Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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ADENOVIRUS VECTORS CONTAINING HETEROLOGOUS TRANSCRIPTION ~
REGULATORY ELEMENTS AND METRODS OF USING SAME
TECHNICAL FIELD
This invention relates to cell transfection using adenovirus vectors,
providing
replication-competent adenovirus vectors and methods of their use. More
specifically, it
relates to cell-specific replication of adenovirus vectors in cells through
the use of eell-
specific, non-adenoviral transcriptional regulatory elements.
BACKGROUND ART
In spite of numerous advances in medical research, cancer remains the second
leading cause of death in the United States. In the industrialized nations,
roughly one in
five persons will die of cancer. Traditional modes of clinical care, such as
surgical
resection, radiotherapy and chemotherapy, have a significant failure rate,
especially for
solid tumors. Neoplasia resulting in benign tumors can usually be completely
cured by
removing the mass surgically. If a tumor becomes malignant, as manifested by
invasion of
surrounding tissue, it becomes much more difficult to eradicate. Once a
malignant tunior
metastasizes, it is much less likely to be eradicated.
Excluding basal cell carcinoma, there are over one nullion new cases of cancer
per
year in the United States alone, and cancer accounts for ovei- one half
million deatlis per
year in t'llis country. In the world as a whole, the five most common cancers
are those of
tunw, stomach_ bt-east, colon/rectum, and uterine cen,ix, and the total number
of new cases
per year is over 6 rnillion.
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Lung cancer is one of the most refractory of solid tumors because inoperable
cases
are up to 60% and the 5-year survival is only 13%. In particular,
adenocarcinomas, which
comprise about one-half of the total lung cancer cases, are mostly chemo-
radioresistant.
Gastric (i.e., stomach) carcinoma is one of the most prevalent forms of
cancers in East
Asia, including Japan and Korea. Although extensive surgical operations have
been
combined with chemotherapy and immunotherapy, the mortality of gastric cancer
is still
high, due to carcinomatous peritonitis and liver metastasis at advanced
stages. Colorectal
cancer is the third most common cancer and the second leading cause of cancer
deaths.
Pancreatic cancer is virtually always fatal. Thus, current treatment prospects
for many
patients with these carcinomas are unsatisfactory, and the prognosis is poor.
Hepatocellular carcinoma (HCC or malignant hepatoma) is one of the most
common cancers in the world, and is especially problematic in Asia. Treatment
prospects
for patients with hepatocellular carcinoma are dim. Even with improvements in
therapy
and availability of liver transplant, only a minority of patients are cured by
removal of the
tumor either by resection or transplantation. For the majority of patients,
the current
treatments remain unsatisfactory, and the prognosis is poor.
Breast cancer is one of the most common cancers in the United States, with an
annual incidence of about 182,000 new cases and nearly 50,000 deaths. In the
industrial
nations, approximately one in eight women can expect to develop breast cancer.
The
mortality rate for breast cancer has remained unchanged since 1930. It has
increased an
average of 0.2% per year, but decreased in women under 65 years of age by an
average of
0.3% per year. See e.g., Marchant (1994) Contemporary Management of Breast
Disease II: Breast Cancer, in: Obstetrics and Gynecology Clinics ofNorth
America
21:555-560; and Colditz (1993) Cancer Suppl. 71:1480-1489.
Despite ongoing improvement in the understanding of the disease, breast cancer
has
remained resistant to medical intervention. Most clinical initiatives are
focused on early
diagnosis, followed by conventional forms of intervention, particularly
surgery and
chemotherapy. Such interventions are of limited success, particularly in
patients where the
tumor has undergone metastasis. There is a pressing need to improve the
arsenal of therapies
available to provide more precise and more effective treatment in a less
invasive way.
Prostate cancer is the fastest growing neoplasm in men with an estimated
244,000
new cases in the United States being diagnosed in 1995, of which approximately
44,000
deaths will result. Prostate cancer is now the most frequently diagnosed
cancer in men.
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Prostate cancer is latent; many men carry prostate cancer cells without overt
signs of
disease. It is associated with a high morbidity. Cancer metastasis to bone
(late stage) is
common and is almost always fatal.
Current treatments include radical prostatectomy, radiation therapy, hormonal
ablation and chemotherapy. Unfortunately, in approximately 80% of cases,
diagnosis of
prostate cancer is established when the disease has already metastasized to
the bones, thus
limiting the effectiveness of surgical treatments. Hormonal therapy frequently
fails with
time with the development of hormone-resistant tumor cells. Although
chemotherapeutic
agents have been used in the treatment of prostate cancer, no single agent has
demonstrated superiority over its counterparts, and no drug combination seems
particularly
effective. The generally drug-resistant, slow-growing nature of most prostate
cancers
makes them particularly unresponsive to standard chemotherapy.
A major, indeed the overwhelming, obstacle to cancer therapy is the problem of
selectivity; that is, the ability to inhibit the multiplication of tumor
cells, while leaving
unaffected the function of normal cells. For example, in prostate cancer
therapy, the
therapeutic ratio, or ratio of tumor cell killing to normal cell killing of
traditional tumor
chemotherapy, is only 1.5:1. Thus, more effective treatment methods and
pharmaceutical
compositions for therapy and prophylaxis of neoplasia are needed.
Of particular interest is development of more specific, targeted forms of
cancer
therapy, especially for cancers that are difficult to treat successfully. In
contrast to
conventional cancer therapies, which result in relatively non-specific and
often serious
toxicity, more specific treatment modalities attempt to inhibit or kill
malignant cells
selectively while leaving healthy cells intact.
One possible treatment approach for many of these cancers is gene therapy,
whereby a gene of interest is introduced into the malignant cell. See, for
gene therapy for
prostate cancer, Boulikas (1997) Anticancer Res. 17:1471-1505. The gene of
interest may
encode a protein which converts into a toxic substance upon treatment with
another
compound, or an enzyme that converts a prodrug to a drug. For example,
introduction of
the herpes simplex gene encoding thymidine kinase (HSV-tk) renders cells
conditionally
sensitive to ganciclovir. Zjilstra et al. (1989) Nature 342: 435; Mansour et
al. (1988)
Nature 336: 348; Johnson et al. (1989) Science 245: 1234; Adair et al. (1989)
Proc. Natl.
Acad. Sci. USA 86: 4574; Capecchi (1989) Science 244: 1288. Alternatively, the
gene of
interest may encode a compound that is directly toxic, such as diphtheria
toxin. For these
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treatments to be rendered specific to cancer cells, the gene of interest can
be under control
of a transcriptional regulatory element (TRE) that is specifically (i.e.,
preferentially)
activated in the cancer cells. Cell or tissue specific expression can be
achieved by using a
TRE with cell-specific enhancers and/or promoters. See generally Huber et al.
(1995) Adv.
Drug Delivery Reviews 17:279-292.
A variety of viral and non-viral (e.g., liposomes) vehicles, or vectors, have
been
developed to transfer these genes. Of the viruses proposed for gene transfer,
adenoviruses
are among the most easily produced and purified. Adenovirus also has the
advantage of
effecting high efficiency of transduction and does not require cell
proliferation for efficient
transduction of cell. In addition, adenovirus can infect a wide variety of
cells in vitro and
in vivo. For general background references regarding adenovirus and
development of
adenoviral vector systems, see Graham et al. (1973) Virology 52:456-467;
Takiff et al.
(1981) Lancet 11:832-834; Berkner et al. (1983) Nucleic Acid Research 11: 6003-
6020;
Graham (1984) EMBO J 3:2917-2922; Bett et al. (1993) J. Virology 67:5911-5921;
and
Bett et al. (1994) Proc. Natl. Acad. Sci. USA 91:8802-8806.
When used as gene transfer vehicles, adenovirus vectors are often designed to
be
replication-defective and are thus deliberately engineered to fail to
replicate in the target
cells of interest. In these vehicles, the early adenovirus gene products E 1 A
and/or E 1 B are
deleted and provided in trans by the packaging cell line 293. Graham et al.
(1987) J. Gen.
Viro136:59-72; Graham (1977) J. Genetic Virology 68:937-940. The gene to be
transduced is commonly inserted into adenovirus in the E 1 A and E 1 B region
of the virus
genome. Bett et al. (1994). Replication-defective adenovirus vectors as
vehicles for
efficient transduction of genes have been described by, inter alia, Stratford-
Perricaudet
(1990) Human Gene Therapy 1:241-256; Rosenfeld (1991) Science 252:431-434;
Wang et
al. (1991) Adv. Exp. Med. Biol. 309:61-66; Jaffe et al. (1992) Nat. Gen. 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
Le 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; Bett et al. (1994).
The virtually exclusive focus in development of adenoviral vectors for gene
therapy is use of adenovirus merely as a vehicle for introducing the gene of
interest, not as
an effector in itself. Replication of adenovirus has been viewed as an
undesirable result,
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largely due to the host immune response. In the treatment of cancer by
replication-defective adenoviruses, the host immune response limits the
duration of repeat
doses at two levels. First, the capsid proteins of the adenovirus delivery
vehicle itself are
immunogenic. Second, viral late genes are frequently expressed in transduced
cells,
eliciting cellular immunity. Thus, the ability to repeatedly administer
cytokines, tumor
suppressor genes, ribozymes, suicide genes, or genes which convert prodrug to
an active
drug has been limited by the immunogenicity of both the gene transfer vehicle
and the
viral gene products of the transfer vehicle as well as the transient nature of
gene
expression.
Adenoviruses generally undergo an effective lytic replication cycle following
infection of a host cell. In addition to lysing the infected cell, the
replicative process of
adenovirus blocks the transport and translation host cell mRNA thus inhibiting
protein
synthesis of the infected cell. For a review of adenoviruses and adenovirus
replication, see
Shenk, T. and Horwitz, M.S., Virology, third edition, Fields, B.N. et al.,
eds., Raven Press
Limited, New York (1996), Chapters 67 and 68, respectively.
Taking advantage of the cytotoxic effects associated with adenovirus
replication,
replication-competent adenovirus vectors have recently been described as
agents for
effecting selective cell growth inhibition. See Henderson et al., U.S. Patent
No. 5,698,443;
Hallenbeck et al., WO 96/17053. In such systems, a cell-specific
transcriptional regulatory
element (TRE) controls the expression of a gene essential for viral
replication, and thus,
viral replication is limited to a cell population in which the TRE is
functional. For
example, an attenuated, replication-competent adenovirus (CN706) has been
generated by
inserting the prostate-specific antigen (PSA) promoter and enhancer (PSE-TRE)
upstream
of the E1A transcription unit in adenovirus serotype 5 (Ad5). CN706
demonstrates
selective cytotoxicity toward PSA expressing cells in vitro and in vivo.
Rodriguez et al.
(1997)- Cancer Res. 57:2559-2563.
In sum, there is a need for vector constructs that are capable of eliminating
essentially all cancerous cells in a minimum number of administrations before
specific
immunological response against the vector prevents further treatment.
Particularly, there
is a continuing serious need for improved replication-competent adenovirus
vectors in
which cell-specific replication can be further elevated, while minimizing the
extent of
replication in non-target (i.e., non-cancerous cells). The present invention
provides
selectively replicating adenovirus vectors that can be employed in these
contexts.
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SUMMARY OF THE INVENTION
The present invention provides an adenovirus vector comprising a first
adenovirus
gene under transcriptional control of a first heterologous transcriptional
regulatory element
(TRE) and at least a second gene under transcriptional control of a second
heterologous
TRE, wherein the first heterologous TREs is cell-specific, the first
heterologous TRE is
different from the second heterologous TRE and the heterologous TREs are
functional in
the same cell.
In one aspect, the invention provides an adenovirus vector in which the cell
specific heterologous TRE controls the transcription of a gene essential for
adenovirus
replication.
In another aspect, the invention provides an adenovirus vector in which the
second
heterologous TRE controls the transcription of a transgene.
In some embodiments, the invention provides adenovirus vector(s) complexed
with
a hydrophilic polymer ("masking agent") to create a masked adenovirus. The
hydrophilic
polymer is attached (covalently or non-covalently) to the capsid proteins of
the adenovirus,
particularly the hexon and fiber proteins. In preferred embodiments, the
adenovirus
vectors of the instant invention are complexed with masking agents to create
masked
adenovirus vectors. In further preferred embodiments, the masking agent is
polyethyleneglycol (PEG) covalently linked to an adenovirus vector of the
instant
invention.
The invention further provides host cells containing the adenovirus vectors of
the
invention.
Further provided are methods of using the adenoviral vectors of the invention.
In
one aspect, methods are provided for using the adenovirus vectors described
herein which
entail_introducing these vector(s) into a cell.
In another aspect, methods are provided for conferring selective cytoxicity on
a cell
which allows the heterologous TREs to function that entail contacting the
cells with an
adenovirus vector described herein, wherein the adenovirus vector enters the
cell.
In another aspect, methods are provided for suppressing tumor growth,
comprising
contacting a target cell with an adenovirus vector described herein such that
the adenovirus
vector enters the cell.
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In another aspect, methods are provided for modifying the genotype of a target
cell,
comprising contacting the cell with an adenovirus vector described herein,
wherein the
adenovirus vector enters the cell.
In yet another aspect, methods are provided for propagating the adenovirus
vectors
of the invention, comprising combining the adenovirus vectors with cells which
allow the
heterologous TREs to function, such that the adenovirus vector enters the cell
and is
propagated.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 depicts schematic diagrams of examples of adenovirus vectors in which
various genes are under transcriptional control of various heterologous TREs.
The
heterologous TREs contained in an adenovirus vector are different from each
other and
from the endogenous adenovirus TREs. All of the heterologous TREs in an
adenovirus
vector are functional in the same cell and at least one of the TREs is cell
specific.
Figure 2 (A)-(C) are schematic diagrams of examples of adenovirus vectors in
which the ElA and E1B genes are under transcriptional control of prostate cell
specific
heterologous TREs. CN702 is an example of a wild-type Adenovirus type 5 and
CN706
contains a single gene (E1A) under control of a single prostate cell specific
TRE, one
derived from the PSA gene.
Figure 3 shows the cytopathic effects of CN702 and CN764 at various
multiplicities of infection on human microvascular endothelial cells.
Figure 4 is a bar graph showing the number of plaque-forming units, expressed
as
percentage of plaques obtained with wild-type adenovirus, obtained when either
LNCaP
cells (hatched bars) or HMVEC cells (bars with square pattern) were infected
with
adenoviral vectors CN739, CN764, CN765 or CN770.
- Figure 5 is a line graph illustrating the one-step growth curve of an
adenovirus,
CN739, in which multiple adenoviral early genes are placed under control of
prostate cell
specific heterologous TREs, in prostate (LNCaP) and non-prostate cells
(HMVEC).
Figure 6 is a line graph illustrating the efficacy in treating a prostate
cancer tumor
in mice of an adenovirus, CN739, in which multiple adenoviral early genes are
placed
under control of prostate cell specific heterologous TREs.
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Figure 7 is a line graph showing serum PSA levels in mice treated with an
adenovirus, CN739, in which multiple adenoviral early genes are placed under
control of
prostate cell specific heterologous TREs.
Figure 8 (A)-(B) are schematic diagrams of examples of adenovirus vectors in
which the E 1 A and E 1 B genes are under transcriptional control of cell
specific
heterologous TREs. Each adenovirus vector contains at least two different
heterologous
TREs, both of which are functional in the same cell.
Figure 9 depicts schematic diagrams of examples of adenovirus vectors in which
the E 1 A, E 1 B, and E4 genes are under transcriptional control of cell
specific heterologous
TREs. Each adenovirus vector contains three different heterologous TREs, all
of which
are functional in the same cell.
Figure 10 is a graph depicting cytotoxicity of an adenoviral vector containing
the
coding sequence for adenoviral death protein (ADP), CN751 (solid squares),
compared to
control CN702 (solid circles), Rec 700 (solid triangles) and mock infection
(Xs).
Figure 11 is a graph comparing extracellular virus yield of CN751 (solid
squares)
and CN702 (solid circles).
Figure 12 is a graph comparing tumor volume in mice harboring LNCaP tumor
xenografts challenged with CN751 ("H"), CN702 ("J"), or buffer ("B").
Figure 13 is a schematic depiction of a method for covalent pegylation of an
adenovirus. In this method, succinimidyl succinamide is used to covalently
attach
methoxy-PEG to adenovirus. The pegylated adenovirus is separated from the
reaction
components by ion exchange chromatography.
Figure 14 shows a half-tone reproduction of sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) gel (mobility shift) of
pegylated
adenovirus proteins. Lanes 1 and 2 are non-pegylated CN706 (control), lanes 3
through 6
are CN706 pegylated under various pH and temperature conditions (lane 3, pH
7.6, room
temperature (RT); lane 4, pH 7.6, 4 C; lane 5, pH 8.2, RT; lane 6, pH 8.2, 4
C).
Figure 15 is a chromatogram of ion exchange chromatography analysis of
pegylated adenovirus (PEG-706) mixed with control adenovirus CN706.
MODES FOR CARRYING OUT THE INVENTION
We have discovered and constructed replication-competent adenovirus vectors
containing heterologous cell-specific transcriptional regulatory elements
(TREs) which can
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preferentially replicate in cells that allow function of said TREs and have
developed
methods of using these adenovirus vectors. The adenovirus vectors of this
invention
comprise a first adenovirus gene under the transcriptional control of a cell-
specific
heterologous TRE and at least one other gene, such as an adenoviral gene or a
transgene,
under control of another heterologous TRE which is different from the first
TRE, where
the heterologous TREs are functional in the same cell but are not the same in
polynucleotide sequence (i.e., have different polynucleotide sequences).
Preferably, at
least two of the heterologous TREs in an adenovirus vector are cell specific
for the same
cell. Preferably, the adenovirus gene is one that enhances cell death, more
preferably one
that is essential for adenovirus replication. Preferably, at least one of the
adenovirus genes
necessary for cell replication is an early gene. Preferably, the genes under
transcriptional
control of the heterologous TREs are necessary for replication. By providing
for cell-
specific transcription through the use of multiple heterologous TREs, the
invention
provides adenovirus vectors that can be used for cell-specific cytotoxic
effects due to
selective replication.
The adenovirus vectors of the invention replicate preferentially in TRE
functional
cells (i.e., at a higher yield than in TRE non-functional cells). This
replication preference
is indicated by comparing the level of replication (i.e., titer) in cells in
which the TRE is
active to the level of replication in cells in which the TRE is not active.
The replication
preference is even more significant, as the adenovirus vectors of the
invention actually
replicate at a significantly lower rate in TRE non-functional cells than wild
type virus.
Comparison of the adenovirus titer of a TRE active cell type to the titer of a
TRE inactive
cell type provides a key indication that the overall replication preference is
enhanced due
to the replication in TRE active cells as well as depressed replication in TRE
inactive cells.
This is especially useful in the cancer context, in which targeted cell
killing is desirable.
The adenovirus vectors of this invention, with the inclusion of at least two
different
heterologous TREs, are more stable and provide even more cell specificity with
regard to
replication than previously described adenovirus vectors. Adenovirus vectors
have been
constructed in which each of the E 1 A and E 1 B genes have been placed under
transcriptional control of two different heterologous TREs, for example, TREs
from the
PSA gene (PSE-TRE) and the probasin gene (PB-TRE) and TREs from the PSA gene
and
the hKLK2 gene (hKLK2-TRE).
