U.S. patent application number 10/691045 was filed with the patent office on 2004-07-29 for cell-specific adenovirus vectors comprising an internal ribosome entry site.
Invention is credited to Henderson, Daniel R., Li, Yuanhao, Little, Andrew S., Yu, De-Chao.
Application Number | 20040146489 10/691045 |
Document ID | / |
Family ID | 27668261 |
Filed Date | 2004-07-29 |
United States Patent
Application |
20040146489 |
Kind Code |
A1 |
Yu, De-Chao ; et
al. |
July 29, 2004 |
Cell-specific adenovirus vectors comprising an internal ribosome
entry site
Abstract
Disclosed herein are replication-competent adenovirus vectors
comprising co-transcribed first and second genes under
transcriptional control of a heterologous, target cell-specific
transcriptional regulatory element (TRE), wherein the second gene
is under translational control of an internal ribosome entry site.
Methods for the preparation and use of such vectors are also
provided. The vectors provide target cell-specific virus
replication in applications such as cancer therapy and gene
therapy.
Inventors: |
Yu, De-Chao; (Foster City,
CA) ; Li, Yuanhao; (Palo Alto, CA) ; Little,
Andrew S.; (Balboa Island, CA) ; Henderson, Daniel
R.; (Palo Alto, CA) |
Correspondence
Address: |
BOZICEVIC, FIELD & FRANCIS LLP
200 MIDDLEFIELD RD
SUITE 200
MENLO PARK
CA
94025
US
|
Family ID: |
27668261 |
Appl. No.: |
10/691045 |
Filed: |
October 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10691045 |
Oct 21, 2003 |
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09814351 |
Mar 21, 2001 |
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6692736 |
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60192156 |
Mar 24, 2000 |
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Current U.S.
Class: |
424/93.2 ;
435/235.1; 435/456; 514/44R |
Current CPC
Class: |
C12N 2710/10322
20130101; C12N 2710/10343 20130101; C12N 2830/008 20130101; C12N
2830/00 20130101; A61K 48/00 20130101; C12N 2840/206 20130101; C12N
15/86 20130101; C12N 2830/001 20130101; C12N 2830/85 20130101 |
Class at
Publication: |
424/093.2 ;
514/044; 435/456; 435/235.1 |
International
Class: |
A61K 048/00; C12N
007/00; C12N 015/861 |
Claims
What is claimed is:
1. A replication-competent adenovirus vector comprising first and
second genes co-transcribed as a single mRNA wherein the first and
the second genes are under transcriptional control of a
heterologous, target cell-specific transcriptional regulatory
element (TRE), wherein the second gene has a mutation in or
deletion of its endogenous promoter and is under translational
control of an internal ribosome entry site (IRES) and wherein said
vector exhibits greater specificity for the target cell than an
adenovirus vector comprising a target cell-specific TRE operably
linked to a gene and lacking an IRES.
2. The vector of claim 1, wherein at least one of said first and
second genes is an adenovirus gene.
3. The vector of claim 2, wherein both of said first and said
second genes are adenovirus genes.
4. The vector of claim 2, wherein at least one of said first and
said second adenovirus gene is essential for viral replication.
5. The vector of claim 4, wherein the adenovirus gene essential for
viral-replication is an adenovirus early gene.
6. The vector of claim 5, wherein the adenovirus early gene
includes E1A, E1B, E2, or E4.
7. The vector of claim 4, wherein the adenovirus gene essential for
viral replication is an adenovirus late gene.
8. The vector of claim 3, wherein both said first and said second
adenovirus genes are essential for viral replication.
9. The vector of claim 8, wherein at least one of said first and
said second adenovirus genes is an adenovirus early gene.
10. The vector of claim 8, wherein at least one of said first and
said second adenovirus genes is an adenovirus late gene.
11. The vector of claim 8, wherein said first adenovirus gene is
E1A and said second adenovirus gene is E1B.
12. The vector of claim 11 wherein E1A has its endogenous promoter
deleted.
13. The vector of claim 11 wherein E1A has an inactivation of E1A
enhancer I.
14. The vector of claim 11 wherein E1B has an inactivation of its
endogenous promoter.
15. The vector of claim 11 wherein E1B has a deletion of the 19-kDa
region.
16. The vector of claim 11 wherein E1A has an inactivation of its
endogenous promoter and E1B has an inactivation of its endogenous
promoter.
17. The vector of claim 16 wherein E1B has a deletion of the 19-kDa
region.
18. The vector of claim 16 wherein E1A has an inactivation of E1A
enhancer I.
19. The vector of claim 1, wherein the internal ribosome entry site
(IRES) is from EMCV.
20. The vector of claim 1 wherein the IRES is from VEGF.
21. The vector of claim 1 wherein the IRES includes the 5'UTR of
HCV; the 5' UTR of BiP; or the 5'UTR of PDGF.
22. The vector of claim 1, wherein the TRE is specific for a target
cell that is a cancer cell.
23. The vector of claim 22 wherein the cancer cell includes a
prostate cancer cell, a breast cancer cell, a hepatoma cell, a
melanoma cell, a bladder cell or a colon cancer cell.
24. The vector of claim 22, wherein the TRE includes the probasin
(PB) TRE, the prostate-specific antigen (PSA) TRE, the mucin (MUC1)
TRE, the .alpha.-fetoprotein (AFP) TRE, the hKLK2 TRE, the
tyrosinase TRE, the human uroplakin II (hUPII) TRE or the
carcinoembryonic antigen (CEA) TRE.
25. The vector of claim 9 wherein said first adenovirus gene has a
deletion of its endogenous promoter.
26. The vector of claim 25 wherein said first adenovirus gene is
E1A.
27. The vector of claim 9 wherein said first and/or said second
adenovirus gene has a deletion of an enhancer region.
28. The vector of claim 27 wherein said first gene is E1A and said
enhancer is E1A enhancer I.
29. The vector of claim 1 wherein said TRE has an endogenous
silencer element deleted.
30. The vector of claim 1 wherein said adenovirus vector comprises
an E3 region.
31. The adenovirus vector of claim 11 wherein said adenovirus
comprises an E3 region.
32. The adenovirus vector of claim 11 further comprising a
transgene.
33. An adenovirus vector comprising a gene under transcriptional
control of a melanocyte-specific TRE.
34. The vector of claim 33 wherein said gene is an adenoviral
gene.
35. The vector of claim 34 wherein said adenoviral gene is a gene
essential for replication.
36. The vector of claim 32 wherein said transgene is co-transcribed
with said first and said second gene and said transgene is under
the translation control of a separate internal ribosome entry site
(IRES).
37. The vector of claim 36 wherein said IRES is from EMCV.
38. The vector of claim 36 wherein said IRES is from VEGF.
39. The vector of claim 30 further comprising an adenovirus death
protein gene (ADP).
40. The vector of claim 32 wherein said transgene is a cytotoxic
gene.
41. The vector of claim 1 wherein said first adenovirus gene is
essential for viral replication and said second adenovirus gene is
the adenovirus death protein gene (ADP).
42. The vector of claim 41 wherein said first adenovirus gene is
E1A.
43. The vector of claim 42 wherein E1A has a deletion of its
endogenous promoter.
44. The vector of claim 42 wherein said E1A has a deletion of E1A
enhancer I.
45. The vector of claim 1 wherein said first gene is essential for
viral replication and said second gene is E3.
46. The vector of claim 45 wherein said first gene is E1A.
47. A composition comprising a vector according to claim 1.
48. The composition of claim 47 further comprising a
pharmaceutically acceptable excipient.
49. A composition comprising a vector according to claim 30.
50. The composition of claim 49 further comprising a
pharmaceutically acceptable excipient.
51. A host cell comprising the vector of claim 1.
52. A host cell comprising the vector of claim 30.
53. An adenovirus vector comprising E1B under transcriptional
control of a heterologous, target cell specific TRE, wherein E1B
has a deletion of part or all of the 19-kDa region.
54. A host cell comprising the adenovirus vector of claim 53.
55. A method for propagating a replication-competent adenovirus
vector comprising a target cell-specific TRE, said method
comprising combining an adenovirus vector of claim 1 with mammalian
cells that permit the function of a target cell-specific TRE, such
that the adenovirus vector enters the cell, whereby said adenovirus
vector is propagated.
56. A method for conferring selective cytotoxicity in target cells,
comprising contacting the cells with an adenovirus vector of claim
41 whereby the vector enters the cell.
57. A method for modifying the genotype of a target cell,
comprising contacting the cell with an adenovirus vector of claim
1, wherein the vector enters the cell.
58. A method for suppressing tumor cell growth, comprising
contacting a tumor cell with an adenovirus vector of claim 41 such
that the adenovirus vector enters the tumor cell and exhibits
selective cytotoxicity for the tumor cell.
Description
TECHNICAL FIELD
[0001] This invention relates to new replication competent
adenovirus vectors comprising an internal ribosome entry site which
replicate preferentially in target cells. The present invention
also relates to cell transduction using adenovirus vectors
comprising an internal ribosome entry site.
BACKGROUND
[0002] Diseases involving altered cell proliferation, particularly
hyperproliferation, constitute an important health problem. For
example, despite 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 surgical removal
of the tumor mass. If a tumor becomes malignant, as manifested by
invasion of surrounding tissue, it becomes much more difficult to
eradicate. Once a malignant tumor metastasizes, it is much less
likely to be eradicated.
[0003] Excluding basal cell carcinoma, there are over one million
new cases of cancer per year in the United States alone, and cancer
accounts for over one half million deaths per year in this country.
In the world as a whole, the five most common cancers are those of
lung, stomach, breast, colon/rectum, and uterine cervix, and the
total number of new cases per year is over 6 million.
[0004] In the United States, transitional cell carcinoma (TCC)
accounts for 90 to 95 percent of all tumors of the bladder.
Squamous cell carcinoma (SCC) represents 5 to 10 percent, and
adenocarcinoma approximately 1 to 2 percent. Squamous cell and
adenomatous elements are often found in association with
transitional cell tumors, especially with high grade tumors.
Bladder cancer is generally divided into superficial and invasive
disease. A critical factor is the distinction between those tumors
that are confined to the mucosa and those that have penetrated the
basement membrane and extended into the lamina propria. The term
"superficial bladder tumor" is generally used to represent a tumor
that has not invaded the muscularis. Invasive tumors are described
as those that have invaded the muscularis propria, the perivesical
fibroadipose tissue, or adjacent structures. Carcinoma in situ
(CIS) is a high grade and aggressive manifestation of TCC of the
bladder that has a highly variable course.
[0005] A number of urothelial cell-specific proteins have been
described, among which are the uroplakins. Uroplakins (UP),
including UPIa and UPIb (27 and 28 kDa, respectively), UPII (15
kDa), and UPIII (47 kDa), are members of a group of integral
membrane proteins that are major proteins of urothelial plaques.
These plaques cover a large portion of the apical surface of
mammalian urothelium and may play a role as a permeability barrier
and/or as a physical stabilizer of the urothelial apical surface.
Wu et al. (1994) J. Biol. Chem. 269:13716-13724. UPs are
bladder-specific proteins, and are expressed on a significant
proportion of urothelial-derived tumors, including about 88% of
transitional cell carcinomas. Moll et al. (1995) Am. J. Pathol.
147:1383-1397; and Wu et al. (1998) Cancer Res. 58:1291-1297. The
control of the expression of the human UPII has been studied, and a
3.6-kb region upstream of the mouse UPII gene has been identified
which can confer urothelial-specific transcription on heterologous
genes (Lin et al. (1995) Proc. Natl. Acad. Sci. USA 92:679-683).
See also, U.S. Pat. Nos. 5,824,543 and 6,001,646.
[0006] Melanoma, a malignant neoplasm derived from melanocytes of
the skin and other sites, has been increasing in incidence
worldwide. The American Joint Committee on Cancer recognizes five
different forms of extraocular melanoma occurring in humans:
lentigo maligna melanoma; radial spreading; nodular; acral
lentiginous; and unclassified. Known melanoma-associated antigens
can be classified into three main groups: tumor-associated
testis-specific antigens MAGE, BAGE, GAGE, and PRAME; melanocyte
differentiation antigens tyrosinase, Melan-A/MART-1 (for Melanoma
Antigen Recognized by T cells), gp100, tyrosinase related protein-1
(TRP-1), tyrosinase related protein-2 (TRP-2); and mutated or
aberrantly expressed antigens MUM-1, cyclin-dependent kinase 4
(CDK4), beta-catenin, gp100-in4, p15, and
N-acetylglucosaminyltransferase V. See, for example, Kirkin et al.
(1998) Exp. Clin. Immunogenet. 15:19-32. Tyrosinase, TRP-1, and
TRP-2 are enzymes involved in melanin biosynthesis and are
specifically expressed in melanocytes. Antigenic epitopes of MART-1
have been studied extensively, with the aim of developing a
melanoma vaccine. An immunodominant epitope, MART-1(27-35) has been
reported to be recognized by a majority of CD8+ cytotoxic T cell
clones generated to MART-1. These MART-[(27-35)-specific CTLs
specifically lyse autologous tumor cell lines expressing the
epitope. Faure and Kourilsky (1998) Crit. Rev. Immunol. 18:77-86.
However, others have reported that presence of such CTLs is not
accompanied by a significant clinical response. Rivoltini et al.
(1998) Crit. Rev. Immunol. 18:55-63.
[0007] 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 without affecting the functions of
normal cells. For example, in traditional chemotherapy of prostate
cancer, the therapeutic ratio, (i.e., the ratio of tumor cell
killing to normal cell killing) is only 1.5:1. Thus, more effective
treatment methods and pharmaceutical compositions for therapy and
prophylaxis of neoplasia are needed.
[0008] Accordingly, the development of more specific, targeted
forms of cancer therapy, especially for cancers that are difficult
to treat successfully, is of particular interest. In contrast to
conventional cancer therapies, which result in relatively
non-specific and often serious toxicity, more specific treatment
modalities, which inhibit or kill malignant cells selectively while
leaving healthy cells intact, are required.
[0009] Gene therapy, whereby a gene of interest is introduced into
a malignant cell, has been attempted as an approach to treatment of
many cancers. See, for example, Boulikas (1997) Anticancer Res.
17:1471-1505, for a description of gene therapy for prostate
cancer. A gene of interest can encode a protein which is converted
into a toxic substance upon treatment with another compound, or it
can encode an enzyme that converts a prodrug to a drug. For
example, introduction of the herpes simplex virus 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, a gene of interest can
encode a compound that is directly toxic, such as, for example,
diphtheria toxin. To render these treatments specific to cancer
cells, the gene of interest is placed under control of a
transcriptional regulatory element (TRE) that is specifically
(i.e., preferentially) active 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.
[0010] A number of viral vectors and non-viral delivery systems
(e.g., liposomes), have been developed for gene transfer. Of the
viruses proposed for gene transfer, adenoviruses are among the most
easily produced and purified. Adenovirus also has the advantage of
a high efficiency of transduction (i.e., introduction of the gene
of interest into the target cell) and does not require cell
proliferation for efficient transduction. 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.
[0011] Adenoviruses generally undergo a 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 cellular
protein synthesis. 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.
[0012] When used for gene transfer, adenovirus vectors are often
designed to be replication-defective and are thus deliberately
engineered to fail to replicate in the target cell. In these
vectors, the early adenovirus gene products E1A and/or E1B are
often deleted, and the gene to be transduced is commonly inserted
into the E1A and/or E1B region of the deleted virus genome. Bett et
al. (1994) supra. Such vectors are propagated in packaging cell
lines such as the 293 line, which provides E1A and E1B functions in
trans. Graham et al. (1987) J. Gen. Virol 36:59-72; Graham (1977)
J. Gen. Virol. 68:937-940. The use of replication-defective
adenovirus vectors as vehicles for efficient transduction of genes
has 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) Nature 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; and Bett et al. (1994) supra.
[0013] 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 a 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, coupled with the transient
nature of gene expression. Despite these limitations, development
of adenoviral vectors for gene therapy has focused almost
exclusively on the use of the virus as a vehicle for introducing a
gene of interest, not as an effector in itself. In fact,
replication of adenovirus vectors has been viewed as an undesirable
result, largely due to the host immune response.
[0014] More recently, however, the use of adenovirus vectors as
effectors has been described. International Patent Application Nos.
PCT/US98/04080, PCT/US98/04084, PCT/US98/04133, PCT/US98/04132,
PCT/US98/16312, PCT/US95/00845, PCT/US96/10838, PCT/EP98/07380 and
U.S. Pat. No. 5,998,205. Adenovirus E1A and E1B genes are disclosed
in Rao et al. (1992, Proc. Natl. Acad. Sci. USA vol. 89:
7742-7746).
[0015] Replication-competent adenovirus vectors, which take
advantage of the cytotoxic effects associated with adenovirus
replication, have recently been described as agents for effecting
selective cell growth inhibition. In such systems, a cell-specific
transcriptional regulatory element (TRE) is used to control the
expression of a gene essential for viral replication, thus limiting
viral replication to cells in which the TRE is functional. See, for
example International Patent Application No. PCT/EP99/07380,
Henderson et al., U.S. Pat. No. 5,698,443; Hallenbeck et al.,
PCT/US95/15455 and U.S. Pat. No. 5,998,205; Rodriguez et al. (1997)
Cancer Res. 57:2559-2563.
[0016] PCT publication PCT/US98/04080 discloses
replication-competent, target cell-specific adenovirus vectors
comprising heterologous TREs, such as those regulating expression
of prostate-specific antigen (PSA), probasin (PB),
.alpha.-fetoprotein (AFP), kallikrien (hKLK2), mucin (MUC1) and
carcinoembryonic antigen (CEA). PCT/US98/04084 discloses
replication-competent adenovirus vectors comprising an
.alpha.-fetoprotein (AFP) TRE that replicate specifically in cells
expressing AFP, such as hepatoma cells.
[0017] Internal ribosome entry sites (IRES) are sequences which
initiate translation from an internal initiation codon (usually
AUG) within a bi-or multi-cistronic RNA transcript continuing
multiple protein coding regions. IRES have been characterized in
encephalomyocarditis virus and related picornaviruses. See, for
example, Jackson et al. (1995) RNA 1: 985-1000 and Herman (1989)
Trends in Biochemical Sciences 14(6): 219-222. IRES sequences are
also detected in mRNAs from other viruses such as cardiovirus,
rhinovirus, aphthovirus, hepatitis C virus (HCV), Friend murine
leukemia virus (FrMLV) and Moloney murine leukemia virus (MoMLV).
The presence of IRES in cellular RNAs has also been described.
Examples of cellular mRNAs containing IRES include those encoding
immunoglobulin heavy-chain binding protein (BiP), vascular
endothelial growth factor (VEGF), fibroblast growth factor 2,
insulin-like growth factor, translational initiation factor eIF4G,
and the yeast transcription factors TFIID and HAP4. See, for
example, Macejak et al. (1991) Nature 353:90-94; Oh et al. (1992)
Genes Dev. 6:1643-1653; Vagner et al. (1995) Mol. Cell. Biol.
15:35-44; He et al. (1996) Proc. Natl. Acad. Sci USA 93:7274-7278;
He et al. (1996) Gene 175:121-125; Tomanin et al. (1997) Gene
193:129-140; Gambotto et al. (1999) Cancer Gene Therapy 6:45-53;
Qiao et al. (1999) Cancer Gene Therapy 6:373-379. Expression
vectors containing IRES elements have been described. See, for
example, International Patent Application No. PCT/US98/03699 and
International Patent Application No. PCT/EP98/07380.
[0018] Thus, there is a continuing need for improved
replication-competent adenovirus vectors in which cell-specific
replication can be further enhanced, while minimizing the extent of
replication in non-target (i.e., non-cancerous cells).
[0019] The disclosure of all patents and publications cited herein
are incorporated by reference in their entirety.
SUMMARY OF THE INVENTION
[0020] The present invention provides improved replication
competent adenovirus vectors comprising co-transcribed first and
second genes under transcriptional control of a heterologous,
target cell-specific transcriptional regulatory element (TRE),
wherein the second gene is under translational control of an
internal ribosome entry site (IRES). In one embodiment, the first
and second genes are co-transcribed as a single mRNA and the second
gene has a mutation in or deletion of its endogenous promoter. The
present invention further provides host cells and methods using the
adenovirus vectors.
[0021] In one aspect, the first and/or second genes are adenovirus
genes and in another aspect, the first and/or second adenovirus
genes are essential for viral replication. An essential gene can be
an early viral gene, including for example, E1A; E1B; E2; and/or
E4, or a late viral gene. In another aspect an early gene is
E3.
[0022] In one embodiment, the first gene is an adenovirus gene and
the second gene is a therapeutic gene. In another embodiment, both
genes are adenovirus genes. In an additional embodiment, the first
adenovirus gene is E1A, and the second adenovirus gene is E1B.
Optionally, the endogenous promoter for one of the co-transcribed
adenovirus gene essential for viral replication, such as for
example, E1A, is deleted and/or mutated such that the gene is under
sole transcriptional control of a target cell-specific TRE.
[0023] In another aspect, the present invention provides adenovirus
vectors comprising an adenovirus gene essential for viral
replication under control of a target cell-specific TRE, wherein
said adenovirus gene has a mutation of or deletion in its
endogenous promoter. In one embodiment, the adenovirus gene is
essential for viral replication. In another embodiment, the
adenovirus gene is E1A wherein the E1A promoter is deleted and
wherein the E1A gene is under transcriptional control of a
heterologous cell-specific TRE. In another embodiment, the
adenovirus gene is E1B wherein the E1B promoter is deleted and
wherein the E1B gene is under transcriptional control of a
heterologous cell-specific TRE.
[0024] In another aspect, the present invention provides adenovirus
vectors comprising E1B under control of a target cell-specific TRE,
wherein said E1B has a deletion in or mutation of the 19-kDa region
of E1B, that encodes a product shown to inhibit apoptosis.
[0025] In other embodiments, an enhancer element for the first
and/or second adenovirus genes is inactivated. The present
invention provides an adenovirus vector comprising E1A wherein an
E1A enhancer is inactivated. In yet other embodiments, the present
invention provides an adenovirus vector comprising E1A wherein the
E1A promoter is inactivated and E1A enhancer I is inactivated. In
further embodiments, the present invention provides an adenovirus
vector comprising a TRE which has its endogenous silencer element
inactivated.
[0026] Any TRE which directs cell-specific expression can be used
in the disclosed vectors. In one embodiment, TREs include, for
example, TREs specific for prostate cancer cells, breast cancer
cells, hepatoma cells, melanoma cells, bladder cells and/or colon
cancer cells. In another embodiment, the TREs include, probasin
(PB) TRE; prostate-specific antigen (PSA) TRE; mucin (MUC1) TRE;
.alpha.-fetoprotein (AFP) TRE; hKLK2 TRE; tyrosinase TRE; human
uroplakin II TRE (hUPII) and carcinoembryonic antigen (CEA) TRE. In
other embodiments, the target cell-specific TRE is a cell
status-specific TRE. In yet other embodiments, the target
cell-specific TRE is a tissue specific TRE.
[0027] In additional embodiments, the adenovirus vector comprises
at least one additional co-transcribed gene under the control of
the cell-specific TRE. In another embodiment, an additional
co-transcribed gene is under the translational control of an
IRES.
[0028] In another aspect of the present invention, adenovirus
vectors further comprise a transgene such as, for example, a
cytotoxic gene. In one embodiment, the transgene is under the
transcriptional control of the same TRE as the first gene and
second genes and optionally under the translational control of an
internal ribosome entry site. In another embodiment, the transgene
is under the transcriptional control of a different TRE that is
functional in the same cell as the TRE regulating transcription of
the first and second genes and optionally under the translational
control of an IRES.
[0029] The present invention also provides compositions comprising
the replication-competent adenovirus vectors described herein. In
one embodiment, the compositions further comprise a
pharmaceutically acceptable excipient. The present invention also
provides kits comprising the replication-competent adnenovirus
vectors described herein.
[0030] Host cells comprising the disclosed adenovirus vectors are
also provided. Host cells include those used for propagation of a
vector and those into which a vector is introduced for therapeutic
purposes.
[0031] In another aspect, methods are provided for propagating
replication-competent adenovirus vectors of the present invention
specific for mammalian cells which permit the function of a target
cell-specific TRE, said method comprising combining an adenovirus
vector(s) described herein with mammalian cells that permit the
function of a target cell-specific TRE, such that the adenovirus
vector(s) enters the cell, whereby said adenovirus is
propagated.
[0032] In another aspect, methods are provided for conferring
selective cytotoxicity in target cells, comprising contacting the
cells with an adenovirus vector(s) described herein, whereby the
vector enters the cell.
[0033] The invention further provides methods of suppressing tumor
cell growth, more particularly a target tumor cell, comprising
contacting a tumor cell with an adenovirus vector(s) of the
invention such that the adenovirus vector enters the tumor cell and
exhibits selective cytotoxicity for the tumor cell.
[0034] In another aspect, methods are provided for detecting a cell
which allows the function of a target cell-specific TRE, which
comprise contacting a cell in a biological sample with an
adenovirus vector(s) of the invention, and detecting replication of
the adenovirus vector(s), if any.
[0035] In another aspect, methods are provided for modifying the
genotype of a target cell, comprising contacting the cell with an
adenovirus vector as described herein, wherein the adenovirus
vector enters the cell.
[0036] The present invention provides an adenovirus vector
comprising an adenovirus gene, wherein said adenovirus gene is
under transcriptional control of a melanocyte-specific TRE. In
another embodiment, a melanocyte-specific TRE is human. In another
embodiment, a melanocyte-specific TRE comprises a
melanocyte-specific promoter and a heterologous enhancer. In other
embodiments, a melanocyte-specific TRE comprises a
melanocyte-specific promoter. In other embodiments, a
melanocyte-specific TRE comprises a melanocyte-specific enhancer
and a heterologous promoter. In other embodiments, a
melanocyte-specific TRE comprises a melanocyte-specific promoter
and a melanocyte-specific enhancer.
[0037] In some embodiments, the adenovirus gene under
transcriptional control of a melanocyte-specific TRE is an
adenovirus gene essential for replication. In some embodiments, the
adenoviral gene essential for replication is an early gene. In
another embodiment, the early gene is E1A. In another embodiment,
the early gene is E1B. In yet another embodiment, both E1A and E1B
are under transcriptional control of a melanocyte-specific TRE. In
further embodiments, the adenovirus gene essential for replication
is E1B, and E1B has a deletion in the 19-kDa region.
[0038] In some embodiments, the melanocyte-specific TRE is derived
from the 5' flanking region of a tyrosinase gene. In other
embodiments, the melanocyte-specific TRE is derived from the 5'
flanking region of a tyrosinase related protein-1 gene. In other
embodiments, the melanocyte-specific TRE is derived from the
5'-flanking region of a tyrosinase related protein-2 gene. In other
embodiments, the melanocyte-specific TRE is derived from the 5'
flanking region of a MART-1 gene. In other embodiments, the
melanocyte-specific TRE is derived from the 5'-flanking region of a
gene which is aberrantly expressed in melanomas.
[0039] In other embodiments, the invention provides an adenovirus
vector comprising (a) an adenovirus gene under transcriptional
control of a melanocyte-specific TRE; and (b) an E3 region. In some
of these embodiments the E3 region is under transcriptional control
of a melanocyte-specific TRE.
[0040] In another aspect, the invention provides a host cell
comprising the melanocyte specific adenovirus vector(s) described
herein.
[0041] In another aspect, the invention provides pharmaceutical
compositions comprising a melanocyte specific adenovirus vector(s)
described herein.
[0042] In another aspect, the invention provides kits which contain
a melanocyte adenoviral vector(s) described herein.
[0043] In another aspect, methods are provided for conferring
selective cytoxicity in target cells (i.e., cells which permit or
induce a melanocyte-specific TRE to function), comprising
contacting the cells with an adenovirus vector(s) described herein,
whereby the vector enters the cell.
[0044] In another aspect, methods are provided for propagating an
adenovirus specific for melanocytes, said method comprising
combining an melanocyte specific adenovirus vector(s) described
herein with melanocytes, whereby said adenovirus is propagated.
[0045] The invention further provides methods of suppressing
melanoma cell growth, comprising contacting a melanoma cell with a
melanocyte specific adenoviral vector of the invention such that
the adenoviral vector enters the melanoma cell and exhibits
selective cytotoxicity for the melanoma cell.
[0046] In another aspect, methods are provided for detecting
melanocytes, including melanoma cells, in a biological sample,
comprising contacting cells of a biological sample with a
melanocyte adenovirus vector(s) described herein, and detecting
replication of the adenovirus vector, if any.
BRIEF DESCRIPTION OF THE DRAWINGS
[0047] FIG. 1 is a schematic of plasmid construct CP627 as
described in Example 1.
[0048] FIGS. 2A-2B is a series of schematic depictions of various
adenoviruses described herein.
[0049] FIG. 3 depicts the replication efficiency of different
viruses as described in Example 4.
[0050] FIGS. 4A and 4B show viral yield for different
liver-specific vectors in different cell types.
[0051] FIG. 5 is a schematic representation of adenovirus vectors
comprising AFP-TRE with and without IRES.
[0052] FIG. 6 depicts an E3 region.
[0053] FIG. 7 is a schematic representation of adenovirus vectors
described herein.
[0054] FIG. 8 depicts in vivo antitumor activity of CV890
containing an IRES. This figure depicts the results of a HepG2
Xenograph treated with CV790 or CV890.
[0055] FIG. 9 depicts an ADP nucleotide and amino acid
sequence.
[0056] FIG. 10 depicts an IC.sub.50 isobologram of doxorubicin and
CV 890 on Hep3B cells at day 5.
[0057] FIG. 11 depicts in vivo efficacy of CV890 with doxorubicin.
Hep3B nude mouse xenografts were grouped (n=6) and treated with
CV890 alone (1.times.10.sup.11 particles/dose, iv), doxorubicin
alone (10 mg/kg, ip), CV890 and doxorubicin combination
(1.times.10.sup.11 particles of CV890 through tail vein and 10
mg/kg doxorubicin ip), or vehicle control. Tumor size was measured
weekly and the tumor volume were normalized as 100% at the day of
treatment. Error bars represent the standard error of the mean.
[0058] FIG. 12 shows the virus yield of CV802, CV882 and CV884 in
cell lines.
MODES FOR CARRYING OUT THE INVENTION
[0059] We have discovered and constructed improved adenovirus
vectors comprising co-transcribed first and second genes under
transcriptional control of a heterologous, target cell-specific
transcriptional regulatory element (TRE), wherein the second gene
is under translational control of an internal ribosome entry site
(IRES). In one embodiment, the first and second genes are
co-transcribed as a single mRNA and the second gene has a mutation
in or deletion of its endogenous promoter. In another embodiment,
at least one of the genes is an adenovirus gene and in yet another
embodiment, both genes are adenovirus genes, including adenovirus
genes that are essential for viral replication. The adenovirus
vector may comprise a gene that contributes to cytotoxicity
(whether direct and/or indirect), and/or causes cell death. An
example of an adenovirus gene that contributes to cytotoxicity
includes, but is not limited to, the adenovirus death protein
gene.
[0060] In some aspects of the present invention, an adenovirus
vector comprising co-transcribed first and second genes under
transcriptional control of a target cell-specific TRE, wherein the
second gene is under translational control of an IRES, exhibits
greater specificity for the target cell than an adenovirus vector
comprising a target cell-specific TRE operably linked to a gene and
lacking an IRES. In some embodiments, specificity is conferred by
preferential transcription and/or translation of the first and
second genes due to the presence of a target cell specific TRE. In
other embodiments, specificity is conferred by preferential
replication of the adenovirus vectors in target cells due to the
target cell-specific TRE driving transcription of a gene essential
for replication.
[0061] Also disclosed herein are IRES containing adenovirus vectors
comprising an adenovirus gene essential for viral replication
wherein said essential gene has a mutation in or deletion of its
endogenous promoter. In an embodiment disclosed herein, the
adenovirus vectors comprise the adenovirus early gene E1A which has
a deletion of its endogenous promoter. In another embodiment
disclosed herein, the adenovirus vectors comprise the adenovirus
early gene E1B which has a deletion of its endogenous promoter. In
other embodiments disclosed herein, the 19-kDa region of E1B is
deleted.
[0062] In another aspect, the adenovirus vectors disclosed herein
comprise an adenovirus gene essential for viral replication wherein
said essential gene has a mutation in or deletion of its endogenous
enhancer. In one embodiment, the adenovirus vector comprises the
adenovirus early gene E1A which has a mutation of or deletion in
its endogenous promoter. In one embodiment, the adenovirus vector
comprises the adenovirus early gene E1A which has a mutation of or
deletion in E1A enhancer 1. In a further embodiment, the adenovirus
vector comprises the adenovirus early gene E1A which has a mutation
of or deletion in its endogenous promoter and a mutation of or
deletion in the E1A enhancer. In an additional embodiment, the
adenovirus vector comprises the adenovirus early gene E1A which has
a mutation of or deletion in its endogenous promoter and the
adenovirus early gene E1B which has a mutation of or deletion in
its endogenous promoter. In an additional embodiment, the
adenovirus vector comprises the adenovirus early gene E1A, which
has a mutation of or deletion in its endogenous promoter and a
mutation of or deletion in the E1A enhancer I, and the adenovirus
early gene E1B which has a mutation of or deletion in its
endogenous promoter. In other embodiments disclosed herein, the
19-kDa region of E1B is deleted.
[0063] The replication-competent adenovirus vectors of the present
invention take advantage of what has been heretofore considered an
undesirable aspect of adenovirus 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,
it is noted 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.
