgttgct gagtcctggc tatgaaccag acactgtgaa agcctttggg atattttgcc 3360
catgcttggg caagcttata tagtttgctt cataaaactc tatttcagtt cttcataact 3420
aatacttcat gactattgct tttcaggtat tccttcataa caaatacttt ggctttcata 3480
tatttgagta aagtccccct tgaggaagag tagaagaact gcactttgta aatactatcc 3540
tggaatccaa acggatagac aaggatggtg ctacctcttt ctggagagta cgtgagcaag 3600
gcctgttttg ttaacatgtt ccttaggaga caaaacttag gagagacacg catagcagaa 3660
aatggacaaa aactaacaaa tgaatgggaa ttgtacttga ttagcattga agaccttgtt 3720
tatactatga taaatgtttg tatttgctgg aagtgctact gacggtaaac cctttttgtt 3780
taaatgtgtg ccctagtagc ttgcagtatg atctattttt taagtactgt acttagctta 3840
tttaaaaatt ttatgtttaa aattgcatag tgctctttca ttgaagaagt tttgagagag 3900
agatagaatt aaattcactt atcttaccat ctagagaaac ccaatgttaa aactttgttg 3960
tccattattt ctgtctttta ttcaacattt tttttagagg gtgggaggaa tacagaggag 4020
gtacaatgat acacaaatga gagcactctc catgtattgt tttgtcctgt ttttcagtta 4080
acaatatatt atgagcatat ttccatttca ttaaatattc ttccacaaag ttattttgat 4140
ggctgtatat caccctactt tatgaatgta ccatattaat ttatttcctg gtgtgggtta 4200
tttgatttta taatcttacc tttagaataa tgaaacacct gtgaagcttt agaaaatact 4260
ggtgcctggg tctcaactcc acagattctg atttaactgg tctgggttac agactaggca 4320
ttgggaattc aaaaagttcc cccagtgatt ctaatgtgta gccaagatcg ggaacccttg 4380
tagacaggga tgataggagg tgagccactc ttagcatcca tcatttagta ttaacatcat 4440
catcttgagt tgctaagtga atgatgcacc tgacccactt tataaagaca catgtgcaaa 4500
taaaattatt ataggacttg gtttattagg gcttgtgctc taagttttct atgttaagcc 4560
atacatcgca tactaaatac tttaaaatgt accttattga catacatatt aagtgaaaag 4620
tgtttctgag ctaaacaatg acagcataat tatcaagcaa tgataatttg aaatgaattt 4680
attattctgc aacttaggga caagtcatct ctctgaattt tttgtacttt gagagtattt 4740
gttatatttg caagatgaag agtctgaatt ggtcagacaa tgtcttgtgt gcctggcata 4800
tgataggcat ttaatagttt taaagaatta atgtatttag atgaattgca taccaaatct 4860
gctgtctttt ctttatggct tcattaactt aatttgagag aaattaatta ttctgcaact 4920
tagggacaag tcatgtcttt gaatattctg tagtttgagg agaatatttg ttatatttgc 4980
aaaataaaat aagtttgcaa gttttttttt tctgccccaa agagctctgt gtccttgaac 5040
ataaaataca aataaccgct atgctgttaa ttattggcaa atgtcccatt ttcaacctaa 5100
ggaaatacca taaagtaaca gatataccaa caaaaggtta ctagttaaca ggcattgcct 5160
gaaaagagta taaaagaatt tcagcatgat tttccatatt gtgcttccac cactgccaat 5220
5224
aaca
<210> 6
<211> 822
<212> DNA
<213> Homo Sapiens
<400> 6
gcattgctgt gaactctgta cttaggacta aactttgagc aataacacac atagattgag 60
gattgtttgc tgttagcata caaactctgg ttcaaagctc ctctttattg cttgtcttgg 120
aaaatttgct gttcttcatg gtttctcttt tcactgctat ctatttttct caaccactca 180
catggctaca ataactgtct gcaagcttat gattcccaaa tatctatctc tagcctcaat 240
cttgttccag aagataaaaa gtagtattca aatgcacatc aacgtctcca cttggagggc 300
ttaaagacgt ttcaacatac aaaccgggga gttttgcctg gaatgtttcc taaaatgtgt 360
cctgtagcac atagggtcct cttgttcctt aaaatctaat tacttttagc ccagtgctca 420
tcccacctat ggggagatga gagtgaaaag ggagcctgat taataattac actaagtcaa 480
taggcataga gccaggactg tttgggtaaa ctggtcactt tatcttaaac taaatatatc 540
caaaactgaa catgtactta gttactaagt ctttgacttt atctcattca taccactcag 600
ctttatccag gccacttatg agctctgtgt ccttgaacat aaaatacaaa taaccgctat 660
gctgttaatt attggcaaat gtcccatttt caacctaagg aaataccata aagtaacaga 720
CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
tataccaaca aaaggttact agttaacagg cattgcctga aaagagtata aaagaatttc 780
agcatgattt tccatattgt gcttccacca ctgccaataa ca 822
<210> 7
<211> 472
<212> DNA
<213> Homo Sapiens
<400> 7
agccaccacc cagtgagcct ttttctagcc cccagagcca cctctgtcac cttcctgttg 60
ggcatcatcc caccttccca gagccctgga gagcatgggg agacccggga ccctgctggg 120
tttctctgtc acaaaggaaa ataatccccc tggtgtgaca gacccaagga cagaacacag 180