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We have found, for example, that adenoviruses containing the PSE-TRE and the
PB-TRE and adenovirus containing the PSE-TRE and the hKLK2-TRE appear to
possess a
stable genome, exhibit higher levels of cell specificity with regard to
replication than
CN706, an adenovirus with the PSE-TRE controlling the E 1 A gene, and
replicate as
efficiently as CN706 in prostate cells. As shown herein, in vitro and in vivo
results
indicate the CN739 (adenovirus with a PB-TRE controlling the E1A gene and a
PSE-TRE
controlling the E 1 B gene, described below) and CN764 (adenovirus with a PSE-
TRE
controlling the E 1 A gene and an hKLK2-TRE controlling the E 1 B gene,
described below)
have a 10 to 100-fold higher cell specificity in replication than CN706, while
retaining
similar prostate tumor killing capacity. Thus, this invention provides an even
more
attenuated replication competent virus by controlling replication genes with
two different
heterologous, cell-specific TREs.
Previous attempts to achieve this level of specificity through the
construction of
adenovirus vectors with the same heterologous TRE controlling transcription of
two
adenoviral vector genes appear to have resulted in unstable genomes and
undesirable
polynucleotide sequence rearrangements. Examination of such adenoviruses, with
techniques such as restriction enzyme digestion and Southern blot analysis,
revealed
alterations in the size of adenoviral polynucleotide fragments after viral
replication.
Without wishing to be bound by theory, such genome instability may be the
result of
homologous recombination through the duplicated TRE sequences.
The adenovirus vectors provided by the present invention, in which two
different
heterologous TREs are used to control replication, achieve a stability of the
viral genome
and an even higher level of target cell specificity than previously described
adenoviruses.
The invention uses and takes advantage of what has been considered an
undesirable
aspect of adenoviral vectors, namely, their replication and possible
concomitant
immunogenicity. Runaway infection is prevented due to the cell-specific
requirements for
viral replication. Without wishing to be bound by any particular theory, the
inventors note
that production of adenovirus proteins can serve to activate and/or stimulate
the immune
system, either generally or specifically toward target cells producing
adenoviral proteins
which can be an important consideration in the cancer context, where patients
are often
moderately to severely immunocompromised.
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General Techniques
The practice of the present invention will employ, unless otherwise indicated,
conventional techniques of molecular biology (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., 1989); "Oligonucleotide
Synthesis" (M.J. Gait, ed., 1984); "Animal Cell Culture" (R.I. Freshney, ed.,
1987);
"Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental
Immunology" (D.M. Weir & C.C. Blackwell, eds.); "Gene Transfer Vectors for
Mammalian Cells" (J.M. Miller & M.P. Calos, eds., 1987); "Current Protocols in
Molecular Biology" (F.M. Ausubel et al., eds., 1987); "PCR: The Polymerase
Chain
Reaction", (Mullis et al., eds., 1994); and "Current Protocols in Immunology"
(J.E.
Coligan et al., eds., 1991).
For techniques related to adenovirus, see, inter alia, Feigner and Ringold
(1989)
Nature 337:387-388; Berkner and Sharp (1983) Nucl. Acids Res. 11:6003-6020;
Graham
(1984) EMBO J. 3:2917-2922; Bett et al. (1993) J. Virology 67:5911-5921; Bett
et al.
(1994) Proc. Natl. Acad. Sci. USA 91:8802-8806.
Definitions
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 indicated otherwise.
The term "polynucleotide" as used herein refers to a polymeric form of
nucleotides
of any length, either ribonucleotides or deoxynucleotides. Thus, this term
includes single-,
double- and triple-stranded DNA, as well as single- and double-stranded RNA,
RNA-DNA
hybrids, or a polymer comprising purine and pyrimidine bases, or other
natural,
chemically, biochemically modified, non-natural or derivatized nucleotide
bases. The
backbone of the polynucleotide can comprise sugars and phosphate groups (as
may
typically be found in RNA or DNA), or modified or substituted sugar or
phosphate groups.
Alternatively, the backbone of the polynucleotide can comprise a polymer of
synthetic
subunits such as phosphoramidates and thus can be a oligodeoxynucleoside
phosphoramidate (P-NH2) or a mixed phosphoramidate- phosphodiester oligomer.
Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-8; Chaturvedi et al.
(1996) Nucleic
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Acids Res. 24: 2318-23; Schultz et al. (1996) Nucleic Acids Res. 24: 2966-73.
A
phosphorothioate linkage can be used in place of a phosphodiester linkage.
Braun et al.
(1988) J. Immunol. 141: 2084-9; Latimer et al. (1995) Mol. Immunol. 32: 1057-
1064.
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. In addition, a double-stranded polynucleotide can be obtained from
the
single-stranded polynucleotide product of chemical synthesis either by
synthesizing the
complementary strand and annealing the strands under appropriate conditions,
or by
synthesizing the complementary strand de novo using a DNA polymerase with an
appropriate primer.
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.
As used herein, a "transcriptional regulatory element", or "TRE" is a
polynucleotide sequence, preferably a DNA sequence, that regulates (i.e.,
controls)
transcription of an operably-linked polynucleotide sequence by an RNA
polymerase to
form RNA. As used herein, a TRE increases transcription of an operably linked
polynucleotide sequence in a host cell that allows the TRE to function. The
TRE
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 TRE may
be in
any orientation and/or distance from the coding sequence of interest, and
comprise
multimers of the foregoing, as long as the desired transcriptional activity is
obtained. As
discussed herein, a TRE may or may not lack a silencer element.
An "enhancer" is a term well understood in the art and is a polynucleotide
sequence derived from a gene which increases transcription of a gene which is
operably-
linked to a promoter to an extent which is greater than the transcription
activation effected
by the promoter itself 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
what has been stated, i.e., it increases transcription of a gene which is
operably linked to a
promoter to an extent which is greater than the increase in transcription
effected by the
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promoter itself when operably linked to the gene, i.e., it increases
transcription from the
promoter.
A polynucleotide or polynucleotide region has a certain percentage (for
example,
80%, 85%, 90%, or 95%) of "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 art, for example those described in Current
Protocols in
Molecular Biology (F.M. Ausubel et al., eds., 1987) Supplement 30, section
7.7.18, Table
7.7.1. A preferred alignment program is ALIGN Plus (Scientific and Educational
Software, Pennsylvania).
In the context of adenovirus vector(s), a first heterologous TRE is
"different" from
a second (or another) heterologous TRE when the polynucleotide sequence
identity
between the two heterologous TREs is less than about 95%, preferably less than
about
90%, preferably less than about 85%, preferably less than about 75%.
Generally,
"different TREs" are derived from the transcriptional regulatory regions of
different genes.
"Different TREs" may also be derived from the transcriptional regulatory
region of the
same gene, as long as the sequence identity between them is less than the
values listed
above (i.e., less than about 95%, preferably less than about 90%, preferably
less than about
85%, preferably less than about 75%). Although two different TREs are not
identical in
polynucleotide sequence, they may be functional in the same cell and may also
have the
same cell-specificity.
As used herein, a TRE 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 said
gene. For
example, as used herein, a "human glandular kallikrein transcriptional
regulatory element",
or "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-TRE to function, such as a cell (preferably a mammalian cell,
even more
preferably a human cell) that expresses androgen receptor. An hKLK2-TRE 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).
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As used herein, a "probasin (PB) transcriptional regulatory element", 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 a PB-
TRE to function, such as a cell (preferably a mammalian cell, even more
preferably a
human cell) that expresses androgen receptor. A PB-TRE 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).
As used herein, a "prostate-specific antigen (PSA) transcriptional regulatory
element", or "PSA-TRE", or "PSE-TRE" is polynucleotide sequence, preferably a
DNA
sequence, which selectively increases transcription of an operably linked
polynucleotide
sequence in a host cell that allows a PSA-TRE to function, such as a cell
(preferably a
mammalian cell, even more preferably a human cell) that expresses androgen
receptor. A
PSE-TRE 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).
As used herein, a "carcinoembryonic antigen (CEA) transcriptional regulatory
element", or "CEA-TRE" is polynucleotide sequence, preferably a DNA sequence,
which
selectively increases transcription of an operably linked polynucleotide
sequence in a host
cell that allows a CEA-TRE to function, such as a cell (preferably a mammalian
cell, even
more preferably a human cell) that expresses CEA. The CEA-TRE 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.
As used herein, an "a-fetoprotein (AFP) transcriptional regulatory element",
or
"AFP-TRE" is polynucleotide sequence, preferably a DNA sequence, which
selectively
increases transcription (of an operably linked polynucleotide sequence) in a
host cell that
allows an AFP-TRE to function, such as a cell (preferably a mammalian cell,
even more
preferably a human cell) that expresses AFP. The AFP-TRE 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.
As used herein, an "a mucin gene (MUC) transcriptional regulatory element", or
"MUCI -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 MUCI-TRE to function, such as a cell (preferably a
mammalian
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cell, even more preferably a human cell) that expresses MUC 1. The MUCI -TRE
is
responsive to transcription factors and/or co-factor(s) associated with MIJC i-
producing
cells and comprises at least a portion of the MUCl promoter and/or enhancer.
A"cell-specific TRE" is preferentially functional in a specific type of cell
relative
to other types of cells of different functionality. A cell-specific TRE may or
may not be
tumor cell specific.
As used herein, the term "target cell-specific" is intended to mean that the
TRE
sequences to which a gene essential for replication of an adenoviral vector is
operably
linked, or to which a transgene is operably linked, functions specifically in
that target cell
so that replication proceeds in that target cell, or so that a transgene
polynucleotide is
expressed in that target cell. This can occur by virtue of the presence in
that target cell,
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 "target cell-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.
The activity of a TRE generally depends upon the presence of transcriptional
regulatory factors and/or the absence of transcriptional regulatory
inhibitors.
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. As discussed herein, a TRE can be of varying lengths, and
of varying
sequence composition. By transcriptional activation, it is 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.
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 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 TRE of interest as tested in a target cell type.
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A "functionally-preserved" variant of a TRE is a TRE which differs from
another
TRE, but still retains ability to increase transcription of an operably linked
polynucleotide,
especially cell-specific transcription activity. The difference in a TRE can
be due to
differences in linear sequence, arising from, for example, single or multiple
base
mutation(s), addition(s), deletion(s), and/or modification(s) of the bases.
The difference
can also arise from changes in the sugar(s), and/or linkage(s) 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 the 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 TRE 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
TRE functional and TRE non-functional cells. Additionally, one of skill in the
art would
recognize that point mutations and deletions can be made to a TRE sequence
without
altering the ability of the sequence to regulate transcription.
"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 which 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 TRE is operably linked to a coding segment if the
TRE
promotes transcription of the coding sequence. Operably 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.
As used herein, "a cell which allows a TRE to function" or a cell in which the
function of a TRE is "sufficiently preserved", or "a cell in which a TRE is
functional" is a
cell in which the TRE, when operably linked to a promoter (if not included in
the TRE)
and a reporter gene, increases expression of the reporter gene at least about
2-fold,
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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 reporter gene when not operably linked to
said TRE.
Methods for measuring levels (whether relative or absolute) of expression are
known in the
art and are described herein.
A "target cell" is any cell that allows a heterologous TRE to function.
Preferably, a
target cell is a mammalian cell, more preferably a human cell.
As used herein, "neoplastic cells", "neoplasia", "tumor", "tumor cells",
"cancer",
and "cancer cells" (used interchangably) 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 benign or
malignant.
"Androgen receptor", or AR as used herein refers to a protein whose function
is to
specifically bind to androgen and, as a consequence of the specific binding,
recognize and
bind to an androgen response element (ARE), following which the AR is capable
of
regulating transcriptional activity. The AR is a nuclear receptor that, when
activated,
binds to cellular androgen-responsive element(s). In normal cells the AR is
activated by
androgen, but in non-normal cells (including malignant cells) the AR may be
activated by
non-androgenic agents, including hormones other than androgens. Encompassed in
the
term "androgen receptor" are mutant forms of an androgen receptor, as long as
the
function is sufficiently preserved. Mutants include androgen receptors with
amino acid
additions, insertions, truncations and deletions, as long as the function is
sufficiently
preserved. In this context, a functional androgen receptor is one that binds
both androgen
and, upon androgen binding, an ARE.
- An "adenovirus vector" or "adenoviral vector" (used interchangeably) is a
term
well understood in the art and generally comprises a polynucleotide (defined
herein)
comprising all or a portion of an adenovirus genome. For the purpose of the
present
invention, an adenovirus vector contains a heterologous TRE operably linked to
a
polynucleotide. The operably linked polynucleotide can be adenoviral or
heterologous.
An adenovirus is exemplified by, but not limited to, Ad2, Ad5, Ad12, and Ad40.
The
terms "vector", "polynucleotide vector", "construct", "polynucleotide
construct" and
"vector construct" are used interchangeably herein. An adenoviral vector of
this invention
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may be in any of several forms, including, but not limited to, naked DNA, DNA
encapsulated in an adenovirus coat, DNA packaged in another viral or viral-
like form
(such as herpes simplex, and AAV), DNA encapsulated in liposomes, DNA
complexed
with polylysine, complexed with synthetic polycationic molecules, conjugated
with
transferrin, complexed with compounds such as PEG to immunologically "mask"
the
molecule and/or increase half-life, or conjugated to a non-viral protein. For
purposes of
this invention, adenovirus vectors are replication-competent in a target cell.
"Heterologous" means derived from a genotypically distinct entity from that of
the
rest of the entity to which it is being compared.
A "heterologous" TRE is one which is not associated with or derived from a
wild-
type adenovirus. Examples of heterologous TREs are the albumin promoter or
enhancer
and other viral promoters and enhancers, such as SV40. Examples of preferred
heterologous TREs are provided herein.
A "heterologous gene" or "transgene" is any gene that is not present in wild-
type
adenovirus. Preferably, the transgene will also not be expressed or present in
the target
cell prior to introduction by the adenovirus vector. Examples of preferred
transgenes are
provided below.
An "endogenous" promoter, enhancer, or TRE is native to or derived from
adenovirus.
"Replication" and "propagation" are used interchangeably and refer to the
ability of
an adenovirus 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
adenovirus proteins and is generally directed to reproduction of adenovirus.
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 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
vector to replicate in a cell.
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-
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translationally modified through reactions that include glycosylation,
acetylation and
phosphorylation.
A "host cell" includes an individual cell or cell culture which can be or has
been a
recipient of an adenoviral vector of this invention. Host cells include
progeny of a single
host cell, and the progeny may not necessarily be completely identical (in
morphology or
in 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 vivo
with an adenoviral vector of this invention.
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
aberrantly compromised (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 which 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 an adenoviral vector of the present invention on a
cell which
allows a TRE to fftznction when compared to the cytotoxicity conferred by an
adenoviral
vector of the present invention on a cell which does not allow, or is less
permissive for, the
same TRE to function. Such cytoxicity 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 adenovirus 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
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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.
An "individual" is a vertebrate, preferably a mammal, more preferably a human.
Mammals include, but are not limited to, 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 an adenoviral 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.
A "masked adenovirus" is an adenovirus which has been complexed with a
hydrophilic polymer ("masking agent"). The adenovirus may be any adenovirus,
including
naturally occurring isolates of adenovirus or engineered adenovirus vectors
such as those
disclosed in the instant application. Masking agents are preferably of low
immunogenicity. Examples of acceptable hydrophilic polymers include:
polyethylene/polypropylene copolymers, polyacrylic acid analogues, sugar
polymers such
as cellulose, polyformaladehyde, poly(N-vinylpyrollidone), polyethylene glycol
(PEG),
and the like. The hydrophilic polymer may be complexed by covalent or non-
covalent
attachment to the capsid proteins of the virus, particularly the hexon and
fiber capsid
proteins. A preferred hydrophilic polymer is PEG, and a preferred masked
adenovirus is
PEG covalently linked to adenovirus ("covalently pegylated adenovirus").
"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
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slowed or lengthened, as compared to not administering adenoviral vectors of
the present
invention.
Adenoviral vectors comprising heterologous TREs
The present invention provides adenoviral vectors which comprise a first
adenovirus gene under transcriptional control of a heterologous (i.e., non-
adenovirus) TRE
and at least a second gene under transcriptional control of a second
heterologous TRE,
wherein the two heterologous TREs are different from each other (in nucleotide
sequence)
but are functional in the same cell. In addition, at least the first
heterologous TRE is cell-
specific. Preferably, the adenovirus gene is one that contributes to
cytotoxicity (whether
direct and/or indirect), more preferably one that contributes to and/or causes
cell death, and
even more preferably the first adenoviral gene is essential from adenovirus
replication.
Examples of an adenovirus gene that contributes to cytotoxicity include, but
are not
limited to, the adenovirus death protein gene. See Fig.l for diagrammatic
examples.
Because the adenovirus vector(s) is selectively (i.e. preferentially)
replication-
competent for propagation in target cells which allow the function of the
heterologous
TREs, these cells will be preferentially subject to the cytotoxic and/or
cytolytic effects of
adenoviral proliferation. Preferably, target cells are neoplastic cells,
although any cell for
which it is desirable and/or tolerable to sustain this cytotoxic activity may
be a target cell.
By combining the adenovirus vector(s) with the mixture of target and non-
target cells, in
vitro or in vivo, the adenovirus vector(s) preferentially replicates in the
target cells. Once
the target cells are destroyed due to selective cytotoxic and/or cytolytic
replication, the
adenovirus vector(s) replication is significantly reduced, lessening the
probability of
runaway infection and undesirable bystander effects. In vitro cultures may be
retained to
continually monitor the mixture (such as, for example, a biopsy or other
appropriate
biological sample) for occurrence (i.e. presence) and/or recurrence of the
target cell, e.g., a
cancer cell in which the cell specific TRE is functional.
To ensure cytotoxicity further, one or more transgenes having a cytotoxic
effect
may also be present and under the selective transcriptional control of a
heterologous TRE.
Additionally, or alternatively, an adenovirus gene that contributes to
cytotoxicity and/or
cell death (such as the adenovirus death protein (ADP) gene) may be included
in the
adenoviral vector, either free of, or under, the selective transcriptional
control of a
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heterologous TRE. In these embodiments, one may provide higher confidence that
the
target cells will be destroyed.
In one embodiment, the invention provides an adenovirus vector comprising an
adenovirus gene essential for replication under transcriptional control of a
heterologous,
cell specific TRE, wherein the TRE selectively regulates the expression of
said gene in a
target cell. Further, the adenovirus vector comprises a second gene under
transcriptional
control of a second heterologous TRE, wherein the first and second
heterologous TREs are
functional in the same cell and the TREs are different from each other.
Cell, or tissue, specific transcriptional regulatory elements are well known
in the
art. TREs may be derived from the transcriptional regulatory sequences of a
single gene or
from different genes and combined to produce a functional TRE. A cell-specific
TRE is
preferentially functional in a limited population (or type) of cells, e.g.,
prostate cells or
liver cells.
As is known in the art, activity of TREs can be inducible. Inducible TREs
generally exhibit low activity in the absence of the inducer, and are up-
regulated in the
presence of the inducer. Inducible TREs may be preferred when expression is
desired only
at certain times or at certain locations, or when it is desirable to titrate
the level of
expression using an inducing agent. For example, transcriptional activity from
the PSE-
TRE, PB-TRE and hKLK2-TRE is inducible by androgen, as described herein.
Accordingly, in one embodiment, a heterologous TRE of the adenovirus vector is
inducible.
As mentioned, TRE can also comprise multimers. For example, a TRE can
comprise a tandem series of at least two, at least three, at least four, at
least five promoter
fragments. Alternatively, a TRE could have one or more promoter regions along
with one
or more enhancer regions. These multimers may also contain promoter and/or
enhancer
sequences from different genes. In addition, the promoter and enhancer
components of a
TRE may be in any orientation and/or distance from the coding sequence of
interest, as
long as the desired cell-specific transcriptional activity is obtained.