This type of immune stimulation can be an important consideration
in the cancer context, where patients are often moderately to
severely immunocompromised.
[0064] The adenovirus vectors of the present invention comprising
an intergenic IRES element(s) which links the translation of two or
more genes, reflects an improvement over vector constructs which
use identical control regions to drive expression of two or more
desired genes in that any potential for homologous recombination
based on the presence of homologous control regions in the vector
is removed. As demonstrated herein, adenovirus vectors comprising
an IRES are stable and in some embodiments provide better
specificity than vectors not containing an IRES. Another advantage
of an adenovirus vector comprising an intergenic IRES is that the
use of an IRES rather than a second TRE may provide additional
space in the vector for an additional gene(s) such as a therapeutic
gene.
[0065] Thus, the adenovirus vectors comprising a second gene under
control of an IRES retain a high level of target cell specificity
and remain stable in the target cell. Accordingly, in one aspect of
the invention, the viral vectors disclosed herein comprise at least
one IRES within a multicistronic transcript, wherein production of
the multicistronic transcript is regulated by a heterologous,
target cell-specific TRE. For adenovirus vectors comprising a
second gene under control of an IRES, it is preferred that the
endogenous promoter of a gene under translational control of an
IRES be deleted so that the endogenous promoter does not interfere
with transcription of the second gene. It is preferred that the
second gene be in frame with the IRES if the IRES contains an
initiation codon. If an initiation codon, such as ATG, is present
in the IRES, it is preferred that the initiation codon of the
second gene is removed and that the IRES and the second gene are in
frame. Alternatively, if the IRES does not contain an initiation
codon or if the initiation codon is removed from the IRES, the
initiation codon of the second gene is used. In one embodiment, the
adenovirus vectors comprises the adenovirus essential genes, E1A
and E1B genes, under the transcriptional control of a heterologous,
cell-specific TRE, and an IRES introduced between E1A and E1B.
Thus, both E1A and E1B are under common transcriptional control,
and translation of E1B coding region is obtained by virtue of the
presence of the IRES. In one embodiment, E1A has its endogenous
promoter deleted. In another embodiment, E1A has an endogenous
enhancer deleted and in yet an additional embodiment, E1A has its
endogenous promoter deleted and E1A enhancer I deleted. In another
embodiment, E1B has its endogenous promoter deleted. In other
embodiments disclosed herein, the 19-kDa region of E1B is
deleted.
[0066] To provide cytotoxicity to target cells, one or more
transgenes having a cytotoxic effect may be present in the vector.
Additionally, or alternatively, an adenovirus gene that contributes
to cytotoxicity and/or cell death, such as the adenovirus death
protein (ADP) gene, can be included in the vector, optionally under
the selective transcriptional control of a heterologous TRE and
optionally under the translational control of an IRES.
[0067] Examples of target cells include neoplastic cells, although
any cell for which it is desirable and/or tolerable to sustain a
cytotoxic activity can be a target cell. By combining an adenovirus
vector(s) comprising a target cell-specific TRE with a mixture of
target and non-target cells, in vitro or in vivo, the vector(s)
preferentially replicates in the target cells, causing cytotoxic
and/or cytolytic effects. Once the target cells are destroyed due
to selective cytotoxic and/or cytolytic activity, replication of
the vector(s) is significantly reduced, lessening the probability
of runaway infection and undesirable bystander effects. In vitro
cultures can be retained to continually monitor the mixture (such
as, for example, a biopsy or other appropriate biological sample)
for the presence of the undesirable target cell, e.g., a cancer
cell in which the target cell-specific TRE is functional. The
adenovirus vectors of the present invention can also be used in ex
vivo procedures wherein desirable biological samples comprising
target cells are removed from the animal, subjected to exposure to
an adenovirus vector of the present invention comprising a target
cell-specific TRE and then replaced within the animal.
[0068] General Techniques
[0069] 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. Wei & 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 and annual updates); PCR: The Polymerase
Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in
Immunology (J. E. Coligan et al., eds., 1991 and annual
updates).
[0070] For techniques related to adenovirus, see, inter alia,
Felgner 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.
[0071] Definitions
[0072] As used herein, an "internal ribosome entry site" or "IRES"
refers to an element that promotes direct internal ribosome entry
to the initiation codon, such as ATG, of a cistron (a protein
encoding region), thereby leading to the cap-independent
translation of the gene. Jackson R J, Howell M T, Kaminski A (1990)
Trends Biochem Sci 15(12):477-83) and Jackson R J and Kaminski, A.
(1995) RNA 1(10):985-1000). The present invention encompasses the
use of any IRES element which is able to promote direct internal
ribosome entry to the initiation codon of a cistron. "Under
translational control of an IRES" as used herein means that
translation is associated with the IRES and proceeds in a
cap-independent manner. Examples of "IRES" known in the art
include, but are not limited to IRES obtainable from picornavirus
(Jackson et al., 1990, Trends Biochem Sci 15(12):477-483); and IRES
obtainable from viral or cellular mRNA sources, such as for
example, immunogloublin heavy-chain binding protein (BiP), the
vascular endothelial growth factor (VEGF) (Huez et al. (1998) Mol.
Cell. Biol. 18(11):6178-6190), the fibroblast growth factor 2, and
insulin-like growth factor, the translational initiation factor
eIF4G, yeast transcription factors TFIID and HAP4. IRES have also
been reported in different viruses such as cardiovirus, rhinovirus,
aphthovirus, HCV, Friend murine leukemia virus (FrMLV) and Moloney
murine leukemia virus (MoMLV). As used herein, "IRES" encompasses
functional variations of IRES sequences as long as the variation is
able to promote direct internal ribosome entry to the initiation
codon of a cistron. In preferred embodiments, the IRES is
mammalian. In other embodiments, the IRES is viral or protozoan. In
one illustrative embodiment disclosed herein, the IRES is
obtainable from encephelomycarditis virus (ECMV) (commercially
available from Novogen, Duke et al. (1992) J. Virol
66(3):1602-1609). In another illustrative embodiment disclosed
herein, the IRES is from VEGF. Table I and Table II disclose a
variety of IRES sequences useful in the present invention.
[0073] A "multicistronic transcript" refers to an mRNA molecule
which contains more than one protein coding region, or cistron. A
mRNA comprising two coding regions is denoted a "bicistronic
transcript." The "5'-proximal" coding region or cistron is the
coding region whose translation initiation codon (usually AUG) is
closest to the 5'-end of a multicistronic mRNA molecule. A
"5'-distal" coding region or cistron is one whose translation
initiation codon (usually AUG) is not the closest initiation codon
to the 5' end of the mRNA. The terms "5'-distal" and "downstream"
are used synonymously to refer to coding regions that are not
adjacent to the 5' end of a mRNA molecule.
[0074] As used herein, "co-transcribed" means that two (or more)
coding regions of polynucleotides are under transcriptional control
of single transcriptional control element.
[0075] A "gene" refers to a coding region of a polynucleotide. A
"gene" may or may not include non-coding sequences and/or
regulatory elements.
[0076] As used herein, a "transcription response element" or
"transcriptional regulatory element", or "TRE" is a polynucleotide
sequence, preferably a DNA sequence, which increases transcription
of an operably linked polynucleotide sequence in a host cell that
allows that TRE to function. A TRE can comprise an enhancer and/or
a promoter. A "transcriptional regulatory sequence" is a TRE. A
"target cell-specific transcriptional response element" or "target
cell-specific TRE" is a polynucleotide sequence, preferably a DNA
sequence, which is preferentially functional in a specific type of
cell, that is, a target cell. Accordingly, a target cell-specific
TRE transcribes an operably linked polynucleotide sequence in a
target cell that allows the target cell-specific TRE to function.
The term "target cell-specific", as used herein, is intended to
include cell type specificity, tissue specificity, developmental
stage specificity, and tumor specificity, as well as specificity
for a cancerous state of a given target cell. "Target cell-specific
TRE" includes cell type-specific and cell status-specific TRE, as
well as "composite" TREs. The term "composite TRE" includes a TRE
which comprises both a cell type-specific and a cell
status-specific TRE. A target cell-specific TRE can also include a
heterologous component, including, for example, an SV40 or a
cytomegalovirus (CMV) promoter(s). An example of a target cell
specific TRE which is tissue specific is a CMV TRE which contains
both promoter(s) and enhancer(s).
[0077] As described in more detail herein, a target cell-specific
TRE can comprise any number of configurations, including, but not
limited to, a target cell-specific promoter; and target
cell-specific enhancer; a heterologous promoter and a target
cell-specific enhancer; a target cell-specific promoter and a
heterologous enhancer; a heterologous promoter and a heterologous
enhancer; and multimers of the foregoing. The promoter and enhancer
components of a target cell-specific TRE may be in any orientation
and/or distance from the coding sequence of interest, as long as
the desired target cell-specific transcriptional activity is
obtained. 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.,
operably linked to) the target cell-specific TRE. As discussed
herein, a target cell-specific TRE can be of varying lengths, and
of varying sequence composition. As used herein, the term "cell
status-specific TRE" is preferentially functional, i.e., confers
transcriptional activation on an operably linked polynucleotide in
a cell which allows a cell status-specific TRE to function, i.e., a
cell which exhibits a particular physiological condition,
including, but not limited to, an aberrant physiological state.
"Cell status" thus refers to a given, or particular, physiological
state (or condition) of a cell, which is reversible and/or
progressive. The physiological state may be generated internally or
externally; for example, it may be a metabolic state (such as in
response to conditions of low oxygen), or it may be generated due
to heat or ionizing radiation. "Cell status" is distinct from a
"cell type", which relates to a differentiation state of a cell,
which under normal conditions is irreversible. Generally (but not
necessarily), as discussed herein, a cell status is embodied in an
aberrant physiological state, examples of which are given
below.
[0078] A "functional portion" of a target cell-specific TRE is one
which confers target cell-specific transcription on an operably
linked gene or coding region, such that the operably linked gene or
coding region is preferentially expressed in the target cells.
[0079] By "transcriptional activation" or an "increase in
transcription," it is intended that transcription is increased
above basal levels in the target cell (i.e., 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 fold 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 (i.e., a different
cell type), or the level of activity (if any) of a reporter
construct lacking a target cell-specific TRE as tested in a target
cell line.
[0080] A "functionally-preserved variant" of a target cell-specific
TRE is a target cell-specific TRE which differs from another target
cell-specific TRE, but still retains target cell-specific
transcription activity, although the degree of activation may be
altered (as discussed below). The difference in a target
cell-specific TRE can be due to differences in linear sequence,
arising from, for example, single 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 target cell-specific TRE. For example,
certain point mutations within sequences of TREs have been shown to
decrease transcription factor binding and stimulation of
transcription. See Blackwood, et al. (1998) Science 281:60-63 and
Smith et al. (1997) J. Biol. Chem. 272:27493-27496. One of skill in
the art would recognize that some alterations of bases in and
around transcription factor binding sites are more likely to
negatively affect stimulation of transcription 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.
[0081] 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, such as
a prostate cell. 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).
[0082] 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,
more preferably a human cell, even more preferably a prostate 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).
[0083] As used herein, a "prostate-specific antigen (PSA)
transcriptional regulatory element", or "PSA-TRE", or "PSE-TRE" is
a polynucleotide sequence, preferably a DNA sequence, which
selectively increases transcription of an operably linked
polynucleotide sequence in a host cell that allows a PSA-TRE to
function, such as a cell (preferably a mammalian cell, more
preferably a human cell, even more preferably a prostate cell) that
expresses androgen receptor. A PSA-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).
[0084] As used herein, a "carcinoembryonic antigen (CEA)
transcriptional regulatory element", or "CEA-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 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.
[0085] As used herein, an ".alpha.-fetoprotein (AFP)
transcriptional regulatory element", or "AFP-TRE" is a
polynucleotide sequence, preferably a DNA sequence, which
selectively increases transcription (of an operably linked
polynucleotide sequence) in a host cell that allows an AFP-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.
[0086] As used herein, an "a mucin gene (MUC) transcriptional
regulatory element", or "MUC1-TRE" is a polynucleotide sequence,
preferably a DNA sequence, which selectively increases
transcription (of an operably-linked polynucleotide sequence) in a
host cell that allows a MUC1-TRE to function, such as a cell
(preferably a mammalian cell, even more preferably a human cell)
that expresses MUC1. The MUC1-TRE is responsive to transcription
factors and/or co-factor(s) associated with MUC 1-producing cells
and comprises at least a portion of the MUC1 promoter and/or
enhancer.
[0087] As used herein, a "urothelial cell-specific transcriptional
response element", or "urothelial cell-specific TRE" is
polynucleotide sequence, preferably a DNA sequence, which increases
transcription of an operably linked polynucleotide sequence in a
host cell that allows a urothelial-specific TRE to function, i.e.,
a target cell. A variety of urothelial cell-specific TREs are
known, are responsive to cellular proteins (transcription factors
and/or co-factor(s)) associated with urothelial cells, and comprise
at least a portion of a urothelial-specific promoter and/or a
urothelial-specific enhancer. Methods are described herein for
measuring the activity of a urothelial cell-specific TRE and thus
for determining whether a given cell allows a urothelial
cell-specific TRE to function.
[0088] As used herein, a "melanocyte cell-specific transcriptional
response element", or "melanocyte cell-specific TRE" is
polynucleotide sequence, preferably a DNA sequence, which increases
transcription of an operably linked polynucleotide sequence in a
host cell that allows a melanocyte-specific TRE to function, i.e.,
a target cell. A variety of melanocyte cell-specific TREs are
known, are responsive to cellular proteins (transcription factors
and/or co-factor(s)) associated with melanocyte cells, and comprise
at least a portion of a melanocyte-specific promoter and/or a
melanocyte-specific enhancer. Methods are described herein for
measuring the activity of a melanocyte cell-specific TRE and thus
for determining whether a given cell allows a melanocyte
cell-specific TRE to function.
[0089] An "E1B 19-kDa region" (used interchangeably with "E1B
19-kDa genomic region") refers to the genomic region of the
adenovirus E1B gene encoding the E1B 19-kDa product. According to
wild-type Ad5, the E1B 19-kDa region is a 261 bp region located
between nucleotide 1714 and nucleotide 2244. The E1B 19-kDa region
has been described in, for example, Rao et al., Proc. Natl. Acad.
Sci. USA, 89:7742-7746. The present invention encompasses deletion
of part or all of the E1B 19-kDa region as well as embodiments
wherein the E1B 19-kDa region is mutated, as long as the deletion
or mutation lessens or eliminates the inhibition of apoptosis
associated with E1B-19 kDa.
[0090] As used herein, a target cell-specific TRE can comprise any
number of configurations, including, but not limited to, a target
cell-specific promoter; a target cell-specific enhancer; a target
cell-specific promoter and a target cell-specific enhancer; a
target cell-specific promoter and a heterologous enhancer; a
heterologous promoter and a target cell-specific enhancer; and
multimers of the foregoing. The promoter and enhancer components of
a target cell-specific TRE may be in any orientation and/or
distance from the coding sequence of interest, as long as the
desired target cell-specific transcriptional activity is obtained.
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.,
operably linked to) the target cell-specific TRE.
[0091] "Replicating preferentially", as used herein, means that the
adenovirus replicates more in a target cell than a non-target cell.
Preferably, the adenovirus replicates at a significantly higher
rate in target cells than non target cells; preferably, at least
about 2-fold higher, preferably, at least about 5-fold higher, more
preferably, at least about 10-fold higher, still more preferably at
least about 50-fold higher, even more preferably at least about
100-fold higher, still more preferably at least about 400- to
500-fold higher, still more preferably at least about 1000-fold
higher, most preferably at least about 1.times.10.sup.6 higher.
Most preferably, the adenovirus replicates solely in the target
cells (that is, does not replicate or replicates at a very low
levels in non-target cells).
[0092] As used herein, the term "vector" refers to a polynucleotide
construct designed for transduction/transfection of one or more
cell types. Vectors may be, for example, "cloning vectors" which
are designed for isolation, propagation and replication of inserted
nucleotides, "expression vectors" which are designed for expression
of a nucleotide sequence in a host cell, or a "viral vector" which
is designed to result in the production of a recombinant virus or
virus-like particle, or "shuttle vectors", which comprise the
attributes of more than one type of vector.
[0093] An "adenovirus vector" or "adenoviral vector" (used
interchangeably) comprises a polynucleotide construct of the
invention. A polynucleotide construct of this invention may be in
any of several forms, including, but not limited to, 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,
and complexed with compounds such as PEG to immunologically "mask"
the molecule and/or increase half-life, and conjugated to a
nonviral protein. 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. For purposes of this invention, adenovirus vectors are
replication-competent in a target cell.
[0094] The terms "polynucleotide" and "nucleic acid", used
interchangeably herein, refer to a polymeric form of nucleotides of
any length, either ribonucleotides or deoxyribonucleotides. These
terms include a single-, double- or triple-stranded DNA, genomic
DNA, cDNA, RNA, DNA-RNA hybrid, or a polymer comprising purine and
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 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) Molec.
Immunol. 32: 1057-1064. 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.
Reference to a polynucleotide sequence (such as referring to a SEQ
ID NO) also includes the complement sequence.
[0095] The following are non-limiting examples of polynucleotides:
a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA,
ribozymes, cDNA, recombinant polynucleotides, branched
polynucleotides, plasmids, vectors, isolated DNA of any sequence,
isolated RNA of any sequence, nucleic acid probes, and primers. A
polynucleotide may comprise modified nucleotides, such as
methylated nucleotides and nucleotide analogs, uracyl, other sugars
and linking groups such as fluororibose and thioate, and nucleotide
branches. The sequence of nucleotides may be interrupted by
non-nucleotide components. A polynucleotide may be further modified
after polymerization, such as by conjugation with a labeling
component. Other types of modifications included in this definition
are caps, substitution of one or more of the naturally occurring
nucleotides with an analog, and introduction of means for attaching
the polynucleotide to proteins, metal ions, labeling components,
other polynucleotides, or a solid support. Preferably, the
polynucleotide is DNA. As used herein, "DNA" includes not only
bases A, T, C, and G, but also includes any of their analogs or
modified forms of these bases, such as methylated nucleotides,
internucleotide modifications such as uncharged linkages and
thioates, use of sugar analogs, and modified and/or alternative
backbone structures, such as polyamides.
[0096] 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. A
preferred alignment program is ALIGN Plus (Scientific and
Educational Software, Pennsylvania), preferably using default
parameters, which are as follows: mismatch=2; open gap=0; extend
gap=2.
[0097] "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
initiation of, or promotes, transcription. "Operably linked" refers
to a juxtaposition wherein the elements are in an arrangement
allowing them to function.
[0098] An "E3 region" (used interchangeably with "E3") is a term
well understood in the art and means the region of the adenoviral
genome that encodes the E3 products (discussed herein). Generally,
the E3 region is located between about 28583 and 30470 of the
adenoviral genome. The E3 region has been described in various
publications, including, for example, Wold et al. (1995) Curr.
Topics Microbiol. Immunol. 199:237-274.
[0099] A "portion" of the E3 region means less than the entire E3
region, and as such includes polynucleotide deletions as well as
polynucleotides encoding one or more polypeptide products of the E3
region.
[0100] As used herein, "cytotoxicity" is a term well understood in
the art and refers to a state in which a cell's usual biochemical
or biological activities are compromised (i.e., inhibited). These
activities include, but are not limited to, metabolism; cellular
replication; DNA replication; transcription; translation; uptake of
molecules. "Cytotoxicity" includes cell death and/or cytolysis.
Assays are known in the art which indicate cytotoxicity, such as
dye exclusion, .sup.3H-thymidine uptake, and plaque assays.
[0101] The term "selective cytotoxicity", as used herein, refers to
the cytotoxicity conferred by an adenovirus vector of the present
invention on a cell which allows or induces a target cell-specific
TRE to function (a target cell) when compared to the cytotoxicity
conferred by an adenoviral vector of the present invention on a
cell which does not allow a target cell-specific TRE to function (a
non-target cell). Such cytotoxicity may be measured, for example,
by plaque assays, by 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.
[0102] In the context of adenovirus, a "heterologous
polynucleotide" or "heterologous gene" or "transgene" is any
polynucleotide or 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.
[0103] In the context of adenovirus, a "heterologous" promoter or
enhancer is one which is not associated with or derived from an
adenovirus gene.
[0104] In the context of adenovirus, an "endogenous" promoter,
enhancer, or TRE is native to or derived from adenovirus. In the
context of promoter, an "inactivation" means that there is a
mutation of or deletion in part or all of the of the endogenous
promoter, ie, a modification or alteration of the endogenous
promoter, such as, for example, a point mutation or insertion,
which disables the function of the promoter.
[0105] In the context of a target cell-specific TRE, a
"heterologous" promoter or enhancer is one which is derived from a
gene other than the gene from which a reference target
cell-specific TRE is derived.
[0106] "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. 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 .sup.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.
[0107] As used herein, the terms "neoplastic cells", "neoplasia",
"tumor", "tumor cells", "cancer" and "cancer cells", (used
interchangeably) 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 (i.e., de-regulated cell division). Neoplastic cells
can be malignant or benign.
[0108] A "host cell" includes an individual cell or cell culture
which can be or has been a recipient of an adenoviral vector(s) 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 or in
vitro with an adenoviral vector of this invention.
[0109] "Replication" and "propagation" are used interchangeably and
refer to the ability of an adenovirus vector of the invention to
reproduce or proliferate. These terms are 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.
[0110] An "ADP coding sequence" is a polynucleotide that encodes
ADP or a functional fragment thereof. In the context of ADP, a
"functional fragment" of ADP is one that exhibits cytotoxic
activity, especially cell lysis, with respect to adenoviral
replication. Ways to measure cytotoxic activity are known in the
art and are described herein.
[0111] A polynucleotide that "encodes" an ADP polypeptide is one
that can be transcribed and/or translated to produce an ADP
polypeptide or a fragment thereof. The anti-sense strand of such a
polynucleotide is also said to encode the sequence.
[0112] An "ADP polypeptide" is a polypeptide containing at least a
portion, or region, of the amino acid sequence of an ADP and which
displays a function associated with ADP, particularly cytotoxicity,
more particularly, cell lysis. As discussed herein, these functions
can be measured using techniques known in the art. It is understood
that certain sequence variations may be used, due to, for example,
conservative amino acid substitutions, which may provide ADP
polypeptides.
[0113] "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, such as those
characterized by amino acid additions, insertions, truncations and
deletions, 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.
[0114] A polynucleotide sequence that is "depicted in" a SEQ ID NO
means that the sequence is present as an identical contiguous
sequence in the SEQ ID NO. The term encompasses portions, or
regions of the SEQ ID NO as well as the entire sequence contained
within the SEQ ID NO.
[0115] A "biological sample" encompasses a variety of sample types
obtained from an individual and can be used in a diagnostic or
monitoring assay. The definition encompasses blood and other liquid
samples of biological origin, solid tissue samples such as a biopsy
specimen or tissue cultures or cells derived therefrom, and the
progeny thereof. The definition also includes samples that have
been manipulated in any way after their procurement, such as by
treatment with reagents, solubilization, or enrichment for certain
components, such as proteins or polynucleotides. The term
"biological sample" encompasses a clinical sample, and also
includes cells in culture, cell supernatants, cell lysates, serum,
plasma, biological fluid, and tissue samples.
[0116] An "individual" is a vertebrate, preferably a mammal, more
preferably a human. Mammals include, but are not limited to, farm
animals, sport animals, rodents, primates, and pets.
[0117] An "effective amount" is an amount sufficient to effect
beneficial or desired results, including 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.
[0118] A given TRE is "derived from" a given gene if it is
associated with that gene in nature.
[0119] "Expression" includes transcription and/or translation.
[0120] As used herein, the term "comprising" and its cognates are
used in their inclusive sense; that is, equivalent to the term
"including" and its corresponding cognates.
[0121] "A," "an" and "the" include plural references unless the
context clearly dictates otherwise.
[0122] Internal Ribosome Entry Site (IRES)
[0123] IRES elements were first discovered in picornavirus mRNAs
(Jackson R J, Howell M T, Kaminski A (1990) Trends Biochem Sci
15(12):477-83) and Jackson R J and Kaminski, A. (1995) RNA
1(10):985-1000). The present invention provides improved adenovirus
vectors comprising co-transcribed first and second genes under
transcriptional control of a heterologous, target cell-specific
TRE, and wherein the second gene (i.e., coding region) is under
translational control of an internal ribosome entry site (IRES).
Any IRES may be used in the adenovirus vectors of the invention, as
long as they exhibit requisite function in the vectors. Example of
IRES which can be used in the present invention include those
provided in Table I and referenced in Table II. Examples of IRES
elements include the encephelomycarditis virus (EMCV) which is
commercially available from Novagen (Duke et al. (1992) J. Virol
66(3):1602-9) the sequence for which is depicted in Table 1 (SEQ ID
NO:1). Another example of an IRES element disclosed herein is the
VEGF IRES (Huez et al. (1998) Mol Cell Biol 18(11):6178-90). This
IRES has a short segment and the sequence is depicted in Table 1
(SEQ ID NO:2).
[0124] The IRES promotes direct internal ribosome entry to the
initiation codon of a downstream cistron, leading to
cap-independent translation. Thus, the product of a downstream
cistron can be expressed from a bicistronic (or multicistronic)
mRNA, without requiring either cleavage of a polyprotein or
generation of a monocistronic mRNA. Therefore, in one illustrative
embodiment of the present invention, an adenovirus vector
comprising E1B under translational control of an IRES allows
translation of E1B from a bicistronic E1A-E1B mRNA under control of
a target cell-specific TRE. FIG. 7 provides a schematic
representation of adenovirus constructs of the present
invention.
[0125] Internal ribosome entry sites are approximately 450
nucleotides in length and are characterized by moderate
conservation of primary sequence and strong conservation of
secondary structure. The most significant primary sequence feature
of the IRES is a pyrimidine-rich site whose start is located
approximately 25 nucleotides upstream of the 3' end of the IRES.
See Jackson et al. (1990).
[0126] Three major classes of picornavirus IRES have been
identified and characterized: (1) the cardio- and aphthovirus class
(for example, the encephelomycarditis virus, Jang et al. (1990)
Gene Dev 4:1560-1572); (2) the entero- and rhinovirus class (for
example, polioviruses, Borman et al. (1994) EMBO J. 13:314903157);
and (3) the hepatitis A virus (HAV) class, Glass et al. (1993)
Virol 193:842-852). For the first two classes, two general
principles apply. First, most of the 450-nucleotide sequence of the
IRES functions to maintain particular secondary and tertiary
structures conducive to ribosome binding and translational
initiation. Second, the ribosome entry site is an AUG triplet
located at the 3' end of the IRES, approximately 25 nucleotides
downstream of a conserved oligopyrimidine tract. Translation
initiation can occur either at the ribosome entry site
(cardioviruses) or at the next downstream AUG (entero/rhinovirus
class). Initiation occurs at both sites in aphthoviruses.
[0127] HCV and pestiviruses such as bovine viral diarrhea virus
(BVDV) or classical swine fever virus (CSFV) have 341 nt and 370 nt
long 5'-UTR respectively. These 5'-UTR fragments form similar RNA
secondary structures and can have moderately efficient IRES
function (Tsukiyama-Kohara et al. (1992) J. Virol. 66:1476-1483;
Frolov I et al., (1998) RNA 4:1418-1435). Table I depicts the
5'-UTR region from HCV genome sequence (GenBank accession
D14853).
[0128] Leishmania RNA virus 1 (LRV1) is a double-stranded RNA
virus. Its 128 nt long 5'-UTR has IRES activity to facilitate the
cap-independent translation, (Maga et al. (1995) Mol Cell Biol
15:4884-4889). This fragment also forms conserved stemloop
secondary structure and at least the front part is essential.
[0129] Recent studies showed that both Friend-murine leukemia virus
(MLV) 5'-UTR and rat retrotransposon virus-like 30S (VL30)
sequences contain IRES structure of retroviral origin (Torrent et
al. (1996) Hum Gene Ther 7:603-612). These fragments are also
functional as packing signal when used in retrovirus derived
vectors. Studies of avian reticuloendotheliosis virus type A
(REV-A) show that its IRES maps downstream of the
packaging/dimerization (E/DLS) sequence and the minimal IRES
sequence appears to be within a 129 nt fragment (452-580) of the 5'
leader, immediately upstream of the gag AUG codon (Lopez-Lastra et
al. (1997) Hum Gene Ther 8:1855-1865).
[0130] In eukaryotic cells, translation is normally initiated by
the ribosome scanning from the capped mRNA 5' end, under the
control of initiation factors. However, several cellular mRNAs have
been found to have IRES structure to mediate the cap-independent
translation (van der Velde, et al. (1999) Int J Biochem Cell Biol.
31:87-106). Examples are immunoglobulin heavy-chain binding protein
(BiP) (Macejak et al. (1991) Nature 353:90-94), antennapedia mRNA
of Drosophilan (Oh et al. (1992) Gene and Dev 6:1643-1653),
fibroblast growth factor-2 (FGF-2) (Vagner et al. (1995) Mol Cell
Biol 15:35-44), platelet-derived growth factor B (PDGF-B)
(Bernstein et al. (1997) J Biol Chem 272:9356-9362), insulin-like
growth factor II (Teerink et al. (1995) Biochim Biophys Acta
1264:403-408), and the translation initiation factor eIF4G (Gan et
al. (1996) J Biol Chem 271:623-626). Table 1 depicts the
5'-noncoding region for BiP and PDGF. Recently, vascular
endothelial growth factor (VEGF) was also found to have IRES
element (Stein et al. (1998) Mol Cell Biol 18:3112-3119; Huez et
al. (1998) Mol Cell Biol 18:6178-6190).
[0131] Apart from the oligopyrimidine tract, nucleotide sequence
per se does not appear to be important for IRES function. Without
wishing to be bound by theory, a possible explanation for the
function of an IRES is that it forms secondary and/or tertiary
structures which orient particular single-stranded regions of its
sequence in a three-dimensional configuration that is conducive to
interaction with a mammalian ribosome (either ribosomal protein
and/or ribosomal RNA components) and/or initiation factor(s) and/or
RNA binding proteins which interact with ribosomes and/or
initiation factors. It is also possible that the three-dimensional
structure of the IRES is determined or stabilized by one or more
RNA-binding proteins. Thus it is possible to devise synthetic IRES
sequences having similar single-stranded regions in a similar
three-dimensional configuration.
[0132] In certain cases, one or more trans-acting cellular proteins
may be required for IRES function. For example, the HAV and
entero/rhinovirus IRESes function inefficiently in vitro in
reticulocyte lysates. Supplementation of a reticulocyte lysate with
a cytoplasmic extract from HeLa, Krebs II ascites, or L-cells
restores activity of entero/rhinovirus IRESes. See, for example,
Brown et al. (1979) Virology 97:396-405; and Dorner et al. (1984)
J. Virol. 50:507-514. Activity of the HAV IRES in vitro is
stimulated by liver cytoplasmic extracts. Glass et al. (1993)
Virology 193:1047-1050. These observations indicate that
cell-specific translational regulation can be achieved through the
use of a cell-specific IRES. Furthermore, coordinated cell-specific
transcriptional and translational regulatory elements can be
included in a vector to further increase cell specificity of viral
replication. For example, the combination of an AFP-TRE and a
HAV-IRES can be used to direct preferential replication of a vector
in hepatic cells. Thus, in one illustrative embodiment, a vector
comprises an AFP-TRE regulating the transcription of a bicistronic
E1A-E1B mRNA in which E1B translation is regulated by an ECMV IRES.
In another illustrative embodiment, the vector comprises a
probasin-TRE regulating the transcription of a bicistronic E1A-E1B
mRNA in which E1B translation is regulated by an ECMV IRES. In yet
another illustrative embodiment, a vector comprises a CMV-TRE
regulating the transcription of a bicistronic E1A-E1B mRNA in which
E1B translation is regulated by an ECMV IRES.
[0133] Examples of IRES which can be used in the present invention
include those provided in Table 1 and Table 2. An IRES sequence
which may be used in the present invention may be tested as
follows. A test vector is produced having a reporter gene, such as
luciferase, for example, placed under translational control of an
IRES to be tested. A desired cell type is transfected with the
vector containing the desired IRES-reporter gene and an assay is
performed to detect the presence of the reporter gene. In one
illustrative example, the test vector comprises a co-transcribed
chloramphenicol transferase (CAT) and luciferase encoding gene
transcriptionally driven by a CMV promoter wherein the luciferase
encoding gene is translationally driven by an IRES to be tested.
Host cells are transiently transfected with the test vector by
means known to those of skill in the art and assayed for the
presence of luciferase.
[0134] IRES may be prepared using standard recombinant and
synthetic methods known in the art, and as described in the
Examples. For cloning convenience, restriction sites may be
engineered into the ends of the IRES fragments to be used.
[0135] Transcriptional Response Elements (TREs)
[0136] The adenovirus vectors of the invention comprise target cell
specific TREs which direct preferential expression of an
operatively linked gene (or genes) in a particular target cell. A
TRE can be tissue-specific, tumor-specific, developmental
stage-specific, cell status specific, etc., depending on the type
of cell present in the tissue or tumor.