cagaggtcag cactggggaa gacaggttgt cctcccaggg gatgggggtc catccacctt 240
gccgaaaaga tttgtctgag gaactgaaaa tagaagggaa aaaagaggag ggacaaaaga 300
ggcagaaatg agaggggagg ggacagagga cacctgaata aagaccacac ccatgaccca 360
cgtgatgctg agaagtactc ctgccctagg aagagactca gggcagaggg aggaaggaca 420
gcagaccaga cagtcacagc agccttgaca aaacgttcct ggaactcaag ca 472
<210> 8
<211> 858
<212> DNA
<213> Homo Sapiens
<400> 8
cgagcggccc ctcagcttcg gcgcccagcc ccgcaaggct cccggtgacc actagagggc 60
gggaggagct cctggccagt ggtggagagt ggcaaggaag gaccctaggg ttcatcggag 120
cccaggttta ctcccttaag tggaaatttc ttcccccact cctccttggc tttctccaag 180
gaggga~ccc aggctgctgg aaagtccggc tggggcgggg actgtgggtt caggggagaa 240
cggggtgtgg aacgggacag ggagcggtta gaagggtggg gctattccgg gaagtggtgg 300
ggggagggag cccaaaacta gcacctagtc cactcattat ccagccctct tatttctcgg 360
ccgctctgct tcagtggacc cggggagggc ggggaagtgg agtgggagac ctaggggtgg 420
gcttcccgac cttgctgtac aggacctcga cctagctggc tttgttcccc atccccacgt 480
tagttgttgc cctgaggcta aaactagagc ccaggggccc caagttccag actgcccctc 540
ccccctcccc cggagccagg gagtggttgg tgaaaggggg aggccagctg gagaacaaac 600
gggtagtcag ggggttgagc gattagagcc cttgtaccct acccaggaat ggttggggag 660
gaggaggaag aggtaggagg taggggaggg ggcggggttt tgtcacctgt cacctgctcg 720
ctgtgcctag ggcgggcggg cggggagtgg ggggaccggt ataaagcggt aggcgcctgt 780
gcccgctcca cctctcaagc agccagcgcc tgcctgaatc tgttctgccc cctccccacc 840
catttcacca ccaccatg 858
<210> 9
<211> 454
<212> DNA
<213> Homo Sapiens
<400> 9
aagcttccac aagtgcattt agcctctcca gtattgctga tgaatccaca gttcaggttc 60
aatggcgttc aaaacttgat caaaaatgac cagactttat attcttacac caacatctat 120
ctgattggag gaatggataa tagtcatcat gtttaaacat ctaccattcc agttaagaaa 180
atatgatagc atcttgttct tagtcttttt cttaataggg acataaagcc cacaaataaa 240
aatatgcctg aagaatggga caggcattgg gcattgtcca tgcctagtaa agtactccaa 300
gaacctattt gtatactaga tgacacaatg tcaatgtctg tgtacaactg ccaactggga 360
tgcaagacac tgcccatgcc aatcatcctg aaaagcagct ataaaaagca ggaagctact 420
ctgcaccttg tcagtgaggt ccagatacct acag 454
adenovirus death protein
<210> 10
<211> 307
<212> DNA
<213> adenovirus
<220>
<221> CDS
CA 02516652 2005-08-19
WO 2005/007832 PCT/US2004/005518
<222> (2)..(304)
<400> 10
g atg acc ggc tca acc atc gcg ccc aca acg gac tat cgc aac acc act 49
Met Thr Gly Ser Thr Ile Ala Pro Thr Thr Asp Tyr Arg Asn Thr Thr
1 5 10 15
get acc gga cta aca tct gcc cta aat tta ccc caa gtt cat gcc ttt 97
Ala Thr Gly Leu Thr Ser Ala Leu Asn Leu Pro G1n Val His Ala Phe
20 25 30
gtc aat gac tgg gcg agc ttg gac atg tgg tgg ttt tcc ata gcg ctt 145
Val Asn Asp Trp Ala Ser Leu Asp Met Trp Trp Phe Ser Ile Ala Leu
35 40 45
atg ttt gtt tgc ctt att att atg tgg ctt att tgt tgc cta aag cgc 193
Met Phe Val Cys Leu Ile Ile Met Trp Leu Ile Cys Cys Leu Lys Arg
50 55 60
aga cgc gcc aga ccc ccc atc tat agg cct atc att gtg ctc aac cca 241
Arg Arg Ala Arg Pro Pro Ile Tyr Arg Pro Ile Ile Val Leu Asn Pro
65 70 75 80
cac aat gaa aaa att cat aga ttg gac ggt ctg aaa cca tgt tct ctt 289
His Asn Glu Lys Ile His Arg Leu Asp Gly Leu Lys Pro Cys Ser Leu
85 90 95
ctt tta cag tat gat taa 307
Leu Leu Gln Tyr Asp
100
<210> 11
<211> 101
<212> PRT
<213> adenovirus
<400> 11
Met Thr Gly Ser Thr Ile Ala Pro Thr Thr Asp Tyr Arg Asn Thr Thr
1 5 10 15
Ala Thr Gly Leu Thr Ser Ala Leu Asn Leu Pro Gln Val His Ala Phe
20 25 30
Val Asn Asp Trp Ala Ser Leu Asp Met Trp Trp Phe Ser Ile Ala Leu
35 40 45
Met Phe Va1 Cys Leu 21e Ile Met Trp Leu Ile Cys Cys Leu Lys Arg
50 55 60
Arg Arg Ala Arg Pro Pro 21e Tyr Arg Pro Ile Ile Val Leu Asn Pro
65 70 75 80
His Asn G1u Lys Ile His Arg Leu Asp Gly Leu Lys Pro Cys Ser Leu
85 90 95
Leu Leu Gln Tyr Asp
100
11