A TRE used in the present invention may or may not lack a silencer. The
presence
of a silencer (i.e., a negative regulatory element known in the art) can
assist in shutting off
transcription (and thus replication) in non-permissive cells. Thus, presence
of a silencer
can confer enhanced cell-specific replication by more effectively preventing
adenoviral
vector replication in non-target cells. Alternatively, lack of a silencer may
assist in
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effecting replication in target cells, thus conferring enhanced cell-specific
replication due
to more effective replication in target cells.
When used in an adenovirus vector of the present invention, the heterologous
TREs
have a different polynucleotide sequence from each other. Accordingly, in a
given
adenovirus vector, the sequence identity between the two heterologous TREs is
less than
about 95%, preferably less than about 90%, more preferably less than about
85%, more
preferably less than 80%, even more preferably less than about 75%. Despite
the
difference in sequence identity, the heterologous TREs of a given adenovirus
vector are
functional in the same cell. The difference(s) may arise from, for example,
varying the
sequence of a TRE derived from a single gene. It is possible to generate such
TREs using
standard methods in the art and/or allowing sequences to alter during the
course of
propagation. The difference(s) may also arise from TREs that are derived from
different
genes.
As is readily appreciated by one skilled in the art, a TRE is a polynucleotide
sequence, and, as such, can exhibit function over a variety of sequence
permutations.
Methods of nucleotide substitution, addition, and deletion are known in the
art, and readily
available functional assays (such as the CAT or luciferase reporter gene
assay) allow one
of ordinary skill to determine whether a sequence variant exhibits requisite
cell-specific
transcription function.
Hence, functionally preserved variants of TREs may also be used including
nucleic
acid substitutions, additions, and/or deletions. Accodingly, variants of TREs
must retain
function in the target cell but need not be of maximal function.
It is possible that certain base modifications will result in enhanced
expression
levels and/or cell-specificity. Nucleic acid sequence deletions or additions
within a TRE
may decrease or increase transcription if they bring transcription regulatory
protein
binding sites too close or too far away or rotate them so they are on opposite
sides of the
DNA helix, as is known in the art. Thus, while the inventors are not wishing
to be bound
by a single theory, it is possible that certain modifications will result in
modulated
resultant expression levels, including enhanced cell-specific expression
levels.
Achievement of enhanced expression levels may be especially desirable in the
case of
more aggressive forms of neoplastic growth, or when a more rapid and/or
aggressive
pattern of cell killing is warranted (due to an immunocompromised condition of
the
individual, for example).
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Transcriptional activity (including decrease or enhancement) 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., operably linked to) a TRE. As discussed
herein, a TRE can
be of varying lengths, and of varying sequence composition. By transcriptional
activity, it
is intended that transcription due to the presence of the enhancer is
increased above basal
levels (i.e., promoter alone; lacking enhancer) 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, 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. Basal levels
are
generally the level of activity, if any, in a non-target cell, or the level of
activity (if any) of
a reporter construct lacking a TRE as tested in a non-target cell.
Maximal transcriptional activation activity of a TRE may not always be
necessary
to achieve a desired result. The level of induction afforded by a fragment of
a TRE may be
sufficient in certain applications to achieve a desired result. For example,
if used for
treatment or palliation of a disease state, less-than-maximal responsiveness
may be
sufficient for the desired result, if, for example, the target cells are not
especially virulent
and/or the extent of disease is relatively confined.
The size of the heterologous TREs will be determined in part by the capacity
of the
adenoviral vector, which in turn depends upon the contemplated form of the
vector (see
below). Generally minimal sizes are preferred, as this provides potential room
for
insertion of other sequences which may be desirable, such as transgenes
(discussed below)
or other additional regulatory sequences. However, if no additional sequences
are
contemplated, or if, for example, an adenoviral vector will be maintained and
delivered
free of any viral packaging constraints, larger DNA sequences may be used as
long as the
resultant adenoviral vector is rendered replication-competent.
If no adenovirus sequences have been deleted, an adenoviral vector can be
packaged with extra sequences totaling up to about 5% of the genome size, or
approximately 1.8 kb. If non-essential sequences are removed from the
adenovirus
genome, an additional 4.6 kb of insert can be tolerated (i.e., a total of
about 1.8 kb plus 4.6
kb, which is about 6.4 kb). Examples of non-essential adenoviral sequences
that can be
deleted are E3 and E4 (as long as the E4 ORF6 is maintained).
24
r
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In order to minimize non-specific replication, endogenous (i.e., adenovirus)
TREs
should preferably be removed. This would also provide more room for inserts in
an
adenoviral vector, which may be of special concern if an adenoviral vector
will be
packaged as a virus (see below). Even more importantly, deletion of endogenous
TREs
would prevent a possibility of a recombination event whereby a heterologous
TRE is
deleted and the endogenous TRE assumes transcriptional control of its
respective
adenovirus coding sequences (thus allowing non-specific replication). In one
embodiment,
an adenoviral vector of the invention is constructed such that the endogenous
transcription
control sequences of adenoviral genes are deleted and replaced by a
heterologous TREs.
However, endogenous TREs may be maintained in the adenovirus vector(s),
provided that
sufficient cell-specific replication preference is preserved. These
embodiments can be
constructed by providing heterologous TREs intervening between the endogenous
TRE
and the replication gene coding segment. Requisite cell-specific replication
preference is
indicated by conducting assays that compare replication of the adenovirus
vector in a cell
which allows function of the heterologous TREs with replication in a cell
which does not.
Generally, it is intended that replication is 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, 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. The acceptable differential can be determined empirically (using, for
example,
Northern assays or other known in the art) and will depend upon the
anticipated use of the
adenoviral vector and/or the desired result.
(a) Exemplary heterologous TREs
Cell, or tissue, specific transcriptional regulatory elements are well known
in the
art. Methods to identify such elements are also well known in the art. The
cell specific
TREs provided below are illustrative examples and not meant to limit the
invention.
In one embodiment, the invention includes adenovirus vectors wherein the
heterologous TREs are prostate cell specific. For example, TREs that function
preferentially in prostate cells and can be used in the present invention to
target adenovirus
replication to prostate neoplasia, include, but are not limited to, TREs
derived from the
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prostate-specific antigen gene (PSA-TRE), the glandular kallikrein-1 gene
(from the
human gene, hKLK2-TRE), and the probasin gene (PB-TRE). All three of these
genes are
preferentially expressed in prostate cells and the expression is androgen-
inducible.
Generally, expression of genes responsive to androgen induction requires the
presence of
an androgen receptor (AR).
PSA is synthesized exclusively by normal, hyperplastic, and malignant
prostatic
epithelia; hence, its tissue-specific expression has made it an excellent
biomarker for
benign prostatic hyperplasia (BPH) and prostatic carcinoma (CaP). Normal serum
levels
of PSA are typically below 5 ng/ml, with elevated levels indicative of BPH or
CaP.
Lundwall et al. (1987) FEBS Lett. 214: 317; Lundwall (1989) Biochem. Biophys.
Res.
Comm. 161: 1151; and Riegmann et al. (1991) Molec. Endocrin. 5: 1921.
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. Biol. Chem. 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 yield
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 TRE depicted in (SEQ ID NO: 1) is the same as that given in
GenBank Accession No. U37672, and published. Schuur et al. (1996). A variant
PSA-
TRE nucleotide sequence is depicted in (SEQ ID NO:2). This is the PSA-TRE
contained
within CN706 clone 35.190.13. CN706 is an adenoviral vector in which the E 1 A
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).
CN706 was passaged through 293 and LNCaP cells. A clone, designated 35.190.13
was
isolated. The structure of this clone was confirmed by PCR, restriction
endonuclease
digestion and Southem blotting. Both DNA strands of the CN706 clone 35.190.13
were
sequenced between positions 1 and 3537. Seven single base pair changes were
found in
the PSE, compared to the sequence reported by Schuur et al. (1996). These
point
mutations are not in the ARE and are thus not likely to affect the function of
the enhancer.
One mutation was found in the PSA promoter region, but is not likely to affect
gene
26
. . . . ... ...... ... .. . r . . . . . . 1
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expression from this promoter. In addition to these mutations, a missense
mutation was
found in the first exon of E1A. This C to G transition at position 3032
results in a Glu to
Arg change in the E 1 A protein sequence. This mutation does not appear to
diminish E 1 A
function.
Human glandular kallikrein (hKLK2, encoding the hK2 protein) is expressed
exclusively in the prostate and its expression is up-regulated by androgens
primarily by
transcriptional activation. Wolf et al. (1992) Molec. Endocrinol. 6:753-762.
Morris
(1989) Clin. Exp. Pharm. Physiol. 16:345-351; Qui et al. (1990) J. Urol.
144:1550-1556;
Young et al. (1992) Biochem. 31:818-824. 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.
Circulating hK2
in different relative proportions to PSA has been detected in the serum of
patients with
prostate cancer. Charlesworth et al. (1997) Urology 49:487-493. Expression of
hK2 has
been detected in each of 257 radical prostatectomy specimens analyzed. Darson
et al.
(1997) Urology 49:857-862. The intensity and extent of hK2 expression,
detected using
specific antibodies, increased from benign epithelium to high-grade prostatic
intraepithelial neoplasia (PIN) and adenocarcinoma, whereas PSA and prostate
acid
phosphatase displayed an inverse pattern of immunoreactivity. Darson et al.
(1997).
Indeed, it has been reported that a certain percentage of PSA-negative tumors
have
detectable hK2. Tremblay et al. (1997) Am. J. Pathol. 150:455-459.
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.
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. Murtha et al. (1993) Biochem. 32:6459-6464. 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 NO: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
27
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WO 98/39464 PCT/US98/04080
transcription start site): about nt -3993 to about nt -3643 (nt 8021 to 8371
of SEQ ID
NO:3), about nt -4814 to about nt -3643 (nt 7200 to 8371 of SEQ ID NO:3),
about nt -
5155 to about nt -3387 (nt 6859 to 8627 of SEQ ID NO:3), about nt -6038 to
about nt -
2394 (nt 5976 to 9620 of SEQ ID NO: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-TRE can then be operably linked to a heterologous
polynucleotide to
confer hKLK2-TRE-specific transcriptional regulation on the linked gene, thus
increasing
its expression.
The rat probasin (PB) gene encodes a nuclear and secreted protein, probasin,
that is
only expressed in the dorsolateral prostate. Dodd et al. (1983) J. Biol. Chem.
258:10731-
10737; Matusik et al. (1986) Biochem. Cell. Biol. 64: 601-607; and Sweetland
et al. (1988)
Mol. Cell. Biochem. 84: 3-15. The dorsolateral lobes of the murine prostate
are considered
the most homologous to the peripheral zone of the human prostate, where
approximately
68% of human prostate cancers are thought to originate.
A PB-TRE 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 information to direct
development and
hormone -regulated expression of an operably linked heterologous gene
specifically to the
prostate in transgenic mice. Greenberg et al. (1994) Mol. Endocrinol. 8:230-
239.
Thus, TREs derived from prostate cell specific TREs, including, but not
limited to,
those described herein, may be used in the present invention to generate
stable adenovirus
vectors that preferentially replicate in cells in which the TREs are
functional, such as cells
expressing an AR, particularly cells derived from prostate neoplasia.
Accordingly, and by
way of example, the invention includes adenovirus vectors in which the first
heterologous
TRE is a PSA-TRE (e.g., PSE-TRE) and the second heterologous TRE is a PB-TRE,
in
which the first heterologous TRE is a PB-TRE and the second heterologous TRE
is a PSA-
TRE (e.g., PSE-TRE); in which the first heterologous TRE is an hKLK2-TRE and
the
second heterologous TRE is a PSA-TRE (e.g., PSE-TRE), in which the first
heterologous
TRE is a PSA-TRE (e.g., PSE-TRE) and the second heterologous TRE is an hKLK2-
TRE,
in which the first heterologous TRE is an hKLK2-TRE and the second
heterologous TRE
28
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is a PB-TRE, in which the first heterologous TRE is a PB-TRE and the second
heterologous TRE is an hKLK2-TRE, as described above. See Examples 1-4, Fig.
2.
In the present invention, replication-competent adenovirus vectors directed at
specific target cells may also be generated with the use of TREs that are
preferentially
functional in the target tumor cells. Non-limiting examples of tumor cell-
specific
heterologous TREs, and non-limiting examples of respective potential target
cells, include
TREs from the following genes: a-fetoprotein (AFP) (liver cancer), mucin-like
glycoprotein DF3 (MUCI )(breast carcinoma), carcinoembryonic antigen (CEA)
(colorectal, gastric, pancreatic, breast, and lung cancers), plasminogen
activator urokinase
(uPA) and its receptor gene (breast, colon, and liver cancers), E2F1 (cell
cycle S-phase
specific promoter) (tumors with disrupted retinoblastoma gene function), HER-
2/neu (c-
erbB2/neu) (breast, ovarian, stomach, and lung cancers).
In the present invention, tumor-specific TREs may be used in conjunction with
tissue-specific TREs from the following exemplary genes (tissue in which the
TREs are
specifically functional are in parentheses): hypoxia responsive element,
vascular
endothelial growth factor receptor (endothelium), albumin (liver), factor VII
(liver), fatty
acid synthase (liver), Von Willebrand factor (brain endothelium), alpha-actin
and myosin
heavy chain (both in smooth muscle), synthetast I (small intestine), Na-K-Cl
transporter
(kidney). Additional tissue specific TREs are known in the art.
Accordingly, in one embodiment, the cell specific, heterologous TRE is tumor
cell
specific. Preferably, both heterologous TREs are tumor cell specific and
functional in the
same cell. In another embodiment, one of the first heterologous TREs is tumor
cell
specific and the second heterologous TRE is tissue specific, whereby both TREs
are
function in the same cell.
AFP is an oncofetal protein, the expression of which is primarily restricted
to
developing tissues of endodermal origin (yolk sac, fetal liver, and gut),
although the level
of its expression varies greatly depending on the tissue and the developmental
stage. AFP
is of clinical interest because 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 but not in surrounding normal liver. Thus, the AFP gene appears to
be
regulated to hepatoma cell-specific expression:
29
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Cell-specific TREs from the AFP gene have been identified. For example, the
cloning and characterization of human AFP-specific enhancer activity is
described in
Watanabe et al. (1987) J. Biol. Chem. 262:4812-4818. 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 NO:5).
The AFP enhancer region in human 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, as depicted in SEQ ID NO:6, yields a fully functional AFP-
TRE. Ido et
al. (1995) describe a 259 bp promoter fragment (nt -230 to nt +29) that is
specific for
HCC. Cancer 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 CAAT boxes. Preferably, the AFP-TRE
contains at least one enhancer region. More preferably, the AFP-TRE contains
both
enhancer regions.
Suitable target cells for adenoviral vectors containing AFP-TREs are any cell
type
that allow an AFP-TRE 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
(Kawamoto et al. (1992) Hepatogastroenterology 39:282-286), primary gall
bladder tumor
(Katsuragi et al. (1989) Rinsko Hoshasen 34:371-374), uterine endometrial
adenocarcinoma cells (Koyama et al. (1996) Jpn. J. Cancer Res. 87:612-617),
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 produce 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-TRE (i.e., allow an AFP-TRE to function).
The protein urokinase plasminogen activator (uPA) and its cell surface
receptor,
urokinase plasminogen activator receptor (uPAR), are expressed in many of the
most
CA 02283231 1999-09-02
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frequently occurring neoplasia and appear to represent important proteins in
cancer
metastasis. Both proteins are implicated in breast, colon, prostate, liver,
renal, lung and
ovarian cancer. Transcriptional regulatory elements that regulate uPA and uPAR
transcription have been extensively studied. Riccio et al. (1985) Nucleic
Acids Res.
13:2759-2771; Cannio et al., (199 1) Nucleic Acids Res. 19:2303-2308.
Thus, cell-specific TREs, including but not limited to those described herein,
could
be used in the present invention to generate stable adenovirus vectors that
preferentially
replicate in cells in which the TREs are functional, such as cells derived
from liver
neoplasia. In one embodiment, the invention includes adenovirus vectors
wherein at least
one of the heterologous TREs are liver cell-specific. Accordingly, and by way
of example,
the invention includes adenovirus vectors in which the first heterologous TRE
is an AFP-
TRE and the second heterologous TRE is an uPA-TRE, in which the first
heterologous
TRE is an uPA-TRE and the second heterologous TRE is an AFP-TRE, in which the
first
heterologous TRE is an AFP-TRE and the second heterologous TRE is an albumin-
TRE,
in which the first heterologous TRE is an albumin-TRE and the second
heterologous TRE
is an AFP-TRE, as described above.
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. CEA is of clinical interest because circulating CEA can be detected
in the great
majority of patients with CEA-positive tumors. In lung cancer, about 50% of
total cases
have circulating CEA, with high concentrations of CEA (greater than 20 ng/ml)
often
detected in adenocarcinomas. Approximately 50% of patients with gastric
carcinoma are
serologically positive for CEA.
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.. Schrewe et al. (1990) Mol.
Cell. Biol.
10:2738-2748. In addition, cell-specific enhancer regions have been found.
WO/95/14100. 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. Richards et al. (1995); WO
95/14100. Further
31
.. ...._. . ~, ~......,.- ~
CA 02283231 1999-09-02
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characterization of the 5' flanking region of the CEA gene by Richards et al.
(1995)
indicated 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 LoVo and SW1463 cells. Richards et al. (1995) also
localized the promoter region 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
NO:7). Any 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 may be
used in the
invention as long as requisite desired functionality is displayed in the
adenovirus vector.
The cloning and characterization of CEA sequences have been described in the
literature
and are thus made available for practice of this invention and need not be
described in
detail herein.
The protein product of the MUCI gene (known as mucin or MUC 1 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. Zotter et al. (1988) Cancer Rev. 11-12: 55-101;
and Girling
et al. (1989) Int. J. Cancer 43: 1072-1076. However, mucin is overexpressed in
75-90%
of human breast carcinomas. Kufe et al. (1984) Hybridoma 3: 223-232. For
reviews, see
Hilkens (1988) Cancer Rev. 11-12: 25-54; and Taylor-Papadimitriou, et al.
(1990) J.
Nucl. Med. Allied Sci. 34: 144-150. Mucin protein expression correlates with
the degree
of breast tumor differentiation. Lundy et al. (1985) Breast Cancer Res. Treat.
5: 269-276.
This overexpression appears to be controlled at the transcriptional level.
Overexpression of the MUCl gene in human breast carcinoma cells MCF-7 and
ZR-75-1 appears to be regulated at the transcriptional level. Kufe et al.
(1984); Kovarik
(1993) J. Biol. Chem. 268:9917-9926; and Abe et al. (1990) J. Cell. Physiol.
143: 226-
231. The regulatory sequences of the MUCl gene have been cloned, including the
approximately 0.9 kb upstream of the transcription start site which contains a
TRE that
appears to be involved in cell-specific transcription, depicted in SEQ ID
NO:8. Abe et al.
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(1993) Proc. Natl. Acad. Sci. USA 90: 282-286; Kovarik et al. (1993); and
Kovarik et al.
(1996) J. Biol. Chem. 271:1 8 1 40-1 8 1 47.
Any MUCI-TREs used in the present invention are derived from mammalian cells,
including but not limited to, human cells. Preferably, the MUCI-TRE is human.
In one
embodiment, the MUCI-TRE may contain the entire 0.9 kb 5' flanking sequence of
the
MUCI gene. In other embodiments, the MUCI-TREs comprise the following
sequences
(relative to the transcription start site of the MUCI 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-erbB2/neu gene has been
associated
with many human cancers, including breast, ovarian, uterine, prostate, stomach
and lung
cancers. The clinical consequences of the c-erbB2/neu protein over-expression
have been
best studied in breast and ovarian cancer. c-erbB2/neu protein over-expression
occurs in
to 40% of intraductal carcinomas of the breast and 30% of ovarian cancers, and
is
associated with a poor prognosis in subcategories of both diseases. Human, rat
and mouse
20 c-erbB2/neu TREs have been identified and shown to confer c-erbB2/neu
expressing cell
specific activity. Tal et al. (1987) Mol. Cell. Biol. 7:2597-2601; Hudson et
al. (1990) J.