[0137] Cell- and tissue-specific transcriptional regulatory
elements, as well as methods for their identification, isolation,
characterization, genetic manipulation and use for regulation of
operatively linked coding sequences, are well known in the art. A
TRE can be derived from the transcriptional regulatory sequences of
a single gene, or sequences from different genes can be 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. Accordingly, in some embodiments,
the TRE used is preferentially functional in any of the following
cell types: prostate; liver; breast; urothelial cells (bladder);
colon; lung; ovarian; pancreas; stomach; and uterine. In other
embodiments, in accordance with cell status, the TRE is functional
in or during: low oxygen conditions (hypoxia); certain stages of
cell cycle, such as S phase; elevated temperature; ionizing
radiation.
[0138] As is known in the art, activity of TREs can be inducible.
Inducible TREs generally exhibit low activity in the absence of
inducer, and are up-regulated in the presence of inducer. Inducers
include, for example, nucleic acids, polypeptides, small molecules,
organic compounds and/or environmental conditions such as
temperature, pressure or hypoxia. 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 and in PCT/US98/04080. Accordingly,
in one embodiment of the present invention, an adenovirus vector
comprises an inducible heterologous TRE.
[0139] TRE multimers are also useful in the disclosed vectors. For
example, a TRE can comprise a tandem series of at least two, at
least three, at least four, or at least five promoter fragments.
Alternatively, a TRE can comprise one or more promoter regions
along with one or more enhancer regions. TRE multimers can also
comprise promoter and/or enhancer sequences from different genes.
The promoter and enhancer components of a TRE can be in any
orientation with respect to each other and can be in any
orientation and/or any distance from the coding sequence of
interest, as long as the desired cell-specific transcriptional
activity is obtained.
[0140] The disclosed vectors are designed such that replication is
preferentially enhanced in target cells in which the TRE(s) is
(are) functional. More than one TRE can be present in a vector, as
long as the TREs are functional in the same target cell. However,
it is important to note that a given TRE can be functional in more
than one type of target cell. For example, the CEA-TRE functions
in, among other cell types, gastric cancer cells, colorectal cancer
cells, pancreatic cancer cells and lung cancer cells.
[0141] A TRE for use in the present vectors may or may not comprise
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-target cells. Thus, presence of a
silencer can confer enhanced cell-specific vector replication by
more effectively preventing replication in non-target cells.
Alternatively, lack of a silencer may stimulate replication in
target cells, thus conferring enhanced target cell-specificity.
[0142] 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 regulatory function. Hence, functionally preserved
variants of TREs, comprising nucleic acid substitutions, additions,
and/or deletions, can be used in the vectors disclosed herein.
Accordingly, variant TREs retain function in the target cell but
need not exhibit maximal function. In fact, maximal transcriptional
activation activity of a TRE may not always be necessary to achieve
a desired result, and the level of induction afforded by a fragment
of a TRE may be sufficient for certain applications. For example,
if used for treatment or palliation of a disease state,
less-than-maximal responsiveness may be sufficient if, for example,
the target cells are not especially virulent and/or the extent of
disease is relatively confined.
[0143] Certain base modifications may result in enhanced expression
levels and/or cell-specificity. For example, nucleic acid sequence
deletions or additions within a TRE can move transcription
regulatory protein binding sites closer or farther away from each
other than they exist in their normal configuration, or rotate them
so they are on opposite sides of the DNA helix, thereby altering
spatial relationship among TRE-bound transcription factors,
resulting in a decrease or increase in transcription, as is known
in the art. Thus, while not wishing to be bound by theory, the
present disclosure contemplates the possibility that certain
modifications of a TRE will result in modulated expression levels
as directed by the TRE, including enhanced cell-specificity.
Achievement of enhanced expression levels may be especially
desirable in the case of more aggressive forms of neoplastic
growth, and/or when a more rapid and/or aggressive pattern of cell
killing is warranted (for example, in an immunocompromised
individual).
[0144] Transcriptional activity directed by a TRE (including both
inhibition and 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 and/or
of a protein product encoded by the sequence under control of
(i.e., operably linked to) a TRE.
[0145] As discussed herein, a TRE can be of varying lengths, and of
varying sequence composition. The size of a heterologous TRE will
be determined in part by the capacity of the viral vector, which in
turn depends upon the contemplated form of the vector (see infra).
Generally minimal sizes are preferred for TREs, as this provides
potential room for insertion of other sequences which may be
desirable, such as transgenes (discussed infra) and/or additional
regulatory sequences. In a preferred embodiment, such an additional
regulatory sequence is an IRES. 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 TRE sequences can be used as long as the resultant
adenoviral vector remains replication-competent.
[0146] An adenoviral vector can be packaged with extra sequences
totaling up to about 5% of the genome size, or approximately 1.8
kb, without requiring deletion of viral sequences. If non-essential
sequences are removed from the adenovirus genome, an additional 4.6
kb of insert can be tolerated (i.e., for a total insertion capacity
of about 6.4 kb). Examples of non-essential adenoviral sequences
that can be deleted are E3, and E4 sequences other than those which
encode E4 ORF6.
[0147] To minimize non-specific replication, endogenous (e.g.,
adenovirus) TREs are preferably removed from the vector. Besides
facilitating target cell-specific replication, removal of
endogenous TREs also provides greater insert capacity in a vector,
which may be of special concern if an adenoviral vector is to be
packaged within a virus particle. Even more importantly, deletion
of endogenous TREs prevents the 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 is constructed such that the
endogenous transcription control sequences of adenoviral genes are
deleted and replaced by one or more heterologous TREs. However,
endogenous TREs can be maintained in the adenovirus vector(s),
provided that sufficient cell-specific replication preference is
preserved. These embodiments are constructed by inserting
heterologous TREs between an endogenous TRE and a replication gene
coding segment. Requisite cell-specific replication preference is
determined 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.
[0148] Generally, a TRE will increase replication of a vector in a
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, compared
to basal levels of replication in the absence of a TRE. The
acceptable differential can be determined empirically (by
measurement of mRNA levels using, for example, RNA blot assays,
RNase protection assays or other assays known in the art) and will
depend upon the anticipated use of the vector and/or the desired
result.
[0149] Replication-competent adenovirus vectors directed at
specific target cells can be generated using TREs that are
preferentially functional in a target cell. In one embodiment of
the present invention, the target cell is a tumor cell.
Non-limiting examples of tumor cell-specific heterologous TREs, and
their respective target cells, include: probasin (PB), target cell,
prostate cancer (PCT/US98/04132); .alpha.-fetoprotein (AFP), target
cell liver cancer (PCT/US98/04084); mucin-like glycoprotein DF3
(MUC1), target cell, breast carcinoma (PCT/US98/04080);
carcinoembryonic antigen (CEA), target cells, colorectal, gastric,
pancreatic, breast, and lung cancers (PCT/US98/04133); plasminogen
activator urokinase (uPA) and its receptor gene, target cells,
breast, colon, and liver cancers (PCT/US98/04080); E2F] (cell cycle
S-phase specific promoter); target cell, tumors with disrupted
retinoblastoma gene function, and HER-2/neu (c-erbB2/neu), target
cell, breast, ovarian, stomach, and lung cancers (PCT/US98/04080);
tyrosinase, target cell, melanoma cells as described herein and
uroplakins, target cell, bladder cells as described herein. Methods
for identification, isolation, characterization and utilization of
additional target cell-specific TREs are readily available to those
of skill in the art.
[0150] In addition, tumor-specific TREs can 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), synthetase I (small intestine) Na.sup.+--K.sup.+--Cl.sup.-
transporter (kidney). Additional tissue-specific TREs are known in
the art.
[0151] In one embodiment of the present invention, a target
cell-specific, heterologous TRE is tumor cell-specific. A vector
can comprise a single tumor cell-specific TRE or multiple
heterologous TREs which are tumor cell-specific and functional in
the same cell. In another embodiment, a vector comprises one or
more heterologous TREs which are tumor cell-specific and
additionally comprises one or more heterologous TREs which are
tissue specific, whereby all TREs are functional in the same
cell.
[0152] Prostate-Specific TREs
[0153] In one embodiment, adenovirus vectors comprise heterologous
TREs that are prostate cell specific. For example, TREs that
function preferentially in prostate cells and can be used to target
adenovirus replication to prostate neoplasia, include, but are not
limited to, TREs derived from the prostate-specific antigen gene
(PSA-TRE) (Henderson U.S. Pat. No. 5,698,443); the glandular
kallikrein-1 gene (from the human gene, hKLK2-TRE) (PCT
US98/16312), and the probasin gene (PB-TRE) (PCT[US98/04132). All
three of these genes are preferentially expressed in prostate cells
and their expression is androgen-inducible. Generally, expression
of genes responsive to androgen induction is mediated by an
androgen receptor (AR).
[0154] Prostate-Specific Antigen (PSA)
[0155] PSA is synthesized exclusively in prostatic epithelial cells
and is synthesized in these cells whether they are normal,
hyperplastic, or malignant. This tissue-specific expression of PSA
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.
[0156] The region of the PSA gene that provides androgen-dependent
cell specificity, particularly in prostate cells, involves
approximately 6.0 kilobases (kb). 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. Within these enhancer
sequences is an androgen response element (ARE) a sequence which
binds androgen receptor. The sequence coordinates of the PSA
promoter are from about nt -540 to nt+8 relative to the
transcription start site. Juxtapositioning of the enhancer and
promoter yields a fully functional, minimal prostate-specific TRE
(PSA-TRE). Other portions of this approximately 6.0 kb region of
the PSA gene can be used in the vectors described herein, as long
as requisite functionality is maintained.
[0157] Human Glandular Kallikrein (hKLK2)
[0158] Human glandular kallikrein (hKLK2, encoding the hK2 protein)
is expressed exclusively in the prostate and its expression is
up-regulated by androgens, primarily through 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; and 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 indicate that hK2 antigen may be a
significant marker for 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, was observed to increase from
benign epithelium to high-grade prostatic intraepithelial neoplasia
(PIN) and adenocarcinoma.
[0159] The activity of the hKLK2 promoter has been described and a
region up to nt -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 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 to levels approximately
30-fold to approximately 100-fold greater than the level of
transcription in the absence of androgen. This induction is
generally independent of the orientation and position of the
enhancer sequences. Enhancer activity has also been demonstrated in
the following regions (all relative to the transcription start
site): about nt -3993 to about nt -3643, about nt -4814 to about nt
-3643, about nt -5155 to about nt -3387, about nt -6038 to about nt
-2394.
[0160] Thus, a hKLK2 enhancer can be operably linked to an hKLK2
promoter or a heterologous promoter to form a hKLK2 transcriptional
regulatory element (hKLK2-TRE). A 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.
[0161] Probasin
[0162] The rat probasin (PB) gene encodes an androgen and
zinc-regulated protein first characterized in the dorsolateral
prostate of the rat. 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.
[0163] A PB-TRE has been shown to exist 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. This minimal promoter sequence from the PB gene appears to
provide sufficient information to direct prostate-specific
developmental- and hormone-regulated expression of an operably
linked heterologous gene in transgenic mice. Greenberg et al.
(1994) Mol. Endocrinol. 8:230-239.
[0164] Alpha-Fetoprotein
[0165] .alpha.-fetoprotein (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. High serum AFP levels in patients appear to be due to AFP
expression in hepatocellular carcinoma (HCC), but not in
surrounding normal liver. Thus, expression of the AFP gene appears
to be characteristic of hepatoma cells. An AFP-TRE is described in
for example PCT/US98/04084.
[0166] According to published reports, the AFP-TRE is responsive to
cellular proteins (transcription factors and/or co-factor(s))
associated with AFP-producing cells, such as AFP-binding protein
(see, for example, U.S. Pat. No. 5,302,698) and comprises at least
a portion of an AFP promoter and/or an AFP enhancer. 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. A 5' AFP regulatory region (containing the promoter,
putative silencer, and enhancer) is contained within approximately
5 kb upstream from the transcription start site.
[0167] Within the AFP regulatory region, a human AFP enhancer
region is located between about nt -3954 and about nt -3335,
relative to the transcription start site of the AFP gene. The human
AFP promoter encompasses a region from about nt -174 to about
nt+29. Juxtapositioning of these two genetic elements, yields a
fully functional AFP-TRE. Ido et al. (1995) Cancer Res.
55:3105-3109 describe a 259 bp promoter fragment (nt -230 to nt+29)
that is specific for expression in HCC cells. The AFP enhancer,
located between nt -3954 and nt -3335 relative to the transcription
start site, contains two regions, denoted A and B. 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.
[0168] Suitable target cells for 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 (HCC) 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 as target cells for an AFP-TRE are hepatocellular
carcinoma cells and any of their metastases.
[0169] AFP production can be measured (and hence AFP-producing
cells can be identified) using immunoassays standard in the art,
such as RIA, ELISA or protein immunoblotting (Western blots) to
determine levels of AFP protein production; and/or RNA blotting
(Northern blots) to determine AFP mRNA levels. 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).
[0170] See also co-owned PCT WO98/39465 regarding AFP-TREs. As
described in more detail therein, an AFP-TRE can comprise any
number of configurations, including, but not limited to, an AFP
promoter; an AFP enhancer; an AFP promoter and an AFP enhancer; an
AFP promoter and a heterologous enhancer; a heterologous promoter
and an AFP enhancer; and multimers of the foregoing. The promoter
and enhancer components of an AFP-TRE can be in any orientation
and/or distance from the coding sequence of interest, as long as
the desired AFP cell-specific transcriptional activity is obtained.
An adenovirus vector of the present invention can comprise an
AFP-TRE endogenous silencer element or the AFP-TRE endogenous
silencer element can be deleted.
[0171] Urokinase Plasminogen Activator
[0172] The protein urokinase plasminogen activator (uPA) and its
cell surface receptor, urokinase plasminogen activator receptor
(uPAR), are expressed in many of the most frequently-occurring
neoplasms and appear to represent important proteins in cancer
metastasis. Both proteins are implicated in breast, colon,
prostate, liver, renal, lung and ovarian cancer. Sequence elements
that regulate uPA and uPAR transcription have been extensively
studied. Riccio et al. (1985) Nucleic Acids Res. 13:2759-2771;
Cannio et al. (1991) Nucleic Acids Res. 19:2303-2308.
[0173] Carcinoembryonic Antigen (CEA)
[0174] CEA is a 180,000 Dalton, tumor-associated, glycoprotein
antigen present on endodermally-derived neoplasms 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.
[0175] The 5'-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
transcriptional start site in the 5' flanking region of the gene,
was shown to confer cell-specific activity by virtue of providing
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. See PCT/GB/02546 The CEA promoter, putative silencer, and
enhancer elements appears to be contained within a region that
extends approximately 14.5 kb upstream from the transcription start
site. Richards et al. (1995); PCT/GB/02546. Further
characterization of the 5'-flanking region of the CEA gene by
Richards et al. (1995) supra indicated that two upstream regions
(one between about -13.6 and about -10.7 kb, and the other between
about -6.1 and about-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) supra also localized the promoter region between
about nt -90 and about nt+69 relative to the transcriptional start
site, with the region between about nt -41 and about nt -18 being
essential for expression. PCT/GB/02546 describes a series of
5'-flanking CEA fragments which confer cell-specific activity,
including fragments comprising the following sequences: 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; and nt -6100 to nt -3800, with
all coordinates being relative to the transcriptional start point.
In addition, cell-specific transcription activity is conferred on
an operably linked gene by the CEA fragment from nt -402 to
nt+69.
[0176] CEA-TREs for use in the vectors disclosed herein are derived
from mammalian cells, including, but not limited to, human cells.
Thus, any of the CEA-TREs can be used as long as the requisite
desired functionality is displayed by the vector.
[0177] Mucin
[0178] The protein product of the MUC1 gene (known as mucin, MUC1
protein; episialin; polymorphic epithelial mucin or PEM; EMA; DF3
antigen; NPGP; PAS-O; or CA15.3 antigen) is normally expressed
mainly at the apical surface of epithelial cells lining the glands
or ducts of the stomach, pancreas, lungs, trachea, kidney, uterus,
salivary glands, and mammary glands. 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.
[0179] Overexpression of the MUC1 gene in human breast carcinoma
cells MCF-7 and ZR-75-1 appears to occur at the transcriptional
level. Kufe et al. (1984) supra; Kovarik (1993) J. Biol. Chem.
268:9917-9926; and Abe et al. (1990) J. Cell. Physiol. 143:226-231.
The regulatory sequences of the MUC1 gene have been cloned,
including the approximately 0.9 kb upstream of the transcription
start site which contains a TRE that appears to be involved in
cell-specific transcription. Abe et al. (1993) Proc. Natl. Acad.
Sci. USA 90:282-286; Kovarik et al. (1993) supra; and Kovarik et
al. (1996) J. Biol. Chem. 271:18140-18147.
[0180] MUC1-TREs are derived from mammalian cells, including but
not limited to, human cells. Preferably, the MUC1-TRE is human. In
one embodiment, the MUC1-TRE contains the entire 0.9 kb 5' flanking
sequence of the MUC1 gene. In other embodiments, MUC1-TREs comprise
the following sequences (relative to the transcription start site
of the MUC1 gene) operably-linked to a promoter: about nt -725 to
about nt+31, about nt -743 to about nt+33, about nt -750 to about
nt+33, and about nt -598 to about nt+485.
[0181] c-erbB2/HER-2/neu
[0182] 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 and, 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 overexpression of the
c-erbB2/neu protein have been best studied in breast and ovarian
cancer. c-erbB2/neu protein over-expression occurs in 20 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.
[0183] Human, rat and mouse c-erbB2/neu TREs have been identified
and shown to confer transcriptional activity specific to
c-erbB2/neu-expressing cells. 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; and Scott et al.
(1994) J. Biol. Chem. 269:19848-19858.
[0184] Melanocyte-Specific TRE
[0185] It has been shown that some genes which encode melanoma
proteins are frequently expressed in melanoma/melanocytes, but
silent in the majority of normal tissues. A variety of
melanocyte-specific TRE are known, are responsive to cellular
proteins (transcription factors and/or co-factor(s)) associated
with melanocytes, and comprise at least a portion of a
melanocyte-specific promoter and/or a melanocyte-specific enhancer.
Known transcription factors that control expression of one or more
melanocyte-specific genes include the microphthalmia associated
transcription factor MITF. Yasumoto et al. (1997) J. Biol. Chem.
272:503-509. Other transcription factors that control expression of
one or more melanocyte specific genes include MART-1/Melan-A,
gp100, TRP-1 and TRP-2 Methods are described herein for measuring
the activity of a melanocyte-specific TRE and thus for determining
whether a given cell allows a melanocyte-specific TRE to
function.
[0186] In some embodiments, the melanocyte-specific TREs used in
this invention are derived from mammalian cells, including but not
limited to, human, rat, and mouse. Any melanocyte-specific TREs may
be used in the adenoviral vectors of the invention. Rodent and
human 5' flanking sequences from genes expressed specifically or
preferentially in melanoma cells 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 following
are some examples of melanocyte-specific TREs which can be used. A
promoter and other control elements in the human tyrosinase gene 5'
flanking region have been described and sequences have been
deposited as GenBank Accession Nos. X16073 and D10751. Kikuchi et
al. (1989) Biochim. Biophys. Acta 1009:283-286; and Shibata et al.
(1992) J. Biol. Chem. 267:20584-20588. A cis-acting element has
been defined that enhances melanocyte-specific expression of human
tyrosinase gene. This element comprises a 20-bp sequence known as
tyrosinase distal element (TDE), contains a CATGTG motif, and lies
at positions about -1874 to about -1835 relative to the human
tyrosinase gene transcription start site. Yasumoto et al. (1994)
Mol. Cell. Biol. 14:8058-8070. A promoter region comprising
sequences from about -209 to +61 of the human tyrosinase gene was
found to direct melanocyte-specific expression. Shibata (1992).
Similarly, the mouse tyrosinase 5' flanking region has been
analyzed and a sequence deposited as GenBank Accession Nos. D00439
and X51743. Kluppel et al. (1991) Proc. Natl. Acad. Sci. USA
88:3777-3788. A minimal promoter has been identified for the mouse
TRP-1 gene, and was reported to encompass nucleotides -44 to +107
relative to the transcription start site. Lowings et al. (1992)
Mol. Cell. Biol. 12:3653-3662. Two regulatory regions required for
melanocyte-specific expression of the human TRP-2 gene have been
identified. Yokoyama et al. (1994) J. Biol. Chem. 269:27080-27087.
A human MART-I promoter region has been described and deposited as
GenBank Accession No. U55231. Melanocyte-specific promoter activity
was found in a 233-bp fragment of the human MART-1 gene 5' flanking
region. Butterfield et al. (1997) Gene 191:129-134. A
basic-helix-loop-helix/leucine zipper-containing transcription
factor, MITF (microphthalmia associated transcription factor) was
reported to be involved in transcriptional activation of tyrosinase
and TRP-1 genes. Yasumoto et al. (1997) J. Biol. Chem.
272:503-509.
[0187] In some embodiments, a melanocyte-specific TRE comprises
sequences derived from the 5' flanking region of a human tyrosinase
gene depicted in Table 3. In some of these embodiments, the
melanocyte-specific TRE comprises tyrosinase nucleotides from about
-231 to about +65 relative to the transcription start site (from
about nucleotide 244 to about nucleotide 546 of SEQ ID NO:10) and
may further comprise nucleotides from about -1956 to about -1716
relative to the human tyrosinase transcription start site (from
about nucleotide 6 to about nucleotide 243 of SEQ ID NO:10). A
melanocyte-specific TRE can comprise nucleotides from about---231
to about +65 juxtaposed to nucleotides from about -1956 to about
-1716. It has been reported that nucleotides from about -1956 to
about -1716 relative to the human tyrosinase transcription start
site can confer melanocyte-specific expression of an operably
linked reporter gene with either a homologous or a heterologous
promoter. Accordingly, in some embodiments, a melanocyte-specific
TRE comprises nucleotides from about -1956 to about -1716 operably
linked to a heterologous promoter.
[0188] A melanocyte-specific TRE can also comprise multimers. For
example, a melanocyte-specific TRE can comprise a tandem series of
at least two, at least three, at least four, or at least five
tyrosinase promoter fragments. Alternatively, a melanocyte-specific
TRE could have one or more tyrosinase promoter regions along with
one or more tyrosinase enhancer regions. These multimers may also
contain heterologous promoter and/or enhancer sequences.
[0189] Cell Status-Specific TREs
[0190] Cell status-specific TREs for use in the adenoviral vectors
of the present invention can be derived from any species,
preferably a mammal. A number of genes have been described which
are expressed in response to, or in association with, a cell
status. Any of these cell status-associated genes may be used to
generate a cell status-specific TRE.
[0191] An example of a cell status is cell cycle. An exemplary gene
whose expression is associated with cell cycle is E2F-1, a
ubiquitously expressed, growth-regulated gene, which exhibits peak
transcriptional activity in S phase. Johnson et al. (1994) Genes
Dev. 8:1514-1525. The RB protein, as well as other members of the
RB family, form specific complexes with E2F-1, thereby inhibiting
its ability to activate transcription. Thus, E2F-1-responsive
promoters are down-regulated by RB. Many tumor cells have disrupted
RB function, which can lead to de-repression of E2F-1-responsive
promoters, and, in turn, de-regulated cell division.
[0192] Accordingly, in one embodiment, the invention provides an
E3-containing adenoviral vector in which an adenoviral gene
(preferably a gene necessary for replication) is under
transcriptional control of a cell status-specific TRE, wherein the
cell status-specific TRE comprises a cell cycle-activated TRE. In
one embodiment, the cell cycle-activated TRE is an E2 .mu.l
TRE.
[0193] Another group of genes that are regulated by cell status are
those whose expression is increased in response to hypoxic
conditions. Bunn and Poyton (1996) Physiol. Rev. 76:839-885; Dachs
and Stratford (1996) Br. J. Cancer 74:5126-5132; Guillemin and
Krasnow (1997) Cell 89:9-12. Many tumors have insufficient blood
supply, due in part to the fact that tumor cells typically grow
faster than the endothelial cells that make up the blood vessels,
resulting in areas of hypoxia in the tumor. Folkman (1989) J. Natl.
Cancer Inst. 82:4-6; and Kallinowski (1996) The Cancer J. 9:37-40.
An important mediator of hypoxic responses is the transcriptional
complex HIF-1, or hypoxia inducible factor-1, which interacts with
a hypoxia-responsive element (HRE) in the regulatory regions of
several genes, including vascular endothelial growth factor, and
several genes encoding glycolytic enzymes, including enolase-1.
Murine HRE sequences have been identified and characterized. Firth
et al. (1994) Proc. Natl. Acad. Sci. USA 91:6496-6500. An HRE from
a rat enolase-1 promoter is described in Jiang et al. (1997) Cancer
Res. 57:5328-5335. An HRE from a rat enolase-1 promoter is depicted
in Table 3.
[0194] Accordingly, in one embodiment, an adenovirus vector
comprises an adenovirus gene, preferably an adenoviral gene
essential for replication, under transcriptional control of a cell
status-specific TRE comprising an HRE. In one embodiment, the cell
status-specific TRE comprises the HRE depicted in Table 3.
[0195] Other cell status-specific TREs include heat-inducible
(i.e., heat shock) promoters, and promoters responsive to radiation
exposure, including ionizing radiation and UV radiation. For
example, the promoter region of the early growth response-1 (Egr-1)
gene contains an element(s) inducible by ionizing radiation.
Hallahan et al. (1995) Nat. Med. 1:786-791; and Tsai-Morris et al.
(1988) Nucl. Acids. Res. 16:8835-8846. Heat-inducible promoters,
including heat-inducible elements, have been described. See, for
example Welsh (1990) in "Stress Proteins in Biology and Medicine",
Morimoto, Tisseres, and Georgopoulos, eds. Cold Spring Harbor
Laboratory Press; and Perisic et al. (1989) Cell 59:797-806.
Accordingly, in some embodiments, the cell status-specific TRE
comprises an element(s) responsive to ionizing radiation. In one
embodiment, this TRE comprises a 5' flanking sequence of an Egr-1
gene. In other embodiments, the cell status-specific TRE comprises
a heat shock responsive element.
[0196] The cell status-specific TREs listed above are provided as
non-limiting examples of TREs that would function in the instant
invention. Additional cell status-specific TREs are known in the
art, as are methods to identify and test cell status specificity of
suspected cell status-specific TREs.
[0197] Urothelial Cell-Specific TREs
[0198] Any urothelial cell-specific TRE may be used in the
adenoviral vectors of the invention. A number of urothelial
cell-specific proteins have been described, among which are the
uroplakins. Uroplakins (UP), including UPIa and UPIb (27 and 28
kDa, respectively), UPII (15 kDa), and UPIII (47 kDa), are members
of a group of integral membrane proteins that are major proteins of
urothelial plaques. These plaques cover a large portion of the
apical surface of mammalian urothelium and may play a role as a
permeability barrier and/or as a physical stabilizer of the
urothelial apical surface. Wu et al. (1994) J. Biol. Chem.
269:13716-13724. UPs are bladder-specific proteins, and are
expressed on a significant proportion of urothelial-derived tumors,
including about 88% of transitional cell carcinomas. Moll et al.
(1995) Am. J. Pathol. 147:1383-1397; and Wu et al. (1998) Cancer
Res. 58:1291-1297. The control of the expression of the human UPII
has been studied, and a 3.6-kb region upstream of the mouse UPII
gene has been identified which can confer urothelial-specific
transcription on heterologous genes (Lin et al. (1995) Proc. Natl.
Acad. Sci. USA 92:679-683).
[0199] Preferred urothelial cell-specific TREs include TREs derived
from the uroplakins UPIa, UPIb, UPII, and UPIII, as well as
urohingin. A uroplakin TRE may be from any species, depending on
the intended use of the adenovirus, as well as the requisite
functionality is exhibited in the target or host cell.
Significantly, adenovirus constructs comprising a urothelial
cell-specific TREs have observed that such constructs are capable
of selectively replicating in urothelial cells as opposed to smooth
muscle cells, which adjoin urothelial cells in the bladder.
[0200] Uroplakin
[0201] Urothelial-specific TREs derived from the hUPII gene are
described herein. Accordingly, in some embodiments, an adenovirus
vector of the invention comprises an adenovirus gene, preferably an
adenoviral gene essential for replication, under transcriptional
control of a urothelial cell-specific TRE which comprises the 2.2
kb sequence from the 5' flanking region of hUPII gene, as shown in
Table 3. In other embodiments, an adenovirus vector of the
invention comprises an adenovirus gene, preferably an adenoviral
gene essential for replication, under transcriptional control of a
urothelial cell-specific TRE which comprises a 1.8 kb sequence from
the 5' flanking region of hUPII gene, from nucleotides 430 to 2239
as shown in Table 3. In other embodiments, the urothelial
cell-specific TRE comprises a functional portion of the 2.2 kb
sequence depicted in Table 3, or a functional portion of the 1.8 kb
sequence of nucleotides 430 to 2239 of the sequence depicted in
Table 3, such as a fragment of 2000 bp or less, 1500 bp or less, or
1000 bp or less, 600 bp less, or at least 200 bp which includes the
200 bp fragment of the hUPII 5'-flanking region.
[0202] A 3.6 kb 5'-flanking sequence located from the mouse UPII
(mUPI) gene which confers urothelial cell-specific transcription on
heterologous genes is one urothelial cell-specific TRE useful in
vectors of the instant invention (Table 3). Smaller TREs (i.e.,
3500 bp or less, more preferably less than about 2000 bp, 1500 bp,
or 1000 bp) are preferred. Smaller TREs derived from the mUPII 3.6
kb fragment are one group of preferred urothelial cell-specific
TREs. In particular, Inventors have identified an approximately 600
bp fragment from the 5' flanking DNA of the mUPII gene, which
contains 540 bp of 5' untranslated region (UTR) of the mUPII gene,
that confers urothelial cell-specific expression on heterologous
genes.
[0203] Accordingly, in some embodiments, an adenovirus vector of
the invention comprises an adenovirus gene, preferably an
adenoviral gene essential for replication, under transcriptional
control of a urothelial cell-specific TRE which comprises the 3.6
kb sequence from the 5' flanking region of mouse UPII gene, as
shown in Table 3. In other embodiments, the urothelial
cell-specific TRE comprises a functional portion of the 3.6 kb
sequence depicted in Table 3, such as a fragment of 3500 bp or
less, 2000 bp or less, 1500 bp or less, or 1000 bp or less which
includes the 540 bp fragment of 5' UTR. The urothelial
cell-specific TRE may also be a sequence which is substantially
identical to the 3.6 kb mUPII 5'-flanking region or any of the
described fragments thereof.
[0204] As an example of how urothelial cell-specific TRE activity
can be determined, a 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 is inserted into
a vector containing an appropriate reporter gene, including, but
not limited to, chloramphenicol acetyl transferase (CAT),
.beta.-galactosidase (encoded by the lacZ gene), luciferase
(encoded by the luc gene), a green fluorescent protein, alkaline
phosphatase, 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 target cell-specific TRE using transfection methods known
in the art, such as calcium phosphate precipitation,
electroporation, liposomes (lipofection) and DEAE dextran. Suitable
host cells include any urothelial cell type, including but not
limited to, KU-1, MYP3 (a non-tumorigenic rat urothelial cell
line), 804G (rat bladder carcinoma cell line), cultured human
urothelial cells (HUC), HCV-29, UM-UC-3, SW780, RT4, HL60, KG-1,
and KG-1A. Non-urothelial cells, such as LNCaP, HBL-100, HLF, HLE,
3T3, Hep3B, HuH7, CADO-LC9, and HeLa are used as a control. Results
are obtained by measuring the level of expression of the reporter
gene using standard assays. Comparison of expression between
urothelial cells and control indicates presence or absence of
transcriptional activation.
[0205] Comparisons between or among various urothelial
cell-specific TREs can be assessed by measuring and comparing
levels of expression within a single urothelial cell line. It is
understood that absolute transcriptional activity of a urothelial
cell-specific TRE will depend on several factors, such as the
nature of the target cell, delivery mode and form of the urothelial
cell-specific 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.
[0206] Alternatively a putative urothelial cell-specific TRE can be
assessed for its ability to confer adenoviral replication
preference for cells that allow a urothelial cell-specific TRE to
function. For this assay, constructs containing an adenovirus gene
essential to replication operatively linked to a putative
urothelial cell-specific TRE are transfected into urothelial cells.
Viral replication in those cells is compared, for example, to viral
replication by wild type adenovirus in those cells and/or viral
replication by the construct in non-urothelial cells.
[0207] TRE Configurations
[0208] A TRE as used in the present invention can be present in a
variety of configurations. A TRE can comprise multimers. For
example, a TRE can comprise a tandem series of at least two, at
least three, at least four, or at least five target cell-specific
TREs. These multimers may also contain heterologous promoter and/or
enhancer sequences.
[0209] Optionally, a transcriptional terminator or transcriptional
"silencer" can be placed upstream of the target cell-specific TRE,
thus preventing unwanted read-through transcription of the coding
segment under transcriptional control of the target cell-specific
TRE. Also, optionally, the endogenous promoter of the coding
segment to be placed under transcriptional control of the target
cell-specific TRE can be deleted.
[0210] A target cell-specific TRE may or may not lack a silencer.
The presence of a silencer (i.e., a negative regulatory element)
may assist in shutting off transcription (and thus replication) in
non-permissive cells (i.e., a non-target cell). Thus, presence of a
silencer may confer enhanced target cell-specific replication by
more effectively preventing adenoviral vector replication in
non-target cells. Alternatively, lack of a silencer may assist in
effecting replication in target cells, thus conferring enhanced
target cell-specific replication due to more effective replication
in target cells.