Biol. Chem. 265:4389-4393; Grooteclaes et al. (1994) Cancer Res. 54:4193-4199;
Ishii
et al. (1987) Proc. Natl. Acad. Sci. USA 84:4374-4378; Scott et al. (1994) J.
Biol. Chem.
269:19848-19858.
Thus, TREs derived from breast cell-specific TREs, including, but not limited
to,
those described herein, may be used in the present invention to generate
stable adenovirus
vectors that preferentially replicate in cells in which the TREs are
functional, particularly
cells derived from breast neoplasia. In one embodiment, the invention includes
adenovirus
vectors wherein the heterologous TREs are breast cell-specific. Accordingly,
and by way
of example, the invention includes adenovirus vectors in which the first
heterologous TRE
is a CEA-TRE and the second heterologous TRE is a MUCI -TRE, in which the
first
heterologous TRE is a MUCI -TRE and the second heterologous TRE is a CEA-TRE,
in
which the first heterologous TRE is a MUCI -TRE and the second heterologous
TRE is a
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HER-TRE, in which the first heterologous TRE is a HER-TRE and the second
heterologous TRE is an MUCI-TRE, in which the first heterologous TRE is a MUCI-
TRE
and the second heterologous TRE is a uPA-TRE, in which the first heterologous
TRE is a
uPA-TRE and the second heterologous TRE is a MUCI -TRE, in which the first
heterologous TRE is a uPA-TRE and the second heterologous TRE is an HER-TRE,
in
which the first heterologous TRE is an HER-TRE and the second heterologous TRE
is a
uPA-TRE, as described above.
Thus, TREs derived from colon cell-specific TREs, including, but not limited
to,
those described herein, may be used in the present invention to generate
stable adenovirus
vectors that preferentially replicate in cells in which the TREs are
functional, particularly
cells derived from colon neoplasia. In one embodiment, the invention includes
adenovirus
vectors wherein the heterologous TREs are colon cell-specific. Accordingly,
and by way
of example, the invention includes adenovirus vectors in which the first
heterologous TRE
is a CEA-TRE and the second heterologous TRE is a uPA-TRE, in which the first
heterologous TRE is a uPA-TRE and the second heterologous TRE is a CEA-TRE, as
described above.
As described above, some of the exemplary TREs are specific for more than one
cell-type and thus, more than one type of neoplasia. Accordingly, adenovirus
vectors of
the present invention, as exemplified above, may be useful in the treatment of
more than
one type of neoplasm, as can be determined by the information provided herein.
The TREs listed above are provided as non-limiting examples of TREs that would
function in the instant invention. Additional cell-specific TREs are known in
the art, as are
methods to identify and test cell specificity of suspected TREs. Further, and
as noted
above, the invention does not require that the TREs be derived from different
genes. As
long as the TRE sequences are sufficiently different, and the requisite
funetionality is
diplayed, the different TREs may be derived from the same gene.
For example, activity of a TRE can be determined as follows. A TRE
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 sequence(s) 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),
(3-galactosidase (encoded by the lacZ gene), luciferase (encoded by the luc
gene), alkaline
34
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phosphatase, green fluorescent protein, and horse radish 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 TRE 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, TRE activity may be measured by detection and/or
quantitation of
reporter gene-derived mRNA or protein product. 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 TRE
activity is determined in each cell type and compared to that of a reporter
gene construct
without the TRE. A TRE is cell specific when it is preferentially functional
in a specific
type of cell over a different type of cell.
For example, the specificity of PB-TRE activity for prostate cell that express
the
androgen receptor (AR) was demonstrated as follows. The region of the PB 5'-
flanking
DNA (nt -426 to nt +28) (SEQ ID NO:4) 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-TRE was intact. Further, the overall luciferase
activity recovered in
the cellular extracts 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-TRE expression is preferentially functional in PSA-producing, AR-producing
prostate
carcinoma cells as compared to PSA-deficient, AR-deficient prostate carcinoma
cells and
that PB-TRE is capable of mediating specific expression in cells producing the
androgen
receptor.
(b) Exemplary genes under transcriptional control of the heterologous TREs
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Any of the various serotypes of adenovirus can be used, such as Ad2, Ad5, Ad
12,
and Ad40. For purposes of illustration the serotype adenovirus 5(Ad5) is
exemplified
herein.
In some embodiments, a cell specific, heterologous TRE is used to control
transcription of an adenovirus gene and a second heterologous TRE, different
from (i.e.,
not the same as) the first heterologous TRE, is used to control transcription
of a second
gene to provide an adenovirus vector so that replication-competence is
preferentially
achievable in target cells which allow for function of the cell-specific TRE.
In addition,
the two heterologous TREs are functional in the target cell. Preferably, the
first
adenovirus gene is essential for adenoviral replication, even more preferably,
both genes
are essential for adenoviral replication. Preferably, at least one of the
genes is an early
gene, such as E 1 A, E 1 B, E2, or E4. (E3 is not essential for viral
replication.) Preferably,
both genes under control of the heterologous TREs are early genes. More
preferably, the
early gene under cell-specific TRE control is E1A and/or E1B. Examples 1, 2
and 5
provide a more detailed description of such constructs.
The E 1 A gene is expressed immediately after viral infection (0-2 hours) 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 E1B, E2,
E3, E4, 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. Flint (1982) Biochem. Biophys. Acta 651:175-208;
Flint (1986)
Advances Virus Research 31:169-228; Grand (1987) Biochem. J. 241:25-38. In the
absence of a functional E 1 A gene, viral infection does not proceed, because
the gene
products necessary for viral DNA replication are not produced. Nevins (1989)
Adv. Virus
Res. 31:35-81. The transcription start site of Ad5 El A is at nt 498 and the
ATG start site
of the E 1 A protein is at nt 560 in the virus genome.
The E1B protein functions in trans and is necessary for transport of late mRNA
from the nucleus to the cytoplasm. Defects in E1B expression result in poor
expression of
late viral proteins and an inability to shut off host cell protein synthesis.
The promoter of
E1 B has been implicated as the defining element of difference in the host
range of Ad40
and Ad5: clinically Ad40 is an enterovirus, whereas Ad5 causes acute
conjunctivitis.
Bailey et al. (1993) Virology 193:631; Bailey et al. (1994) Virology 202:695-
706. The
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E 1 B promoter of Ad5 consists of a single high-affinity recognition site for
Spi and a
TATA box.
Accordingly, in one embodiment, the adenovirus E 1 A gene is under
transcriptional
control of the cell specific, heterologous TRE. In another embodiment, the
adenovirus
E 1 B gene is under transcriptional control of the cell specific, heterologous
TRE. In
another embodiment, both the adenovirus E 1 A and E 1 B genes are under
transcriptional
control of two different heterologous TREs, preferably both TREs are cell
specific.
The E2 region of adenovirus codes for proteins related to replication of the
adenoviral genome, including the 72 kD DNA-binding protein, the 80 kD
precursor
terminal protein and the viral DNA polymerase. The E2 region of Ad5 is
transcribed in a
rightward orientation from two promoters, termed E2 early and E2 late, mapping
at 76.0
and 72.0 map units, respectively. While the E21ate promoter is transiently
active during
late stages of infection and is independent of the E 1 A transactivator
protein, the E2 early
promoter is crucial during the early phases of viral replication.
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 Micro. and 1mm. (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 for 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 codon 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 binding sites E2F and ATF. Therefore, insertion
of a
heterologous TRE having Spel ends into the Spel site in the plus strand would
disrupt the
endogenous E2 early promoter of Ad5 and should allow TRE-regulated expression
of E2
transcripts.
The E4 gene has a number of transcription products. The E4 region codes for
two
polypeptides which are responsible for stimulating the replication of viral
genomic DNA
and for stimulating late gene expression. The protein products of open reading
frames
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(ORFs) 3 and 6 can both perform these functions by binding the 55-kD protein
from E 1 B
and heterodimers of E2F-1 and DP-1. The ORF 6 protein requires interaction
with the EIB
55-kD protein for activity while the ORF 3 protein does not. In the absence of
functional
protein from ORF 3 and ORF 6, plaques are produced with an efficiency less
than 10-6 that
of wild type virus. To restrict further the viral replication to target cells,
E4 ORFs 1-3 can
be deleted, making viral DNA replication and late gene synthesis dependent on
E4 ORF 6
protein. By combining such a mutant with sequences in which the E1B region is
regulated
by a target cell-specific TRE, a virus can be obtained in which both the E1B
function and
E4 function are dependent on the target cell-specific TRE driving EIB.
The major late genes relevant to the subject invention are L1, L2 and L3,
which
encode proteins of the Ad5 virus virion. All of these genes (typically coding
for structural
proteins) are probably required for adenoviral replication. The late genes are
all under the
control of the major late promoter (MLP), which is located in Ad5 at +5986 to
+6048.
In one embodiment, the adenovirus E4 gene is under transcriptional control of
the
cell specific, heterologous TRE. In another embodiment, an adenovirus late
gene is under
transcriptional control of the cell specific, heterologous TRE. In another
embodiment, one
early gene and one late gene are under transcriptional control of differrent
heterologous
TREs.
In addition to conferring selective cytotoxic and/or cytolytic activity by
virtue of
preferential replication competence in cells which allow function of the
heterologous
TREs, the adenovirus vectors of this invention can further include a
heterologous
polynucleotide (transgene) under the control of a heterologous TRE. 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 and/or rate of cytotoxic activity,
due to, for
example, the relatively refractory nature or particular aggressiveness of the
target cell.
This could be accomplished by coupling the cell-specific replicative cytotoxic
activity with
cell-specific expression of, for example, HSV-tk and/or cytosine deaminase
(cd), which
renders cells capable of metabolizing 5-fluorocytosine (5-FC) to the
chemotherapeutic
agent 5-fluorouracil (5-FU). Using these types of transgenes may also confer a
bystander
effect.
Other desirable transgenes that may be introduced via an adenovirus vector(s)
include genes encoding cytotoxic proteins, such as the A chains of diphtheria
toxin, ricin
or abrin (Palmiter et al. (1987) Cell 50: 435; Maxwell et al. (1987) Mol.
Cell. Biol. 7:
38
, ~
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1576; Behringer et al. (1988) Genes Dev. 2: 453; Messing et al. (1992) Neuron
8: 507;
Piatak et al. (1988) J. Biol. Chem. 263: 4937; Lamb et al. (1985) Eur. J.
Biochem. 148:
265; Frankel et al. (1989) Mol. Cell. Biol. 9: 415), genes encoding a factor
capable of
initiating apoptosis, sequences encoding antisense transcripts or ribozymes,
which among
other capabilities may be directed to mRNAs encoding proteins essential for
proliferation,
such as structural proteins, or transcription factors; viral or other
pathogenic proteins,
where the pathogen proliferates intracellularly, genes that encode an
engineered
cytoplasmic variant of a nuclease (e.g. RNase A) or protease (e.g. awsin,
papain,
proteinase K, carboxypeptidase, etc.), or encode the Fas gene, and the like.
Other genes of
interest include potential therapeutic genes such as cytokines, antigens,
transmembrane
proteins, and the like, such as IL-1, -2, -6, -12, GM-CSF, G-CSF, M-CSF, IFN-
a, -P, -y,
TNF-a, -(3, TGF-a, -0, NGF, and the like. The positive effector genes could be
used in an
early phase, followed by cytotoxic activity due to replication.
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, the invention provides adenovirus vectors in which a first
adenovirus
gene is in under transcriptional control of a first heterologous cell-specific
TRE and a
polynucleotide sequence encoding an ADP under control of a second heterologous
TRE,
which is different from the first TRE but functional in the same cell as the
first TRE,
preferably the first 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 NO:9 and
SEQ
ID NO: 10, 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
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inserted in other locations of the adenovirus genome, such as the E4 region.
Alternatively,
the ADP coding sequence could be operably linked to a heterologous TRE,
including, but
not limited to, another viral TRE, a tissue specific TRE such as that of AFP,
CEA, hKLK2,
MUCI, PSE and PB.
It is understood that the present invention does not exclude adenovirus
vectors
containing additional genes under control of the heterologous TREs.
Accordingly, the
invention provides adenoviral vectors comprising a third gene under
transcriptional control
of a third heterologous TRE where all of the heterologous TREs are different
from each
other in polynucleotide sequence but all are functional in the same cell.
Preferably, the
third gene is one that contributes to cytotoxicity (whether direct and/or
indirect), more
preferably one thatcontributes to and/or enhances cell death, and even more
preferably the
third gene is essential from adenovirus replication. Preferably the third
heterologous TRE
is target cell specific. For example, an adenovirus vector may contain a PB-
TRE, a PSE-
TRE and an hKLK2-TRE, each prostate cell specific and each controlling the
transcription
of a different gene.
As is known in the art and described herein, the ability of enhancers to
increase
transcription of an operably linked gene is independent of its orientatoin and
distance
relative to the gene. Accordingly, the invention provides adenoviral vectors
comprising at
least an additional gene (beyond the first and the second genes) under
transcriptional
control of the second heterologous TRE. Preferably, the additional gene is one
that
contributes to cytotoxicity (whether direct and/or indirect), more preferably
one that
enhances cell death, and even more preferably the third gene is essential from
adenovirus
replication.
(c) Delivery of adenoviral vectors to cells
The adenoviral 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 vector(s) more immunogenic); packaged into other particulate viral
forms such
as HSV or AAV; complexed with agents to enhance or dampen an immune response;
_. _ _ ,
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complexed with agents that facilitate in vivo transfection, such as DOTMATM,
DOTAPTM,
and polyamines.
If an adenoviral vector is packaged into an adenovirus, the adenovirus itself
may be
selected to further enhance targeting. For example, adenovirus fibers mediate
primary
contact with cellular receptor(s) aiding in tropism. See, e.g., Amberg 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
subgenus (or
subgenera) could be used to further increase cell-specificity of cytotoxicity
and/or
cytolysis.
The adenoviral vectors may be delivered to the target cell in a variety of
ways,
including, but not limited to, liposomes, general transfection methods that
are well known
in the art (such as calcium phosphate precipitation and electroporation), and
direct
injection, and intravenous infusion. The means of delivery will depend in
large part on the
particular adenoviral vector (including its form) as well as the type and
location of the
target cells (i.e., whether the cells are in vitro or in vivo).
If used in a packaged adenovirus, adenovirus vectors may be administered in an
appropriate physiologically acceptable carrier at a dose of about 104 to about
1014. The
multiplicity of infection will generally be in the range of about 0.001 to
100. If
administered as a polynucleotide construct (i.e., not packaged as a virus)
about 0.01 g to
1000 g of an adenoviral vector can be administered. The adenoviral vectors
may be
administered one or more times, depending upon the intended use and the immune
response potential of the host or may be administered as multiple simultaneous
injections.
If an immune response is undesirable, the immune response may be diminished by
employing a variety of immunosuppressants, so as to permit repetitive
administration,
without a strong immune response. If packaged as another viral form, such as
HSV, an
amount 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, an adenovirus vector(s) is complexed to a hydrophilic
polymer to create a masked adenovirus. The hydrophilic polymer is attached
(covalently
or non-covalently) to the capsid proteins of the adenovirus, particularly the
hexon and fiber
proteins. In preferred embodiments, the adenovirus vectors of the instant
invention a
complexed with masking agents to create masked adenovirus vectors. Masked
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adenoviruses are advantageous due to (a) the masking of the adenovirus surface
to
adenovirus neutralizing antibodies or opsinins which are in circulation, and
(b) increasing
systemic circulation time of adenovirus particles by reduction of non-specific
clearance
mechanism in the body (i.e., macrophages, etc.). In the in vivo context, the
systemic
delivery of a masked adenovirus results in a longer circulation of viral
particles, less
immunogenicity, and increased biodistribution with a decrease in clearance by
the liver
and spleen. Extensive research has been done on modification of proteins and
lipids with
hydrophilic polymers (especially PEG), but the inventors are unaware of any
other use of
masking agents in conjunction with adenovirus or adenovirus constructs.
Accordingly, the invention provides an adenovirus complexed with a masking
agent. Preferably, the masking agent is PEG. A schematic of one method of
making a
masked adenovirus is depicted in Figure 13 (see also Example 7). A preferred
embodiment is a masked adenovirus comprising an adenovirus vector(s) described
herein,
with a more preferred embodiment comprising a pegylated adenovirus comprising
an
adenovirus described herein. The invention also provides methods to make and
use these
masked adenoviruses, which are evident from the description herein.
The masking agent may be of various molecular weights, as long as the desired
complex and requisite functionality is obtained. Most masking agents obtained
from
commercial sources are normally polydisperse in relation to the stated
molecular weight
(i.e., the masking agent is supplied in a distribution of molecular weights
surrounding the
nominal molecular weight). Masking agents useful in masking adenoviruses
according to
the instant invention may have nominal weights of about 2000 to about 50,000;
preferably,
about 2500 to about 30,000; preferably, about 3000 to about 25,000; more
preferably,
about 5000 to about 20,000. Preferably, the nominal molecular weight is less
than about
20,000, more preferably less than about 10,000, more preferably less than
about 7500,
more preferably less than about 5000. Preferably, the masking agent is PEG
with a
nominal molecular weight of less than about 5000 Da. Mixtures of different
weight
masking agents are also contemplated.
The masking may be covalently or non-covalently attached. In the case of non-
covalent attachment, the attachment may be via electrostatic, hydrophobic, or
affinity
interactions. The masking agents used for non-covalent attachment may be
modified
masking agents (i.e., the masking agent is synthesized or modified to contain
particular
chemical moieties not normally found in the masking agent). Masking agents
useful for
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electrostatic attachment to adenoviral vectors will be masking agents which
contain, or
have been modified or synthesized to contain, charged moieties which bind to
the
adenovirus surface by electrostatic interaction. Negatively charged masking
agents
include masking agents which contain phosphate groups, sulfate groups, carboxy
groups,
and the like. Quaternary amine groups are useful as positively charged
moieties for
electrostatic, non-covalent attachment of masking agents to adenovirus.
Masking agents
containing or modified or synthesized to contain hydrophobic groups, such as
lipids (e.g.,
phosphatidylethanolamine and the like) and other hydrophobic groups (such as
phenyl
groups or long alkyl chains), can be complexed to adenoviral vectors by
hydrophobic
interaction with stable hydrophobic regions on the virus. Affinity masking
agents can be
made using any small molecule, peptide or protein which binds to adenovirus.
The affinity
and hydrophobic moieties may be attached to the masking agent by any method
known in
the art, preferably by chemical crosslinking with a chemical crosslinker.
If the masking agent is covalently attached, a chemical crosslinker is
preferably
used to covalently bond the masking agent to the adenovirus. The crosslinker
may be any
crosslinker capable of creating a covalent linkage or bridge between the
masking agent and
the adenovirus. Direct crosslinking, in which the adenovirus, masking agent
and a
separate crosslinker molecule are reacted, may be employed to created
covalently masked
adenovirus, using any chemical crosslinker known in the art which will create
crosslinks
between the masking agent and protein. Either the masking agent or the
adenovirus may
be modified prior to the crosslinking reaction, so that the chemical
crosslinker will react
with the two molecules (e.g., the masking agent may be modified to add amine
groups,
allowing it to be crosslinked to the adenovirus by crosslinking agents which
react with
amines).