[0211] It is also understood that the invention includes a target
cell-specific TRE regulating the transcription of a bicistronic
mRNA in which translation of the second mRNA is associated by an
IRES. An adenovirus vector may further include an additional
heterologous TRE which may or may not be operably linked to the
same gene(s) as the target cell-specific TRE. For example a TRE
(such as a cell type-specific or cell status-specific TRE) may be
juxtaposed to a second type of target-cell-specific TRE.
"Juxtaposed" means a target cell-specific TRE and a second TRE
transcriptionally control the same gene. For these embodiments, the
target cell-specific TRE and the second TRE may be in any of a
number of configurations, including, but not limited to, (a) next
to each other (i.e., abutting); (b) both 5' to the gene that is
transcriptionally controlled (i.e., may have intervening sequences
between them); (c) one TRE 5' and the other TRE 3' to the gene.
[0212] As is readily appreciated by one skilled in the art, a
target cell-specific 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
target cell-specific transcription function. Hence, the invention
also includes functionally-preserved variants of the TRE nucleic
acid sequences disclosed herein, which include nucleic acid
substitutions, additions, and/or deletions. The variants of the
sequences disclosed herein may be 80%, 85%, 90%, 95%, 98%, 99% or
more identical, as measured by, for example, ALIGN Plus (Scientific
and Educational Software, Pennsylvania), preferably using efault
parameters, which are as follows: mismatch=2; open gap=0; extend
gap=2 to any of the urothelial cell-specific TRE sequences
disclosed herein. Variants of target cell-specific TRE sequences
may also hybridize at high stringency, that is at 68.degree. C. and
0.1.times.SSC, to any of the target cell-specific TRE sequences
disclosed herein.
[0213] In terms of hybridization conditions, the higher the
sequence identity required, the more stringent are the
hybridization conditions if such sequences are determined by their
ability to hybridize to a sequence of TRE disclosed herein.
Accordingly, the invention also includes polynucleotides that are
able to hybridize to a sequence comprising at least about 15
contiguous nucleotides (or more, such as about 25, 35, 50, 75 or
100 contiguous nucleotides) of a TRE disclosed herein. The
hybridization conditions would be stringent, i.e., 80.degree. C.
(or higher temperature) and 6M SSC (or less concentrated SSC).
Another set of stringent hybridization conditions is 68.degree. C.
and 0.1.times.SSC. For discussion regarding hybridization
reactions, see below.
[0214] Hybridization reactions can be performed under conditions of
different "stringency". Conditions that increase stringency of a
hybridization reaction of widely known and published in the art.
See, for example, Sambrook et al. (1989) at page 7.52. Examples of
relevant conditions include (in order of increasing stringency):
incubation temperatures of 25.degree. C., 37.degree. C., 50.degree.
C. and 68.degree. C.; buffer concentrations of 10.times.SSC,
6.times.SSC, 1.times.SSC, 0.1.times.SSC (where SSC is 0.15 M NaCl
and 15 mM citrate buffer) and their equivalents using other buffer
systems; formamide concentrations of 0%, 25%, 50%, and 75%;
incubation times from 5 minutes to 24 hours; 1, 2, or more washing
steps; wash incubation times of 1, 2, or 15 minutes; and wash
solutions of 6.times.SSC, 1.times.SSC, 0.1.times.SSC, or deionized
water. An exemplary set of stringent hybridization conditions is
68.degree. C. and 0.1.times.SSC.
[0215] "T.sub.m" is the temperature in degrees Celcius at which 50%
of a polynucleotide duplex made of complementary strands hydrogen
bonded in anti-parallel direction by Watson-Crick base pairing
dissociates into single strands under conditions of the experiment.
T.sub.m may be predicted according to a standard formula, such
as:
T.sub.m=81.5+16.6 log[X.sup.+]+0.41(% G/C)-0.61 (% F)-600/L
[0216] where [X.sup.+] is the cation concentration (usually sodium
ion, Na.sup.+) in mol/L; (% G/C) is the number of G and C residues
as a percentage of total residues in the duplex; (% F) is the
percent formamide in solution (wt/vol); and L is the number of
nucleotides in each strand of the duplex.
[0217] While not wishing to be bound by a single theory, the
inventors note that it is possible that certain modifications will
result in modulated resultant expression levels, including enhanced
expression levels. Achievement of modulated resultant expression
levels, preferably enhanced expression levels, may be especially
desirable in the case of certain, more aggressive forms of cancer,
or when a more rapid and/or aggressive pattern of cell killing is
warranted (due to an immunocompromised condition of the individual,
for example).
[0218] Determination of TRE Activity
[0219] Activity of a TRE can be determined, for example, 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), .beta.-galactosidase (encoded by the lacZ gene), luciferase
(encoded by the luc gene), alkaline phosphatase (AP), green
fluorescent protein (GFP), and horseradish peroxidase (HRP). 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, DEAE dextran-mediated transfer, particle bombardment or
direct injection. TRE activity is measured by detection and/or
quantitation of reporter gene-derived mRNA and/or protein. Reporter
protein product can be detected directly (e.g., immunochemically)
or through its enzymatic activity, if any, using an appropriate
substrate. Generally, to determine cell specific activity of a TRE,
a TRE-reporter gene construct is 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 lacking the TRE.
A TRE is determined to be cell-specific if it is preferentially
functional in one cell type, compared to a different type of
cell.
[0220] Adenovirus Early Genes
[0221] The adenovirus vectors of the invention comprise two or more
genes which are co-transcribed under the control of a target
cell-specific TRE wherein the second gene is under translational
control of an IRES. One or more of the genes can be an adenovirus
gene, preferably an adenovirus gene essential for replication. Any
gene that is essential for adenovirus replication, such as E1A,
E1B, E2, E4 or any of the late genes, is useful. The adenovirus may
also comprise E3. In addition, one or more of the genes can be a
transgene or heterologous gene. Any of the various adenovirus
serotypes can be used, such as, for example, Ad2, Ad5, Ad12 and
Ad40. For purposes of illustration, the Ad5 serotype is exemplified
herein.
[0222] The E1A gene is expressed immediately (between 0 and 2
hours) after viral infection, before any other viral genes. E1A
protein is a trans-acting positive 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 also expressed during early times after Ad5
infection. Flint (1982) Biochem. Biophys. Acta 651:175-208; Flint
(1986) Advances Virus Research 31:169-228; and Grand (1987)
Biochem. J. 241:25-38. In the absence of a functional E1A gene,
viral infection does not proceed, because the gene products
necessary for viral DNA replication are not produced. Nevins (1989)
Adv. Virus Res. 31:35-81. The transcription start site of Ad5 E1A
is at coordinate 498 and the ATG start site of the E1A protein is
at coordinate 560 in the virus genome.
[0223] The E1B protein is necessary in trans 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 E1B 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 E1B
promoter of Ad5 consists of a single high-affinity recognition site
for Sp1 and a TATA box, and extends from Ad5 nt 1636 to 1701.
[0224] Adenovirus E1B 19-kDa (19K) protein is a potent inhibitor of
apoptosis and cooperates with E1A to produce oncogenic
transformation of primary cells (Rao, et al., 1992, Cell Biology,
89:7742-7746). During productive adenovirus infection, E1A
stimulates host cell DNA synthesis, thereby causing cells to
aberrantly go through the cell cycle. In response to cell cycle
deregulation, the host cell undergoes apoptosis. As a defense
mechanism, the E1B 19-kDa protein inhibits this E1A-induced
apoptosis and allows assembly of viral progeny to be completed
before the cell commits suicide. E1B 19-kDa conducts anti-apoptotic
function by multiple mechanisms. E1B 19-kDa inhibits the apoptosis
of multiple stimuli, including E1a, p53 and TNF, for example.
According to wild-type Ad5, the E1B 19-kDa region is located
between nucleotide 1714 and nucleotide 2244. The E1B 19-kDa region
has been described in, for example, Rao et al., Proc. Natl. Acad.
Sci. USA, 89:7742-7746.
[0225] In a preferred embodiment, expression of the E1A and E1B
regions of the Ad genome is facilitated in a cell-specific fashion
by placing a cell-specific TRE upstream of E1A and a internal
ribosome entry site between E1A and E1B.
[0226] The E2 region of adenovirus encodes 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
E2 late promoter is transiently active during late stages of
infection and is independent of the E1A transactivator protein, the
E2 early promoter is crucial during the early phases of viral
replication.
[0227] The E2 early promoter of Ad5 is located between nucleotides
27,050 and 27,150, and consists of a major and a minor
transcription initiation site (the latter accounting for about 5%
of 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 E2 promoter architecture see
Swaminathan et al. (1995) Curr. Topics in Micro. and Imm. 199 part
3:177-194.
[0228] 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 by
only a few base pairs with sequences on the counterstrand which
encode a 33 kD protein. Notably, an SpeI restriction site (Ad5
position 27,082) 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 box from the upstream E2F
and ATF binding sites. Therefore, insertion of a heterologous TRE
having SpeI ends into the SpeI site disrupts the endogenous E2
early promoter of Ad5 and allows TRE-regulated expression of E2
transcripts.
[0229] An E3 region refers to the region of the adenoviral genome
that encodes the E3 products. The E3 region has been described in
various publications, including, for example, Wold et al. (1995)
Curr. Topics Microbiol. Immunol. 199:237-274. Generally, the E3
region is located between about 28583 and about 30470 of the
adenoviral genome. An E3 region for use in the present invention
may be from any adenovirus serotype. An E3 sequence is a
polynucleotide sequence that contains a sequence from an E3 region.
In some embodiments, the sequence encodes ADP. In other
embodiments, the sequence encodes other than ADP and excludes a
sequence encoding only ADP. As is well known in the art, the ADP
coding region is located in the E3 region within the adenoviral
genome from about 29468 bp to about 29773 bp; including the Y
leader, the location of ADP is from about 28375 bp to about 29773
bp for Ad5. Other ADP regions for other serotypes are known in the
art. An E3 sequence includes, but is not limited to, deletions;
insertions; fusions; and substitutions. An E3 sequence may also
comprise an E3 region or a portion of the E3 region. It is
understood that, as an "E3 sequence" is not limited to an "E3
region", alternative references herein to an "E3 region" or "E3
sequence" do not indicate that these terms are interchangeable.
Assays for determining a functional E3 sequence for purposes of
this invention are described herein.
[0230] The E4 gene has a number of transcription products and
encodes two polypeptides (the products of open reading frames
(ORFs) 3 and 6) which are responsible for stimulating the
replication of viral genomic DNA and stimulating late gene
expression, through interaction with heterodimers of cellular
transcription factors E2F-1 and DP-1. The ORF 6 protein requires
interaction with the E1B 55 kD protein for activity while the ORF 3
protein does not. In the absence of functional ORF 3- and ORF
6-encoded proteins, efficiency of plaque formation is less than
10.sup.-6 that of wild type virus.
[0231] To further increase cell-specificity of replication, it is
possible to take advantage of the interaction between the E4 ORF 6
gene product and the E1B 55 kD protein. For example, if E4 ORFs 1-3
are deleted, viral DNA replication and late gene synthesis becomes
dependent on E4 ORF6 protein. By generating such a deletion in a
vector in which the E1B region is regulated by a cell-specific TRE,
a virus is obtained in which both E1B and E4 functions are
dependent on the cell-specific TRE which regulates E1B.
[0232] Late genes relevant to the disclosed vectors are L1, L2 and
L3, which encode proteins of the virion. All of these genes
(typically coding for structural proteins) are probably required
for adenoviral replication. All late genes are under the control of
the major late promoter (MLP), which is located in Ad5 between
nucleotides 5986 and 6048.
[0233] In one embodiment, an adenovirus early gene is under
transcriptional control of a cell specific, heterologous TRE. In
additional embodiments, the early gene is selected from the group
including E1A, E1B, E2, E3, E4. In another embodiment, an
adenovirus late gene is under transcriptional control of a cell
specific, heterologous TRE. In further embodiments, two or more
early genes are under the control of heterologous TREs that
function in the same target cell. The heterologous TREs can be the
same or different, or one can be a variant of the other. In
additional embodiments, two or more late genes are under the
control of heterologous TREs that function in the same target cell.
The heterologous TREs can be the same or different, or one can be a
variant of the other. In yet another embodiment, one or more early
gene(s) and one or more late gene(s) are under transcriptional
control of the same or different heterologous TREs, wherein the
TREs function in the same target cell.
[0234] In some embodiments of the present invention, the adenovirus
vector comprises the essential gene E1A and the E1A promoter is
deleted. In other embodiments, the adenovirus vector comprises the
essential gene E1A and the E1A enhancer I is deleted. In yet other
embodiments, the E1A promoter is deleted and E1A enhancer I is
deleted. In other embodiments, an internal ribosome entry site
(IRES) is inserted upstream of E1B (so that E1B is translationally
linked), and a target cell-specific TRE is operably linked to E1A.
In still other embodiments, an (IRES) is inserted upstream of E1B
(so that E1B is translationally linked), and target cell-specific
TRE is operably linked to E1A, which may or may not maintain the
E1A promoter and/or enhancer I (i.e., the E1A promoter and/or
enhancer I may be, but not necessarily be, deleted). In yet other
embodiments, the 19-kDa region of E1B is deleted.
[0235] For adenovirus vectors comprising a second gene under
control of an IRES, it is preferred that the endogenous promoter of
a gene under translational control of an IRES be deleted so that
the endogenous promoter does not interfere with transcription of
the second gene. It is preferred that the second gene be in frame
with the IRES if the IRES contains an initiation codon. If an
initiation codon, such as ATG, is present in the IRES, it is
preferred that the initiation codon of the second gene is removed
and that the IRES and second gene are in frame. Alternatively, if
the IRES does not contain an initiation codon or if the initiation
codon is removed from the IRES, the initiation codon of the second
gene is used.
[0236] Adenovirus Death Protein (ADP) Gene and Gene Product
[0237] In the construction of adenovirus vectors, the E3 region is
often deleted to facilitate insertion of one or more TREs and/or
transgenes. In some embodiments, however, the adenovirus death
protein (ADP), encoded within the E3 region, is retained in an
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. (1992) J.
Virol. 66:3633; and Tollefson et al. (1996) J. Virol. 70:2296.
Thus, inclusion of an ADP gene in a viral vector can render the
vector more potent, making possible more effective treatment and/or
a lower dosage requirement.
[0238] An ADP coding sequence is obtained preferably from Ad2
(since this is the strain in which the ADP has been most 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 placed in
operative linkage to the ADP coding sequence. The ADP coding
sequence (with or without the Y leader) is then introduced into an
adenoviral genome, for example, in the E3 region, where expression
of the ADP coding sequence will be driven by the MLP. The ADP
coding sequence can, of course, also be inserted in other locations
of the adenovirus genome, such as the E4 region. Alternatively, the
ADP coding sequence can be operably linked to a heterologous TRE,
including, but not limited to, another viral TRE or a target
cell-specific TRE (see infra). In another embodiment, the ADP gene
is present in a viral genome such that it is transcribed as part of
a multi-cistronic mRNA in which its translation is associated with
an IRES.
[0239] E3-Containing Target Cell-Specific Adenoviral Vectors
[0240] In some embodiments, the adenovirus vectors contain an E3
region, or a portion of an E3 region. Inclusion of the E3 region of
adenovirus can enhance cytotoxicity of the target cell-specific
adenoviral vectors of the present invention. Adenoviral vectors
containing an E3 region may maintain their high level of
specificity and can be (a) significantly more cytotoxic; (b)
produce higher virus yield including extracellular virus yield; (c)
form larger plaques; (d) produce rapid cell death; and (e) kill
tumors more efficiently in vivo than vectors lacking the E3 region.
The adenoviral vectors of this invention may contain the E3 region
or a portion of the E3 region. It is understood that, as inclusion
of E3 confers observable and measurable functionality on the
adenoviral vectors, for example, increased replication and
production, functionally equivalent (in which functionality is
essentially maintained, preserved, or even enhanced or diminished)
variants of E3 may be constructed. For example, portions of E3 may
be used. A portion may be, non-inclusively, either of the
following: (a) deletion, preferably at the 3' end; (b) inclusion of
one or more various open reading frames of E3. Five proteins which
are encoded by the Ad-E3 region have been identified and
characterized: (1) a 19-kDa glycoprotein (gp19k) is one of the most
abundant adenovirus early proteins, and is known to inhibit
transport of the major histocompatibility complex class I molecules
to the cell surface, thus impairing both peptide recognition and
clearance of Ad-infected cells by cytotoxic T lymphocytes (CTLs);
(2) E3 14.7k protein and the E3 10.4k/14.5k complex of proteins
inhibit the cytotoxic and inflammatory responses mediated by tumor
necrosis factor (TNF); (3) E3 10.4k/14.5k protein complex down
regulates the epidermal growth factor receptor, which may inhibit
inflammation and activate quiescent infected cells for efficient
virus replication; (4) E3 11.6k protein (adenoviral death protein,
ADP) from adenovirus 2 and 5 appears to promote cell death and
release of virus from infected cells. The functions of three
E3-encoded proteins--3.6k, 6.7k and 12.5k--are unknown. A ninth
protein having a molecular weight of 7.5 kDa has been postulated to
exist, but has not been detected in cells infected with wild-type
adenovirus. Wold et al. (1995) Curr. Topics Microbiol. Immunol.
199:237-274. The E3 region is schematically depicted in FIG. 6.
These intact, portions, or variants of E3 may be readily
constructed using standard knowledge and techniques in the art.
Preferably, an intact E3 region is used.
[0241] In the adenovirus vectors of the present invention, E3 may
or may not be under transcriptional control of native adenoviral
transcriptional control element(s). The E3 promoter is located
within the coding sequence for virion protein VIII, an essential
protein which is highly conserved among adenovirus serotypes. In
some embodiments, E3 is under transcriptional control of a
heterologous TRE, including, but not limited to, a target
cell-specific TRE. Accordingly, in one embodiment, the invention
provides an adenoviral vector, preferably replication competent,
that comprises E3 region (or a portion of E3) under transcriptional
control of a target cell-specific TRE. In other embodiments, the E3
region is under transcriptional control of a native adenoviral TRE,
and the vector further comprises an adenoviral gene essential for
replication under transcriptional control of a target cell-specific
TRE. In other embodiments, the E3 region is under transcriptional
control of a target cell-specific TRE, and the vector further
comprises an adenoviral gene essential for replication under
transcriptional control of a target cell-specific TRE.
[0242] Transgenes Under Transcriptional Control of a Target
Cell-Specific TRE
[0243] Various other replication-competent adenovirus vectors can
be made according to the present invention in which, in addition to
having a single or multiple adenovirus gene(s) under control of a
target cell-specific TRE, a transgene(s) is/are also under control
of a target cell-specific TRE and optionally under translational
control of an IRES. Transgenes include, but are not limited to,
therapeutic transgenes and reporter genes.
[0244] Reporter Genes
[0245] For example, a target cell-specific TRE can be introduced
into an adenovirus vector immediately upstream of and operably
linked to an early gene such as E1A or E1B, and this construct may
further comprise a second co-transcribed gene under translational
control of an IRES. The second gene may be a reporter gene. The
reporter gene can encode a reporter protein, including, but not
limited to, chloramphenicol acetyl transferase (CAT),
.beta.-galactosidase (encoded by the lacZ gene), luciferase,
alkaline phosphatase, a green fluorescent protein, and horse radish
peroxidase. For detection of a putative cancer cell(s) in a
biological sample, the biological sample may be treated with
modified adenoviruses in which a reporter gene (e.g., luciferase)
is under control of a target cell-specific TRE. The target
cell-specific TRE will be transcriptionally active in cells that
allow the target cell-specific TRE to function, and luciferase will
be produced. This production will allow detection of target cells,
including cancer cells in, for example, a human host or a
biological sample. Alternatively, an adenovirus can be constructed
in which a gene encoding a product conditionally required for
survival (e.g., an antibiotic resistance marker) is under
transcriptional control of a target cell-specific TRE. When this
adenovirus is introduced into a biological sample, the target cells
will become antibiotic resistant. An antibiotic can then be
introduced into the medium to kill the non-cancerous cells.
[0246] Therapeutic Transgenes
[0247] Transgenes also include genes which may confer a therapeutic
effect, such as enhancing cytotoxicity so as to eliminate unwanted
target cells. In this way, various genetic capabilities may be
introduced into target cells, particularly cancer 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
cancerous target cell. This could be accomplished by coupling the
target cell-specific 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.
[0248] 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: 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 cytokines, antigens, transmembrane
proteins, and the like, such as IL-1, -2, -6, -12, GM-CSF, G-CSF,
M-CSF, IFN-.alpha., -.beta., -.chi., TNF-.alpha., -.beta.,
TGF-.alpha., -.beta., NGF, and the like. The positive effector
genes could be used in an earlier phase, followed by cytotoxic
activity due to replication.
[0249] Host Cells
[0250] 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
sequences requisite for maintenance in that host, such as
appropriate replication origin(s), are present. For convenience,
selectable markers are also provided. Host systems are known in the
art and need not be described in detail herein. Prokaryotic host
cells include bacterial cells, for example, E. coli, B. subtilis,
and mycobacteria. Among eukaryotic host cells are yeast, insect,
avian, plant, C. elegans (or nematode) and mammalian host cells.
Examples of fungi (including yeast) host cells are S. cerevisiae,
Kluyveromyces lactis (K. lactis), species of Candida including C.
albicans and C. glabrata, Aspergillus nidulans, Schizosaccharomyces
pombe (S. pombe), Pichia pastoris, and Yarrowia lipolytica.
Examples of mammalian cells are cultured human target cells (HUC),
KU-1, MYP3 (a non-tumorigenic rat target cell line), 804G (rat
bladder carcinoma cell line), HCV-29, UM-UC-3, SW780, RT4, HL60,
KG-1, and KG-1A. COS cells, mouse L cells, LNCaP cells, Chinese
hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, and
African green monkey cells. Xenopus laevis oocytes, or other cells
of amphibian origin, may also be used.
[0251] Compositions and Kits
[0252] 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. Compositions
can comprise an adenoviral vector(s) of the invention and a
suitable solvent, such as a physiologically acceptable buffer.
These are well known in the art. In other embodiments, 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 or
local administration to individuals in unit dosage forms, sterile
parenteral solutions or suspensions, sterile non-parenteral
solutions or oral solutions or suspensions, oil in water or water
in oil emulsions and the like. Formulations for parenteral and
nonparenteral drug delivery are known in the art and are set forth
in Remington's Pharmaceutical Sciences, 19th Edition, Mack
Publishing (1995). Compositions also include lyophilized and/or
reconstituted forms of the adenoviral vectors (including those
packaged as a virus, such as adenovirus) of the invention.
[0253] The present invention also encompasses kits containing an
adenoviral vector(s) 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 bladder cancer cells in a suitable
biological sample, such as biopsy specimens.
[0254] 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.
[0255] Preparation of the Adenovirus Vectors of the Invention
[0256] The adenovirus vectors of this invention can be prepared
using recombinant techniques that are standard in the art.
Generally, a target cell-specific TRE is inserted 5' to the
adenoviral gene of interest, preferably an adenoviral replication
gene, more preferably one or more early replication genes (although
late gene(s) can be used). A target cell-specific TRE 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 a target cell-specific
TRE. Accordingly, convenient restriction sites for annealing (i.e.,
inserting) a target cell-specific TRE can be engineered onto the 5'
and 3' ends of a UP-TRE using standard recombinant methods, such as
PCR.
[0257] 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.
[0258] Adenoviral vectors containing all replication-essential
elements, with the desired elements (e.g., E1A) under control of a
target cell-specific TRE, are conveniently prepared by homologous
recombination or in vitro ligation of two plasmids, one providing
the left-hand portion of adenovirus and the other plasmid providing
the right-hand region, one or more of which contains at least one
adenovirus gene under control of a target cell-specific 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 a target cell-specific TRE 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 also 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.
[0259] For convenience, plasmids are available that provide the
necessary portions of adenovirus. Plasmid pXC.1 (McKinnon (1982)
Gene 19:33-42) contains the wild-type left-hand end of Ad5. pBHG10
(Bett et al. (1994); 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 target cell-specific
TRE without deleting the endogenous enhancer/promoter. The gene for
E3 is located on the opposite strand from E4 (r-strand). pBHG11
provides an even larger E3 deletion (an additional 0.3 kb is
deleted). Bett et al. (1994). Alternatively, the use of pBHGE3
(Microbix Biosystems, Inc.) provides the right hand end of Ad5,
with a full-length of E3.
[0260] For manipulation of the early genes, the transcription start
site of Ad5 E1A is at 498 and the ATG start site of the E1A coding
segment is at 560 in the virus genome. This region can be used for
insertion of a target cell-specific TRE. A restriction site may be
introduced by employing polymerase chain reaction (PCR), where the
primer that is employed may be limited to the Ad5 genome, or may
involve a portion of the plasmid carrying the Ad5 genomic DNA. For
example, where pBR322 is used, the primers may use the EcoRI site
in the pBR322 backbone and the XbaI site at nt 1339 of Ad5. By
carrying out the PCR in two steps, where overlapping primers at the
center of the region introduce a nucleotide sequence change
resulting in a unique restriction site, one can provide for
insertion of target cell-specific TRE at that site.
[0261] A similar strategy may also be used for insertion of a
target cell-specific TRE element to regulate E1B. The E1B promoter
of Ad5 consists of a single high-affinity recognition site for Sp1
and a TATA box. This region extends from Ad5 nt 1636 to 1701. By
insertion of a target cell-specific TRE in this region, one can
provide for cell-specific transcription of the E1B gene. By
employing the left-hand region modified with the cell-specific
response element regulating E1A, as the template for introducing a
target cell-specific TRE to regulate E1B, the resulting adenovirus
vector will be dependent upon the cell-specific transcription
factors for expression of both E1A and E1B. In additional
embodiments, the 19-kDa region of E1B is deleted.
[0262] Similarly, a target cell-specific TRE can 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
Micro. and Immunol. (1995) 199(part 3):177-194.
[0263] 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 SpeI 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 target cell-specific TRE having SpeI ends into the
SpeI site in the 1-strand would disrupt the endogenous E2 early
promoter of Ad5 and should allow target cell-restricted expression
of E2 transcripts.
[0264] For E4, one must use the right hand portion of the
adenovirus genome. The E4 transcription start site is predominantly
at about nt 35605, the TATA box at about nt 35631 and the first
AUG/CUG of ORF I is at about nt 35532. Virtanen et al. (1984) J.
Virol. 51: 822-831. Using any of the above strategies for the other
genes, a UP-TRE may be introduced upstream from the transcription
start site. For the construction of full-length adenovirus with a
target cell-specific TRE inserted in the E4 region, the
co-transfection and homologous recombination are performed in W162
cells (Weinberg et al. (1983) Proc. Natl. Acad. Sci. 80:5383-5386)
which provide E4 proteins in trans to complement defects in
synthesis of these proteins.
[0265] Adenoviral constructs containing an E3 region can be
generated wherein homologous recombination between an E3-containing
adenoviral plasmid, for example, BHGE3 (Microbix Biosystems Inc.,
Toronto) and a non-E3-containing adenoviral plasmid, is carried
out.
[0266] Alternatively, an adenoviral vector comprising an E3 region
can be introduced into cells, for example 293 cells, along with an
adenoviral construct or an adenoviral plasmid construct, where they
can undergo homologous recombination to yield adenovirus containing
an E3 region. In this case, the E3-containing adenoviral vector and
the adenoviral construct or plasmid construct contain complementary
regions of adenovirus, for example, one contains the left-hand and
the other contains the right-hand region, with sufficient sequence
overlap as to allow homologous recombination.
[0267] Alternatively, an E3-containing adenoviral vector of the
invention can be constructed using other conventional methods
including standard recombinant methods (e.g., using restriction
nucleases and/or PCR), chemical synthesis, or a combination of any
of these. Further, deletions of portions of the E3 region can be
created using standard techniques of molecular biology.
[0268] Insertion of an IRES into a vector is accomplished by
methods and techniques that are known in the art and described
herein supra, including but not limited to, restriction enzyme
digestion, ligation, and PCR. A DNA copy of an IRES can be obtained
by chemical synthesis, or by making a cDNA copy of, for example, a
picornavirus IRES. See, for example, Duke et al. (1995) J. Vvirol.
66(3):1602-9) for a description of the EMCV IRES and Huez et al.
(1998), Mol. Cell. Biol. 18(11):6178-90) for a description of the
VEGF IRES. The internal translation initiation sequence is inserted
into a vector genome at a site such that it lies upstream of a
5'-distal coding region in a multicistronic mRNA. For example, in a
preferred embodiment of an adenovirus vector in which production of
a bicistronic E1A-E1B mRNA is under the control of a target
cell-specific TRE, the E1B promoter is deleted or inactivated, and
an IRES sequence is placed between E1A and E1B. IRES sequences of
cardioviruses and certain aphthoviruses contain an AUG codon at the
3' end of the IRES that serves as both a ribosome entry site and as
a translation initiation site. Accordingly, this type of IRES is
introduced into a vector so as to replace the translation
initiation codon of the protein whose translation it regulates.
However, in an IRES of the entero/rhinovirus class, the AUG at the
3' end of the IRES is used for ribosome entry only, and translation
is initiated at the next downstream AUG codon. Accordingly, if an
entero/rhinovirus IRES is used in a vector for translational
regulation of a downstream coding region, the AUG (or other
translation initiation codon) of the downstream gene is retained in
the vector construct.
[0269] Methods of packaging polynucleotides into adenovirus
particles are known in the art and are also described in co-owned
PCT PCT/US98/04080.
[0270] Delivery of Adenovirus Vectors
[0271] The adenoviral vectors can be used in a variety of forms,
including, but not limited to, naked polynucleotide (usually DNA)
constructs. Adenoviral vectors can, alternatively, comprise
polynucleotide constructs that are complexed with agents to
facilitate entry into cells, such as cationic liposomes or other
cationic 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 (such as PEG) to enhance or
dampen an immune response; complexed with agents that facilitate in
vivo transfection, such as DOTMA.TM., DOTAP.TM., and
polyamines.
[0272] If an adenoviral vector comprising an adenovirus
polynucleotide is packaged into a whole adenovirus (including the
capsid), the adenovirus itself may also 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.
[0273] 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, electroporation, 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).
[0274] If used in packaged adenoviruses, adenovirus vectors may be
administered in an appropriate physiologically acceptable carrier
at a dose of about 10.sup.4 to about 10.sup.14. 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 .mu.g to about 1000 .mu.g of an adenoviral vector
can be administered. The adenoviral vector(s) 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.
[0275] Methods Using the Adenovirus Vectors of the Invention
[0276] 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 using the
adenoviral vectors described above.
[0277] In one embodiment, methods are provided for conferring
selective cytotoxicity in cells that allow a target cell-specific
TRE to function, preferably target cells, comprising contacting
such cells with an adenovirus vector described herein. Cytotoxicity
can be measured using standard assays in the art, such as dye
exclusion, .sup.3H-thymidine incorporation, and/or lysis.
[0278] In another embodiment, methods are provided for propagating
an adenovirus specific for cells which allow a target cell-specific
TRE to function, preferably target cells, preferably cancer cells.
These methods entail combining an adenovirus vector with the cells,
whereby said adenovirus is propagated.
[0279] Another embodiment provides methods for killing cells that
allow a target cell-specific TRE to function in a mixture of cells,
comprising combining the mixture of cells with an adenovirus vector
of the present invention. The mixture of cells is generally a
mixture of normal cells and cancerous cells that allow a target
cell-specific TRE to function, and can be an in vivo mixture or in
vitro mixture.
[0280] The invention also includes methods for detecting cells
which allow a target cell-specific TRE to function, such as cancer
cells, 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. In one method, cells of a biological sample are
contacted with an adenovirus vector, and replication of the
adenoviral vector is detected. Alternatively, the sample can be
contacted with an adenovirus in which a reporter gene is under
control of a target cell-specific TRE. When such an adenovirus is
introduced into a biological sample, expression of the reporter
gene indicates the presence of cells that allow a target
cell-specific TRE to function. Alternatively, an adenovirus can be
constructed in which a gene conditionally required for cell
survival is placed under control of a target cell-specific TRE.
This gene may encode, for example, antibiotic resistance. Later the
biological sample is treated with an antibiotic. The presence of
surviving cells expressing antibiotic resistance indicates the
presence of cells capable of target cell-specific TRE function. A
suitable biological sample is one in which cells that allow a
target cell-specific TRE to function, such as cancer cells, may be
or are suspected to be present. Generally, in mammals, a suitable
clinical sample is one in which cancerous cells that allow a target
cell-specific TRE to function, such as carcinoma 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, cells that
allow a target cell-specific TRE to function can be detected using
in vitro assays that detect adenoviral proliferation, which are
standard in the art. Examples of such standard assays include, but
are not limited to, burst assays (which measure virus yield) and
plaque assays (which measure infectious particles per cell).
Propagation can also be detected by measuring specific adenoviral
DNA replication, which are also standard assays.
[0281] 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.
[0282] The invention further provides methods of suppressing tumor
cell growth, preferably a tumor cell that allows a target
cell-specific TRE to function, 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. For these methods, the adenoviral vector may or
may not be used in conjunction with other treatment modalities for
tumor suppression, such as chemotherapeutic agents (such as those
listed below), radiation and/or antibodies.
[0283] 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
that allow a target cell-specific TRE to function. Tumor cell
markers include, but are not limited to, CK-20. 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 from the individual to be
tested, and a suitable assay, such as an ELISA, is performed on the
biological sample. For these methods, the adenoviral vector may or
may not be used in conjunction with other treatment modalities for
tumor suppression, such as chemotherapeutic agents (such as those
listed below), radiation and/or antibodies.