Preferably, either the masking agent or the adenovirus is first activated by
reaction
with a crosslinking agent. Unreacted crosslinker is then removed from the
masking agent
or adenovirus. The activation reaction preferably results in one or two
molecules of
crosslinking agent per molecule of masking agent, more preferably a single
molecule of
crosslinking agent per-molecule of masking agent. The activated masking agent
or
adenovirus is then mixed with adenovirus (if the masking agent is activated)
or masking
agent (if the adenovirus is activated) under the appropriate reaction
conditions to form
masked adenovirus. Preferably, the masking agent is activated, then reacted
with
adenovirus.
43
._._ ._. ... _._.____.~......,..~._..____.....
.~....~~,. __ _ _ . .._....w....._~....~
__ ..._....W~-.,_,.W..~.~,...___w.~....~..~
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The preferred masking agent is PEG. Preferred activated PEGs include, but are
not
limited to: nucleophilic crosslinking PEGs such as end terminal amine PEG, PEG
amino
acid esters, PEG hydrazine hydrochloride, thiol PEGs, and the like; carboxyl
PEGs
including succinate PEG, carboxymethylated PEG, PEG-propionic acid, and PEG
amino
acids; sulfhydryl-selective PEGs such as PEG-maleimide, PEG-orthopyridyl-
disulfide and
the like; heterofunctional PEGs including amines and acids PEG, NHS-maleimide
PEG
and NHS-vinylsulfone PEG; PEG silanes; biotin PEGs; Vinyl derivatives of PEG
such as
allyl PEG, PEG acrylate, PEG methacrylate, and the like; and electrophilic
active PEGs,
including PEG succinimidyl succinate, PEG succinimidyl succinamide, PEG
succinimidyl
proprionate, succinimidyl ester of carboxymethylated PEG, PEG2-NHS,
succinimidyl
esters of amino acid PEGs, pendant modified PEG NHS esters (such as those
available
from Innophase, Inc.), PEG-glycidyl ether (epoxide), PEG-oxycarbonylimidazole,
PEG
nitrophenyl carbonates, PEG trichlorophenyl carbonates, PEG treslate, PEG-
aldehyde,
PEG-isocyanate, copolymers of PEG allyl ether and maleic anhydride, PEG
vinylsulfone,
and other activated PEGs as will be apparent to one of skill in the art. The
activated PEG
is preferably PEG-N-hydroxysuccinimidyl succinamide or PEG-succinimidyl
succinate,
more preferably PEG-N-hydroxysuccinimidyl succinamide.
Host Cells
The present invention also provides host cells comprising (i.e., transformed
with)
the adenoviral vectors described herein. Both prokaryotic and eukaryotic host
cells can be
used 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.
Host systems are known in the art and need not be described in detail herein.
Suitable host
cells also include any cells that produce proteins and other factors necessary
for expression
of the gene under control of the heterologous TREs, such factors necessary for
said
expression are produced naturally or recombinantly.
Suitable host cells for the adenovirus include any eukaryotic cell type that
allows
function of the heterologous TREs, preferably mammalian. For example, if the
heterologous TRE(s) used is prostate cell-specific, the cells are preferably
prostate cells,
for example LNCaP cells. The prostate cells used may or may not be producing
an
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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 DU145 can
be
used, provided an androgen receptor-encoding expression vector is introduced
into the
cells along with the adenovirus. If the heterologous TRE(s) used is derived
from the AFP
gene, for example, suitable host cells include any cell type that produces
AFP, including
but not limited to, Hep3B, HepG2, HuH7, HuHI/C12. Activity of a given TRE in a
given
cell can be assessed by measuring the level of expression of a operably-linked
reporter
gene using standard assays. The comparison of expression between cells in
which the
TRE 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 cell line. It is understood
that absolute
transcriptional activity of a TRE 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
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 adenoviral 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
can
comprise an effective amount of an adenoviral vector of this 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
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and the like. Formulations for parenteral and nonparenteral drug delivery are
known in the
art and are set forth in Remington :s Pharmaceutical Sciences, 18"' Edition,
Mack
Publishing (1990). Compositions also include lyophilized and/or reconstituted
forms of
the adenoviral vectors (including those packaged as a virus, such as
adenovirus) of the
invention.
Kits
The present invention also encompasses kits containing an adenoviral vector of
this
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 someone to detect the presence of target cells in a
suitable biological
sample, such as biopsy specimens.
The kits of the invention comprise an adenoviral vector 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.
Preparation of the adenovirus vectors of the invention
The adenovirus vectors of this invention can be prepared using recombinant
techniques that are standard in the art. Generally, heterologous TREs are
inserted 5' to the
adenoviral genes of interest, preferably one or more early genes (although
late gene(s) may
be used). Heterologous 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 adeno-DNA sequence or
introduced by
methods such as PCR or site-directed mutagenesis, provide an insertion site
for the
heterologous TREs. Accordingly, convenient restriction sites for annealing
(i.e., inserting)
heterologous TREs can be engineered onto the 5' and 3' ends of the
heterologous TRE
using standard recombinant methods, such as PCR.
Polynucleotides used for making adenoviral vectors of this invention may be
obtained using standard methods in the art such as chemical synthesis
recombinant
methods and/or obtained from biological sources.
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Adenovirus vectors containing all replication-essential elements, with the
desired
elements (e.g., ElA) under control of heterologous TREs, are conveniently
prepared by
homologous recombination or in vitro ligation of two plasmids, one providing
the left-
hand portion of adenovirus and the other providing the right-hand portion. The
resultant
adenovirus vector contains at least two different heterologous TREs, with at
least one of
the heterologous TREs cell-specific and one adenovirus gene under control of a
first
heterologous TRE and a second gene under control of a second heterologous TRE.
If
homologous recombination is used, the two plasmids should share at least about
500 bp of
sequence overlap. 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
heterologous
TREs for propagation of the adenovirus. Plasmids are generally introduced into
a suitable
host cell such as 293 cells using appropriate means of transduction, such as
cationic
liposomes. Alternatively, in vitro ligation of the right and left-hand
portions of the
adenovirus genome can be used to construct recombinant adenovirus derivative
containing
all the replication-essential portions of adenovirus genome. Berkner et al.
(1983) Nucleic
Acid Research 11:6003-6020; Bridge et al. (1989) J. Virol. 63:631-638.
For convenience, plasmids are available that provide the necessary portions of
adenovirus. Plasmid pXC.l (McKinnon (1982) Gene 19:33-42) contains the wild-
type
left-hand end of Ad5, from Adenovirus 5 nt 22 to 5790. pBHG10 (Bett et al.
(1994) Proc.
Natl. Acad. Sci. USA 91:8802-8806; Microbix Biosystems Inc., Toronto) provides
the
right-hand end of Ad5, with a deletion in E3. The deletion in E3 provides room
in the
virus to insert a 3-kb of TRE sequence or transgene sequence without deleting
the
endogenous enhancer-promoter. The gene for E3 is located on the opposite
strand from E4
(r-strand). pBHG11 [Bett et al. (1994)] provides an even larger E3 deletion
(an additional
0.3 kh is deleted).
For manipulation of the early genes, the transcription start site of Ad5 E 1 A
is at nt
498 and the ATG start site of this gene's coding segment is at nt 560 in the
virus genome.
This region can be used for insertion of a heterologous TRE. A restriction
site may be
3i) introduced by employing 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 used, the primers may use the EcoRl site in the
pBR322
backbone and the Xbal site at nt 1339 of Ad5. By carrying out the PCR in two
steps,
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where overlapping primers at the center of the region introduce a 30 sequence
change
resulting in a unique restriction site, one can provide for insertion of a
heterologous TRE at
that site.
A similar strategy may be used for insertion of a heterologous TRE to regulate
E 1 B. The E 1 B promoter of Ad5 consists of a single high-affinity
recognition site for Spl
and a TATA box. This region extends from Ad5 nt 1636 to 1701. By insertion of
a cell-
specific, heterologous TRE in this region, one can provide for cell-specific
transcription of
the E 1 B gene. By employing the left-hand region modified with a cell-
specific TRE
regulating E 1 A as the template for introducing a second, different cell-
specific TRE to
regulate E 1 B, the resulting adenovirus vector will be dependent upon the
cell-specific
transcription factors for expression of both EIA and E1B. Examples 1, 2 and 5
provide a
more detailed description of how such constructs can be prepared.
Similarly, a heterologous TRE may be inserted upstream of the E2 gene to make
its
expression cell-specific. 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 Microbiol. 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 codon 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
heterologous TRE having Spel ends into the Spel site in the plus-strand would
disrupt the
endogenous E2 early promoter of Ad5 and should allow TRE 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/CUG of ORF I is at nt 35532. Virtanen et al. (1984) J. Virol. 51:
822-831.
Using any of the above strategies for the other genes, a heterologous TRE may
be
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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 W 162
cells (Weinberg 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.
The methods of preparation of masked adenovirus vary according to masking
agent
and the mode of attachment (covalent or non-covalent). Masked adenovirus with
non-
covalently attached masking agent is prepared by thoroughly and intimately
mixing the
adenovirus and the masking agent. Covalent crosslinking is achieved by mixing
the
reaction components under conditions appropriate to the crosslinking reagent.
Chemical
crosslinking protocols are well known in the art. The exact reaction
conditions for any
given chemical crosslinker will vary according to the chemistry of the
crosslinker and the
modifications (if any) to the PEG and the adenovirus, as will be apparent to
one of skill in
the art. When the masking agent is PEG, the molar ratio of adenovirus to PEG
is
preferably between 1:1 x 106 and 1:1 x 107, more preferably about 1:4 x 106.
For the
preferred activated PEG, PEG-NHS-succinamide, the activated PEG is preferably
between
0.5 to 5 mM in the crosslinking reaction, more preferably about 2 mM.
Preferably
adenovirus in the crosslinking reaction is between 106 and 1012, more
preferably about 5 x
109. Using the preferred activated PEG, the pH of the crosslinking reaction is
preferably
between about 7 and 9, more preferably between about 7.5 and 8, and the
reaction is
preferably run for 10 to 30 minutes at a temperature ranging from about 4 C
to room
temperature (about 20 C).
Following the crosslinking reaction, the masked adenovirus is separated from
the
reaction components. The separation may be accomplished by any method known to
one
of skill in the art, including chromatographic methods such as size exclusion
chromatography, ion exchange chromatography or hydrophobic interaction
chromatography, electrophoretic methods, or filtration methods such as
dialysis,
diafiltration or ultrafiltration.
Methods using the adenovirus vectors of the invention
The subject 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
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using the adenoviral vectors described above. In one embodiment, methods for
using
adenovirus vectors comprise introducing an adenovirus vector into a cell,
preferably a
eukaryotic cell, more preferably a mammalian cell.
In one embodiment, methods for using adenovirus vectors comprise introducing
an
adenovirus vector into a target cell, preferably a neoplastic cell. In another
embodiment,
methods for using adenovirus vectors comprise introducing an adenovirus vector
into a
prostate cell. In another embodiment, methods for using adenovirus vectors
comprise
introducing an adenovirus vector into a liver cell. In another embodiment,
methods for
using adenovirus vectors comprise introducing an adenovirus vector into a
breast cancer
cell. In another embodiment, methods for using adenovirus vectors comprise
introducing
an adenovirus vector into a colon cancer cell.
In one embodiment, methods are provided for conferring selective cytotoxicity
in
cells which allow function of the target cell-specific TRE, comprising
contacting cells with
an adenovirus vector described herein, such that the adenovirus vector(s)
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. See Example 3,
Fig. 3.
In another embodiment, methods are provided for propagating an adenovirus
specific for cells which allow function of the heterologous TREs, preferably
eukaryotic
cells, more preferably mammalian cells. These methods entail combining an
adenovirus
vector with mammalian cells which allow function of the heterologous TREs,
whereby
said adenovirus is propagated.
Another embodiment provides methods of killing cells that allow a heterologous
TRE to function comprising combining the mixture of cells with an adenovirus
vector of
the present invention. The mixture of cells is generally a mixture of
cancerous cells in
which the heterologous 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 the
heterologous
TREs are 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 adenovirus vector, and replication of the
adenoviral 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
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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 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 adenoviral
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 adenovirus vector described
herein, wherein
the adenoviral vector enters the cell.
The invention further provides methods of suppressing tumor cell growth,
comprising contacting a tumor cell with an adenoviral vector of the invention
such that the
adenoviral vector enters the tumor cell and exhibits selective cytotoxicity
for the tumor
cell. As used herein, "tumor cells" and "tumor" 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. 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 adenoviral vector
described herein.
See Example 4, Fig.6.
The invention also provides methods of lowering the levels of a tumor cell
marker
in an individual, comprising administering to the individual an adenoviral
vector of the
present invention, wherein the adenoviral vector is selectively cytotoxic
toward cells
producing the tumor cell marker. Tumor cell markers include, but are not
limited to, PSA,
carcinoembryonic antigen and hK2. Methods of measuring the levels of a tumor
cell
marker are known to those of ordinary 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 marker. In general, a biological sample
is obtained
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from the individual to be tested, and a suitable assay, such as an ELISA, is
performed on
the biological sample. See Example 4, Fig. 7.
The invention also provides methods of treatment, in which an effective amount
of
an adenoviral vector(s) described herein is administered to an individual. For
example,
treatment using an adenoviral vector(s) in which at least one heterologous TRE
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 adenoviral vector(s) 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 comprising an
adenoviral
vector(s) is administered. Pharmaceutical compositions are described above.
The amount of adenoviral vector(s) to be administered will depend on several
factors, such as route of administration, the condition of the individual, the
degree of
aggressiveness of the disease, the particular heterologous TREs employed, and
the
particular vector construct (i.e., which adenovirus genes are under
heterologous TRE
control).
If administered as a packaged adenovirus, from about 104 to about 1014,
preferably
from about 104 to about 1012, more preferably from about 104 to about 1010. If
administered as a polynucleotide construct (i.e., not packaged as a virus),
about 0.01 gg to
about 100 g can be administered, preferably 0.1 gg to about 500 g, more
preferably
about 0.5 gg to about 200 gg. More than one adenoviral vector can be
administered, either
simultaneously or sequentially. Administrations are typically given
periodically, while
monitoring any response. Administration can be given, for example,
intratumorally,
intravenously or intraperitoneally.
The adenoviral vectors of the invention can be used alone or in conjunction
with
other active agents, such as chemotherapeutics, that promote the desired
objective.
The following examples are provided to illustrate but not limit the invention.
EXAMPLES
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EXAMPLE 1
Generation of adenovirus vector constructs in which a first adenovirus gene is
under
transcriptional control of an hKLK2-TRE and a second adenovirus gene is under
transcriptional control of a PSE-TRE
1.1 Construction of adenovirus constructs in which expression of one
adenovirus gene is
controlled by an hKLK2-TRE
To generate hKLK2-TRE adenovirus constructs, four hKLK2-TRE fragments which
contain at least the hKLK2 minimal promoter were amplified using the DNA
sequence
from approximately 12,000 bp lying upstream of the first exon in hKLK2 (SEQ ID
NO:3)
and synthetic oligonucleotides as described below. The four constructs were
generated by
ligating these fragments into pGEM-T vector.
= CN294 is a pGEM-T vector derivative containing an hKLK2 full promoter. The
fragment was amplified by PCR with oligonucleotide 42.100.1: 5'-GAT CAC CGG
TGT CCA CGG CCA GGT GGT GC-3' (SEQ ID NO:11) (PinAl site underlined),
which is complementary to the 5'-untranslated region (UTR) of the first exon
in
hKLK2, in combination with 42.100.2 (5'-GAT CAC CGG TGC TCA CGC CTG TAA
TCT CAT CAC-3'; SEQ ID NO:12; PinAl site underlined). 42.100.2 corresponds to
the upstream region of the hKLK2 promoter.
= CN296 is a pGEM-T vector derivative containing an hKLK2 fragment from nt -
2247 to
nt +33, which was amplified by PCR with oligonucleotides 42.100.1 and 42.100.3
(5'-
GAT CAC CGG TGG TTT GGG ATG GCA TGG CTT TGG-3'; SEQ ID NO:13,
PinAl site underlined). 42.100.3 corresponds to a region approximately 2300 bp
upstream of hKLK2.
= CN317 is a pGEM-T derivative containing the hKLK2 minimal promoter. A PCR
fragment corresponding to the hKLK2 5'-UTR from nt -323 to nt +33 was
amplified
with two synthetic oligonucleotides; 42.100.1 and 43.121.1 (5'-GAT CAC CGG TAA
AGA ATC AGT GAT CAT CCC AAC-3'; SEQ ID NO:14, PinAl site underlined).
= CN3 10 is a pGEM-T vector derivative containing an hKLK2 full promoter and
is
identical to CN294 except Eagl sites flank the insert. The fragment was
amplified by
PCR with oligonucleotide 42.174.1 (5'-GAT CCG GCC GTG GTG CTC ACG CCT
GTA ATC-3'; SEQ ID NO:15, Eagl site underlined) in combination with 42.174.2
(5'-
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GAT CCG GCC GTG TCC ACG GCC AGG TGG TGC AG-3'; SEQ ID NO:16; Eagl
site underlined).
hKLK2 promoter-driven ElA Ad5 plasmid CN303
CN303 was produced by inserting the hKLK2 promoter just upstream of the EIA
coding segment in a derivative of pXC- 1, a plasmid containing the left hand
end of' the
Ad5 genome, as follows.
= CN124 is a derivative of construct pXC-1 which contains the wild-type left
hand end
of Ad5, including both E 1 A and E 1 B(McKinnon (1982) Gene 19:33-42). CN124
also has, among other alterations, an artificial PinAl site at Ad5 nt 547
(just upstream
of the E 1 A transcriptional start at nt 560 and the E I A coding segment
beginning with
ATG at 610). CN124 was linearized with PinAl and dephosphorylated with calf
intestinal alkaline phosphatase (New England Biolabs).
= CN294 was digested with PinAl to free the hKLK2 promoter. The hKLK2 promoter
was then ligated into the PinAl linearized CN124, producing CN303. CN304 is
similar to CN303 except for the hKLK2 promoter fragment is in the reverse
orientation.
Thus, construct CN303 contains the hKLK2 promoter inserted upstream of and
operably linked to the E 1 A coding segment in the Adenovirus 5 genome.
hKLK2 promoter-driven EIB Ad5 plasmid CN316
CN316 was produced by inserting the hKLK2-promoter just upstream of the E 1 B
coding segment in a derivative of pXC-l, a plasmid containing the left hand
end of the
Ad5 genome, as follows.
= CN 124, described above, also contains an artificial Eagl site at Ad5 nt
1682, just
upstream of the E 1 B coding segment. The hKLK2 promoter was excised from CN3
10
with EagI and inserted into CN 124 digested with EagI to produce CN316. CN316
contains the hKLK2 promoter immediately upstream of and operably linked to the
E 1 B
coding segment.
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1. 2 Construction of adenovirus constructs in which expression of one
adenovirus
replication gene is controlled by an hKLK2-TRE and expression of another
adenovirus
replication gene is controlled by a PSE-TRE
Ad5 construct comprising an hKLK2-TRE driven EIA and a PSE-TRE driven EIB
(CN301)
CN301 was generated from CN125 by inserting an hKLK2 promoter upstream of
the E I A gene as follows.
= CN125 is a pXC-1 derivative in which expression of the E1B gene is driven by
PSE.
A PinAI site lies upstream of the El A gene, whose expression is driven by its
wild-
type promoter. CN 125 was created by inserting PSE as an EagI fragment from
construct CN 105 into the Eagl site immediately upstream of the E 1 B gene in
CN 124.
CN105 contains the PSE region from -5322 to -3875 relative to the PSA
transcription
start site.