[0284] The invention also provides methods of treatment, in which
an effective amount of an adenoviral vector(s) described herein is
administered to an individual. Treatment using an adenoviral
vector(s) is indicated in individuals with cancer as described
above. Also indicated are individuals who are considered to be at
risk for developing cancer (including single cells), such as those
who have had disease which has been resected and those who have had
a family history of cancer. 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) in a pharmaceutically acceptable excipient is
administered. Pharmaceutical compositions are described above. For
these methods, the adenoviral vector may or may not be used in
conjunction with other treatment modalities for tumor suppression,
such as chemotherapeutic agents (such as those listed below),
radiation and/or antibodies.
[0285] 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 target cell-specific TRE employed, and the
particular vector construct (i.e., which adenovirus gene(s) is
under target cell-specific TRE control) as well as whether the
adenoviral vector is used in conjunction with other treatment
modalities.
[0286] If administered as a packaged adenovirus, from about
10.sup.4 to about 10.sup.14, preferably from about 10.sup.4 to
about 10.sup.12, more preferably from about 10.sup.4 to about
10.sup.10. If administered as a polynucleotide construct (i.e., not
packaged as a virus), about 0.01 .mu.g to about 100 .mu.g can be
administered, preferably 0.1 .mu.g to about 500 .mu.g, more
preferably about 0.5 .mu.g to about 200 .mu.g. 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.
[0287] 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. Examples of chemotherapeutics
which are suitable for suppressing bladder tumor growth are BGC
(bacillus Calmett-Guerin); mitomycin-C; cisplatin; thiotepa;
doxorubicin; methotrexate; paclitaxel (TAXOL.TM.); ifosfamide;
gallium nitrate; gemcitabine; carboplatin; cyclosphasphamid;
vinblastine; vincristin; fluorouracil; etoposide; bleomycin.
Examples of combination therapies include (CISCA (cyclophosphamide,
doxorubicin, and cisplatin); CMV (cisplatin, methotrexate,
vinblastine); MVMJ (methodtrextate, vinblastine, mitoxantrone,
carboplain); CAP (cyclophosphamide, doxorubicin, cisplatin); MVAC
(methotrexate, vinblastine, doxorubicin, cisplatin). Radiation may
also be combined with chemotherapeutic agent(s), for example,
radiation with cisplatin. Administration of the chemotherapeutic
agents is generally intravesical (directly into the bladder) or
intravenous.
[0288] The following examples are provided to illustrate but not
limit the invention.
EXAMPLES
Example 1
Construction of a Replication-Competent Adenovirus Vector
Comprising an AFP-TRE and an EMCV IRES
[0289] The encephalomyocarditis virus (ECMV) IRES as depicted in
Table 1 was introduced between the E1A and E1B regions of a
replication-competent adenovirus vector specific for cells
expressing AFP as follows. Table 1 shows the 519 base pair IRES
segment which was PCR amplified from Novagen's pCITE vector by
primers A/B as listed in Table 4. A 98 base pair deletion in the
E1A promoter region was created in PXC.1, a plasmid which contains
the left-most 16 mu of Ad5. Plasmid pXC.1 (McKinnon (1982) Gene
19:33-42) contains the wild-type left-hand end of Ad5, from
Adenovirus 5 nt 22 to 5790 including the inverted terminal repeat,
the packaging sequence, and the E1a and E1b genes in vector pBR322.
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 resultant
plasmid, CP306 (PCT/US98/16312), was used as the backbone in
overlap PCR to generate CP624. To place a Sal1 site between E1a and
E1b, primers C/D, E/F (Table 4) were used to amplify CP306, plasmid
derived from pXC.1 and lacking the E1a promoter. After first round
PCR using CP306 as template and primers C/D, E/F, the resultant two
DNA fragments were mixed together for another round of overlapping
PCR with primers C/F. The overlap PCR product was cloned by blunt
end ligation to vector. The resultant plasmid, CP624 (Table 5),
contains 100 bp deletion in E1a/E1b intergenic region and
introduces Sal1 site into the junction. On this plasmid, the
endogenous E1a promoter is deleted, and the E1a polyadenylation
signal and the E1b promoter are replaced by the Sal1 site. Next,
the Sal1 fragment of CP625 was cloned into the Sal1site in CP624 to
generate CP627 (Table 5). CP627 has an EMCV IRES connecting
adenovirus essential genes E1a and E1b. In CP627, a series of
different tumor-specific promoters can be placed at the PinA1 site
in front of E1a to achieve transcriptional control on E1
expression.
1TABLE 4 Primer Sequence Note A. 5'-GACGTCGACTAATTCCGGTTATTTTCCA
For PCR EMCV IRES, GTCGAC is a SalI site. B.
5'-GACGTCGACATCGTGTTTTTCAAAGGAA For PCR EMCV IRES, GTCGAC is a SalI
site. C. 5'-CCTGAGACGCCCGACATCACCT- GTG Ad5 sequence to 1314 to
1338. D. 5'-GTCGACCATTCAGCAAACAAAGGCGTTAAC Antisense of Ad5
sequence 1572 to 1586. GTCGAC SalI site. Underline region overlaps
with E. E. 5'-TGCTGAATGGTCGACATGGAGGCTTGGGAG Ad5 sequence 1714 to
1728. GTCGAC is a SalI site. Underline region overlaps with D. F.
5'-CACAAACCGCTCTCCACAGATGCATG Antisense of Ad5 sequence 2070 to
2094.
[0290] For generating a liver cancer-specific virus, an about 0.8
kb AFP promoter fragment as shown in Table 3 was placed into the
PinA1 site of CP627 thereby yielding plasmid CP686. Full-length
viral genomes were obtained by recombination between CP686 and a
plasmid containing a right arm of an adenovirus genome. The right
arms used in virus recombination were pBHGE3 (Microbix Biosystems
Inc.), containing an intact E3 region, and pBHG11 or pBHG10 (Bett
et al. (1994) containing a deletion in the E3 region.
[0291] The virus obtained by recombination of CP686 with a right
arm containing an intact E3 region was named CV890. The virus
obtained by recombination of CP686 with a right arm containing a
deleted E3 region (pBHG 10) was named CV840. The structure of all
viral genomes was confirmed by conducting PCR amplifications that
were diagnostic for the corresponding specific regions.
[0292] Therefore, adenovirus vector designated CV890 comprises 0.8
kb AFP promoter, E1A, a deletion of the E1A promoter, EMCV IRES,
E1B a deletion of the E1B promoter and an intact E3 region.
Adenovirus vector CV840 comprises AFP promoter, E1A, a deletion of
the E1A promoter, EMCV IRES, E1B, a deletion of the E1B promoter
and a deleted E3 region.
2TABLE 5 Plasmid designation Brief description CP306 An E1A
promoter deleted plasmid derived from pXC.1 CP624 Overlap PCR
product from CP306 to generate 100 bp deletion and introduce a Sal1
site at E1A and E1B junction; E1A and E1B promoter deleted in
E1A/E1B intergenic region. CP625 EMCV IRES element ligated to
PCR-blunt vector (Invitrogen pCR .RTM. blunt vector). CP627 IRES
element derived from CP625 by Sal1 digestion and ligated to CP624
Sal1 site placing IRES upstream from E1B. CP628 Probasin promoter
derived from CP251 by PinA1 digestion and cloned into PinA1 site on
CP627. CP629 HCMV IE promoter amplified from pCMV beta (Clontech)
with PinA1 at 5' and 3' ends ligated into CP627 PinA1 site. CP630 A
163 bp long VEGF IRES fragment (Table 1) cloned into the Sal1 site
on CP628. CP686 AFP promoter from CP219 digested with PinA1 and
cloned into PinA1 site on CP627.
Example 2
Construction of a Replication-Competent Adenovirus Vector With a
Probasin TRE and an EMCV IRES
[0293] The probasin promoter as shown in Table 3 was inserted at
the PinAI site of plasmid CP627 (see Example 1) to generate CP628,
which contains a probasin promoter upstream of E1A and an EMCV IRES
between E1A and E1B. Full-length viral genomes were obtained by
recombination between CP628 and a plasmid containing a right arm of
an adenovirus genome. The right arms used in virus recombination
were pBHGE3, containing an intact E3 region, and pBHG11 or pBHG10
containing a deletion in the E3 region. The structure of all viral
genomes was confirmed by conducting PCR amplifications that were
diagnostic for the corresponding specific regions.
[0294] Therefore, adenovirus designated CV 834 comprises probasin
promoter, E1A, a deletion of the E1A promoter, EMCV IRES, E1B, a
deletion of the E1B endogenous promoter and a deleted E3
region.
Example 3
Construction of a Replication-Competent Adenovirus Vector With a
hCMV-TRE and an EMCV IRES
[0295] The hCMV immediate early gene (IE) promoter from plasmid
CP629, originally derived from pCMVBeta (Clonetech, Palo Alto) was
inserted at the PinAI site of plasmid CP627 (see Example 1) to
generate CP629, containing a CMV IE promoter upstream of E1A and an
IRES between E1A and E1B. Full-length viral genomes were obtained
by recombination between CP629 and a plasmid containing a right arm
of an adenovirus genome. The right arms used in virus recombination
were pBHGE3, containing an intact E3 region, and pBHG11 or pBHG10
containing a deletion in the E3 region. The structure of all viral
genomes was confirmed by conducting PCR amplifications that were
diagnostic for the corresponding specific regions.
[0296] Therefore, adenovirus vector designated CV835 comprises
hCMV-IE promoter, E1A, a deletion of the E1A promoter, EMCV IRES,
E1B a deletion in the E1B endogenous promoter and a deleted E3
region. CV835 lacks the hCMV enhancer and is therefore not tissue
specific. By adding the hCMV IE enhancer sequence to CV835, the
vector is made tissue specific.
Example 4
Replication of IRES-Containing Adenovirus Vectors With Different
TREs Controlling E1 Expression
[0297] The viral replication of adenovirus vectors comprising the
probasin promoter (CV836 and CV834) generally considered a weak
promoter, and the human cytomegalovirus immediate early gene
(HCMV-IE) promoter (CV837 and CV835), generally considered a strong
promoter, were characterized in the virus yield assay.
[0298] Probasin promoter containing adenovirus vectors (see
PCT/US98/04132), CV836 and CV834, and HCMV-IE promoter containing
adenovirus vectors, CV837 and CV835, were tested against a panel of
cell lines for viral replication (indicative of lethality) and
specificity. Cell lines 293 (the producer line), LNCap and HepG2
were plated at 0.5.times.10.sup.6 per well in 6 well tissue culture
plates, incubated for 24 hours at 37.degree. C., then infected with
CV836, CV834 or CV837 and CV835 at a multiplicity of infection
(MOI) of 2 plaque forming units per cell (PFU/cell) for 4 hours at
37.degree. C. At the end of the infection period, the medium was
replaced and the cells were incubated at 37.degree. C. for a
further 72 hours before harvesting for a viral yield assay as
described in Yu et al. (1999) Cancer Res. 59:1498-1504. The results
are shown in FIG. 3.
[0299] The data demonstrate that the presence of an IRES element in
the intergenic region between E1A and E1B does not significantly
affect viral replication, as compared to control viruses lacking an
IRES, such as a wild-type AD5 with a deletion in the E3 region. In
CV834, the loss of tissue cytotoxicity could be caused by the
weakness of the probasin promoter in the virus structure.
Example 5
Comparison of Dual TRE Vectors With Single TRE/IRES-Containing
Vectors
[0300] Two liver cancer-specific adenovirus vectors, CV790 and
CV733 (also designated CN790 and CN733, respectively), were
generated and characterized. See PCT/US98/04084. These viruses
contain two AFP TREs, one upstrean of E1A and one upstream of E1B.
They differ in that CV790 contains an intact E3 region, while the
E3 region is deleted in CV733. Replication of these two viruses was
compared with that of the newly generated IRES-containing viruses,
CV890 and CV840 (see Example 1).
[0301] Virus replication was compared, in different cell types,
using a virus yield assay as described in Example 4. Cells were
infected with each type of virus and, 72 hrs after infection, virus
yield was determined by a plaque assay. FIGS. 4A and 4B show viral
yield for different viruses in different cell types. The results
indicate that vectors containing an IRES between E1A and E1B (CV890
and CV840), in which E1B translation is regulated by the IRES,
replicate to similar extents as normal adenovirus and viruses with
dual AFP TREs, in AFP-producing cells such as 293 cells and
hepatoma cells. In SK-Hep-1 (liver cells), PA-1 (ovarian carcinoma)
and LNCaP cells (prostate cells) the IRES-containing viruses do not
replicate as well as dual TRE or wild-type adenoviruses, indicating
that the IRES-containing viruses have higher specificity for
hepatoma cells. Based on these results, it is concluded that
IRES-containing vectors have unaltered replication levels, but are
more stable and have better target cell specificity, compared to
dual-TRE vectors.
Example 6
Uroplakin Adenoviral Constructs Containing an EMCV IRES
[0302] A number of E3-containing viral constructs were prepared
which contained uroplakin II sequences (mouse and/or human) as well
as an EMCV internal ribosome entry site (IRES). The viral
constructs are summarized in Table 6. All of these vectors lacked
an E1A promoter and retained the E1A enhancer.
[0303] The 519 base pair EMCV]RES segment was PCR amplified from
Novagen's pCITE vector by primers A/B:
3 primer A: 5'-GACGTCGACTAATTCCGGTTATTTTCCA primer B
5'-GACGTCGACATCGTGTTTTTCAAAGGAA (GTCGAC is a SalI site).
[0304] The EMCV IRES element was ligated to PCR blunt vector
(Invitrogen pCR.RTM. blunt vector).
[0305] CP1066
[0306] The 1.9 kb-(-1885 to +1) fragment of mouse UPII from CP620
was digested with AflIII (blunted) and HindIII and inserted into
pGL3-Basic from CP620 which had been digested with XhoI (blunted)
and HindIII to generate CP1066.
[0307] CP1086
[0308] The 1.9 kb mouse UPII insert was digested with PinAI and
ligated with CP269 (CMV driving E1A and IRES driving E1B with the
deletions of E1A/E1B endogenous promoter) which was similarly cut
by PinAI.
[0309] CP1087
[0310] The 1 kb (-1128 to +1) human UPII was digested with PinAI
from CP665 and inserted into CP629 which had been cut by PinAI and
purified (to elute CMV).
[0311] CP1088
[0312] The 2.2 kb (-2225 to +1) human UPII was amplified from CP657
with primer 127.2.1 (5'-AGGACCGGTCACTATAGGGCACGCGTGGT-3') PLUS
127.2.2 (5'-AGGACCGGTGGGATGCTGGGCTGGGAGGTGG-3') and digested with
PinAI and ligated with CP629 cut with PinAI.
[0313] CP627 is an Ad5 plasmid with an internal ribosome entry site
(IRES) from encephelomycarditis virus (EMCV) at the junction of E1A
and E1B. First, CP306 (Yu et al., 1999) was amplified with primer
pairs 96.74.3/96.74.6 and 96.74.4/96.74.5.
[0314] The two PCR products were mixed and amplified with primer
pairs 96.74.3 and 96.74.5. The resultant PCR product contains a 100
bp deletion in E1A-E1B intergenic region and a new SaII site at the
junction. EMCV IRES fragment was amplified from pCITE-3a(+)
(Novagen) using primers 96.74.1 and 96.74.2. The Sail fragment
containing IRES was placed into Sail site to generate CP627 with
the bicistronic E1A-IRES-E1B cassette. CP629 is a plasmid with CMV
promoter amplified from pCMVbeta (Clontech) with primer 99.120.1
and 99.120.2 and cloned into PinAI site of CP627.
[0315] CP657 is a plasmid with 2.2 kb 5' flanking region of human
UP II gene in pGL3-Basic (Promega). The 2.2 kb hUPII was amplified
by PCR from GenomeWalker product with primer 100.113.1 and
100.113.2 and TA-cloned into pGEM-T to generate CP655.
[0316] The 2.2 kb insert digested from SacII (blunt-ended) and KpnI
was cloned into pGL3-Basic at HindIII (blunted) and KpnI to create
CP657.
[0317] CP1089
[0318] The 1 kb (-965 to +1) mouse UPII was digested by PinAI from
CP263 and inserted into CN422 (PSE driving E1A and GKE driving E1B
with the deletions of E1A/E1B endogenous promoter) cut by PinAI and
purified and further digested with EagI and ligated with 1 kb
(-1128 to +1) human UPII cut from CP669 with EagI.
[0319] CP1129
[0320] The 1.8 kb hUPII fragment with PinAI site was amplified from
CP657 with primer 127.50.1 and 127.2.2 and cloned into PinAI site
of CP629.
[0321] CP1131
[0322] CP686 was constructed by replacing the CMV promoter in CP629
with an AFP fragment from CP219. A 1.4 kb DNA fragment was released
from CP686 by digesting it with BssHII, filling with Klenow, then
digesting with BglII. This DNA fragment was then cloned into a
similarly cut CP686 to generate CP1199. In CP1199, most of the E1B
19-KDa region was deleted. The 1.8 kb hUPII fragment with PinAI
site was amplified from CP657 by PCR with primer 127.50.1 and
127.2.2 and inserted into similarly digested CP1199 to create
CP1131.
[0323] The plasmids above were all co-transfected with pBHGE3 to
generate CV874 (from CP1086), CV875 (from CP1087), CV876 (from
1088) and CV877 (from CP1089), CV882 (from CP1129) and CV884 (from
CP1131). CP1088, CP1129 and CP 1131 were cotransfected with pBHGE3
for construction of CV876, CV892 and CV884, respectively by
lipofectAMINE (Gibco/BRL) for 11-14 days. pBHGE3 was purchased from
Microbix, Inc., and was described previously. The cells were lysed
by three freeze-thaw cycles and plaqued on 293 cells for a week.
The single plaques were picked and amplified by infection in 293
cells for 3-5 days. The viral DNAs were isolated from the lysates
and the constructs were confirmed by PCR with primer
31.166.1/51.176 for CV876 and primer 127.50.1/51.176 for CV882 and
CV884 at E1 region and primer 32.32.1/2 for all three viruses at E3
region.
4TABLE 6 Name Vector Ad 5 Vector E1A TRE E1B TRE E3 CV874 CP1086
pBHGE3 1.9 kb mUPII IRES intact CV875 CP1087 pBHGE3 1.0 kb hUPII
IRES intact CV876 CP1088 pBHGE3 2.2 kb hUPII IRES intact CV877
CP1089 pBHGE3 1.0 kb mUPII 1.0 kb hUPII intact (E1B promoter
deleted) CV882 CP1129 pBHGE3 1.8 kb hUPII IRES intact CV884 CP1131
pBHGE3 1.8 kb hUPii IRES (E1B intact 19-kDa deleted)
[0324] Viruses are tested and characterized as described above.
[0325] Primer Sequences:
5 96.74.1 GACGTCGACATCGTGTTTTTCAAAGGAA 96.74.2
GACGTCGACTAATTCCGGTTATTTTCCA 96.74.3 CCTGAGACGCCCGACATCACCTGTG
96.74.4 TGCTGAATGGTCGACATGGAGGC- TTGGGAG 96.74.5
CACAACCGCTCTCCACAGATGCATG 96.74.6 GTCGACCATTCAGCAAACAAAGGCGTTAAC
100.113.1 AGGGGTACCCACTATAGGGCACGCGTGGT 100.113.2
ACCCAAGCTTGGGATGCTGGGCTGGGAGGTGG 127.2.2
AGGACCGGTGGGATGCTGGGCTGGGAGGTGG 127.50.1
AGGACCGGTCAGGCTTCACCCCAGACCCAC 31.166.1 TGCGCCGGTGTACACAGGAAGTGA
32.32.1 GAGTTTGTGCCATCGGTCTAC 32.32.2 AATCAATCCTTAGTCCTCCTG 51.176
GCAGAAAAATCTTCCAAACACTCCC 99.120.1 ACGTACACCGGTCGTTACATAACTTAC
99.120.2 CTAGCAACCGGTCGGTTCACTAAACG
Example 7
Construction of a Replication-Competent Adenovirus Vector With a
Tyrosinase TRE and EMCV IRES
[0326] CP621 is a plasmid containing a human tyrosinase enhancer
and promoter elements in a PinAI fragment. This fragment is ligated
to the PinAI site on CP627 to generate CP1078. CP1078 is combined
with pBHGE3 to generate a new melanoma specific virus, CV859. Table
3 depicts the polynucleotide sequence of the PinAI fragment which
contains a tyrosinase promoter and enhancer.
Example 8
Construction of a Replication-Competent Adenovirus Vector With a
Probasin-TRE and a VEGF IRES
[0327] Using a strategy similar to that described in Example 1, the
IRES fragment from the mouse vascular endothelial growth factor
(VEGF) gene is amplified and cloned into CP628 at the SalI site.
Table 1 depicts the IRES fragment obtainable from vascular
endothelial growth factor (VEGF) mRNA. In order to clone this
fragment into the E1a/E1b intergenic region, two pieces of long
oligonucleotide are synthesized. The sense oligonucleotide is shown
in the Table, whereas the second piece is the corresponding
antisense one. After annealing the two together to create a duplex,
the duplex is subjected to SalI digestion and the resulting
fragment is cloned into the SalI site on CP628. The resulting
plasmid, CP630, has a probasin promoter in front of E1a and an VEGF
IRES element in front of E1b. This plasmid is used to construct a
prostate cancer-specific virus comprising the VEGF IRES
element.
Example 9
Construction of a Replication-Competent Adenovirus Vector With an
AFP-TRE and a VEGF IRES
[0328] Using a strategy similar to Example 1, a PinAI fragment
which contains AFP TRE can be obtained. This AFP TRE is cloned into
the PinAI site in front of E1A on CP628 yielding plasmid CP1077.
This plasmid has the AFP TRE for E1 transcriptional control and the
VEGF IRES element before E1b. CP1077 can be recombined with pBHGE3
to generate a liver-specific adenovirus, designated as CV858.
Example 10
Construction of a Replication-Competent Adenovirus Vector With a
hKLK2-TRE and a EMCV IRES
[0329] Using a strategy similar to Example 1, the TRE fragment from
human glandular kallikrein II as shown in Table 3 was cloned into
the PinAI site in CP627. The resultant plasmid, CP1079, is
cotransfected with pBHGE3 to create CV860.
Example 11
Treatment of Hep3B Tumor Xenografts With Replication-Competent
Hepatoma Specific CV790 and Doxorubicin and Hepatoma Specific CV890
and Doxorubicin
[0330] CV790 is an AFP producing hepatocellular carcinoma specific
adenovirus, with E1A and E1B under the control of an identical AFP
promoter and enhancer (822 base pair promoter shown in Table 3)
with an E3 region. The CV890 adenovirus construct is also a
hepatoma or liver-specific adenoviral mutant with the E1A and E1B
genes under transcriptional control of 822 bp AFP promoter (827 bp
including nucleotides for restriction site), wherein E1B is under
translational control of EMCV IRES and having an intact E3 region.
The structure of CV890 therefore reads as AFP/E1A, IRES/E1B, E2,
E3, E4. In vivo studies of the efficacy of combinations of CV790
and doxorubicin and CV890 and doxorubicin were performed according
to the protocols described in detail in Example 4, with minor
alterations which are described below.
[0331] Xenografts in the study of CV790 and CV890 combined with
chemotherapeutic agents utilized liver carcinoma Hep3B cells.
Virus, CV790 or CV890, was administered by a single intravenous
injection of 1.times.10 1 particles through the tail veins of the
nude mice. One day after virus delivery, a single dose of
doxorubicin was given to each animal by i.p. injection. The
doxorubicin dose was 10 mg/kg for both doxorubicin alone and
doxorubicin combined with virus treatments. Tumor volume was
measured once a week for six weeks. Tumors were measured weekly in
two dimensions by external caliper and volume was estimated by the
formula [length (mm).times.width (mm).sub.2]/2.
[0332] FIG. 8 depicts the anti-tumor activity of CV890 containing
an IRES as compared to CV 790 containing dual TREs. As FIG. 8
demonstrates, relative tumor volume was less with administration of
CV890 than administration of CV790.
[0333] Furthermore, both CV790/doxorubicin and CV890/doxorubicin
treatment of the hepatoma showed synergistic results. Four days
after treatment with either CV790/doxorubicin or CV890/doxorubicin
the relative tumor volume was less than 10%. Unlike mice treated
with either virus alone or doxorubicin alone, after day 4, the
relative tumor volume did not increase for either the either
CV790/doxorubicin or CV890/doxorubicin treated mice. At day 6 in
the control mice, the relative tumor volume was approximately 1000%
in the CV790 study and approximately 600% in the CV890 study. The
relative tumor volumes of mice treated with virus alone were 250%
(CV790) and 520% (CV890) while the relative tumor volumes for mice
treated with doxorubicin alone were 450% with 280% in the CV790
study and 500% in the CV890 study.
Example 12
In Vitro Characterization of Melanocyte-Specific TRE-Containing
Adenoviral Constructs
[0334] An especially useful objective in the development of
melanocyte cell-specific adenoviral vectors is to treat patients
with melanoma. Methods are described below for measuring the
activity of a melanocyte-specific TRE and thus for determining
whether a given cell allows a melanocyte-specific TRE to
function.
[0335] Cells and Culture Methods
[0336] Host cells such as, HepG2 (liver); Lovo (colon); LNCaP
(prostate); PMEL (melanoma); SKMel (melanoma); G361 (melanoma) and
MeWo cells are obtained at passage 9 from the American Type Culture
Collection (Rockville, Md.). MeWo cells are maintained in RPMI 1640
medium (RPMI) supplemented with 10% fetal bovine serum (FBS;
Intergen Corp.), 100 units/mL of penicillin, and 100 units/mL
streptomycin. MeWo cells being assayed for luciferase expression
are maintained in 10% strip-serum (charcoal/dextran treated fetal
bovine serum to remove T3, T4, and steroids; Gemini Bioproduct,
Inc., Calabasas, Calif.) RPMI.
[0337] Transfections of MeWo Cells
[0338] For transfections, MeWo cells are plated out at a cell
density of 5.times.10.sup.5 cells per 6-cm culture dish (Falcon,
N.J.) in complete RPMI. DNAs are introduced into MeWo cells after
being complexed with a 1:1 molar lipid mixture of
N-[1-(2,3-dioleyloxy)propyl-N,N,N-trimethylamm- onium chloride
(DOTAP.TM.; Avanti Polar Lipids, Ala.) and
dioleoyl-phosphatidylethanolamine (DOPE.TM.; Avanti Polar Lipids,
Ala.); DNA/lipid complexes are prepared in serum-free RPMI at a 2:1
molar ratio. Typically, 8 .mu.g (24.2 nmole) of DNA is diluted into
200 .mu.L of incomplete RPMI and added dropwise to 50 mmole of
transfecting, lipids in 200 .mu.L of RPMI with gentle vortexing to
insure homogenous mixing of components. The DNA/lipid complexes are
allowed to anneal at room temperature for 15 minutes prior to their
addition to MeWo cells. Medium is removed from MeWo cells and
replaced with 1 mL of serum-free RPMI followed by the dropwise
addition of DNA/lipid complexes. Cells are incubated with complexes
for 4-5 hours at 37.degree. C., 5% CO.sub.2. Medium was removed and
cells washed once with PBS. The cells were then trypsinized and
resuspended in 10% strip-serum RPMI (phenol red free). Cells were
replated into an opaque 96-well tissue culture plate (Falcon, N.J.)
at a cell density of 40,000 cells/well per 100 .mu.L media and
assayed.
[0339] Plaque Assays
[0340] To determine whether the adenoviral constructs described
above replicate preferentially in melanocytes, plaque assays are
performed. Plaquing efficiency is evaluated in the following cell
types: melanoma cells (MeWo), prostate tumor cell lines (LNCaP),
breast normal cell line (HBL-100), ovarian tumor cell line
(OVCAR-3, SK-OV-3), and human embryonic kidney cells (293). 293
cells serve as a positive control for plaquing efficiency, since
this cell line expresses Ad5 E1A and E1B proteins. For analyzing
constructs comprising a melanocyte-specific TRE, cells that allow a
melanocyte-specific TRE to function, such as the cell lines
provided above and cells that do not allow such function, such as
HuH7, HeLa, PA-1, or G361, are used. The plaque assay is performed
as follows: Confluent cell monolayers are seeded in 6-well dishes
eighteen hours before infection. The monolayers are infected with
10-fold serial dilutions of each virus. After infecting monolayers
for four hours in serum-free media (MEM), the medium is removed and
replaced with a solution of 0.75% low melting point agarose and
tissue culture media. Plaques are scored two weeks after
infection.
Example 13
Construction of a Replication-Competent Adenovirus Vector With a
CEA-TRE and a EMCV IRES
[0341] Using a strategy similar to Example 1, the TRE fragment from
Carcinembryonic antigen (CEA)(Table 3, SEQ ID NO:14) is used to
construct virus designated CV873. A PinAI fragment containing the
CEA-TRE was cloned into the PinAI site in front of E1A CP627 for
the transcriptional control. The resultant plasmid CP1080 is used
together with pBHGE3 to generate CV873.
Example 14
In Vitro and In Vivo Assays of Anti-Tumor Activity
[0342] An especially useful objective in the development of
urothelial cell-specific adenoviral vectors is to treat patients
with bladder cancer. An initial indicator of the feasibility is to
test the vector(s) for cytotoxic activity against cell lines and
tumor xenografts grown subcutaneously in Balb/c nu/nu mice.
[0343] In Vitro Characterization of CV876
[0344] Virus Yield Assay for CV876
[0345] 5.times.10.sup.5293, RT-4, SW780, PA-1, G361, MKNI, HBL-100,
Fibroblast (from lung) and Smooth muscle cells (from bladder) were
plated into each well of six-well plates. Twenty-four hours later,
medium was aspirated and replaced with 1 ml of serum-free RPMI 1640
containing CV802 (wt.Ad5 with E3) or CV876 at a MOI of 2 pfu/cell.
After a 4-h incubation at 37.degree. C., cells were washed with
prewarmed PBS, and 2 ml of complete RPMI 1640 were added to each
well. After an additional 72 h at 37.degree. C., the cells were
scraped into medium and lysed by three freeze-thaw cycles. The
lysates were tested for virus production by triplicate plaque assay
for 8-10 days under semisolid agarose on 293 cells.
[0346] Unlike wt. Ad5, CV802 which grows well in all of the cells
tested, CV876 replicates much better in permissive cells (293, RT-4
and SW780) than in non-permissive cells (PA-1, G361, MKN1, HBL-100
and primary cells) by about 100-10000 fold. Noticeably, the
replication in SW780 for CV876 is about 100 fold less than CV802,
which indicates the limitation of this virus in efficacy.
[0347] Growth Curve Experiment for CV876
[0348] 5.times.10.sup.5 RT-4, PA-1, Smooth muscle and Fibroblast
cells were plated into each well of six-well plates. Twenty-four
hours later, medium was aspirated and replaced with 1 ml of
serum-free RPMI 1640 containing CV802 (wt.Ad5 with 133) or CV876 at
a MOI of 2 pfu/cell. After a 4-h incubation at 37.degree. C., cells
were washed with prewarmed PBS, and 2 ml of complete RPMI 1640 were
added to each well. At different time points of 0, 12, 24, 36, 48,
72, 96 and 120 h, the cells were scraped into medium and lysed by
three freeze-thaw cycles. The lysates were tested for virus
production by triplicate plaque assay for 8-10 days under semisolid
agarose on 293 cells.
[0349] Very similar as in virus yield assay, CV876 replicates well
only in RT-4 but not in primary cells and PA-1 over a 120 h period
of time. However, CV876 does show a delay of replication in RT-4
compared to CV802.
[0350] Cytopathic Effect Assay for CV876
[0351] 5.times.10.sup.5293, RT-4, SW780, PA-1, MKN1 and LNCap were
plated into each well of six-well plates. Twenty-four hours later,
medium was aspirated and replaced with 1 ml of serum-free RPMI 1640
containing CV802 (wt.Ad5 with E3) or CV876 at increasing MOI from
0.001 to 10 (the data shown was at MOI 1). After a 4-h incubation
at 37.degree. C., medium was replaced with 3 ml of complete RPMI
1640 and incubated at 37.degree. C. for 6-8 days when cytopathic
effect was observed for CV802 at MOI 0.01.
[0352] CV802 shows efficacy in all the cells tested while CV876
only kills the permissive cells (293, RT-4 and SW780) but not the
non-permissive cells (PA-1, MKN-1 and LNCap).
[0353] MTT Assay for CV876
[0354] 2.times.10.sup.4293, RT-4, SW780, MKN1, PA-1, HBL-100,
Smooth muscle cells (from bladder) and Fibroblast (from lung) were
plated into each well of 96-well plates. Twenty-four hours later,
the cells were infected with CV802 and CV876 at increasing MOI from
0.001 to 10 in complete RPMI 1640. A rapid colorimetric assay for
cell growth and survival was run at different time point of day 1,
3, 5, 7 and 10. The medium was replaced by 50 ul of MTT at 1 mg/ml
solution, which is converted to an insoluble purple formazan by
dehydrogenase enzymes present in active mitochondria of live cells.
After 3-4 h incubation at 37.degree. C., the solution was replaced
by isopropanol and the plates were incubated at 30.degree. C. for 1
h and read at 560 nm test wavelength and 690 nm reference
wavelength.
[0355] Similar as the results in CPE assay, CV876 shows efficacy
only in permissive cells but not in non-permissive cells. Again, in
RT-4 and SW780, CV876 kills the cells much slower than CV802.
[0356] In Vitro Characterization of CV882
[0357] Virus Yield Assay for CV882
[0358] 5.times.10.sup.5293, RT-4, SW780, G361, LNCap, HBL-100,
MKN1, PA-1, Fibroblast and Smooth muscle cells were plated into
each well of six-well plates. Twenty-four hours later, medium was
aspirated and replaced with 1 ml of serum-free RPMI 1640 containing
CV802 (wt.Ad5 with E3) or CV882 at a MOI of 2 pfu/cell. After a 4-h
incubation at 37.degree. C., cells were washed with prewarmed PBS,
and 2 ml of complete RPMI 1640 were added to each well. After an
additional 72 h at 37.degree. C., the cells were scraped into
medium and lysed by three freeze-thaw cycles. The lysates were
tested for virus production by triplicate plaque assay for 8-10
days under semisolid agarose on 293 cells.
[0359] The replication of CV882 in permissive cells (293, RT-4 and
SW780) is comparable to CV802 (the difference is less than 100
fold) while it shows over 1000-1000000 fold difference in
non-permissive cells (G361, LNCap, HBL-100, MKN1, PA-1 and primary
cells).
[0360] Growth Curve Experiment for CV882
[0361] 5.times.10.sup.5RT-4, PA-1, and Fibroblast cells were plated
into each well of six-well plates. Twenty-four hours later, medium
was aspirated and replaced with 1 ml of serum-free RPMI 1640
containing CV802 (wt.Ad5 with E3) or CV882 at a MOI of 2 pfu/cell.
After a 4-h incubation at 37.degree. C., cells were washed with
prewarmed PBS, and 2 ml of complete RPMI 1640 were added to each
well. At different time points of 0, 12, 24, 36, 48, 72, 96 and 120
h, the cells were scraped into medium and lysed by three,
freeze-thaw cycles. The lysates were tested for virus production by
triplicate plaque assay for 8-10 days under semisolid agarose on
293 cells.
[0362] Very similar as in virus yield assay, CV882 replicates well
only in RT-4 but not in primary cells and PA-1 over a 120 h period
of time. Additionally, CV882 shows better replication in RT-4
compared to CV876.
[0363] Cytopathic Effect Assay for CV882
[0364] 5.times.10.sup.5293, RT-4, SW780, HBL-100, G361, PA-1 and
Fibroblast cells were plated into each well of six-well plates.
Twenty-four hours later, medium was aspirated and replaced with 1
ml of serum-free RPNI 1640 containing CV802 (wt.Ad5 with E3) or
CV882 at increasing MOI from 0.001 to 10 (the data shown was at MOI
1). After a 4-h incubation at 37.degree. C., medium was replaced
with 3 ml of complete RPMI 1640 and incubated at 37.degree. C. for
6-8 days when cytopathic effect was observed for CV802 at MOI
0.01.
[0365] CV802 shows efficacy in all the cells tested while CV882
only kills the permissive cells (293, RT-4 and SW780) but not the
non-permissive cells (HBL-100, G361, PA-1 and Fibroblast
cells).
[0366] MTT Assay for CV882
[0367] 2.times.10.sup.4RT-4, SW780, PA-1, HBL-100, U118 and
Fibroblast were plated into each well of 96-well plates.
Twenty-four hours later, the cells were infected with CV802 and
CV882 at increasing MOI from 0.001 to 10 in complete RPMI 1640. A
rapid colorimetric assay for cell growth and survival was run at
different time points of day 1, 3, 5, 7 and 10. The medium was
replaced by 50 ul of MTT at 1 mg/ml solution, which is converted to
an insoluble purple formazan by dehydrogenase enzymes present in
active mitochondria of live cells. After 3-4 h incubation at
37.degree. C., the solution was replaced by isopropanol and the
plates were incubated at 30.degree. C. for 1 h and read at 560 nm
test wavelength and 690 nm reference wavelength.
[0368] Similar as the results in CPE assay, CV882 shows efficacy
only in permissive cells but not in non-permissive cells.
[0369] In Vitro Characterization of CV884
[0370] Virus Yield Assay for CV884
[0371] 5.times.10.sup.5293, RT-4, SW780, G361, LNCap, HBL-100,
MKN1, PA-1, Fibroblast and Smooth muscle cells were plated into
each well of six-well plates. Twenty-four hours later, medium was
aspirated and replaced with 1 ml of serum-free RPMI 1640 containing
CV802 (wt.Ad5 with E3) or CV984 at a MOI of 2 pfu/cell. After a 4-h
incubation at 37.degree. C., cells were washed with prewarmed PBS,
and 2 ml of complete RPMI 1640 were added to each well. After an
additional 72 h at 37.degree. C., the cells were scraped into
medium and lysed by three freeze-thaw cycles. The lysates were
tested for virus production by triplicate plaque assay for 8-10
days under semisolid agarose on 293 cells.
[0372] The replication of CV884 is very similar as CV802 in
permissive cells (293, RT-4 and SW780) but shows over 1000 fold
difference with CV802 in non-permissive cells (G361, LNCap,
HBL-100, MKN1, PA-1 and primary cells). CV884 shows better efficacy
than CV876 and CV882 without losing much specificity.
[0373] Growth Curve Experiment for CV884
[0374] 5.times.10.sup.5RT-4, PA-1, Smooth muscle and Fibroblast
cells were plated into each well of six-well plates. Twenty-four
hours later, medium was aspirated and replaced with 1 ml of
serum-free RPMI 1640 containing CV802 (wt.Ad5 with E3) or CV884 at
a MOI of 2 pfu/cell. After a 4-h incubation at 37.degree. C., cells
were washed with prewarmed PBS, and 2 ml of complete RPMI 1640 were
added to each well. At different time points of 0, 12, 24, 36, 48,
72, 96 and 120 h, the cells were scraped into medium and lysed by
three freeze-thaw cycles. The lysates were tested for virus
production by triplicate plaque assay for 8-10 days under semisolid
agarose on 293 cells.
[0375] Very similar as in virus yield assay, CV884 replicates very
well only in RT-4 (similar as CV802) but not in primary cells and
PA-1. Again, the replication of CV884 is better than CV882 and
CV876.
[0376] Cytopathic Effect Assay for CV884
[0377] 5.times.10.sup.5293, RT-4, SW780, G361, PA-1 and Fibroblast
cells were plated into each well of six-well plates. Twenty-four
hours later, medium was aspirated and replaced with 1 ml of
serum-free RPMI 1640 containing CV802 (wt.Ad5 with E3) or CV884 at
increasing MOI from 0.001 to 10 (the data shown was at MOI 1).
After a 4-h incubation at 37.degree. C., medium was replaced with 3
ml of complete RPMI 1640 and incubated at 37.degree. C. for 6-8
days when cytopathic effect was observed for CV802 at MOI 0.01.
[0378] CV802 shows efficacy in all the cells tested while CV884
only kills the permissive cells (293, RT-4 and SW780) but not the
non-permissive cells (G361, PA-I and Fibroblast cells).
[0379] MTT Assay for CV884
[0380] 2.times.10.sup.4293, RT-4, SW780, U118, Fibroblast and
Smooth muscle cells were plated into each well of 96-well plates.
Twenty-four hours later, the cells were infected with CV802 and
CV884 at increasing MOI from 0.001 to 10 in complete RPMI 1640. A
rapid colorimetric assay for cell growth and survival was run at
different time points of day 1, 3, 5, 7 and 10. The medium was
replaced by 50 ul of MTT at 1 mg/ml solution which is converted to
an insoluble purple formazan by dehydrogenase enzymes present in
active mitochondria of live cells. After 3-4 h incubation at
37.degree. C., the solution was replaced by isopropanol and the
plates were incubated at 30.degree. C. for 1 h and read at 560 nm
test wavelength and 690 nm reference wavelength.
[0381] Similar as the results in CPE assay, CV884 shows strong
efficacy (similar as wt. Ad5) only in permissive cells but not in
non-permissive cells.
[0382] In Vivo Activity of CV808
[0383] Mice were given subcutaneous (SC) injections of
1.times.10.sup.6 sW780 cells. When tumors grew to about 500
mm.sup.3, CV808 was introduced into the mice (5.times.10.sup.7 PFU
of virus in 0.1 ml PBS and 10% glycerol) intratumorally. Control
mice received vehicle alone. Tumor sizes were measured weekly. The
results are shown in FIG. 11. The data indicate that CV808 was
effective at suppressing tumor growth.
[0384] While it is highly possible 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 adenovirus vector 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 mice bearing bladder epithelial tumors
are inoculated with 108 to 1010 PFU of an adenoviral vector by tail
vein injection, or with buffer used to carry the virus as a
negative control. The effect of IV injection of the adenoviral
vector on tumor size is compared to vehicle treatment.
Example 15
Synergistic Effect of CV 890 With Chemotherapeutics
[0385] Materials and Methods
[0386] Cells. Hepatocellular carcinoma cell lines HepG2, Hep3B,
PLC/PRF/5, SNU449, and Sk-Hep-1, Chang liver cell (human normal
liver cells), as well as other tumor cell lines PA-1 (ovarian
carcinoma), UM-UC-3 (bladder carcinoma), SW 780 (bladder
carcinoma), HBL100 (breast epithelia), Colo 201 (Colon
adenocarcinoma), U 118 MG (glioblastoma) and LNCaP (prostate
carcinoma) were obtained from the American Type Culture Collection.
HuH-7 (liver carcinoma) was a generous gift of Dr. Patricia Marion
(Stanford University). 293 cells (human embryonic kidney containing
the E1 region of Adenovirus) were purchased from Microbix, Inc.
(Toronto, Canada). The primary cells nBdSMC (normal human bladder
smooth muscle cells), nHLFC (normal human lung fibroblast cells),
and nHMEC (normal human mammary epithelial cells) were purchased
from Clonetics (San Diego, Calif.). All tumor cell lines were
maintained in RPMI 1640 (BioWhittaker, Inc.) supplemented with 10%
fetal bovine serum (Irvine Scientific), 100 U/ml penicillin and 100
ug/ml streptomycin. Primary cells were maintained in accordance
with vendor instructions (Clonetics, San Diego). Cells were tested
for the expression of AFP by immunoassay (Genzyme Diagnostics, San
Carlos, Calif.).
[0387] Virus yield and one-step growth curves. Six well dishes
(Falcon) were seeded with 5.times.10.sup.5 cells per well of calls
of interest 24 hrs prior to infection. Cells were infected at an
multiplicity of infection (MOI) of 2 PFU/cell for three hours in
serum-free media. After 3 hours, the virus containing media was
removed, monolayers were washed three times with PBS, and 4 ml of
complete media (RPMI1640+10% FBS) was added to each well. 72 hours
post infection, cells were scraped into the culture medium and
lysed by three cycles of freeze-thaw.
[0388] The one-step growth curves time points were harvested at
various time points after infection. Two independent infections of
each virus cell-combination were titered in duplicate on 293 cells
(Yu et al., 1999, Cancer Research, 59:1498-1504.
[0389] Northern blot analysis. Hep3B or HBL100 cells were infected
at an MOI of 20 PFU/cell (plaque forming unit per cell) with either
CV802 or CV890 and harvested 24 hours post infection. Total cell
RNA was purified using the RNeasy protocol (Qiagen). The Northern
blot was conducted using NorthernMax Plus reagents (Ambion, Austin,
Tex.). 5 ug of RNA was fractionated on a 1% agarose,
formaldehyde-based denaturing gel and transferred to a
BrightStar-Plus (Ambion) positively charged membrane by capillary
transfer. The antisense RNA probes for E1A (adenovirus genome 501
bp to 1141 bp) or E1B (1540 bp-3910 bp) were PCR products cloned in
pGEM-T easy (Promega) and transcription labeled with [.sup.32P]
UTP. Blots were hybridized at 68.degree. C. for 14 hours with
ZipHyb solution and washed using standard methods (Ambion).
Membranes were exposed to BioMax film (Kodak).
[0390] Western blot analysis. Hep3B or HBL100 cells were infected
at MOI of 20 PFU/cell with either CV802 or CV890 and harvested 24
hours post infection. Cells were washed with cold PBS and lysed for
30 min on ice in (50 mM Tris, pH 8.0, 150 mM NaCl, 1% IGEPAL CA360
a NP40 equivalent (Sigma), 0.5% sodium deoxycholate, and protease
inhibitor cocktail from (Roche, Palo Alto, Calif.). After 30 min
centrifugation at 4 C, the supernatant was harvested and the
protein concentration determined with protein assay ESL kit
(Roche). Fifty micrograms of protein per lane were separated on
816% SDS-PAGE and electroblotted onto Hybond ECL membrane (Amersham
Pharmacia, Piscataway, N.J.). The membrane was blocked overnight in
PBST (PBS with 0.1% Tween-20) supplemented with 5% nonfat dry milk.
Primary antibody incubation was done at room temperature for 2-3
hrs with PBST/1% milk diluted antibody, followed by wash and 1 hr
incubation with diluted horseradish peroxidase-conjugated secondary
antibody (Santa Cruz Biotechnology Inc., Santa Cruz, Calif.).
Enhanced chemiluminescence (ECL; Amersham Pharmacia) was used for
the detection. E1A antibody (clone M58) was from NeoMarkers
(Fremont, Calif.), E1B-21 kD antibody was from Oncogene (Cambridge,
Mass.). All antibodies were used according manufacturer's
instruction.
[0391] Cell viability assay and statistical analysis. To determine
the cell killing effect of virus and chemotherapeutic agent in
combination treatment, a cell viability assay was conducted as
previously described with modifications (Denizot, 1986, Journal
Immunology. Methods, 89:271-277). On 96 well plates, cells of
interest were seeded at 10,000 calls per well 48 hr prior to
infection. Cells were then treated with virus alone, drug alone, or
in combination. Cell viability was measured at different time
points by removing the media, adding 50 l of 1 mg/ml solution of
MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium
bromide) (Sigma, St. Louis, Mo.) and incubating for 3 hrs at
37.degree. C. After removing the MTT solution, the crystals
remaining in the wells were solubilized by the addition of 50 l of
isopropanol followed by 30 C incubation for 0.5 hr. The absorbency
was determined on a microplate reader (Molecular Dynamics) at 560
nm (test wavelength) and 690 nm (reference wavelength). The
percentage of surviving cells was estimated by dividing the
OD.sub.550-OD.sub.650 of virus or drug treated cells by the
OD.sub.550-OD.sub.650 of control cells. 6 replica samples were
taken for each time point and each experiment was repeated at least
three times.
[0392] For statistical analysis, CurveExpert (shareware by Daniel
Hyams, version 1.34) was used to plot the dose-response curves for
virus and drugs. Based upon the dose-response curves, the
isobolograms were made according to the original theory of Steel
and Peckham (1993, Int. J. Rad. Onc. Biol. Phys., 5:85) and method
described in Aoe et al. (1999, Anticancer Res. 19:291-299).
[0393] Animal studies. Six to eight week old athymic BALB/C nu/nu
mice were obtained from Simonson Laboratories (Gilroy, Calif.) and
acclimated to laboratory conditions one week prior to tumor
implantation. Xenografts were established by injecting
1.times.10.sup.6 Hep3B, HepG2 or LNCAP cells suspended in 100 l of
RPMI 1640 media subcutaneously. When tumors reached between 200
mm.sup.3 and 300 mm.sup.3, mice were randomized and dosed with 100
l of test article via intratumoral or the tail vein injection.
Tumors were measured in two dimensions by external caliper and
volume was estimated by the formula [length (mm).times.width
(mm).sup.2]/2. Animals were humanely killed when their tumor burden
became excessive. Serum was harvested weekly by retro-orbital
bleed. The level of AFP in the serum was determined by AFP
Immunoassay kit (Genzyme Diagnostics, San Carlos, Calif.). The
difference in mean tumor volume and mean serum AFP concentration
between treatment groups was compared for statistical significance
using the unpaired, two-tailed, t-test.
[0394] Transcription and Translation of E1A/E1B Bicistronic
Cassette of CV890 in Different Cells.
[0395] In wild type adenovirus infection, E1A and E1B genes produce
a family of alternatively spliced products. It has been found that
there are five E1A mRNAs, among them 12S (880 nucleotides, nts) and
13S (1018 nts) mRNAs are the dominant ones that are expressed both
early and late after infection. The 12S and 13S mRNAs encode the
gene product of 243 amino acids (243R) and 289 amino acids (289R)
respectively (reviewed by Shenk, 1996). The two major E1B
transcripts that code for 19 kD and 55 kD proteins are 12S (1031
nts) and 22S (2287 nts) mRNAs. E1B 12S mRNA only codes the 19 kD
product, whereas the 22S mRNA codes for both 19 kD and 55 kD
products due to different initiation sites during translation. In
the current study, the generation of E1A-IRES-E1B bicistronic
cassette was expected to change the pattern of E1A and E1B
transcripts in viral infection. Therefore, Northern blot analysis
was conducted to evaluate the steady-state level of E1A and E1B
transcripts. First, CV802 or CV890 were infected to Hep3B (AFP) or
HBL100 (AFP) cells for 24 hours. The total RNA samples were
separated on agarose gels and processed for Northern blot by
hybridizing to antisense RNA probes. The Northern blot with E1A
probe visualized the 12S and 13S mRNAs in both wild type CV802
infected cells. For CV890, E1A transcripts can only be seen in
Hep3B cells, indicating the conditional transcription of E1A. It is
of interest to find that in CV890, there is only one large
transcript (about 3.51 Kb), whereas the 12S and 13S mRNAs are no
longer present. This large transcript indicates the continuous
transcription of E1A-IRES-E1B bicistronic cassette, suggesting an
alteration of viral E1A splicing pattern in CV890. Transcription of
E1B from CV890 also appears to be AFP-dependent. It is clear that
both 12S and 22S mRNAs of E1B were present in wild type CV802
samples, whereas the 128 mRNA and an enlarged 22S mRNA (3.5 Kb)
appeared in CV890 infected cells. Obviously, the identity of this
enlarged mRNA is the same 3.5 Kb transcript as visualized in E1A
blot, which is from the transcription of E1A/E1B bicistronic
cassette. Therefore, the E1B mRNA is tagged after E1A mRNA in this
large transcript. This large transcript contains all the coding
information for E1A, E1B 19 kD and E1B 55 kD. The mRNA splice
pattern that appears in CV802 is not valid in CV890, the 12S mRNA
with E1B probe disappeared. Meanwhile, in the E1B Northern blot,
due to the selection of our E1B probe (1540 bp-3910 bp), mRNA of
the Adenovirus gene IX (3580 bp-4070 bp), the hexon-associated
protein, was also detected. In CV890 infected Hep3B cells, gene IX
expression is equivalent to that of CV802, whereas in CV890
infected HBL100, its expression was also completely shut down. This
result further demonstrated that the AFP controlled E1A/E1B
expression is the key for late gene expression as well as viral
replication.
[0396] Results of the same samples in the Western blot also
indicate that CV890 has AFP dependent expression of E1A and E1B.
Under our experimental conditions, E1A expression level of CV890 in
Hep3B cells is similar to that of CV802. However, when E1B 19 kD
protein was detected, it was found that the expression level was
much lower than CV802 E1A. Previously, it has been addressed that
IRES-mediated second gene has less expression (Mizuguchi et al.,
2000, Mol. Ther. 1:376-382). Taken together, CV890 infection in
permissive Hep3B cells can produce normal amounts of E1A and lesser
amounts of E1B proteins capable of initiating a normal productive
infection. In AFP.sup.- cells, however, this process was attenuated
due to a lack of E1A and E1B gene transcription and translation.
These data demonstrated that the expression of both E1A and E1B
genes are under the control of AFP TRE and the artificial E1A/E1B
bicistronic cassette is functioning properly in CV890.
[0397] In Vitro Replication Specificity of CV890 in Tumor Cells and
Primary Cells.
[0398] From in vitro comparison of virus yield, CV890 has a better
specificity profile than CV732 (CV732 is an AFP-producing,
cell-specific adenovirus variant in which the E1A gene is under
control of AFP-TRE). In order to gain further insights of using
CV890 in liver cancer therapy, more tumor cell lines and primary
cells were tested to characterize in vitro virus replication.
First, all cells in the study were analyzed for their AFP status by
AFP immune assay. Based on AFP produced in the cells and media, all
the cells were divided into three groups, high (>2.5 g/10.sup.6
cells/10 days), low (<0.6 g/10.sup.6 cells/10 days) and none
(undetectable in our study) (Table 7). It was confirmed that
replication of CV890 in different cell lines correlates well with
the AFP status of the host cell. Among the group of liver cell
lines, CV890 only replicates well in AFP.sup.+ cells, including
Hep3B, HepG2, Huh7, SNU449 and PLC/PRF/5. The amount of AFP
required for the promoter activity seems very low as one of the
hepatoma cell lines, SNU449, a previous reported AFP.sup.- cell
(Park et al., 1995, Int. J. Cancer 62:276-282), produces very low
AFP (about 60 ng/10.sup.6 cells/10 days) compared to other cells.
Nevertheless, even with very low amount of AFP, SNU449 cells can
still support CV890 replication to the extent comparable to cells
producing significantly higher levels of AFP such as HepG2.
Compared to CV802, CV890 is attenuated 5,000 to 100,000 fold in
cells that do not produce AFP, including the hepatoma cell Sk-Hep1
and Chang liver cell, other tumor cells and primary cells. Taken
together the results indicate that CV890 has shown a good
specificity profile from a broad spectrum of tumor cells. Among
them, only the AFP.sup.+ liver cells, AFP production level from
high to low, are permissive for CV890.
[0399] In another experiment, CV890 was compared to CV802 for their
single step growth curves on different cells. Results demonstrated
that CV890 has a similar growth kinetics to wild-type CV802 in
AFP.sup.+ cells except that virus yields are slightly lower (2-8
fold) in low AFP producing cells. In consideration of experimental
error, there is no dramatic difference in the replication of CV890
and CV802 in AFP.sup.+ hepatoma cells. However, the growth curves
of CV890 in AFP.sup.- cells showed clear attenuation. During a 5
day experiment, CV890 failed to replicate in AFP.sup.- cells
including hepatoma cell (Change liver) and primary cells (nHLFC).
From all the in vitro virus replication studies, it is clear that
replication of CV890 is under the tight control of AFP-TRE and this
adenovirus variant has an excellent specificity profile of
preferentially targeting AFP producing hepatocellular carcinoma
cells.
[0400] In Vivo Specificity and Efficacy of CV890.
[0401] CV890 specificity was also evaluated in animals bearing
prostate cancer LNCaP xenografts. In this in vivo test, nude mice
with prostate xenograft were intravenously injected with either
CV890 or CV787, a prostate cancer specific adenovirus variant (Yu
et al., 1999, Cancer Research, 59:4200-4203). Tumor volumes were
documented and indicated that only CV787 had a significant
antitumor efficacy in LNCaP xenografts, while tumors in the animals
treated with CV890 grew, from 400 mm.sup.3 to approximately 1200
mm.sup.3 in six weeks, similar to the group treated with vehicle.
This study indicates that CV890 does not attack LNCaP xenograft and
keeps the good specificity profile under in vivo conditions.
[0402] To evaluate in vivo antitumor efficacy of CV890, different
studies were carried out in the nude mouse model harboring human
hepatoma xenografts. First, BALB/c nu/nu mice with HepG2 or Hep3B
xenografts were established, animals were further challenged with
single dose or multiple doses of CV890 into the tumor mass
(intratumoral administration, IT) or via their tail vein
(intravenous administration, IV). Tumor volume and the level of
serum AFP were monitored weekly after the start of treatment, and
hence the efficacy of the treatment was determined. The in vitro
cytotoxicity study has demonstrated that CV890 has a better
cytolytic effect than CV732. In order to further examine their
antitumor activity, we first conducted animal study to compare
CV890 to CV732. Animals harboring 300 mm.sup.3 Hep3B xenograft were
grouped (n=6) and injected with vehicle alone (control group),
CV890 (1.times.10.sup.11 particles/dose, CV890 group), or CV732
(1.times.10.sup.11 particles/dose, CV732 group). The Hep3B
xenograft is a very aggressive tumor model and tumors grow very
fast. Most animals can not survive long because of excessive tumor
burden. During a six week study, single intravenous administration
of CV890 have shown significant tumor growth inhibition, whereas
control mice had over 10 fold tumor growth at week 5. In the group
treated with CV732, single dose IV injection also reduced the tumor
growth as compared to control group, however, it was much less
effective compared to CV890. For example, the average tumor volume
of the CV890 treated group dropped from 312 mm.sup.3 to 219
mm.sup.3, while tumor volume increased from 308 mm.sup.3 to 1542
mm.sup.3 5 weeks after treatment in control. Both control group and
the CV732 group were terminated at week 5 because excessive tumor
size. Previously, CV732 has been demonstrated to restrict the
hepatoma tumor from growth after 5 doses of intravenous
administration. Similar efficacy can be achieved with just a single
intravenous administration of CV890, indicating that under in vivo
conditions, CV890 has better efficacy than CV732 in hepatoma
xenografts. In this experiment, 4 out of five CV890 treated mice
were tumor free three weeks after treatment. However, in CV732
group, xenografts in two mice stopped growing but none of treated
animals were tumor free through the six-week experiment. There was
no tumor reduction in this group or the control group of animals.
By statistical analysis, the differences in mean relative tumor
volumes and serum AFP concentrations between CV890 treated and
CV732 treated or vehicle treated tumors are significant
(p<0.01)). Taken together, these studies suggest that CV890 has
a significant antitumor activity and its oncolytic efficacy is
better than CV732, an adenovirus variant similar to AvE1a04I, in
which the AFP TRE was applied to control E1A alone (Hallenback et
al, 1999, Hum. Gene. Ther., 10:1721-1733).
[0403] Synergistic Antitumor Efficacy of CV890 in Combination With
Chemotherapeutic Agents
[0404] In this example, different chemotherapeutic agents were
tested in combination with CV890 for their in vitro killing effect
in Hep3B or HepG2 cells. Drug concentrations were optimized to the
extent that they would not generate extensive cytotoxic effect on
their own. Under such conditions, some agents had shown higher cell
killing effect in combination with CV890. Among them, doxorubicin,
a drug currently used in treatment of HCC showed synergistic
cytotoxicity with CV890. In experiments using doxorubicin together
with CV890 on Hep3B cells, doxorubicin at 10 ng/ml did not generate
cytotoxicity, whereas CV890 at an MOI of 0.01 (pfu/cell) only had
about 35% of cell killed at day 9. However, when both were applied
together, 90% cells were killed 9 days after treatment. In order to
determine the potential synergistic effect from the combination
treatment, the MTT cell viability data were subjected to further
statistical analysis. FIG. 10 shows a representative IC.sub.50
isobologram of doxorubicin and CV890 on Hep3B cells at day 5.
First, the dose-response curves of doxorubicin alone or CV890 alone
were made. Based on the original theory of Steel and Peckham (1993)
and method by Aoe et al. (1999), three isoeffect curves (mode I and
mode 2a, 2b) were constructed. From this isobologram, several data
points were in the synergy or additive area, indicating that
combination of CV890 and doxorubicin provides synergistic effect on
killing of Hep3B cells.
[0405] Although CV890 alone has good antitumor activity, we applied
combination therapy with doxorubicin for in vivo evaluation of
synergy. Animals harboring 300 mm.sup.3 Hep3B xenografts were
grouped (n=6) and injected with vehicle alone (control group),
CV890 alone (1.times.10.sup.11 particles/dose, CV890 group),
doxorubicin alone (10 mg/kg, doxorubicin group), or CV890 in
combination with doxorubicin (combination group). FIG. 11 shows
weekly change of the relative tumor size normalized to 100% at day
1. In this experiment, by week six, all animals in the control
group had excessive tumor which has increased by 700% of baseline,
whereas in CV890 group and combination group, animals had either
tumor free or tumor reduction. Of the eight Hep3B xenografts,
treated with CV890, three animals (37.5%) had no palpable tumor at
week 5, another three animals had tumor regressed by more than 60%.
In combination group, four out of eight animals were tumor free
from week 5, another four animals had tumor reduction about 90%.
All the animals in the CV890 and combination group were alive and
tumor was suppressed even ten weeks following treatment whereas the
control animals were sacrificed for excessive tumor burden after
week 6. Furthermore, CV890 also caused a drop in the serum AFP
concentration in these mice. Statistical analysis shows that
differences in mean relative tumor volumes and serum AFP
concentrations between CV890 and vehicle treated group or
combination and doxorubicin treated group are significant at
different times (p<0.005).
[0406] The strong efficacy in the combination treatment shows that
single IV injection of CV890 in combination of doxorubicin can
eradicate aggressive Hep3B xenografts in most of the animals.