= The hKLK2-TRE fragment was freed from CN294 by PinAl digestion and ligated
into
PinAl digested CN125 to create CN301.
The CN301 construct contains the hKLK2 promoter immediately upstream of and
operably-linked to the E1A gene and the PSE-TRE immediately upstream of and
operably-
linked to the E 1 B gene.
Ad5 constructs comprising PSE-TRE driven ElA gene and hKLK2 promoter driven EI
B
gene (CN323)
CN323 was constructed as follows so that the expression of ElA is mediated by
PSE-TRE, and expression of E 1 B is mediated by an hKLK2 promoter.
= CN314 is a plasmid containing a PSE-TRE fragment in pGEM-T vector. This PSE
fragment was amplified from CN706, an adenoviral construct in which a PSE-TRE
(SEQ ID NO:2) drives expression of the ElA transcription unit in Ad5, with two
synthetic oligonucleotides:
51.10.1 (5'-CTC ATT TTC AGT CAC CGG TAA GCT TGG-3'; SEQ ID NO:17) and
51.10.2 (5'-GAG CCG CTC CGA CAC CGG TAC CTC-3'; SEQ ID NO:18).
= The PSE-TRE fragment was isolated by digesting CN314 with PinAl and ligated
into
PinAl digested CN316 (described above).
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The CN323 construct is a plasmid containing PSE-TRE immediately upstream of
and operably-linked to the E 1 A gene and the hKLK2 promoter immediately
upstream of
and operably-linked to the E1B gene.
1. 3 Construction of additional adenoviral constructs comprising a first
adenoviral gene
under transcriptional control of an hKLK2-TRE and a second adenoviral gene
under
transcriptional control of a PSE-TREs
= CN306 was derived from CN124 by removing the endogenous 64-nucleotide E1A
promoter.
= CN421 was constructed by inserting an hKLK2-TRE (comprising an hKLK2
enhancer
from nucleotides -5155 to -3387 relative to the hKLK2 gene transcription start
site
(nucleotides 6859 to 8627 of SEQ ID NO:3) and an hKLK2 minimal promoter as in
CN379 into CN306. The hKLK2-TRE fragment was amplified by PCR from CN379,
digested with PinAl and ligated into similarly cut CN3 06, to produce CN42 1.
= CN438 was constructed by inserting an hKLK2-TRE (comprising an hKLK2
enhancer
from nucleotides -4814 to -3643 relative to the hKLK2 gene transcription start
site
(nucleotides 7200 to 8371 of SEQ ID NO:3) and a minimal hKLK2 promoter as in
CN390 into CN306. The enhancer fragment was amplified by PCR from CN390,
digested with PinAl and ligated into similarly cut CN306, to produce CN438.
= CN321 was created from CN306 by inserting a large PSE-TRE amplified from
CN96
(see US Patent No. 5,698,443; Rodriguez et al. (1997)).
= CN416 was constructed by inserting an hKLK2-TRE (comprising an hKLK2
enhancer
from nucleotides -5155 to -3387 relative to the hKLK2 gene transcription start
site
(nucleotides 6859 to 8627 of SEQ ID NO:3) and an hKLK2 minimal promoter as in
CN379 into CN321. The enhancer fragment was amplified by PCR from CN379,
digested with Eagl and ligated into similarly cut CN321, to generate CN416.
= CN422 was constructed by inserting an hKLK2-TRE (comprising an hKLK2
enhancer
from nucleotides -5155 to -3387 relative to the hKLK2 gene transcription start
site
(nucleotides 6859 to 8627 of SEQ ID NO:3) and an hKLK2 minimal promoter as in
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CN379 into CN369. CN369 is a derivative of CN306 in which the endogenous E1B
promoter was removed. The hKLK2-TRE was amplified from CN379, digested with
EagI, and ligated into similarly cut CN369 to produce CN422.
= CN444 was constructed by replacing the hKLK2-TRE of CN442 with an hKLK2-TRE
comprising an hKLK2 enhancer from nucleotides -4814 to -3643 relative to the
hKLK2
transcription start site (nucleotides 7200 to 8371 of SEQ ID NO:3) and a
minimal
hKLK2 promoter, as in CN390. The hKLK2-TRE was amplified from CN390, digested
with Eagl, and ligated into similarly cut CN369, to produce CN444.
= CN446 is similar to CN444, except that the endogenous E1B promoter was not
removed. The hKLK2-TRE was amplified from CN390, digested with EagI, and
ligated into similarly cut CN321, to produce CN446.
= CN459 and CN460 are similar to CN444, except that each contains an hKLK2-TRE
comprising an hKLK2 enhancer from nucleotides -3993 to -3643 relative to the
hKLK2
transcription start site and a hKLK2 minimal promoter, as in CN396.
= CN463 was constructed by inserting an hKLK2-TRE (comprising an hKLK2
enhancer
from nucleotides -4814 to -3643 relative to the hKLK2 transcription start site
and a
minimal hKLK2 promoter, as in CN390, into CN25 1. The hKLK2-TRE was excised
from CN446 with EagI, and ligated into similarly cut CN25 1, to produce CN463.
1. 4 Generation of recombinant adenoviruses
Adenovirus containing hKLK2-TRE were generated by homologous recombination
in 293 -cells. Briefly, CN301 was co-transfected with BHG10 (which contains
right hand
end of the adenovirus genome), into 293 cells. The cells were overlaid with
media, and
infectious virus generated by in vivo recombination was detected by cytopathic
effect and
isolated. Plaque-purified stocks of an adenovirus vector, designated CN747,
were
established. The structure of the recombinant virus was characterized by PCR,
restriction
endonuclease digestion and Southern blot. CN747 is a full-length Ad5 with the
hKLK2
promoter driving the expression of E 1 A and a PSE-TRE driving expression of E
1 B.
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Virus CN754 were generated with the same approach except that the CN301
plasmid was replaced with CN323. CN754 is a virus whose E 1 A and E 1 B are
under the
control of the PSE-TRE and the hKLK2-TRE, respectively.
Viruses CN753, CN755, CN759, CN761, CN763, CN764, CN765, CN767,
CN768, CN769, CN770, CN772 and CN773 were generated using the method as
described
above, from the parent plasmids CN326, CN328, CN398, CN316, CN421, CN416,
CN422, CN436, CN438, CN444, CN446, CN459, CN460, and CN463, respectively.
These viral constructs are shown schematically in Figures 2A, 2B and 2B.
EXAMPLE 2
Construction of adenovirus vectors in which expression of one viral
replication gene is
controlled by a PSE-TRE and expression of another is controlled by a PB-TRE
2.1 Generation of adenovirus constructs in which expression of one adenovirus
gene is
controlled by a PB-TRE
The 454 nucleotide fragment (nt about -426 to about +28) of the rat PB-TRE,
which contains two androgen response elements (ARE sites) and a promoter
element (SEQ
ID NO:4), was amplified by PCR using rat genomic DNA as template and the
following
oligonucleotides primers:
5'-GATCACCGGTAAGCTTCCACAAGTGCATTTAGCC-3', 42.2.1 (PinAI site
underlined) (SEQ ID NO: 19) and
5'-GATCACCGGTCTGTAGGTATCTGGACCTCACTG-3', 42.2.2 (SEQ ID
NO:20)
or primers:
5'-GATCCGGCCGAAGCTTCCACAAGTGCATTTAGCC-3', 42.2.3 (Eagl site
underlined) (SEQ ID NO:21) and
5'-GATCCGGCCGCTGTAGGTATCTGGACCTCACTG-3'. 42.2.4 (SEQ ID
NO:22)
The oligonucleotides created a unique PinAl (Agel) site (A/CCGGT) or Eagl site
(C/GGCCG) at both ends of the PCR fragments. The PCR fragments were ligated
into the
pGEM-T vector (Promega) to generate plasmids CN249 and CN250. Similarly, CN256
was created using the same strategy but the PB-TRE fragment was ligated into
the pCRT
vector (Invitrogen); CN271 is identical to CN250 but with a HindIII site at
the 5'-end.
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These plasmids provide the PB-TRE DNA fragments for the constructs reported
below.
In some of the adenovirus vectors described below, the endogenous (adenoviral)
TREs
were not deleted; rather, in each construct, the PB-TRE was inserted between
the
endogenous TRE (e.g., the ElA TRE) and its respective coding segment (e.g.,
the ElA
coding segment). In other vectors, the endogenous (Ad5) promoter-enhancer has
been
deleted, and the prostate-specific promoter-enhancer placed immediately
upstream of an
early gene.
PB-TRE-driven EIA Ad5plasmid (CN251)
An adenovirus vector in which expression of early gene EIA is under
transcriptional control of PB-TRE was constructed as follows.
CN 124 was linearized with PinAl and dephosphorylated with calf intestinal
alkaline phosphatase (New England Biolabs). CN249 was digested with PinAl to
free the
PB-TRE fragment. The PB-TRE fragment was then ligated into the PinAl-
linearized
CN124, producing CN25 1. CN253 is similar to CN251 except for the PB-TRE is in
the
reverse orientation.
Thus, construct CN251 contains the PB-TRE inserted upstream of and operably
linked to the EIA coding segment in the Adenovirus 5 genome. The vector CN253
is
similar, but the PB-TRE is in the reverse orientation.
PB-TRE-driven EI B Ad5 plasmid (CN254)
An adenovirus derivative in which the expression of early gene E 1 B is under
transcriptional control of the PB-TRE was constructed as follows.
CN124, which carries the left-end of Ad5, as described above, also contains an
artificial EagI site at Ad5 nt 1682, or just upstream of the E1B coding
segment. The PB-
TRE fragment was excised from CN250 with Eagl and inserted into CN124 digested
with
Eagl. This produced CN254, which contains the PB-TRE immediately upstream of
and
operably linked to the E 1 B coding segment.
CN255 is identical to CN254, but the orientation of the PB-TRE insert is
reversed.
CN275 is the same as CN254, but with a HindIII site at the 5'-end.
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2.2 Generation of adenovirus constructs in which expression of one adenovirus
replication gene is controlled by a PB-TRE and expression of another
adenovirus gene is
controlled by a PSE-TRE.
Adenovirus vector comprising a PB-TRE driven El A and a PSE-TRE driven EI
B(CN257)
An adenovirus vector in which expression of the E1 A gene is under control of
the
PB-TRE and expression of the E1B gene is under control of the prostate
specific antigen
transcriptional regulatory element (PSE-TRE) was constructed as follows. The
PSE-TRE
region has been described in detail in, inter alia, U.S. Patent Nos. 5,648,478
and 5,698,443;
Lundwall (1989) Biochim. Biophys. Res. Commun. 161:1151-1159; and Zhang et al.
(1997) Nucleic Acids Res. 25:3143-50.
The PinAI PB-TRE fragment was inserted into CN125 digested with PinAl, which
cleaves just upstream of ElA, to create construct CN257, which is a plasmid
containing a
PB-TRE operably linked to the E 1 A gene and a PSE-TRE operably linked to the
E 1 B
gene. CN258 is similar to CN257, but with the opposite orientation of the PB-
TRE
fragment.
Ad5 plasmid comprising PSE-TRE driven EIA and PB-TRE driven El B(CN273)
An adenovirus vector was constructed in which expression of E I A is mediated
by a
PSE-TRE and expression of E 1 B is mediated by a PB-TRE.
CN 143 is a pBluescript (Stratagene, La Jolla, California) derivative
containing the
PSE-TRE fragment. This fragment was excised with PinAl and ligated into PinAl-
digested CN254. The final construct is a plasmid containing a PSE-TRE operably
linked
to the E 1 A gene and a PB-TRE operably linked to the E I B gene. CN274 is
similar to
CN273 except for the opposite orientation of PB-TRE.
CN306 was derived from CN124 by removing E1A endogenous promoter of
64 nts. CN321 was created from CN306 with a inserting of large PSE fragment
amplified
from CN96.
CN326 was derived from CN321 by inserting PB-TRE into Eagl site. CN321 is a
plasmid containing PSE (from CN96) at the PinAl site of CN306.
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2.3 Construction of adenovirus constructs in which expression of one
adenovirus
replication gene is controlled by hKLK2-TRE and expression of another
adenovirus
replication gene is controlled by PB-TRE
CN463 was generated from CN251 by inserting an hKLK2-TRE into the Eagl site.
The hKLK2-TRE was excited from plasmid CN446 with Eagl, ligated into a
similarly cut
CN25 1, to produce CN463. The CN463 construct is a plasmid containing a PB-TRE
immediately upstream and operably linked to the E 1 A gene and an hKLK2-TRE
(comprising an hKLK2 enhancer from nucleotides -4814 to -3643 relative to the
hKLK2
transcription start site and minimal hKLK2 promoter) immediately upstream and
operably
linked to the E 1 B gene.
2.4 Generation of adenoviruses that contain two heterologous TREs
Adenoviruses that contain two heterologous TREs were generated by homologous
recombination in 293 cells. Briefly, 5 gg of CN257 and 5 g of BHG10, which
contains
the right hand end of Ad5, was co-transfected into 293 cells. The cells were
overlaid with
medium, and infectious virus, generated by in vivo recombination, was detected
by
cytopathic effect and isolated. Plaque-purified stocks of an adenovirus
vector, designated
CN739, were established. The structure of the recombinant virus was
characterized by
PCR, restriction endonuclease digestion and Southern blot. The viral genome of
CN739
has the E 1 A transcription unit of Ad5 under the control of PB-TRE while E 1
B is under the
control of PSE-TRE.
As shown in Figure 2, adenoviruses CN750, CN753, CN764, CN767, CN770,
CN772, CN774, were generated with the same approach except that CN257 was
replaced
with CN273, CN326, CN416, CN436, CN446, CN459, CN463, respectively.
EXAMPLE 3
In vitro characterization of adenoviral constructs comprising an adenoviral
gene under
transcriptional control ofprostate cell specific heterologous TREs
Plaque assays
To determine whether the adenoviral constructs described in Examples 1 and 2
replicate preferentially in prostate cells, plaque assays were performed. A
plaque assay is
an infectious quantitative assay that quantifies how efficiently a particular
virus produces
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an infection in a cell. Plaquing efficiency was evaluated in the following
cell types:
prostate tumor cell lines (LNCaP, PC-3), breast normal cell line (HBL-100),
breast
carcinoma cell line (MCF-7), ovarian tumor cell lines (OVCAR-3, SK-OV-3),
hepatocarcinoma cell lines (HepG2, SK-Hep-1), and human embryonic kidney cells
(293).
LNCaP cells express both androgen receptor and PSA, while the other cell lines
tested do
not. 293 cells serve as a positive control for plaquing efficiency, since this
cell line
expresses Ad5 E1A and E1B proteins. The plaque assay was performed as follows.
Confluent cell monolayers were seeded in 6-well dishes eighteen hours before
infection.
The monolayers were infected with 10-fold serial dilutions of each virus.
After infecting
monolayers for four hours in serum-free media (MEM), the media was removed and
replaced with a solution of 0.75% low melting point agarose and tissue culture
media.
Plaques were scored two weeks after infection. CN702 has no modifications in
its El
region and is used as a wild type control.
Adenovirus with heterologous TREs plaque assay data
(Percent of wild-type adenovirus (PFU/ml))
Table 1:
293 LNCaP HBL-100 OVCAR-3 SK-OV-3
Viruses
CN702 100 100 100 100 100
CN706 100 23 4.2 5.5 8.9
CN764 100 31 0.25 0.032 0.003
CN769 100 11 0.14 0.015 0.0008
CN770 100 24 0.27 0.036 0.084
CN772 100 29 0.27 0.096 0.21
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Adenovirus with heterologous TREs plaque assay data
(Percent of wild-type adenovirus (PFU/ml))
Table 2:
293 LNCaP HBL-100 OVCAR-3
Viruses
CN702 100 100 l00 100
CN706 100 25 2.7 7.7
CN753 100 33 0.067 0.52
CN755 100 29 0.52 0.6
Adenovirus with heterologous TREs plaque assay data
(Percent of wild-type adenovirus (PFU/ml))
Table 3:
293 LNCaP HBL-100 OVCAR-3
Viruses
CN702 100 100 100 100
CN706 100 33 2.5 3.4
CN739 100 35 0.12 0.0023
CN753 100 41 0.23 0.11
Adenovirus with heterologous TREs plaque assay data
(Percent of wild-type adenovirus (PFU/ml))
Table 4:
293 LNCaP HBL-100 OVCAR-3
Viruses
CN702 100 100 100 100
CN753 100 37 0.15 0.086
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CN765 100 20 0.22 0.012
CN772 100 29 0.27 0.096
Adenovirus with heterologous TREs plaque assay data
(Percent of wild-type adenovirus (PFU/ml))
Table 5:
293 LNCaP HBL-100 OVCAR-3 SK-OV-3
Viruses
CN702 100 100 100 100 100
CN706 100 23 4.2 6.1 8.9
CN764 100 31 0.25 0.032 0.003
CN769 100 11 0.14 0.015 0.0008
CN770 100 24 0.27 0.036 0.084
Adenovirus with heterologous TREs plaque assay data
(Percent of wild-type adenovirus (PFU/ml))
Table 6:
293 LNCaP PC-3 HBL-100 OVCAR-3 HepG2 SK-Hep 1
Viruses
CN702 100 100 100 100 100 100 100
CN706 100 33 74 2.4 1.8 12 1.8
CN739 100 35 46 0.058 0.0057 0.068 0.00
Adenovirus with heterologous TREs plaque assay data
(Percent of wild-type adenovirus (PFU/ml))
Table 7:
293 LNCaP MCF-7
Viruses
CN702 100 100 100
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CN706 100 25 1.5
CN737 100 26 0.11
Tables 1-7 show the results of plaque assays performed with the adenoviral
vectors
described in Examples 1 and 2. The results are expressed as percent of wild-
type
adenovirus plaque-forming units (PFU) per ml. The tables show the average
titer of
duplicate samples for the viruses tested. The titer for a particular virus in
all cell lines was
normalized to its titer on 293 cells. Once the titers on a cell type were
normalized to 293
cells, the normalized numbers of the recombinant viruses were compared to
CN702. A
ratio of less than 100 suggests that the virus tested plaques less efficiently
than CN702.
Conversely, a ratio greater than 100 suggests that the virus plaques more
efficiently than
CN702.
The following observations were made. First, hKLK2-TRE, PSE-TRE and PB-
TRE engineered adenoviruses demonstrate preferential replication in prostate
tumor cells.
Since this carcinoma expresses androgen receptors, the PSE, hKLK2 and PB TREs
contained in the adenoviral vectors should be active in promoting the
transcription of the
adenovirus early genes. The data presented in Tables 1-7 suggest that the
heterologous
TRE containing adenoviral vectors induce cytopathic effects with a slightly
lower
efficiency than wild type adenovirus in prostate tumor cells. Second, hKLK2-
TRE, PSE-
TRE and PB-TRE containing adenoviruses show a dramatically lower plaquing
efficiency
in non-prostate tumor cells when compared to wild type. For example, in the
ovarian
carcinoma cell line OVCAR-3, CN764 and CN739 produced 3,000- and 10,000-fold
less
plaques than wild type Ad5, respectively. The results are similar for these
two viruses in
HBL- 100 cells, where virus replication is also severely compromised. Third,
adenoviral
vectors containing two prostate cell specific heterologous TREs give 10 to 100-
fold (or
more) less plaques in non-prostate cells that an adenoviral vector containing
a single
heterologous prostate cell specific TRE despite the titers of the two types of
adenovirus
- vectors being similar in LNCaP cells. For example, PSE-TRE and hKLK2-TRE
(CN764)
or PSE-TRE and PB-TRE (CN739) adenovirus vectors give 10- to 100-fold less
plaques in
HBL- 100 and OVCAR-3 cells than CN706, although their titers were similar to
CN706 in
LNCaP cells. Thus, adenoviruses engineered with two different prostate cell
specific
TREs were significantly attenuated relative to wild-type adenovirus and CN706
in non-
prostate cells, but they showed similar activity to CN706 in LNCaPs.