6TABLE 7 AFP production in different tumor cells AFP CELLS
(ng/10.sup.6 cells/10 days) Hep3B 2645 High HepG2 3140 HuH7 4585
SNU449 60 Low PLC/PRF/5 600 Chang 0 None SK-Hep1 0 HBL100 0 PA-1 0
LoVo 0
[0407]
7TABLE 1 IRES SEQUENCES SEQ ID NO:1 A 519 base pair IRES obtainable
from encephelomycarditis virus (EMCV). 1
GACGTCGACTAATTCCGGTTATTTTCCACCATATTGCCGTCTTTTGGCAA SalI 51
TGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGAC- GAGCATTCCTAGGG 101
GTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGA- ATGTCGTGAAG 151
GAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCT- GTAGCGAC 201
CCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTG- CGGCC 251
AAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGT- GC 301
CACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAG 351
CGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGG 401
GATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGG 451
TTAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGA SalI 501
AAAACACGATGTCGACGTC SEQ ID NO:2 An IRES obtainable from vascular
endothelial growth factor (VEGF). 1
ACGTAGTCGACAGCGCAGAGGCTTGGGGCAGCCGAGCGGCAGCCAGGCC- C SalI 51
CGGCCCGGGCCTCGGTTCCAGAAGGGAGAGG- AGCCCGCCAAGGCGCGCAA 101
GAGAGCGGGCTGCCTCGCAGTCCGAGCCGGAGAG- GGAGCGCGAGCCGCGC 151
CGGCCCCGGACGGCCTCCGAAACCATGGTCGACACGT- A SalI SEQ ID NO:3 A 5'UTR
region of HCV. 1 GCCAGCCCCCTGATGGGGGCGACACTCCGCCATGA-
ATCACTCCCCTGTGAGGAACTACTG 61 TCTTCACGCAGAAAGCGTCTAGCCATGG-
CGTTAGTATGAGTGTCGTGCAGCCTCCAGGAC 121
CCCCCCTCCCGGGAGAGCCATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAG 181
GACGACCGGGTCCTTTCTTGGATTAACCCGCTCAATGCCTGGAGATTTGGGCGTGCCCCC 241
GCAAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTGCCTGATAG- G 301
GTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACC (341) SEQ ID NO:4 A 5'UTR
region of BiP SEQ ID NO:4 1
CCCGGGGTCACTCCTGCTGGACCTACTCCGACCCCCTAGGCCGGGAGTGAAGGCGGGACT 61
TGTGCGGTTACCAGCGGAAATGCCTCGGGGTCAGAAGTCGCAGGAGAGATAGACAGCTGC 121
TGAACCAATGGGACCAGCGGATGGGGCGGATGTTATCTACCATTGGTGAACGTTAGAAA- C 181
GAATAGCAGCCAATGAATCAGCTGGGGGGGCGGAGCAGTGACGTTTATTGCG- GAGGGGGC 241
CGCTTCGAATCGGCGGCGGCCAGCTTGGTGGCCTGGGCCAATGAA- CGGCCTCCAACGAGC 301
AGGGCCTTCACCAATCGGCGGCCTCCACGACGGGGCTG- GGGGAGGGTATATAAGCCGAGT 361
AGGCGACGGTGAGGTCGACGCCGGCCAAGAC- AGCACAGACAGATTGACCTATTGGGGTGT 421
TTCGCGAGTGTGAGAGGGAAGCGC- CGCGGCCTGTATTTCTAGACCTGCCCTTCGCCTGGT 481
TCGTGGCGCCTTGTGACCCCGGGCCCCTGCCGCCTGCAAGTCGAAATTGCGCTGTGCTCC 541
TGTGCTACGGCCTGTGGCTGGACTGCCTGCTGCTGCCCAACTGGCTGGCAAGATG (595) SEQ
ID NO:5 A 5'UTR of PDGF SEQ ID NO:5 1
GTTTGCACCTCTCCCTGCCCGGGTGCTCGAGCTGCCGTTGCAAAGCCAACTTTGGAAAAA 61
GTTTTTTGGGGGAGACTTGGGCCTTGAGGTGCCCAGCTCCGCGCTTTCCGATTTTGGGGG 121
CTTTCCAGAAAATGTTGCAAAAAAGCTAAGCCGGCGGGCAGAGGAAAACGCCTGTAGCC- G 181
GCGAGTGAAGACGAACCATCGACTGCCGTGTTCCTTTTCCTCTTGGAGGTTG- GAGTCCCC 241
TGGGCGCCCCCACACCCCTAGACGCCTCGGCTGGTTCGCGACGCA- GCCCCCCGGCCGTGG 301
ATGCTGCACTCGGGCTCGGGATCCGCCCAGGTAGCCGG- CCTCGGACCCAGGTCCTGCGCC 361
CAGGTCCTCCCCTGCCCCCCAGCGACGGAGC- CGGGGCCGGGGGCGGCGGCGCCGGGGGCA 421
TGCGGGTGAGCCGCGGCTGCAGAG- GCCTGAGCGCCTGATCGCCGCGGACCTGAGCCGAGC 481
CCACCCCCCTCCCCAGCCCCCCACCCTGGCCGCGGGGGCGGCGCGCTCGATCTACGCGTC 541
CGGGGCCCCGCGGGGCCGGGCCCGGAGTCGGCATG (575)
[0408]
8TABLE 2 LITERATURE REFERENCES FOR IRES IRES Host Example Reference
Picornavirus HAV Glass et al., 1993. Virol 193: 842-852 EMCV Jang
& Wimmer, 1990. Gene Dev 4: 1560-1572 Poliovirus Borman et al.,
1994. EMBO J 13: 3149-3157 HCV and HCV Tsukiyama-Kohara et al.,
1992. J Virol 66: 1476-1483 Leishmania LRV-1 Maga et al., 1995. Mol
Cell Biol 15: 4884-4889 virus Retroviruses MoMLV Torrent et al.,
1996. Hum Gene Ther 7: 603-612 VL30 (Harvey murine sarcoma virus)
REV Lopez-Lastra et al., 1997. Hum Gene Ther 8: 1855-1865
Eukaryotic BiP Macejak & Sarnow, 1991. Nature 353: 90-94 mRNA
antennapedia Oh et al., 1992. Gene & Dev 6: 1643-1653 mRNA
FGF-2 Vagner et al., 1995. Mol Cell Biol 15: 35-44 PDGF-B Bernstein
et al., 1997. J Biol Chem 272: 9356-9362 IGFII Teerink et al.,
1995. Biochim Biophys Acta 1264: 403-408 eIF4G Gan & Rhoads,
1996. J Biol Chem 271: 623-626 VEGF Stein et al., 1998. Mol Cell
Biol 18: 3112-3119; Huez et al., 1998. Mol Cell Biol 18:
6178-6190
[0409]
9TABLE 3 TRE SEQUENCES Nucleotide sequence of a human uroplakin II
5' flanking region. Position +1 (the translational start site) is
denoted with an asterisk. SEQ ID NO:6 (number 1 of SEQ ID NO:6
corresponds to position -2239 with respect to the translational
start site). TCGATAGGTA CCCACTATAG GGCACGCGTG GTCGACGGCC CGGGCTGGTC
1 50 TGGCAACTTC AAGTGTGGGC CTTTCAGACC GGCATCATCA GTGTTACGGG 51 100
GAAGTCACTA GGAATGCAGA ATTGATTGAG CACGGTGGCT CACACCTGTA 101 150
ATCCCAACAC TCTGGGAGGC CAAGGCAGGT GGATCACTTG TGGTCAGGAG 151 200
TTTGAGACCA GCCTGGCCAA CATGGTGAAA CCTCATCTCT ACTAAAAATA 201 250
CAAAAATTAG CTGGGAATGG TGGCACATGC CTATAATCCC AGTTACTCAG 251 300
GAGGCTGAGG CAGGAGAATC ATTTGAACCT GGGAGGCAGA GGTTGCAGTG 301 350
AGCCGAGATC ACGCCACTGC ACTCCAGCCT GGGTGACACA GCGAGACTCT 351 400
GTCTCAAAAA AAAAAAAATG CAGAATTTCA GGCTTCACCC CAGACCCACT 401 450
GCATGACTGC ATGAGAAGCT GCATCTTAAC AAGATCCCTG GTAATTCATA 451 500
CGCATATTAA ATTTGGAGAT GCACTGGCGT AAGACCCTCC TACTCTCTGC 501 550
TTAGGCCCAT GAGTTCTTCC TTTACTGTCA TTCTCCACTC ACCCCAAACT 551 600
TTGAGCCTAC CCTTCCCACC TTGGCGGTAA GGACACAACC TCCCTCACAT 601 650
TCCTACCAGG ACCCTAAGCT TCCCTGGGAC TGAGGAAGAT AGAATAGTTC 651 700
GTGGAGCAAA CAGATATACA GCAACAGTCT CTGTACAGCT CTCAGGCTTC 701 750
TGGAAGTTCT ACAGCCTCTC CCGACAAAGT ATTCCACTTT CCACAAGTAA 751 800
CTCTATGTGT CTGAGTCTCA GTTTCCACTT TTCTCTCTCT CTCTCTCTCT 801 850
CAACTTTCTG AGACAGAGTT TCACTTAGTC GCCCAGGCTG GAGTGCAGGG 851 900
GCACAATCTC GGCTCACTGC AACCTCCACC TCCTGGGTTC AAGTGTTTCT 901 950
CCTGTCTCAG CCTCCCGAGT AGCTGGGATT ACAGGCACAC ACCACCGCGT 951 1000
TAGTTTTTGT ATTTTTGGTA GAGATGGTGT TTCGCCATAT TGGCCAGGCT 1001 1050
GATCTCGAAC TCCTGACCTC AGGTGATCCG CCCACCTCGG CCTCCCAAAG 1051 1100
TGCTGGGATT ACAGGCATGA GCCACCACGC CCGGCTGATC TCTTTTCTAT 1101 1150
TTTAATAGAG ATCAAACTCT CTGTGTTGCC TAGGCTGGTC TTGAACTCCT 1151 1200
GGCCTCGAGT GATCCTCCCA CCTTGGCCTC CCAAAGTGTT GAGATTACAG 1201 1250
GCATGAGCCA CTGTGCCTGG CCTCAGTTCT ACTACAAAAG GAAGCCAGTA 1251 1300
CCAGCTACCA CCCAGGGTGG CTGTAGGGCT ACAATGGAGC ACACAGAACC 1301 1350
CCTACCCAGG GCCCGGAAGA AGCCCCGACT CCTCTCCCCT CCCTCTGCCC 1351 1400
AGAACTCCTC CGCTTCTTTC TGATGTAGCC CAGGGCCGGA GGAGGCAGTC 1401 1450
AGGGAAGTTC TGTCTCTTTT TCATGTTATC TTACGAGGTC TCTTTTCTCC 1451 1500
ATTCTCAGTC CAACAAATGG TTGCTGCCCA AGGCTGACTG TGCCCACCCC 1501 1550
CAACCCCTGC TGGCCAGGGT CAATGTCTGT CTCTCTGGTC TCTCCAGAAG 1551 1600
TCTTCCATGG CCACCTTCGT CCCCACCCTC CAGAGGAATC TGAAACCGCA 1601 1650
TGTGCTCCCT GGCCCCCACA GCCCCTGCCT CTCCCAGAGC AGCAGTACCT 1651 1700
AAGCCTCAGT GCACTCCAAG AATTGAAACC CTCAGTCTGC TGCCCCTCCC 1701 1750
CACCAGAATG TTTCTCTCCC ATTCTTACCC ACTCAAGGCC CTTTCAGTAG 1751 1800
CCCCTTGGAG TATTCTCTTC CTACATATCA GGGCAACTTC CAAACTCATC 1801 1850
ACCCTTCTGA GGGGTGGGGG AAAGACCCCC ACCACATCGG GGGAGCAGTC 1851 1900
CTCCAAGGAC TGGCCAGTCT CCAGATGCCC GTGCACACAG GAACACTGCC 1901 1950
TTATGCACGG GAGTCCCAGA AGAAGGGGTG ATTTCTTTCC CCACCTTAGT 1951 2000
TACACCATCA AGACCCAGCC AGGGCATCCC CCCTCCTGGC CTGAGGGCCA 2001 2050
GCTCCCCATC CTGAAAAACC TGTCTGCTCT CCCCACCCCT TTGAGGCTAT 2051 2100
AGGGCCCAAG GGGCAGGTTG GACTGGATTC CCCTCCAGCC CCTCCCGCCC 2101 2150
CCAGGACAAA ATCAGCCACC CCAGGGGCAG GGCCTCACTT GCCTCAGGAA 2151 2200
CCCCAGCCTG CCAGCACCTA TTCCACCTCC CAGCCCAGCA 2201 2239 Nucleotide
sequence of a mouse uroplakin II 5' flanking region. The
translational start site is denoted with an asterisk. SEQ ID NO:7
(number 1 of SEQ ID NO:7 corresponds to position -3592 with respect
to the translational start site).
CTCGAGGATCTCGGCCCTCTTTCTGCATCCTTGTCCTAAATCATTTTCAT 1 50
ATCTTGCTAGACCTCAGTTTGAGAGAAACGAACCTTCTCATTTTCAAGTT 51 100
GAAAAAAAAAAGAGGTTCAAAGTGGCTCACTCAAAGTTACAAGCCAACAC 101 150
TCACCACTACGAGTACAATGGCCACCATTAGTGCTGGCATGCCCCAGGAG 151 200
ACAGGCATGCATATTATTCTAGATGACTGGGAGGCAGAGGGGTGGCCTAG 201 250
TGAGGTCAGACTGTGGACAGATCAGGCAGATGTGGGTTCTGATCCCAATT 251 300
CCTCAGGCCGCAGAACTACTGTGGTTCAAGAAGGGGACAAAAGGACTGCA 301 350
GTCCGGAACAGGAGGTCCATTTGAGAGCTGACTGAGCAGAAGAGGAAAGT 351 400
GAAGAACTTCTGGGGCAAGAGCTTACCCTACTTTACAGCTTTGTTGTCTT 401 450
CTTTACTCCAGGGGCGTCCCTGGTACTCAGTAAATGTCTGTTGGCTTGAG 451 500
GAACATATGTGTAAGGAGGAAGGAGAGGGAACTTGAGGGAGTTAAGACTC 501 550
AAGAATCAATCAAGGAGAGGACAGCAGAGAAGACAGGGTTTGGGAGAGAG 551 600
ACTCCAGACATTGGCCCTGGTTCCCTTCTTGGCCACTGTGAAACCCTCCA 601 650
GAGGAACTGAGTGCTGTGGCTTTAAATGATCTCAGCACTGTCAGTGAAGC 651 700
GCTCTGCTCAAAGAGTTATCCTCTTGCTCCTGTGCCGGGGCCTCCCCCTC 701 750
CTCTCAGCTCCCAAACCCTTCTCAGCCACTGTGATGGCATAATTAGATGC 751 800
GAGAGCTCAGACCGTCAGGTCTGCTCCAGGAACCACCCATTTTCCCCAAC 801 850
CCCAGAGAAAGGTCCTAGTGGAAAAGTGGGGGCCACTGAAGGGCTGATGG 851 900
GGTTCTGTCCTTTCCCCCATGCTGGGTGGACTTAAAGTCTGCGATGTGTG 900 950
TAGGGGGTAGAAGACAACAGAACCTGGGGGCTCCGGCTGGGAGCAGGAGG 951 1000
AACTCTCACCAGACGATCTCCAAATTTACTGTGCAATGGACGATCAGGAA 1001 1050
ACTGGTTCAGATGTAGCTTCTGATACAGTGGGTCTGAGGTAAAACCCGAA 1051 1100
ACTTAATTTCTTTCAAAAATTTAAAGTTGCATTTATTATTTTATATGTGT 1101 1150
GCCCATATGTGTGCCACAGTGTCTATGTGGAGGTCAGAGGGCAAGTTGTG 1151 1200
GGCATTGGCTCTCTCCTTTCATAATGTGGCTTCTGGGGACCAAAATGTCA 1201 1250
GGCATGGTGGCAAGAGCTTTTACCTGTTGAGCCATCTCATGGTTTCGTAA 1251 1300
AACTTCCTATGACGCTTACAGGTAACGCAGAGACACAGACTCACATTTGG 1301 1350
AGTTAGCAGATGCTGTATTGGTGTAAACACTCATACACAGACACACACAC 1351 1400
ATACTCATACACACACACACACACTTATCACATGCACACACATACTCGTA 1401 1450
TACACACAGACACACACACATGCACTCTCACATTCACATATTCATACACA 1451 1500
TCCACACACACACTCATCCACACACACAGACACACATACTCATCCACACA 1501 1550
CACACACACACATACTCATACACACACACAGACACACATACTCATACACA 1551 1600
CACACAGACACACACATATAATCATACATACACAGACACACTCATACATG 1601 1650
TGCACACACACACTCATCCACACACACACACTCATACACACACACACTCA 1651 1700
TACACACACACACTCATACACACACACACGAGGTTTTTCTCAGGCTGCCT 1701 1750
TTGGGTGGAGACTGGAACTGATTTCTGTTTTTCAGCTCCTTGGCTTTTTG 1751 1800
TCCCTTTAGATGAGATCTCCTCCTCACTTTACACACAGAAAGATCACACA 1801 1850
CGAGGGAGAACTGGCGGTGCGGAAGAGGGCTACACGGTAGGGTGTCAGGG 1851 1900
TCAGGAGATCTTCCTGGCAAGTCTCAAACCTCCACATAGCACAGTGTTTA 1901 1950
CGTGAGGATTTAGGAGGAATCAGGAAGAGGATTGGTTTACTGCAGAGCAG 1951 2000
ACCATATAGGTCCACTCCTAAGCCCCATTTGAAATTAGAAGTGAGACAGT 2001 2050
GTGGGATAAAAAGAGCAGATCTCTGGTCACATTTTTAAAGGGATATGAGG 2051 3000
GTCCTGTGCCTTTAAGCCTTCCCATCTCCCTCCAATCCCCCCTCACCTTC 2101 2150
CCCACCCTAACCCTCCCCAGGTTTCTGGAGGAGCAGAGTTGCGTCTTCTC 2151 2200
CCTGCCCTGCCGAGCTGCTCACTGGCTGCTCTAGAGGCTGTGCTTTGCGG 2201 2250
TCTCCATGGAAACCATTAGTTGCTAAGCAACTGGAGCATCATCTGTGCTG 2251 2300
AGCTCAGGTCCTATCGAGTTCACCTAGCTGAGACACCCACGCCCCTGCAG 2301 2350
CCACTTTGCAGTGACAAGCCTGAGTCTCAGGTTCTGCATCTATAAAAACG 2351 2400
AGTAGCCTTTCAGGAGGGCATGCAGAGCCCCCTGGCCAGCGTCTAGAGGA 2401 2450
GAGGTGACTGAGTGGGGCCATGTCACTCGTCCATGGCTGGAGAACCTCCA 2451 2500
TCAGTCTCCCAGTTAGCCTGGGGCAGGAGAGAACCAGAGGAGCTGTGGCT 2501 2550
GCTGATTGGATGATTTACGTACCCAATCTGTTGTCCCAGGCATCGAACCC 2551 2600
CAGAGCGACCTGCACACATGCCACCGCTGCCCCGCCCTCCACCTCCTCTG 2601 2650
CTCCTGGTTACAGGATTGTTTTGTCTTGAAGGGTTTTGTTGTTGCTACTT 2651 2700
TTTGCTTTGTTTTTTCTTTTTTAACATAAGGTTTCTCTGTGTAGCCCTAG 2701 2750
CTGTCCTGGAACTCACTCTGTAGACCAGGCTGGCCTCAAACTCAGAAATC 2751 2800
CACCTTCCTCCCAAGTGCTGGGATTAAAGGCATTCGCACCATCGCCCAGC 2801 2850
CCCCGGTCTTGTTTCCTAAGGTTTTCCTGCTTTACTCGCTACCCGTTGCA 2851 2900
CAACCGCTTGCTGTCCAAGTCTGTTTGTATCTACTCCACCGCCCACTAGC 2901 2950
CTTGCTGGACTGGACCTACGTTTACCTGGAAGCCTTCACTAACTTCCCTT 2951 3000
GTCTCCACCTTCTGGAGAAATCTGAAGGCTCACACTGATACCCTCCGCTT 3001 3050
CTCCCAGAGTCGCAGTTTCTTAGGCCTCAGTTAAATACCAGAATTGGATC 3051 3100
TCAGGCTCTGCTATCCCCACCCTACCTAACCAACCCCCTCCTCTCCCATC 3101 3150
CTTACTAGCCAAAGCCCTTTCAACCCTTGGGGCTTTTCCTACACCTACAC 3151 3200
ACCAGGGCAATTTTAGAACTCATGGCTCTCCTAGAAAACGCCTACCTCCT 3201 3250
TGGAGACTGACCCTCTACAGTCCAGGAGGCAGACACTCAGACAGAGGAAC 3251 3300
TCTGTCCTTCAGTCGCGGGAGTTCCAGAAAGAGCCATACTCCCCTGCAGA 3301 3350
GCTAACTAAGCTGCCAGGACCCAGCCAGAGCATCCCCCTTTAGCCGAGGG 3351 3400
CCAGCTCCCCAGAATGAAAAACCTGTCTGGGGCCCCTCCCTGAGGCTACA 3401 3450
GTCGCCAAGGGGCAAGTTGGACTGGATTCCCAGCAGCCCCTCCCACTCCG 3451 3500
AGACAAAATCAGCTACCCTGGGGCAGGCCTCATTGGCCCCAGGAAACCCC 3501 3550
AGCCTGTCAGCACCTGTTCCAGGATCCAGTCCCAGCGCAGTA 3551 3592 AFP-TRE. SEQ
ID NO:8. 1
GCATTGCTGTGAACTCTGTACTTAGGACTAAACTTTGAGCAATAACACACATAGATTGAG 61
GATTGTTTGCTGTTAGCATACAAACTCTGGTTCAAAGCTCCTCTTTATTGCTTGTCTTGG 121
AAAATTTGCTGTTCTTCATGGTTTCTCTTTTCACTGCTATCTATTTTTCTCAACCACTC- A 181
CATGGCTACAATAACTGTCTGCAAGCTTATGATTCCCAAATATCTATCTCTA- GCCTCAAT 241
CTTGTTCCAGAAGATAAAAAGTAGTATTCAAATGCACATCAACGT- CTCCACTTGGAGGGC 301
TTAAAGACGTTTCAACATACAAACCGGGGAGTTTTGCC- TGGAATGTTTCCTAAAATGTGT 361
CCTGTAGCACATAGGGTCCTCTTGTTCCTTA- AAATCTAATTACTTTTAGCCCAGTGCTCA 421
TCCCACCTATGGGGAGATGAGAGT- GAAAAGGGAGCCTGATTAATAATTACACTAAGTCAA 481
TAGGCATAGAGCCAGGACTGTTTGGGTAAACTGGTCACTTTATCTTAAACTAAATATATC 541
CAAAACTGAACATGTACTTAGTTACTAAGTCTTTGACTTTATCTCATTCATACCACTCAG 601
CTTTATCCAGGCCACTTATGAGCTCTGTGTCCTTGAACATAAAATACAAATAACCGCTA- T 661
GCTGTTAATTATTGGCAAATGTCCCATTTTCAACCTAAGGAAATACCATAAA- GTAACAGA 721
TATACCAACAAAAGGTTACTAGTTAACAGGCATTGCCTGAAAAGA- GTATAAAAGAATTTC 781
AGCATGATTTTCCATATTGTGCTTCCACCACTGCCAAT- AACA (822) Probasin-TRE SEQ
ID NO:9 -426
5'-AAGCTTCCACAAGTGCATTTAGCCTCTCCAGTATTGCTGATGAATCCACAGT
TCAGGTTCAATGGCGTTCAAAACTTGATCAAAAATGACCAGACTTTATATTTA
CACCAACATCTATCTGATTGGAGGAATGGATAATAGTCATCATGTTTAAACAT
CTACCATTCCAGTTAAGAAAATATGATAGCATCTTGTTCTTAGTCTTTTTCTTA ARE-1
ATAGGGACATAAAGCCCACAAATAAAA- ATATGCCTGAAGAATGGGACAGGC
ATTGGGCATTGTCCATGCCTAGTAAAGTACTCC- AAGAACCTATTTGTATACTA ARE-2
GATGACACAATGTCAATGTCTGTGTACAACTGCCAACTGGGATGCAAGACAC
TGCCCATGCCAATCATCCTGAAAAGCAGCTATAAAAAGCAGGAAGCTACTCT CAAT box TATAA
box +1 +28 GCACCTTGTCAGTAGGTCCAGATACCTACAG-3' Transcription site
Tyrosinase-TRE. SEQ ID NO:10 PinAl end 1
CCGGTTGAAAATGATAAGTTGAATTCTGTCTTCGAGAACATAGAAAAGAA 51
TTATGAAATGCCAACATGTGGTTACAAGTAATGCAGACCCAAGGCTCCCC 101
AGGGACAAGAAGTCTTGTGTTAACTCTTTGTGGCTCTGAAAGAAAGAGAG 151
AGAGAAAAGATTAAGCCTCCTTGTGGAGATCATGTGATGACTTCCTGATT 201
CCAGCCAGAGCGAGCATTTCCATGGAAACTTCTCTTCCTCTTCACTCGAG 251
ATTACTAACCTTATTGTTAATATTCTAACCATAAGAATTAAACTATTAAT 301
GGTGAATAGAGTTTTTCACTTTAACATAGGCCTATCCCACTGGTGGGATA 351
CGAGCCAATTCGAAAGAAAAAGTCAGTCATGTGCTTTTCAGAGGATGAAA 401
GCTTAAGATAAAGACTAAAAGTGTTTGATGCTGGAGGTGGGAGTGGTATT 451
ATATAGGTCTCAGCCAAGACATGTGATAATCACTGTAGTAGTAGCTGGAA 501
AGAGAAATCTGTGACTCCAATTAGCCAGTTCCTGCAGACCTTGTGA PinAl end Human
glandular kallikrein-TRE SEQ ID NO:11 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 ggtatgaagc cctgcctgca ttagctcttt
tccctaatgc tctcactact gccccaccct 3240 caccctgaca gggcaaacag
acaacctaca gaatgggagg aaatttttgc aatctattca 3300 tctgacaaag
gtcaagaata tccagaatct acaaggaact taagcaaatt tttacttttt 3360
aataatagcc actctgactg gcgtgaaatg gtatctcatt gtggttttca tttgaatttc
3420 tctgatgatc agtgacgatg agcatttttt catatttgtt ggctgcttgt
acgtcttttg 3480 agaagtgtct cttcatgcct tttggccact ttaatgggat
tattttttgc tttttagttt 3540 aagttcctta tagattctgg atattagact
tcttattgga tgcatagttt gtgaatactc 3600 tcttccattc tgtaggttgt
ctgtttactc tattgatggc ttcttttgct gtgccgaagc 3660 atcttagttt
aattagaaac cacctgccaa tttttgtttt tgttgcaatt gcttttgggg 3720
acttagtcat aaactctttg ccaaggtctg ggtcaagaag agtatttcct aggttttctt
3780 ctagaatttt gaaagtctga atgtaaacat ttgcattttt aatgcatctt
gagttagttt 3840 ttgtatatgt gaaaggtcta ctctcatttt ctttccctct
ttctttcttt ctttcttttc 3900 tttctttctt tctttctttc tttctttctt
tctttctttc tttctttttg tccttctttc 3960 tttctttctt tctctttctt
tctctctttc tttttttttt ttgatggagt attgctctgt 4020 tgcccaggct
gcagtgcagc ggcacgatct cggctcactg caacctctgc ctcctgggtt 4080
caactgattc tcctgcatca gccttccaag tagctgggat tataggcgcc cgccaccacg
4140 cccgactaat ttttgtattt ttagtagaga cggggttgtg ccatgttggc
caggctggtt 4200 tgaaactcct gacctcaaac gatctgcctg ccttggcctc
ccaaagtgct gggattacag 4260 gtgtgagcca ctgtgcccag ccaagaatgt
cattttctaa gaggtccaag aacctcaaga 4320 tattttggga ccttgagaag
agaggaattc atacaggtat tacaagcaca gcctaatggc 4380 aaatctttgg
catggcttgg cttcaagact ttaggctctt aaaagtcgaa tccaaaaatt 4440
tttataaaag ctccagctaa gctaccttaa aaggggcctg tatggctgat cactcttctt
4500 gctatacttt acacaaataa acaggccaaa tataatgagg ccaaaattta
ttttgcaaat 4560 aaattggtcc tgctatgatt tactcttggt aagaacaggg
aaaatagaga aaaatttaga 4620 ttgcatctga cctttttttc tgaattttta
tatgtgccta caatttgagc taaatcctga 4680 attattttct ggttgcaaaa
actctctaaa gaagaacttg gttttcattg tcttcgtgac 4740 acatttatct
ggctctttac tagaacagct ttcttgtttt tggtgttcta gcttgtgtgc 4800
cttacagttc tactcttcaa attattgtta tgtgtatctc atagttttcc ttcttttgag
4860 aaaactgaag ccatggtatt ctgaggacta gagatgactc aacagagctg
gtgaatctcc 4920 tcatatgcaa tccactgggc tcgatctgct tcaaattgct
gatgcactgc tgctaaagct 4980 atacatttaa aaccctcact aaaggatcag
ggaccatcat ggaagaggag gaaacatgaa 5040 attgtaagag ccagattcgg
ggggtagagt gtggaggtca gagcaactcc accttgaata 5100 agaaggtaaa
gcaacctatc ctgaaagcta acctgccatg gtggcttctg attaacctct 5160
gttctaggaa gactgacagt ttgggtctgt gtcattgccc aaatctcatg ttaaattgta
5220 atccccagtg ttcggaggtg ggacttggtg gtaggtgatt cggtcatggg
agtagatttt 5280 cttctttgtg gtgttacagt gatagtgagt gagttctcgt
gagatctggt catttaaaag 5340 tgtgtggccc ctcccctccc tctcttggtc
ctcctactgc catgtaagat acctgctcct 5400 gctttgcctt ctaccataag
taaaagcccc ctgaggcctc cccagaagca gatgccacca 5460 tgcttcctgt
acagcctgca gaaccatcag ccaattaaac ctcttttctg tataaattac 5520
cagtcttgag tatctcttta cagcagtgtg agaacggact aatacaaggg tctccaaaat
5580 tccaagttta tgtattcttt cttgccaaat agcaggtatt taccataaat
cctgtcctta 5640 ggtcaaacaa ccttgatggc atcgtacttc aattgtctta
cacattcctt ctgaatgact 5700 cctcccctat ggcatataag ccctgggtct
tgggggataa tggcagaggg gtccaccatc 5760 ttgtctggct gccacctgag
acacggacat ggcttctgtt ggtaagtctc tattaaatgt 5820 ttctttctaa
gaaactggat ttgtcagctt gtttctttgg cctctcagct tcctcagact 5880
ttggggtagg ttgcacaacc ctgcccacca cgaaacaaat gtttaatatg ataaatatgg
5940 atagatataa tccacataaa taaaagctct tggagggccc tcaataattg
ttaagagtgt 6000 aaatgtgtcc aaagatggaa aatgtttgag aactactgtc
ccagagattt tcctgagttc 6060 tagagtgtgg gaatatagaa cctggagctt
ggcttcttca gcctagaatc aggagtatgg 6120 ggctgaagtc tgaagcttgg
cttcagcagt ttggggttgg cttccggagc acatatttga 6180 catgttgcga
ctgtgatttg gggtttggta tttgctctga atcctaatgt ctgtccttga 6240
ggcatctaga atctgaaatc tgtggtcaga attctattat cttgagtagg acatctccag
6300 tcctggttct gccttctagg gctggagtct gtagtcagtg acccggtctg
gcatttcaac 6360 ttcatataca gtgggctatc ttttggtcca tgtttcaacc
aaacaaccga ataaaccatt 6420 agaacctttc cccacttccc tagctgcaat
gttaaaccta ggatttctgt ttaataggtt 6480 catatgaata atttcagcct
gatccaactt tacattcctt ctaccgttat tctacaccca 6540 ccttaaaaat
gcattcccaa tatattccct ggattctacc tatatatggt aatcctggct 6600
ttgccagttt ctagtgcatt aacatacctg atttacattc ttttacttta aagtggaaat
6660 aagagtccct ctgcagagtt caggagttct caagatggcc cttacttctg
acatcaattg 6720 agatttcaag ggagtcgcca agatcatcct caggttcagt
gattgctggt agccctcata 6780 taactcaatg aaagctgtta tgctcatggc
tatggtttat tacagcaaaa gaatagagat 6840 gaaaatctag caagggaaga
gttgcatggg gcaaagacaa ggagagctcc aagtgcagag 6900 attcctgttg
ttttctccca gtggtgtcat ggaaagcagt atcttctcca tacaatgatg 6960
tgtgataata ttcagtgtat tgccaatcag ggaactcaac tgagccttga ttatattgga
7020 gcttggttgc acagacatgt cgaccacctt catggctgaa ctttagtact
tagcccctcc 7080 agacgtctac agctgatagg ctgtaaccca acattgtcac
cataaatcac attgttagac 7140 tatccagtgt ggcccaagct cccgtgtaaa
cacaggcact ctaaacaggc aggatatttc 7200 aaaagcttag agatgacctc
ccaggagctg aatgcaaaga cctggcctct ttgggcaagg 7260 agaatccttt
accgcacact ctccttcaca gggttattgt gaggatcaaa tgtggtcatg 7320
tgtgtgagac accagcacat gtctggctgt ggagagtgac ttctatgtgt gctaacattg
7380 ctgagtgcta agaaagtatt aggcatggct ttcagcactc acagatgctc
atctaatcct 7440 cacaacatgg ctacagggtg ggcactacta gcctcatttg
acagaggaaa ggactgtgga 7500 taagaagggg gtgaccaata ggtcagagtc
attctggatg caaggggctc cagaggacca 7560 tgattagaca ttgtctgcag
agaaattatg gctggatgtc tctgccccgg aaagggggat 7620 gcactttcct
tgacccccta tctcagatct tgactttgag gttatctcag acttcctcta 7680
tgataccagg agcccatcat aatctctctg tgtcctctcc ccttcctcag tcttactgcc
7740 cactcttccc agctccatct 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 agatectaga 9300 accacaggaa accaggactg aaaggtgcta
gagaatggcc atatgtcgct gtccatgaaa 9360 tctcaaggac ttctgggtgg
agggcacagg agcctgaact tacgggtttg ccccagtcca 9420 ctgtcctccc
aagtgagtct cccagatacg aggcactgtg ccagcatcag cttcatctgt 9480
accacatctt gtaacaggga ctacccagga ccctgatgaa caccatggtg tgtgcaggaa
9540 gagggggtga aggcatggac tcctgtgtgg tcagagccca gagggggcca
tgacgggtgg 9600 ggaggaggct gtggactggc tcgagaagtg ggatgtggtt
gtgtttgatt tcctttggcc 9660 agataaagtg ctggatatag cattgaaaac
ggagtatgaa gaccagttag aatggagggt 9720 caggttggag ttgagttaca
gatggggtaa aattctgctt cggatgagtt tggggattgg 9780 caatctaaag
gtggtttggg atggcatggc tttgggatgg aaataggttt gtttttatgt 9840
tggctgggaa gggtgtgggg attgaattgg ggatgaagta ggtttagttt tggagataga
9900 atacatggag ctggctattg catgcgagga tgtgcattag tttggtttga
tctttaaata 9960 aaggaggcta ttagggttgt cttgaattag attaagttgt
gttgggttga tgggttgggc 10020 ttgtgggtga tgtggttgga ttgggctgtg
ttaaattggt ttgggtcagg ttttggttga 10080 ggttatcatg gggatgagga
tatgcttggg acatggattc aggtggttct cattcaagct 10140 gaggcaaatt
tcctttcaga cggtcattcc agggaacgag tggttgtgtg ggggaaatca 10200
ggccactggc tgtgaatatc cctctatcct ggtcttgaat tgtgattatc tatgtccatt
10260 ctgtctcctt cactgtactt ggaattgatc tggtcattca gctggaaatg
ggggaagatt 10320 ttgtcaaatt cttgagacac agctgggtct ggatcagcgt
aagccttcct tctggtttta 10380 ttgaacagat gaaatcacat tttttttttc
aaaatcacag aaatcttata gagttaacag 10440 tggactctta taataagagt
taacaccagg actcttattc ttgattcttt tctgagacac 10500 caaaatgaga
tttctcaatg ccaccctaat tctttttttt tttttttttt tttttgagac 10560
acagtctggg tcttttgctc tgtcactcag gctggagcgc agtggtgtga tcatagctca
10620 ctgaaccctt gacctcctgg acttaaggga tcctcctgct tcagcctcct
gagtagatgg 10680 ggctacaggt gcttgccacc acacctggct aattaaattt
tttttttttt tttgtagaga 10740 aagggtctca ctttgttgcc ctggctgatc
ttgaacttct gacttcaagt gattcttcag 10800 ccttggactc ccaaagcact
gggattgctg gcatgagcca ctcaccgtgc ctggcttgca 10860
gcttaatctt ggagtgtata aacctggctc ctgatagcta gacatttcag tgagaaggag
10920 gcattggatt ttgcatgagg acaattctga cctaggaggg caggtcaaca
ggaatccccg 10980 ctgtacctgt acgttgtaca ggcatggaga atgaggagtg
aggaggccgt accggaaccc 11040 catattgttt agtggacatt ggattttgaa
ataataggga acttggtctg ggagagtcat 11100 atttctggat tggacaatat
gtggtatcac aaggttttat gatgagggag aaatgtatgt 11160 ggggaaccat
tttctgagtg tggaagtgca agaatcagag agtagctgaa tgccaacgct 11220
tctatttcag gaacatggta agttggaggt ccagctctcg ggctcagacg ggtataggga
11280 ccaggaagtc tcacaatccg atcattctga tatttcaggg catattaggt
ttggggtgca 11340 aaggaagtac ttgggactta ggcacatgag actttgtatt
gaaaatcaat gattggggct 11400 ggccgtggtg ctcacgcctg taatctcatc