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As indicated above, adenoviruses containing two heterologous TREs demonstrated
an unexpectedly high preferential replication in prostate tumor cells as
compared to wild
type adenovirus and to CN706, in which a PSE-TRE controls EIA. This increase
in
specificity is more than an additive effect of inserting a second prostate-
specific TRE.
Since CN739 and CN764 appeared to be the most specific viruses, they were
further characterized in the following experiments.
Cytopathic effects
To characterize the differential viral replication and cytopathic effects
(CPE), CPE
assays were performed as follows. Cells were infected with virus at increasing
multiplicities of infection (MOI) and monitored for cytopathic effect. Assays
were
terminated when complete cytolysis of the monolayers was observed at an MOI of
0.01
with wild-type adenovirus. One primary, non-immortalized human microvascular
endothelial cell line (hMVEC) was chosen to test its sensitivity to CN764 and
wild-type
adenovirus (CN702) infection, in vitro. As shown in Figure 3, CN702 caused
complete
monolayer cytolysis of hMVECs at MOIs as low as 0.01 within 10 days. In
contrast,
CN764 infected hMVEC monolayers did not show significant cytopathic effects at
the
same time points with MOIs of 10, 1.0, 0.1 and 0.01. Cytolysis of hMVECs
equivalent to
that seen with wild-type adenovirus was only evident at MOIs between 100 and
1000
times as high (MOI>10).
When the CPE assay was performed with CN739, the results were similar to that
of
CN764 as CN739-infected hMVEC monolayers did not show significant cytopathic
effects
at the same time points with MOIs of 10, 1.0, 0.1 and 0.01.
Thus, CN764 and CN739-mediated cytolysis is significantly attenuated relative
to
wild-type adenovirus in primary normal human cells.
Differential viral replication
To determine if levels of virus replication correlate with the cytopathic
effects of
CN739 in prostate tumor cells or human normal cells, virus replication
titration was
carried out on PSA producing prostate tumor cells (LNCaP) and primary human
microvascular endothelial cells (hMVECs). Cells were grown to 70-90%
confluence and
infected with either wild-type adenovirus (CN702) or CN739, CN764, CN765,
CN770 for
90 min at a MOI of 10. Fifty-five hours after infection, the virus was
released from the
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cells by three freeze/thaw cycles, and the resulting supernatant was titered
on 293 cells.
The amount of CN739 produced 56 hours after infection was normalized against
the
amount of wild-type virus produced in the same cell line during the same time
period. The
data, shown in Figure 4, indicate that the titers of adenovirus vectors
containing two
different prostate cell specific TREs were 30% of CN702 titers in LNCaPs, but
were
reduced to less than 1/100those of the wild-type viruses in normal cells.
These data
suggest that CN764-like viruses replicate poorly in primary normal human
cells, and are
somewhat attenuated in prostate cancer cells.
One-step growth curve
The efficiency and kinetics of replication of CN739, CN706 and CN702 were
measured using a one-step growth curve assay in LNCaPs and hMVECs. Replicate
monolayers of LNCaPs and hMVECs were infected at a MOI of 10 to obtain a
synchronous infection of all the cells. Duplicate cultures were harvested at
various times
post-infection. The number of infectious virus was determined by plaque assay
on 293
cells (Figure 5). CN739 and CN706 grew at a similar efficiency in LNCaPs.
However,
under identical conditions, CN739 grew poorly in the hMVECs, producing about
10,000-
fold and 80,000-fold less infectious virus than CN706 and wild-type
adenovirus,
respectively.
Thus, the one-step growth curve demonstrated that an adenovirus containing two
different prostate cell specific TREs, each controlling a different adenoviral
gene, grew
well in prostate tumor cells but grew poorly in non-prostate endothelial
cells, indicating
significantly enhanced specificity for target cells.
Stability of the adenoviruses containing two different heterologous TREs
The use of two different heterologous TREs in the adenovirus vectors appears
to
provide stability to the genome during adenoviral replication. CN739 and CN764
were
plaque purified for three times and their DNA were examined by PCR, as well
as,
Southern blot analysis. By this analysis, the viral genomes do not appear to
have
undergone sequence rearrangement or loss since the fragments were of the
expected sizes.
The results indicate that the adenoviral genome of the adenovirus vectors
containing two
different heterologous TREs are stable.
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EXAMPLE 4
Testing cytotoxic ability of adenovirus vectors on tumor xenografts
An especially useful objective in the development of target cell specific
adenoviral
vectors is to treat patients with neoplasia comprising the target cells. For
example, the
prostate cell specific adenoviral vectors described above may be useful to
treat patients
with prostate carcinoma. An initial indicator of the feasibility is to test
the vectors using a
technique known in the art, such as testing the vectors for cytotoxicity
against neoplastic
cell, such as prostate carcinoma, xenografts grown subcutaneously in Balb/c
nu/nu mice.
To examine the therapeutic efficacy of CN739 in vivo, LNCaP tumor xenografts
were
grown in athymic mice. The tumor cells were injected subcutaneously into each
flank of
each mouse, and after establishment of palpable tumors (mean tumor volume 300
mm),
the tumors were directly injected with CsCI purified CN739 at 2.5 x 108
particles per mm3,
or PBS containing 10% glycerol (vehicle) as a control. Tumor growth was then
followed
for 6 weeks, at which time the mean tumor volume in each group was determined
and
serum samples were collected for PSA analysis on day 0 and weekly thereafter.
The data depicted in Figure 6 show that treatment of LNCaP tumors with CN739
resulted in an 80% reduction in average tumor volume whereas the average tumor
volume
in vehicle-treated group I, at day 28, had increased to 400% of the initial
volume. Four of
seven (57%) animals in CN739-treated group Il were free of palpable tumors at
day 42.
This study demonstrates that a fixed, single dose of an adenovirus containing
two different
prostate cell specific TREs, each operably linked to two different adenoviral
genes,
(CN739) per tumor is efficacious against LNCaP xenografts in vivo.
Effects on serum PSA levels
The serum PSA level is a widely used marker for the diagnosis and management
of
prostate carcinoma. LNCaP cells express and secrete high levels of PSA into
the culture
media and into circulation. An experiment was designed to examine the effects
of
treatment with CN739 on the serum PSA concentration in mice with LNCaP tumor
xenografts.
Following treatment, the average serum PSA level in group I (vehicle only)
increased to approximately 800% of the initial value by day 28, whereas the
average PSA
level in group II (CN706 treatment) and group III (CN739 treatment) remained
essentially
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constant through day 21 and declined to 10% of the initial value by day 35
(Figure 7).
There was a statistically significant difference (p<0.001; T-test) between
group I and
group II on day 14 and thereafter.
These results demonstrate that CN739 treatment is efficacious in the LNCaP
xenograft model when the outcome is measured either by reduction in tumor
growth or
serum PSA concentration. Taken together with the in vitro data, it appears
that an
adenovirus with two different prostate cell specific TREs (CN739) has a
therapeutic
efficacy in vivo similar to that of an adenovirus with a single prostate cell
specific TRE
(CN706) but in addition has a higher cell-specificity than CN706.
While it is likely that a therapeutic based on the viruses described here
would be
given intralesionally (i.e., direct injection), it would also be desirable to
determine if
intravenous (IV) administration of the virus can affect tumor growth. If so,
then it is
conceivable that the virus could be used to treat metastatic tumor deposits
inaccessible to
direct injection. For this experiment, groups of five mice bearing prostate
cancer tumors
are inoculated with 108 pfu of an adenoviral vector of the present invention
by tail vein
injection, or 108 pfu of a replication defective adenovirus (CMV-LacZ) to
control for non-
specific toxic effects of the virus, or with buffer used to carry the virus.
The effect of IV
injection of the adenoviral vector on tumor size is compared to the sham
treatment.
EXAMPLE 5
Construction of other adenovirus constructs comprising a first adenoviral gene
under
control of a cell specifrc heterologous TRE and at least a second gene under
control of a
second heterologous TRE, where both heterologous TREs are functional in the
same cell.
Adenovirus vectors containing heterologous TREs may be generated so as to
target
the adenoviral replication to a variety of neoplasia. The following describes
examples of
cell specific (in some cases, tumor cell specific) TREs that may be used to
control the
expression of adenovirus genes so as to lead to replication of the adenovrus
vectors
preferentially in the target cells as compared to non-target cells. Cell
specific adenovirus
vectors are listed under a type of neoplsia for which the vectors may provide
a treatment.
Several of the adenovirus vectors may be useful in the treatment of more than
one type of
neoplasia because the TREs are functional in more than one type of tumor cell,
as
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described herein. Schematic diagrams of exemplary adenovirus vectors are
depicted in
Fig. 8 (A)-(B).
Generation of liver neoplasia specific adenovirus vectors.
As described above, TREs from the AFP gene have been shown to be specifically
active in liver neoplasia. Human AFP enhancer domains A and B (included in the
region -
3954 bp to -3335 bp relative to the AFP cap site) were PCR amplified from
human
genomic DNA using the following primers:
5' GTGACCGGTGCATTGCTGTGAACTCTGTA 3' (39.055B) (SEQ ID NO:23) and
5' ATAAGTGGCCTGGATAAAGCTGAGTGG 3' (39.044D) (SEQ ID NO:24)
The AFP promoter was amplified from -163 to +34 using the following primers:
5' GTCACCGGTCTTTGTTATTGGCAGTGGT 3' (39.055J) (SEQ ID NO:25)
5' ATCCAGGCCACTTATGAGCTCTGTGTCCTT 3' (29.055M) (SEQ ID NO:26)
The enhancer and promoter segments were annealed, and a fusion construct was
generated using overlap PCR with primers 39.055B and 39.055J. This minimal
enhancer/promoter fragment was digested with PinAl and ligated with CN124
using the
engineered Agel site 5' of the E 1 A cap site to produce CN219. In CN219, the
AFP-TRE
is immeadiately upstream of and operably linked to the E1A coding sequence.
The AFP-TRE described above was amplified with the following primers (Eagl
sites under lined):
5' TATCGGCCGGCATTGCTGTGAACTCT 3' (39.077A) (SEQ ID NO:27) and
5' TTACGGCCGCTTTGTTATTGGCAGTG 3' (39.077C) (SEQ ID NO:28)
The PCR product was digested with EagI and ligated into the Eagl site
immediately
upstream of the EIB gene in CN124 to make CN234. In CN234, the AFP-TRE is
immeadiately upstream of and operably linked to the E 1 B coding sequence.
uPA promoter and the other transcription response element (Riccio et al.
(1985)
Nucleic Acids Res. 13:2759-2771; Cannio et al. (1991) Nucleic Acid.s Res.
19:2303-2308)
are amplified by PCR with Eagl sites at the ends and ligated into EagI
digested CN219, to
generate CN479. CN479 is a construct with the AFP-TRE operably linked to E1A
and
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uPA-TRE operably linked to E1B. Similarly, uPA-TRE is engineered into CN234 at
the
PinAl site, to produce CN480. CN480 is a construct in which E 1 A is under
transcriptional
control of uPA-TRE and E1B is under transcriptional control ofAFP-TRE.
Generation of breast cancer cell specific adenovirus vectors.
As described above, TREs from the MUCI gene have been identified that are
specifically active in breast cancer cells. An adenovirus vector in which
expression of the
E 1 A gene is under control of MUCI -TRE was constructed as follows.
The MUCI-TRE region of SEQ ID NO:29 was amplified from human genomic
DNA by PCR with the following primer pairs:
5' TAA TCC GGA CGG TGA CCA CTA GAG GG 3' (39.088A, SEQ ID NO:30) and
5' TAT TCC GGA TCA CTT AGG CAG CGC TG 3' (39.088B, SEQ ID NO:3 1).
The primers were constructed with BspEl ends, which are compatible with the
Agel site in CN124. As described herein, CN124 has an Agel site at Ad5 nt 547
(just
upstream of the E 1 A transcriptional start at nt 498 and the E 1 A coding
segment beginning
with ATG at 610) and an Eagl site at Ad5 nt 1682, or just upstream of the E1B
coding
segment. The MUCI -TRE PCR product was digested with Bspl and ligated into the
Agel
site of CN124 to make CN226. In CN226, the MUCI-TRE is immeadiately upstream
of
and operably linked to the E 1 A coding sequence.
The MUCI -TRE was amplified from CN226 to include EagI ends with the
following primer pairs:
5' TAA CGG CCG CGG TGA CCA CTA GAG 3' (39.120A, SEQ ID NO:32) and
5' TAT CGG CCG GCA GAA CAG ATT CAG 3' (39.120B, SEQ ID NO:33).
The MUCI-TRE PCR product was digested with EagI and ligated in the Eagl site
CN 124 to make CN292. In CN292, the MUC 1-TRE is immediately upstrem of and
operably linked to the E 1 B gene.
Human HER-2/neu (HER) -TRE (Tal et al. (1987) Mol. Cell. Biol. 7:2597-2601;
Hudson et al. (1990) J. Biol. Chem. 265:4389-4393; Grooteclaes et al. (1994)
Cancer Res.
54:4193-4199; Ishii et al. (1987) Proc. Natl. Acad. Sci. USA 84:4374-4378;
Scott et al.
(1994) J. Biol. Chem. 269:19848-19858) is amplified from human genomic DNA
with an
Eagl site at end, ligated into a Eagl cut CN226, to produce CN481. CN481 is a
construct
in which the MUCI -T RE is operably linkedad to the E 1 A gene and HER-TRE is
operably
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linked to the E1B gene. Similarly, HER-TRE is amplified from CN481 by PCR with
PinAI site at the end, and ligated into a similarly cut CN292; to produce
CN482. CN482 is
a construct in which the HER-TRE is operably linked to the E 1 A gene and the
MUC1-T RE
is operably linked to the E1B gene.
Another set of breast cancer cell specific adenoviral vectors is generated
when the
uPA-TRE is released from CN485 with Eagl and ligated into a similarly cut CN48
1, to
produce CN487. CN487 is a construct in which the MUCI-TRE is operably linked
to the
E 1 A gene and the uPA-TRE is operably linked to the E 1 B gene. CN488 is the
same as
CN487 except the positions of two heterologousTREs are exchanged. CN488 is a
construct in which the uPA-TRE is operably linked to the El A gene and the
MUCI -TRE is
operably linked to the E1B gene.
Another set of breast cancer cell specific adenoviral vectors is generated
when the
CEA-TRE is released from CN484 with Eagl and ligated into a similarly cut
CN487, to
produce CN489. CN489 is a construct in which the MUCI-TRE is operably linked
to the
E1A gene and the CEA-TRE is operably linked to the E1B gene. CN490 is the same
as
CN489 except the positions of two heterologousTREs are exchanged. CN490 is a
construct in which the CEA-TRE is operably linked to the E1A gene and the MUCI-
TRE
is operably linked to the E 1 B gene.
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Generation of colon cancer cell specific adenovirus vectors.
As described above, TREs from the CEA gene have been identified that are
specifically active in a number of neoplasia including colon cancers. An
adenovirus vector
in which expression of the E1A gene is under control of CEA-TRE was
constructed as
follows.
A TRE of the carcinoembryonic antigen (CEA-TRE), about -402 to about +69 bp
relative to the transcriptional start (SEQ ID NO:7), was amplified by PCR from
human
genomic DNA using primers(which introduced Agel sites at the ends):
5' ATT ACC GGT AGC CAC CAC CCA GTG AG 3' (39.174B, SEQ ID NO:34) and
5' TAG ACC GGT GCT TGA GTT CCA GGA AC 3' (39.174D, SEQ ID NO:35).
The CEA-TRE PCR fragment was ligated into pGEM-T vector which had been
linearized with EcoRV and designated CN265. The CEA-TRE was excised from CN265
by digestion with PinAl and was ligated into similarly digested CN124 to
generate CN266.
CN266 is a construct in which the CEA-TRE is operably linked to the E1A gene.
The CEA-TRE was amplified from CN266 by PCR using primers:
5' TAA CGG CCG AGC CAC CAC CCA 3' ( 39.180A, SEQ ID NO:36) and
5' TAT CGG CCG GCT TGA GTT CCA GG 3' (39.180B, SEQ ID NO:37)
The unique restriction site Eagl was introduced by the primer pair at the ends
of the
PCR amplified product. The PCR product was ligated into pGEM-T Vector
(Promega),
and the resultant plasmid designated CN284. The Eagl CEA-TRE fragment was
excised
from CN284 and isolated by gel electrophoresis, and ligated into CN124 which
had been
cut with Eagl to make CN290. CN290 is a construct in which the CEA-TRE is
immediately upstream of and operably linked to the E I B gene.
A uPA-TRE is released from CN479 with Eagl and ligated into a similarly cut
CN266, to produce CN483. CN483 is a construct in which the CEA-TRE is operably
linked to the ElA gene and the uPA-TRE is operably linked to the E1B gene.
Similarly,
CN484 is a construct in which the uPA-TRE is operably linked to the E 1 A gene
and the
CEA-TRE is operably linked to the E 1 B gene.
Generation of colon cancer cell specific adenovirus vectors.
CN485 is a construct in which the HER-TRE is operably linked to the E 1 A gene
and the uPA-TRE is operably linked to the EIB gene. uPA-TRE is released from
CN479
with Eagl and ligated to a similarly cut CN482, to produce CN485.
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CN486 is the same as CN485 except the position of two heterologous TREs are
exchanged. CN486 is a construct in which the uPA-TRE is operably linked to the
E1A
gene and the HER-TRE is operably linked to the E1B gene.
Generation additional adenovirus vectors containing multiple heterologous
TREs.
The invention does not exclude additional genes under transcriptional control
of
heterologous TREs. Accordingly, adenovirus vectors may be generated in which a
third
gene is under trascriptional control of a third heterologous TRE, where all
the TREs are
different from each other and all are functional in the same cell.
An adenovirus vector is generated through the insertion of the PB-TRE, the PSE-
TRE, and the hKLK2-TRE in operable linkage with three genes, for example, the
E 1 A,
E1B, and E4 genes. These prostate cell specific TREs are amplified as
described above
and inserted into the adenovirus vectors as described herein. Fig. 9 depicts
examples of
such adenoviral constructs.
Also depicted in Fig. 9 is an example of a construct in which three genes are
controlled by three other cell specific TREs described above. The adenovirus
construct
CN781, for example, may be useful to target liver neoplasia in which the HER-
TRE is
functional.
Adenoviruses that contain multiple heterologous TREs are generated by
homologous recombination in 293 cells as described above.
EXAMPLE 6
Characterization of an E3 deleted adenovirus, CN751, that contains the
adenovirus death
protein gene
- An Adenovirus death protein mutant, CN75 1, was constructed to test whether
such
a construct may be more effective for cytotoxicity. The Adenovirus death
protein (ADP),
an 11.6 kD Asn-glycosylated integral membrane peptide expressed at high levels
late in
infection, migrates to the nuclear membrane of infected cells and affects
efficient lysis of
the host. The Adenovirus 5(Ad5) E3 region expresses the adp gene.
Construction of CN751
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CN751 was constructed in two parts. First, an E3 deleted platform plasmid that
contains Ad5 sequence 3' from the BamHI site at 21562 bp was generated. The
Ad2 adp
was engineered into the remainder of the E3 region of this plasmid to yield
CN252. An
ADP cassette is constructed using overlap PCR. The Y leader, an important
sequence for
correct expression of some late genes, is PCR-amplified using primers:
5' GCCTTAATTAAAAGCAAACCTCACCTCCG...Ad2 28287 bp (37.124.1)
(SEQ ID NO:38); and
5' GTGGAACAAAAGGTGATTAAAAAATCCCAG...Ad2 28622 bp (3 ) 7.146.1)
(SEQ ID NO:39).
The ADP coding region is PCR amplified using primers:
5' CACCTTTTGTTCCACCGCTCTGCTTATTAC...Ad2 29195 bp (37.124.3)
(SEQ ID NO:40) and
5' GGCTTAATTAACTGTGAAAGGTGGGAGC...Ad2 29872 bp (37.124.4)
(SEQ ID NO:41).
The two fragments were annealed and the overlap product was PCR amplified
using primers 37.124.1 and 37.124.4. The ends of the product were polished
with Klenow
fragment and ligated to BamHl cut pGEM-72 (+) (CN241; Promega, Madison, WI).
The
ADP cassette was excised by digesting CN241 with Pac 1 restriction
endonuclease and
ligated with two vectors, CN247 and CN248 generating plasmids CN252 and CN270,
respectively. CN247 contains a unique PacI site in the E3 region and was
constructed as
follows. A plasmid containing the full length Ad5 genome, TG3602 (Transgene,
France),
was digested with BamHl and religated to yield CN221. The backbone of this
plasmid
(outside of the Ad5 sequence) contained a PacI site that needed to be removed
to enable
further manipulations. This was effected by digesting CN221 with PacI and
polishing the
ends with T4 DNA polymerase, resulting in CN246. CN246 was digested with AscI
and
AvrII (to remove intact E3 region). This fragment was replaced by a similarly
cut
fragment derived from BHG11. The resulting plasmid, CN 247, contained a
deleted E3
region and a PacI site suitable for insertion of the ADP cassette fragment
(described
above). Ligation of CN247 with the ADP cassette generated CN252.
To construct the second part, the 5' Ad5 sequence necessary for CN751 was
obtained by digesting purified CN702 DNA with EcoRl and isolating the left
hand
fragment by gel extraction. After digesting CN252 with EcoRl, the left hand
fragment of
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CN702 and CN252 were ligated. 293 cells were transfected with this ligation
mixture by
using the standard lipofection transfection protocol developed at Calydon,
Inc. and
incubated at 37 C. Ten days later, the cells were harvested, freeze-thawed
three times, and
the supernatant was plaqued on 293 monolayers. Individual plaques were picked
and used
to infect monolayers of 293 cells to grow enough virus to test. After several
days, plate
lysates were screened using a polymerase chain reaction (PCR) based assay to
detect
candidate viruses. One of the plaques that scored positive was designated CN75
1.
Siructural Characterization of 'CN751
The structure of CN751 was confirmed by two methods. First, primers 37.124.1
and 37.124.4 were used to screen candidate viruses by PCR to detect the
presence of the
adp cassette. CN751 produced an extension fragment consistent with the
expected product
(1065bp). Second, CN751 was analyzed by Southern blot. Viral DNA was purified,
digested with PacI, SacI, and Accl/Xhol, and probed with a sequence homologous
to the
ADP coding region. The structure of CN751 matched the expected pattern.
In Vitro Characterization of CN751
Two experiments were conducted to examine the cytotoxicity and virus yield of
CN75 1. In the first study, CN751's cytotoxicity was evaluated in LNCaP cells
by
measuring the accumulation of a cytosolic enzyme, lactate dehydrogenase (LDH),
in the
supernatant over several days. The level of extracellular LDH correlates with
the extent of
cell lysis. Healthy cells release very little, if any, enzyme, whereas dead
cells release large
quantities. LDH was chosen as a marker because it is a stable protein that can
be readily
detected by a simple protocol. CN751's ability to cause cell death was
compared to that of
CN702, a vector lacking the ADP gene, and Rec700, a vector containing the ADP
gene.
Monolayers of LNCaP cells were infected at a multiplicity of infection of one
with
either CN702, Rec700, or CN751 and then seeded in 96 well dishes. Samples were
harvested once a day from one day after infection to five days after infection
and scored
using Promega's Cytotox 96 kit. This assay uses a coupled enzymatic reaction
which
converts a tetrazolium salt to a red formazan product that can be determined
in a plate
reader at 490nm.
Since the absorbance of a sample corresponds to the level of LDH released from
infected cells, a plot of how a sample's absorbance changes with time
describes how
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efficiently the viruses studied induce cell lysis (Figure 10). Each dta point
represents the
average of sixteen separate samples. The results suggest that CN751 kills
cells more
efficiently than the adp- control, CN702, and similarly to the adp+ control,
Rec700. The
concentration of LDH in the supematant increases rapidly from two days and
reaches a
maximum at four days in wells infected with CN75 1. In contrast, LDH
concentration in
the supernatant of CN702 infected cells begins to rise slowly at two days and
continues
until the conclusion of the experiment. Significantly, the amount of LDH
released from
CN751 infected cells at three days is two times that released from CN702
infected cells.
The data demonstrate that adenovirus vectors with adp gene kill cells more
efficiently than
adenovirus vectors that lack the adp gene.
Not only is it important for adnovirus vectors to kill cells efficiently, they
must also
be able to shed progeny that can infect other target cells. Viral vectors that
can shed large
amounts of virus might be better therapeutics than those that shed only small
amounts. A
virus yield assay was undertaken to evaluate whether CN751 can induce the
efficient
release of its progeny from the infected cell. A549 cells were infected at an
MOI of five.
Supernatant was harvested at various times after infection and titered on 293
cells to
determine the virus yield (Figure 11). The data suggests that cells infected
with CN751
shed virus more efficiently than those infected with CN702. At forty-eight
hours post
infection, CN751 infected cells have released ten times more virus than CN702
infected.
At seventy-two hours post infection, CN751 infected cells have released forty
times more
virus. In sum, the virus yield data demonstrate that adenovirus vectors with
the adp gene
release more virus.
In Vivo Characterization of CN751
LNCaP nude mouse xenografts were challenged with a single intratumoral dose (1
- X 104 particles/mm3 tumor) of either CN75 1, a vector containing the ADP
gene, or
CN702, a vector lacking the gene. A third group of tumors was treated with
buffer alone.
The tumors were monitored weekly for six weeks and their relative volume was
graphed
against time. The results are shown in Figure 12. Error bars represent the
standard error
for each sample group. The initial average tumor volume for CN751 treated
animals (n =
14) was 320 mm3 for CN702 treated (n = 14), and 343 mm3 for buffer treated (n
= 8). The
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data suggest that CN751 kills tumor cells more effectively than CN702. On
average,
tumors challenged with CN751 remained the same size throughout the course of
the
experiments while nine out of fourteen tumors (64%) regressed. Those treated
with
CN702 doubled in size. Buffer treated tumors grew to nearly five times their
initial
volume. The Students T-test indicates that the difference in tumor size
between CN751
and CN702 treated tumors was statistically significant from day 7 (p = 0.016)
through the
end of the experiment (p = 0.003).
EXAMPLE 7
Preparation of covalently pegylated adenovirus
A series of experiments were carried out to alter the surface capsids of
adenovirus
by complexing adenovirus with PEG. The objective of PEG complexation masking
of the
adenovirus surface is the following: 1) adenovirus neutralizing antibodies or
opsinins
which are in circulation, and 2) increase systemic circulation time of
adenovirus particles
by reduction of non-specific clearance mechanism in the body (i.e.,
macrophages, etc.).
Surface capsids of wild type adenovirus CN706 were modified through covalent
attachment of PEG to hexon and fiber proteins using N-hydroxysuccinimidyl
succinamide
(NHS). The PEGs (Shearwater Polymers, Inc.) had nominal molecular weight of
5000 Da.
The activated PEG (approximately 2 mM) was reacted with 5 x 109 particles/ml
adenovirus in a tris-HCl buffer (the approximate molar ratio of virus particle
to PEG was
1:4 X 106). Various combinations of pH, temperature, and reaction times were
used.
After the reaction, unreacted activated PEG, unreacted adenovirus, and
pegylated
adenovirus were separated by anionic ion exchange chromatography on Q
Sepharose XL
(Pharmacia), running a 0 to 1.5 M NaCI gradient in 50 mM tris, pH 8Ø The
gradient was
run over 10 column volumes.
Characterization of PEG-CN706
Pegylation of CN706 was verified by SDS-Page. Figure 14 depicts the pegylation
of CN706 and the mobility shift of pegylated proteins. Lanes 1 and 2 are non-
pegylated
CN706 (control), lanes 3 through 6 are pegylated CN706 under several pH and
temperature conditions. Lanes 3 through 6 show the appearance of a second band
above
the hexon proteins of CN706, most likely pegylated hexon, and the loss of the
fiber protein
band. Since no additional bands associated with the virus except that
corresponding to the
78
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CA 02283231 1999-09-02
WO 98/39464 PCT/US98/04080
PEG-hexon protein, the pegylated fiber protein is assumed to be under one of
the
unpegylated proteins on the SDS gel.
Figure 15 is an ion exchange chromatogram showing the change in surface
properties of CN706. Pegylation of CN706 results in its earlier elution from
the Q
Sepharose resin used to capture the virus. This result is most likely due to
PEG rendering
the virus more charge neutral in appearance and hence decreasing its binding
potential to
the ion exchange matrix. A broadening of the virus' chromatogram is expected
since the
pegylation of CN706 occurs to different percentages.
The infectivity of pegylated CN706 was evaluated in an in vitro plaque assay
on
293 cells. Table 8 depicts a 5 to 10-fold reduction in plaquing efficiency of
PEG-CN706
as compared to CN706. This is most likely due to pegylation masking the virus
cells,
decreasing the recognition and endocytosis of the viral particles.
Table 8: Comparison of Plaquing Efficiency of CN706 and PEG-CN706.
Sample Description Number of Plaques (Arbitrary Units)
CN706 15 5
PEG-CN706 4 1
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 specificaliy and individually indicated to be incorporated by
reference.
Although the foregoing has been described in some detail by way of
illustration
and example for purposes of clarity of understanding, it will be apparent to
those skilled in
the art that certain changes and modifications can be practiced. Therefore,
the description
and examples should not be construed as limiting the scope of the invention,
which is
delineated by the appended claims.
79
_.. , .. ._,-.-..... - _- .~___...._. __..__.__...__.__~__...._~..._..,~.~._._
. _.~._~..,_~_ ..~..~.,~....
CA 02283231 1999-11-02
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: Calydon, Inc.
(ii) TITLE OF INVENTION: ADENOVIRUS VECTORS CONTAINING
HETEROLOGOUS TRANSCRIPTION REGULATORY ELEMENTS AND METHODS
OF USING SAME
(iii) NUMBER OF SEQUENCES: 41
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: BORDEN ELLIOT SCOTT & AYLEN
(B) STREET: 1000-60 Queen Street
(C) CITY: Ottawa
(D) PROVINCE: Ontario
(E) COUNTRY: CANADA
(F) POSTAL CODE: K1P 5Y7
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Diskette
(B) COMPUTER: IBM Compatible
(C) OPERATING SYSTEM: Windows
(D) SOFTWARE: FastSEQ for Windows Version 2.Ob
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: 2,283,231
(B) FILING DATE: September 2, 1999
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: USA 60/039,762
(B) FILING DATE: March 3, 1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Joachim T. Fritz
(B) REGISTRATION NUMBER: 4173
(C) REFERENCE/DOCKET NUMBER: PAT 45036W-1
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (613) 237-5160
(B) FACSIMILE: (613) 787-3558
(2) INFORMATION FOR SEQ ID NO:1:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5836 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
CA 02283231 1999-11-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: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
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
81
CA 02283231 1999-11-02
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 AA.F.AAAATTT 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
(2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5835 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
82
CA 02283231 1999-11-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: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
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 AAAAA-A-kAAA 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 AGTAA.GCAAG 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
83
CA 02283231 1999-11-02
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
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
(2) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 12047 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
84
CA 02283231 1999-11-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: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 TGTAGTAACA 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
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
CA 02283231 1999-11-02
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 GACCTCAA.AC 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
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
86
CA 02283231 1999-11-02
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 CCAGCTGGCC 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
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
87
CA 02283231 1999-11-02
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
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 454 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: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
88
CA 02283231 1999-11-02
GAACCTATTT GTATACTAGA TGACACAATG TCAATGTCTG TGTACAACTG CCAACTGGGA 360
TGCAAGACAC TGCCCATGCC AATCATCCTG AAAAGCAGCT ATAAAAAGCA GGAAGCTACT 420
CTGCACCTTG TCAGTGAGGT CCAGATACCT ACAG 454
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 5224 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: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
89
CA 02283231 1999-11-02
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
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 TAAAGATTTC 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
AACA 5224
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 822 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
CA 02283231 1999-11-02
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: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
TATACCAACA AAAGGTTACT AGTTAACAGG CATTGCCTGA AAAGAGTATA AAAGAATTTC 780
AGCATGATTT TCCATATTGT GCTTCCACCA CTGCCAATAA CA 822
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 472 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: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
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 858 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
CGAGCGGCCC CTCAGCTTCG GCGCCCAGCC CCGCAAGGCT CCCGGTGACC ACTAGAGGGC 60
GGGAGGAGCT CCTGGCCAGT GGTGGAGAGT GGCAAGGAAG GACCCTAGGG TTCATCGGAG 120
CCCAGGTTTA CTCCCTTAAG TGGAAATTTC TTCCCCCACT CCTCCTTGGC TTTCTCCAAG 180
GAGGGAACCC AGGCTGCTGG AAAGTCCGGC TGGGGCGGGG ACTGTGGGTT CAGGGGAGAA 240
91
CA 02283231 1999-11-02
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
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 307 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ix) FEATURE:
(A) NAME/KEY: Coding Sequence
(B) LOCATION: 2...304
(D) OTHER INFORMATION:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
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
GCT 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 Gln 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
92
CA 02283231 1999-11-02
CTT TTA CAG TAT GAT TAA 307
Leu Leu Gln Tyr Asp
100
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 101 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
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 Val Cys Leu Ile Ile Met Trp Leu Ile Cys Cys Leu Lys Arg
50 55 60
Arg Arg Ala Arg Pro Pro Ile Tyr Arg Pro Ile Ile Val Leu Asn Pro
65 70 75 80
His Asn Glu Lys Ile His Arg Leu Asp Gly Leu Lys Pro Cys Ser Leu
85 90 95
Leu Leu Gln Tyr Asp
100
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
GATCACCGGT GTCCACGGCC AGGTGGTGC 29
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
93
CA 02283231 1999-11-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
GATCACCGGT GCTCACGCCT GTAATCTCAT CAC 33
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
GATCACCGGT GGTTTGGGAT GGCATGGCTT TGG 33
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
GATCACCGGT AAAGAATCAG TGATCATCCC AAC 33
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
GATCCGGCCG TGGTGCTCAC GCCTGTAATC 30
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 32 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
GATCCGGCCG TGTCCACGGC CAGGTGGTGC AG 32
94
CA 02283231 1999-11-02
(2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
CTCATTTTCA GTCACCGGTA AGCTTGG 27
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
GAGCCGCTCC GACACCGGTA CCTC 24
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
GATCACCGGT AAGCTTCCAC AAGTGCATTT AGCC 34
(2) INFORMATION FOR SEQ ID NO:20:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
GATCACCGGT CTGTAGGTAT CTGGACCTCA CTG 33
(2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
CA 02283231 1999-11-02
(A) LENGTH: 34 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
GATCCGGCCG AAGCTTCCAC AAGTGCATTT AGCC 34
(2) INFORMATION FOR SEQ ID NO:22:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
GATCCGGCCG CTGTAGGTAT CTGGACCTCA CTG 33
(2) INFORMATION FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GTGACCGGTG CATTGCTGTG AACTCTGTA 29
(2) INFORMATION FOR SEQ ID NO:24:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
ATAAGTGGCC TGGATAAAGC TGAGTGG 27
(2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
96
CA 02283231 1999-11-02
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
GTCACCGGTC TTTGTTATTG GCAGTGGT 28
(2) INFORMATION FOR SEQ ID NO:26:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:
ATCCAGGCCA CTTATGAGCT CTGTGTCCTT 30
(2) INFORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
TATCGGCCGG CATTGCTGTG AACTCT 26
(2) INFORMATION FOR SEQ ID NO:28:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
TTACGGCCGC TTTGTTATTG GCAGTG 26
(2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 786 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
97
CA 02283231 1999-11-02
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CGGTGACCAC TAGAGGGCGG GAGGAGCTCC TGGCCAGTGG TGGAGAGTGG CAAGGAAGGA 60
CCCTAGGGTT CATCGGAGCC CAGGTTTACT CCCTTAAGTG GAAATTTCTT CCCCCACTCC 120
TCCTTGGCTT TCTCCAAGGA GGGAACCCAG GCTGCTGGAA AGTCCGGCTG GGGCGGGGAC 180
TGTGGGTTCA GGGGAGAACG GGGTGTGGAA CGGGACAGGG AGCGGTTAGA AGGGTGGGGC 240
TATTCCGGGA AGTGGTGGGG GGAGGGAGCC CAAAACTAGC ACCTAGTCCA CTCATTATCC 300
AGCCCTCTTA TTTCTCGGCC GCTCTGCTTC AGTGGACCCG GGGAGGGCGG GGAAGTGGAG 360
TGGGAGACCT AGGGGTGGGC TTCCCGACCT TGCTGTACAG GACCTCGACC TAGCTGGCTT 420
TGTTCCCCAT CCCCACGTTA GTTGTTGCCC TGAGGCTAAA ACTAGAGCCC AGGGGCCCCA 480
AGTTCCAGAC TGCCCCTCCC CCCTCCCCCG GAGCCAGGGA GTGGTTGGTG AAAGGGGGAG 540
GCCAGCTGGA GAACAAACGG GTAGTCAGGG GGTTGAGCGA TTAGAGCCCT TGTACCCTAC 600
CCAGGAATGG TTGGGGAGGA GGAGGAAGAG GTAGGAGGTA GGGGAGGGGG CGGGGTTTTG 660
TCACCTGTCA CCTGCTCGCT GTGCCTAGGG CGGGCGGGCG GGGAGTGGGG GGACCGGTAT 720
AAAGCGGTAG GCGCCTGTGC CCGCTCCACC TCTCAAGCAG CCAGCGCCTG CCTGAATCTG 780
TTCTGC 786
(2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
TAATCCGGAC GGTGACCACT AGAGGG 26
(2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
TATTCCGGAT CACTTAGGCA GCGCTG 26
(2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
TAACGGCCGC GGTGACCACT AGAG 24
98
CA 02283231 1999-11-02
(2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
TATCGGCCGG CAGAACAGAT TCAG 24
(2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
ATTACCGGTA GCCACCACCC AGTGAG 26
(2) INFORMATION FOR SEQ ID NO:35:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:
TAGACCGGTG CTTGAGTTCC AGGAAC 26
(2) INFORMATION FOR SEQ ID NO:36:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:
TAACGGCCGA GCCACCACCC A 21
(2) INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE CHARACTERISTICS:
99
CA 02283231 1999-11-02
(A) LENGTH: 23 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
TATCGGCCGG CTTGAGTTCC AGG 23
(2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
GCCTTAATTA AAAGCAAACC TCACCTCCG 29
(2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
GTGGAACAAA AGGTGATTAA AAAATCCCAG 30
(2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:
CACCTTTTGT TCCACCGCTC TGCTTATTAC 30
(2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 28 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
100
CA 02283231 1999-11-02
(D) TOPOLOGY: linear
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:
GGCTTAATTA ACTGTGAAAG GTGGGAGC 28
101