actttgggag accgaagtgg gaggatggct 11460 tgatctcaag agttggacac
cagcctaggc aacatggcca gaccctctct ctacaaaaaa 11520 attaaaaatt
agctggatgt ggtggtgcat gcttgtggtc tcagctatcc tggaggctga 11580
gacaggagaa tcggttgagt ctgggagttc aaggctacag ggagctgcga tcacgccgct
11640 gcactccagc ctgggaaaca gagtgagact gtctcagaat ttttttaaaa
aagaatcagt 11700 gatcatccca acccctgttg ctgttcatcc tgagcctgcc
ttctctggct ttgttcccta 11760 gatcacatct ccatgatcca taggccctgc
ccaatctgac ctcacaccgt gggaatgcct 11820 ccagactgat ctagtatgtg
tggaacagca agtgctggct ctccctcccc ttccacagct 11880 ctgggtgtgg
gagggggttg tccagcctcc agcagcatgg ggagggcctt ggtcagcatc 11940
taggtgccaa cagggcaagg gcggggtcct ggagaatgaa ggctttatag ggctcctcag
12000 ggaggccccc cagccccaaa ctgcaccacc tggccgtgga caccggt 12047
HRE-TRE SEQ ID NO:12 ccccgagg cagtgcat gaggctcagg gcgtgcgt
gagtcgcagcgagaccccg gggtgcag gccgga PSA-TRE SEQ ID NO:13 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 aaaaaaaaaa gagatgacct ctcaggctct 720 gaggggaaac
gcctgaggtc tttgagcaag gtcagtcctc tgttgcacag tctccctcac 780
agggtcattg tgacgatcaa atgtggtcac gtgtatgagg caccagcaca tgcctggctc
840 tggggagtgc cgtgtaagtg tatgcttgca ctgctgaatg gctgggatgt
gtcagggatt 900 atcttcagca cttacagatg ctcatctcat cctcacagca
tcactatggg atgggtatta 960 ctggcctcat ttgatggaga aagtggctgt
ggctcagaaa ggggggacca ctagaccagg 1020 gacactctgg atgctgggga
ctccagagac catgaccact caccaactgc agagaaatta 1080 attgtggcct
gatgtccctg tcctggagag ggtggaggtg gaccttcact aacctcctac 1140
cttgaccctc tcttttaggg ctctttctga cctccaccat ggtactagga ccccattgta
1200 ttctgtaccc tcttgactct atgaccccca ccgcccactg catccagctg
ggtcccctcc 1260 tatctctatt cccagctggc cagtgcagtc tcagtgccca
cctgtttgtc agtaactctg 1320 aaggggctga cattttactg acttgcaaac
aaataagcta actttccaga gttttgtgaa 1380 tgctggcaga gtccatgaga
ctcctgagtc agaggcaaag gcttttactg ctcacagctt 1440 agcagacagc
atgaggttca tgttcacatt agtacacctt gcccccccca aatcttgtag 1500
ggtgaccaga gcagtctagg tggatgctgt gcagaagggg tttgtgccac tggtgagaaa
1560 cctgagatta ggaatcctca atcttatact gggacaactt gcaaacctgc
tcagcctttg 1620 tctctgatga agatattatc ttcatgatct tggattgaaa
acagacctac tctggaggaa 1680 catattgtat cgattgtcct tgacagtaaa
caaatctgtt gtaagagaca ttatctttat 1740 tatctaggac agtaagcaag
cctggatctg agagagatat catcttgcaa ggatgcctgc 1800 tttacaaaca
tccttgaaac aacaatccag aaaaaaaaag gtgttactgt ctttgctcag 1860
aagacacaca gatacgtgac agaaccatgg agaattgcct cccaacgctg ttcagccaga
1920 gccttccacc ctttctgcag gacagtctca acgttccacc attaaatact
tcttctatca 1980 catcccgctt ctttatgcct aaccaaggtt ctaggtcccg
atcgactgtg tctggcagca 2040 ctccactgcc aaacccagaa taaggcagcg
ctcaggatcc cgaaggggca tggctgggga 2100 tcagaacttc tgggtttgag
tgaggagtgg gtccaccctc ttgaatttca aaggaggaag 2160 aggctggatg
tgaaggtact gggggaggga aagtgtcagt tccgaactct taggtcaatg 2220
agggaggaga ctggtaaggt cccagctccc gaggtactga tgtgggaatg gcctaagaat
2280 ctcatatcct caggaagaag gtgctggaat cctgaggggt agagttctgg
gtatatttgt 2340 ggcttaaggc tctttggccc ctgaaggcag aggctggaac
cattaggtcc agggtttggg 2400 gtgatagtaa tgggatctct tgattcctca
agagtctgag gatcgagggt tgcccattct 2460 tccatcttgc cacctaatcc
ttactccact tgagggtatc accagccctt ctagctccat 2520 gaaggtcccc
tgggcaagca caatctgagc atgaaagatg ccccagaggc cttgggtgtc 2580
atccactcat catccagcat cacactctga gggtgtggcc agcaccatga cgtcatgttg
2640 ctgtgactat ccctgcagcg tgcctctcca gccacctgcc aaccgtagag
ctgcccatcc 2700 tcctctggtg ggagtggcct gcatggtgcc aggctgaggc
ctagtgtcag acagggagcc 2760 tggaatcata gggatccagg actcaaaagt
gctagagaat ggccatatgt caccatccat 2820 gaaatctcaa gggcttctgg
gtggagggca cagggacctg aacttatggt ttcccaagtc 2880 tattgctctc
ccaagtgagt ctcccagata cgaggcactg tgccagcatc agccttatct 2940
ccaccacatc ttgtaaaagg actacccagg gccctgatga acaccatggt gtgtacagga
3000 gtagggggtg gaggcacgga ctcctgtgag gtcacagcca agggagcatc
atcatgggtg 3060 gggaggaggc aatggacagg cttgagaacg gggatgtggt
tgtatttggt tttctttggt 3120 tagataaagt gctgggtata ggattgagag
tggagtatga agaccagtta ggatggagga 3180 tcagattgga gttgggttag
ataaagtgct gggtatagga ttgagagtgg agtatgaaga 3240 ccagttagga
tggaggatca gattggagtt gggttagaga tggggtaaaa ttgtgctccg 3300
gatgagtttg ggattgacac tgtggaggtg gtttgggatg gcatggcttt gggatggaaa
3360 tagatttgtt ttgatgttgg ctcagacatc cttggggatt gaactgggga
tgaagctggg 3420 tttgattttg gaggtagaag acgtggaagt agctgtcaga
tttgacagtg gccatgagtt 3480 ttgtttgatg gggaatcaaa caatggggga
agacataagg gttggcttgt taggttaagt 3540 tgcgttgggt tgatggggtc
ggggctgtgt ataatgcagt tggattggtt tgtattaaat 3600 tgggttgggt
caggttttgg ttgaggatga gttgaggata tgcttgggga caccggatcc 3660
atgaggttct cactggagtg gagacaaact tcctttccag gatgaatcca gggaagcctt
3720 aattcacgtg taggggaggt caggccactg gctaagtata tccttccact
ccagctctaa 3780 gatggtctta aattgtgatt atctatatcc acttctgtct
ccctcactgt gcttggagtt 3840 tacctgatca ctcaactaga aacaggggaa
gattttatca aattcttttt tttttttttt 3900 tttttttgag acagagtctc
actctgttgc ccaggctgga gtgcagtggc gcagtctcgg 3960 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 CEA TRE SEQ ID NO:14 aagcttttta gtgctttaga
cagtgagctg gtctgtctaa cccaagtgac ctgggctcca 60 tactcagccc
cagaagtgaa gggtgaagct gggtggagcc aaaccaggca agcctaccct 120
cagggctccc agtggccctga gaaccattgg acccaggacc cattacttct agggtaagga
180 aggtacaaac accagatcca accatggtct ggggggacag ctgtcaaatg
cctaaaaata 240 tacctgggag aggagcaggc aaactatcac tgccccaggt
tctctgaaca gaaacagagg 300 ggcaacccaa agtccaaatc caggtgagca
ggtgcaccaa atgcccagag atatgacgag 360 gcaagaagtg aaggaaccac
ccctgcatca aatgttttgc atgggaagga gaagggggtt 420 gctcatgttc
ccaatccagg agaatgcatt tgggatctgc cttcttctca ctccttggtt 480
agcaagacta agcaaccagg actctggatt tggggaaaga cgtttatttg tggaggccag
540 tgatgacaat cccacgaggg cctaggtgaa gagggcagga aggctcgaga
cactggggac 600 tgagtgaaaa ccacacccat gatctgcacc acccatggat
gctccttcat tgctcacctt 660 tctgttgata tcagatggcc ccattttctg
taccttcaca gaaggacaca ggctagggtc 720 tgtgcatggc cttcatcccc
ggggccatgt gaggacagca ggtgggaaag atcatgggtc 780 ctcctgggtc
ctgcagggcc agaacattca tcacccatac tgacctccta gatgggaatg 840
gcttccctgg ggctgggcca acggggcctg ggcaggggag aaaggacgtc aggggacagg
900 gaggaagggt catcgagacc cagcctggaa ggttcttgtc tctgaccatc
caggatttac 960 ttccctgcat ctacctttgg tcattttccc tcagcaatga
ccagctctgc ttcctgatct 1020 cagcctccca ccctggacac agcaccccag
tccctggccc ggctgcatcc acccaatacc 1080 ctgataaccc aggacccatt
acttctaggg taaggagggt ccaggagaca gaagctgagg 1140 aaaggtctga
agaagtcaca tctgtcctgg ccagagggga aaaaccatca gatgctgaac 1200
caggagaatg ttgacccagg aaagggaccg aggacccaag aaaggagtca gaccaccagg
1260 gtttgcctga gaggaaggat caaggccccg agggaaagca gggctggctg
catgtgcagg 1320 acactggtgg ggcatatgtg tcttagattc tccctgaatt
cagtgtccct gccatggcca 1380 gactctctac tcaggcctgg acatgctgaa
ataggacaat ggccttgtcc tctctcccca 1440 ccatttggca agagacataa
aggacattcc aggacatgcc ttcctgggag gtccaggttc 1500 tctgtctcac
acctcaggga ctgtagttac tgcatcagcc atggtaggtg ctgatctcac 1560
ccagcctgtc caggcccttc cactctccac tttgtgacca tgtccaggac cacccctcaa
1620 atcctgagcc tgcaaatacc cccttgctgg gtgggtggat tcagtaaaca
gtgagctcct 1680 atccagccc cagagccacc tctgtcacct tcctgctggg
catcatccca ccttcacaag 1740 cactaaagag catggggaga cctggctagc
tgggtttctg catcacaaag aaaataatcc 1800 cccaggttcg gattcccagg
gctctgtatg tggagctgac agacctgagg ccaggagata 1860 gcagaggtca
gccctaggga gggtgggtca tccacccagg ggacaggggt gcaccagcct 1920
tgctactgaa agggcctccc caggacagcg ccatcagccc tgcctgagag ctttgctaaa
1980 cagcagtcag aggaggccat ggcagtggct gagctcctgc tccaggcccc
aacagaccag 2040 accaacagca caatgcagtc cttccccaac gtcacaggtc
accaaaggga aactgaggtg 2100 ctacctaacc ttagagccat caggggagat
aacagcccaa tttcccaaac aggccagttt 2160 caatcccatg acaatgacct
ctctgctctc attcttccca aaataggacg ctgattctcc 2220 cccaccatgg
atttctccct tgtcccggga gccttttctg ccccctatga tctgggcact 2280
cctgacacac acctcctctc tggtgacata tcagggtccc tcactgtcaa gcagtccaga
2340 aaggacagaa ccttggacag cgcccatctc agcttcaccc ttcctccttc
acagggttca 2400 gggcaaagaa taaatggcag aggccagtga gcccagagat
ggtgacaggc agtgacccag 2460 gggcagatgc ctggagcagg agctggcggg
gccacaggga gaaggtgatg caggaaggga 2520 aacccagaaa tgggcaggaa
aggaggacac aggctctgtg gggctgcagc ccagggttgg 2580 actatgagtg
tgaagccatc tcagcaagta aggccaggtc ccatgaacaa gagtgggagc 2640
acgtggcttc ctgctctgta tatggggtgg gggattccat gccccataga accagatggc
2700 cggggttcag atggagaagg agcaggacag gggatcccca ggataggagg
accccagtgt 2760 ccccacccag gcaggtgact gatgaatggg catgcagggt
cctcctgggc tgggctctcc 2820 ctttgtccct caggattcct tgaaggaaca
tccggaagcc gaccacatct acctggtggg 2880 ttctggggag tccatgtaaa
gccaggagct tgtgttgcta ggaggggtca tggcatgtgc 2940 tgggggcacc
aaagagagaa acctgagggc aggcaggacc tggtctgagg aggcatggga 3000
gcccagatgg ggagatggat gtcaggaaag gctgccccat cagggagggt gatagcaatg
3060 gggggtctgt gggagtgggc acgtgggatt ccctgggctc tgccaagttc
cctcccatag 3120 tcacaacctg gggacactgc ccatgaaggg gcgcctttgc
ccagccagat gctgctggtt 3180 ctgcccatcc actaccctct ctgctccagc
cactctgggt ctttctccag atgccctgga 3240 cagccctggc ctgggcctgt
cccctgagag gtgttgggag aagctgagtc tctggggaca 3300 ctctcatcag
agtctgaaag gcacatcagg aaacatccct ggtctccagg actaggcaat 3360
gaggaaaggg ccccagctcc tccctttccc actgagaggg tcgaccctgg gtggccacag
3420 tgacttctgc gtctgtccca gtcaccctga aaccacaaca aaaccccagc
cccagaccct 3480 gcaggtacaa tacatgtggg gacagtctgt acccagggga
agccagttct ctcttcctag 3540 gagaccgggc ctcagggctg tgcccggggc
aggcgggggc agcacgtgcc tgtccttgag 3600 aactcgggac cttaagggtc
tctgctctgt gaggcacagc aaggatcctt ctgtccagag 3660 atgaaagcag
ctcctgcccc tcctctgacc tcttcctcct tcccaaatct caaccaacaa 3720
ataggtgttt caaatctcat catcaaatct tcatccatcc acatgagaaa gcttaaaacc
3780 caatggattg acaacatcaa gagttggaac aagtggacat ggagatgtta
cttgtggaaa 3840 tttagatgtg ttcagctatc gggcaggaga atctgtgtca
aattccagca tggttcagaa 3900 gaatcaaaaa gtgtcacagt ccaaatgtgc
aacagtgcag gggataaaac tgtggtgcat 3960 tcaaactgag ggatattttg
gaacatgaga aaggaaggga ttgctgctgc acagaacatg 4020 gatgatctca
cacatagagt tgaaagaaag gagtcaatcg cagaatagaa aatgatcact 4080
aattccacct ctataaagtt tccaagagga aaacccaatt ctgctgctag agatcagaat
4140 ggaggtgacc tgtgccttgc aatggctgtg agggtcacgg gagtgtcact
tagtgcaggc 4200 aatgtgccgt atcttaatct gggcaggcct ttcatgagca
cataggaatg cagacattac 4260 tgctgtgttc attttacttc accggaaaag
aagaataaaa tcagccgggc gcggtggctc 4320 acgcctgtaa tcccagcact
ttagaagcct gaggtgggca gattacttga ggtcaggagt 4380 tcaagaccac
cctggccaat atggtgaaac cccggctcta ctaaaaatac aaaaattagc 4440
tgggcatggt ggtgcgcgcc tgtaatccca gctactcggg aggctgaggc tggacaattg
4500 cttggaccca ggaagcagag gttgcagtga gccaagattg tgccactgca
ctccagcttg 4560 ggcaacagag ccagactctg taaaaaaaaa aaaaaaaaaa
aaaaaaagaa agaaagaaaa 4620 agaaaagaaa gtataaaatc tctttgggtt
aacaaaaaaa gatccacaaa acaaacacca 4680 gctcttatca aacttacaca
actctgccag agaacaggaa acacaaatac tcattaactc 4740 acttttgtgg
caataaaacc ttcatgtcaa aaggagacca ggacacaatg aggaagtaaa 4800
actgcaggcc ctacttgggt gcagagaggg aaaatccaca aataaaacat
taccagaagg 4860 agctaagatt tactgcattg agttcattcc ccaggtatgc
aaggtgattt taacacctga 4920 aaatcaatca ttgcctttac tacatagaca
gattagctag aaaaaaatta caactagcag 4980 aacagaagca atttggcctt
cctaaaattc cacatcatat catcatgatg gagacagtgc 5040 agacgccaat
gacaataaaa agagggacct ccgtcacccg gtaaacatgt ccacacagct 5100
ccagcaagca cccgtcttcc cagtgaatca ctgtaacctc ccctttaatc agccccaggc
5160 aaggctgcct gcgatggcca cacaggctcc aacccgtggg cctcaacctc
ccgcagaggc 5220 tctcctttgg ccaccccatg gggagagcat gaggacaggg
cagagccctc tgatgcccac 5280 acatggcagg agctgacgcc agagccatgg
gggctggaga gcagagctgc tggggtcaga 5340 gcttcctgag gacacccagg
cctaagggaa ggcagctccc tggatggggg caaccaggct 5400 ccgggctcca
acctcagagc ccgcatggga ggagccagca ctctaggcct ttcctagggt 5460
gactctgagg ggaccctgac acgacaggat cgctgaatgc acccgagatg aaggggccac
5520 cacgggaccc tgctctcgtg gcagatcagg agagagtggg acaccatgcc
aggcccccat 5580 ggcatggctg cgactgaccc aggccactcc cctgcatgca
tcagcctcgg taagtcacat 5640 gaccaagccc aggaccaatg tggaaggaag
gaaacagcat cccctttagt gatggaaccc 5700 aaggtcagtg caaagagagg
ccatgagcag ttaggaaggg tggtccaacc tacagcacaa 5760 accatcatct
atcataagta gaagccctgc tccatgaccc ctgcatttaa ataaacgttt 5820
gttaaatgag tcaaattccc tcaccatgag agctcacctg tgtgtaggcc catcacacac
5880 acaaacacac acacacacac acacacacac acacacacac acagggaaag
tgcaggatcc 5940 tggacagcac caggcaggct tcacaggcag agcaaacagc
gtgaatgacc catgcagtgc 6000 cctgggcccc atcagctcag agaccctgtg
agggctgaga tggggctagg caggggagag 6060 acttagagag ggtggggcct
ccagggaggg ggctgcaggg agctgggtac tgccctccag 6120 ggagggggct
gcagggagct gggtactgcc ctccagggag ggggctgcag ggagctgggt 6180
actgccctcc agggaggggg ctgcagggag ctgggtactg ccctccaggg agggggctgc
6240 agggagctgg gtactgccct ccagggaggc aggagcactg ttcccaacag
agagcacatc 6300 ttcctgcagc agctgcacag acacaggagc ccccatgact
gccctgggcc agggtgtgga 6360 ttccaaattt cgtgccccat tgggtgggac
ggaggttgac cgtgacatcc aaggggcatc 6420 tgtgattcca aacttaaact
actgtgccta caaaatagga aataacccta ctttttctac 6480 tatctcaaat
tccctaagca caagctagca ccctttaaat caggaagttc agtcactcct 6540
ggggtcctcc catgccccca gtctgacttg caggtgcaca gggtggctga catctgtcct
6600 tgctcctcct cttggctcaa ctgccgcccc tcctgggggt gactgatggt
caggacaagg 6660 gatcctagag ctggccccat gattgacagg aaggcaggac
ttggcctcca ttctgaagac 6720 taggggtgtc aagagagctg ggcatcccac
agagctgcac aagatgacgc ggacagaggg 6780 tgacacaggg ctcagggctt
cagacgggtc gggaggctca gctgagagtt cagggacaga 6840 cctgaggagc
ctcagtggga aaagaagcac tgaagtggga agttctggaa tgttctggac 6900
aagcctgagt gctctaagga aatgctccca ccccgatgta gcctgcagca ctggacggtc
6960 tgtgtacctc cccgctgccc atcctctcac agcccccgcc tctagggaca
caactcctgc 7020 cctaacatgc atctttcctg tctcattcca cacaaaaggg
cctctggggt ccctgttctg 7080 cattgcaagg agtggaggtc acgttcccac
agaccaccca gcaacagggt cctatggagg 7140 tgcggtcagg aggatcacac
gtccccccat gcccagggga ctgactctgg gggtgatgga 7200 ttggcctgga
ggccactggt cccctctgtc cctgagggga atctgcaccc tggaggctgc 7260
cacatccctc ctgattcttt cagctgaggg cccttcttga aatcccaggg aggactcaac
7320 ccccactggg aaaggcccag tgtggacggt tccacagcag cccagctaag
gcccttggac 7380 acagatcctg agtgagagaa cctttaggga cacaggtgca
cggccatgtc cccagtgccc 7440 acacagagca ggggcatctg gaccctgagt
gtgtagctcc cgcgactgaa cccagccctt 7500 ccccaatgac gtgacccctg
gggtggctcc aggtctccag tccatgccac caaaatctcc 7560 agattgaggg
tcctcccttg agtccctgat gcctgtccag gagctgcccc ctgagcaaat 7620
ctagagtgca gagggctggg attgtggcag taaaagcagc cacatttgtc tcaggaagga
7680 aagggaggac atgagctcca ggaagggcga tggcgtcctc tagtgggcgc
ctcctgttaa 7740 tgagcaaaaa ggggccagga gagttgagag atcagggctg
gccttggact aaggctcaga 7800 tggagaggac tgaggtgcaa agagggggct
gaagtagggg agtggtcggg agagatggga 7860 ggagcaggta aggggaagcc
ccagggaggc cgggggaggg tacagcagag ctctccactc 7920 ctcagcattg
acatttgggg tggtcgtgct agtggggttc tgtaagttgt agggtgttca 7980
gcaccatctg gggactctac ccactaaatg ccagcaggac tccctcccca agctctaaca
8040 accaacaatg tctccagact ttccaaatgt cccctggaga gcaaaattgc
ttctggcaga 8100 atcactgatc tacgtcagtc tctaaaagtg actcatcagc
gaaatccttc acctcttggg 8160 agaagaatca caagtgtgag aggggtagaa
actgcagact tcaaaatctt tccaaaagag 8220 ttttacttaa tcagcagttt
gatgtcccag gagaagatac atttagagtg tttagagttg 8280 atgccacatg
gctgcctgta cctcacagca ggagcagagt gggttttcca agggcctgta 8340
accacaactg gaatgacact cactgggtta cattacaaag tggaatgtgg ggaattctgt
8400 agactttggg aagggaaatg tatgacgtga gcccacagcc taaggcagtg
gacagtccac 8460 tttgaggctc tcaccatcta ggagacatct cagccatgaa
catagccaca tctgtcatta 8520 gaaaacatgt tttattaaga ggaaaaatct
aggctagaag tgctttatgc tcttttttct 8580 ctttatgttc aaattcatat
acttttagat cattccttaa agaagaatct atccccctaa 8640 gtaaatgtta
tcactgactg gatagtgttg gtgtctcact cccaacccct gtgtggtgac 8700
agtgccctgc ttccccagcc ctgggccctc tctgattcct gagagctttg ggtgctcctt
8760 cattaggagg aagagaggaa gggtgttttt aatattctca ccattcaccc
atccacctct 8820 tagacactgg gaagaatcag ttgcccactc ttggatttga
tcctccaatt aatgacctct 8880 atttctgtcc cttgtccatt tcaacaatgt
gacaggccta agaggtgcct tctccatgtg 8940 atttttgagg agaaggttct
caagataagt tttctcacac ctctttgaat tacctccacc 9000 tgtgtcccca
tcaccattac cagcagcatt tggacccttt ttctgttagt cagatgcttt 9060
ccacctcttg agggtgtata ctgtatgctc tctacacagg aatatgcaga ggaaatagaa
9120 aaagggaaat cgcattacta ttcagagaga agaagacctt tatgtgaatg
aatgagagtc 9180 taaaatccta agagagccca tataaaatta ttaccagtgc
taaaactaca aaagttacac 9240 taacagtaaa ctagaataat aaaacatgca
tcacagttgc tggtaaagct aaatcagata 9300 tttttttctt agaaaaagca
ttccatgtgt gttgcagtga tgacaggagt gcccttcagt 9360 caatatgctg
cctgtaattt ttgttccctg gcagaatgta ttgtcttttc tccctttaaa 9420
tcttaaatgc aaaactaaag gcagctcctg ggccccctcc ccaaagtcag ctgcctgcaa
9480 ccagccccac gaagagcaga ggcctgagct tccctggtca aaataggggg
ctagggagct 9540 taaccttgct cgataaagct gtgttcccag aatgtcgctc
ctgttcccag gggcaccagc 9600 ctggagggtg gtgagcctca ctggtggcct
gatgcttacc ttgtgccctc acaccagtgg 9660 tcactggaac cttgaacact
tggctgtcgc ccggatctgc agatgtcaag aacttctgga 9720 agtcaaatta
ctgcccactt ctccagggca gatacctgtg aacatccaaa accatgccac 9780
agaaccctgc ctggggtcta caacacatat ggactgtgag caccaagtcc agccctgaat
9840 ctgtgaccac ctgccaagat gcccctaact gggatccacc aatcactgca
catggcaggc 9900 agcgaggctt ggaggtgctt cgccacaagg cagccccaat
ttgctgggag tttcttggca 9960 cctggtagtg gtgaggagcc ttgggaccct
caggattact ccccttaagc atagtgggga 10020 cccttctgca tccccagcag
gtgccccgct cttcagagcc tctctctctg aggtttaccc 10080 agacccctgc
accaatgaga ccatgctgaa gcctcagaga gagagatgga gctttgacca 10140
ggagccgctc ttccttgagg gccagggcag ggaaagcagg aggcagcacc aggagtggga
10200 acaccagtgt ctaagcccct gatgagaaca gggtggtctc tcccatatgc
ccataccagg 10260 cctgtgaaca gaatcctcct tctgcagtga caatgtctga
gaggacgaca tgtttcccag 10320 cctaacgtgc agccatgccc atctacccac
tgcctactgc aggacagcac caacccagga 10380 gctgggaagc tgggagaaga
catggaatac ccatggcttc tcaccttcct ccagtccagt 10440 gggcaccatt
tatgcctagg acacccacct gccggcccca ggctcttaag agttaggtca 10500
cctaggtgcc tctgggaggc cgaggcagga gaattgcttg aacccgggag gcagaggttg
10560 cagtgagccg agatcacacc actgcactcc agcctgggtg acagaatgag
actctgtctc 10620 aaaaaaaaag agaaagatag catcagtggc taccaagggc
taggggcagg ggaaggtgga 10680 gagttaatga ttaatagtat gaagtttcta
tgtgagatga tgaaaatgtt ctggaaaaaa 10740 aaatatagtg gtgaggatgt
agaatattgt gaatataatt aacggcattt aattgtacac 10800 ttaacatgat
taatgtggca tattttatct tatgtatttg actacatcca agaaacactg 10860
ggagagggaa agcccaccat gtaaaataca cccaccctaa tcagatagtc ctcattgtac
10920 ccaggtacag gcccctcatg acctgcacag gaataactaa ggatttaagg
acatgaggct 10980 tcccagccaa ctgcaggtgc acaacataaa tgtatctgca
aacagactga gagtaaagct 11040 gggggcacaa acctcagcac tgccaggaca
cacacccttc tcgtggattc tgactttatc 11100 tgacccggcc cactgtccag
atcttgttgt gggattggga caagggaggt cataaagcct 11160 gtccccaggg
cactctgtgt gagcacacga gacctcccca cccccccacc gttaggtctc 11220
cacacataga tctgaccatt aggcattgtg aggaggactc tagcgcgggc tcagggatca
11280 caccagagaa tcaggtacag agaggaagac ggggctcgag gagctgatgg
atgacacaga 11340 gcagggttcc tgcagtccac aggtccagct caccctggtg
taggtgcccc atccccctga 11400 tccaggcatc cctgacacag ctccctcccg
gagcctcctc ccaggtgaca catcagggtc 11460 cctcactcaa gctgtccaga
gagggcagca ccttggacag cgcccacccc acttcactct 11520 tcctccctca
cagggctcag ggctcagggc tcaagtctca gaacaaatgg cagaggccag 11580
tgagcccaga gatggtgaca gggcaatgat ccaggggcag ctgcctgaaa cgggagcagg
11640 tgaagccaca gatgggagaa gatggttcag gaagaaaaat ccaggaatgg
gcaggagagg 11700 agaggaggac acaggctctg tggggctgca gcccaggatg
ggactaagtg tgaagacatc 11760 tcagcaggtg aggccaggtc ccatgaacag
agaagcagct cccacctccc ctgatgcacg 11820 gacacacaga gtgtgtggtg
ctgtgccccc agagtcgggc tctcctgttc tggtccccag 11880 ggagtgagaa
gtgaggttga cttgtccctg ctcctctctg ctaccccaac attcaccttc 11940
tcctcatgcc cctctctctc aaatatgatt tggatctatg tccccgccca aatctcatgt
12000 caaattgtaa accccaatgt tggaggtggg gccttgtgag aagtgattgg
ataatgcggg 12060 tggattttct gctttgatgc tgtttctgtg atagagatct
cacatgatct ggttgtttaa 12120 aagtgtgtag cacctctccc ctctctctct
ctctctctta ctcatgctct gccatgtaag 12180 acgttcctgt ttccccttca
ccgtccagaa tgattgtaag ttttctgagg cctccccagg 12240 agcagaagcc
actatgcttc ctgtacaact gcagaatgat gagcgaatta aacctctttt 12300
ctttataaat tacccagtct caggtatttc tttatagcaa tgcgaggaca gactaataca
12360 atcttctact cccagatccc cgcacacgct tagccccaga catcactgcc
cctgggagca 12420 tgcacagcgc agcctcctgc cgacaaaagc aaagtcacaa
aaggtgacaa aaatctgcat 12480 ttggggacat ctgattgtga aagagggagg
acagtacact tgtagccaca gagactgggg 12540 ctcaccgagc tgaaacctgg
tagcactttg gcataacatg tgcatgaccc gtgttcaatg 12600 tctagagatc
agtgttgagt aaaacagcct ggtctggggc cgctgctgtc cccacttccc 12660
tcctgtccac cagagggcgg cagagttcct cccaccctgg agcctcccca ggggctgctg
12720 acctccctca gccgggccca cagcccagca gggtccaccc tcacccgggt
cacctcggcc 12780 cacgtcctcc tcgccctccg agctcctcac acggactctg
tcagctcctc cctgcagcct 12840 atcggccgcc cacctgaggc ttgtcggccg
cccacttgag gcctgtcggc tgccctctgc 12900 aggcagctcc tgtcccctac
accccctcct tccccgggct cagctgaaag ggcgtctccc 12960 agggcagctc
cctgtgatct ccaggacagc tcagtctctc acaggctccg acgcccccta 13020
tgctgtcacc tcacagccct gtcattacca ttaactcctc agtcccatga agttcactga
13080 gcgcctgtct cccggttaca ggaaaactct gtgacaggga ccacgtctgt
cctgctctct 13140 gtggaatccc agggcccagc ccagtgcctg acacggaaca
gatgctccat aaatactggt 13200 taaatgtgtg ggagatctct aaaaagaagc
atatcacctc cgtgtggccc ccagcagtca 13260 gagtctgttc catgtggaca
caggggcact ggcaccagca tgggaggagg ccagcaagtg 13320 cccgcggctg
ccccaggaat gaggcctcaa cccccagagc ttcagaaggg aggacagagg 13380
cctgcaggga atagatcctc cggcctgacc ctgcagccta atccagagtt cagggtcagc
13440 tcacaccacg tcgaccctgg tcagcatccc tagggcagtt ccagacaagg
ccggaggtct 13500 cctcttgccc tccagggggt gacattgcac acagacatca
ctcaggaaac ggattcccct 13560 ggacaggaac ctggctttgc taaggaagtg
gaggtggagc ctggtttcca tcccttgctc 13620 caacagaccc ttctgatctc
tcccacatac ctgctctgtt cctttctggg tcctatgagg 13680 accctgttct
gccaggggtc cctgtgcaac tccagactcc ctcctggtac caccatgggg 13740
aaggtggggt gatcacagga cagtcagcct cgcagagaca gagaccaccc aggactgtca
13800 gggagaacat ggacaggccc tgagccgcag ctcagccaac agacacggag
agggagggtc 13860 cccctggagc cttccccaag gacagcagag cccagagtca
cccacctccc tccaccacag 13920 tcctctcttt ccaggacaca caagacacct
ccccctccac atgcaggatc tggggactcc 13980 tgagacctct gggcctgggt
ctccatccct gggtcagtgg cggggttggt ggtactggag 14040 acagagggct
ggtccctccc cagccaccac ccagtgagcc tttttctagc ccccagagcc 14100
acctctgtca ccttcctgtt gggcatcatc ccaccttccc agagccctgg agagcatggg
14160 gagacccggg accctgctgg gtttctctgt cacaaaggaa aataatcccc
ctggtgtgac 14220 agacccaagg acagaacaca gcagaggtca gcactgggga
agacaggttg tcctcccagg 14280 ggatgggggt ccatccacct tgccgaaaag
atttgtctga ggaactgaaa atagaaggga 14340 aaaaagagga gggacaaaag
aggcagaaat gagaggggag gggacagagg acacctgaat 14400 aaagaccaca
cccatgaccc acgtgatgct gagaagtact cctgccctag gaagagactc 14460
.vertline..sup..fwdarw. transcription start site agggcagagg
gaggaaggac agcagaccag acagtcacag cagccttgac aaaacgttcc 14520
tggaactcaa gctcttctcc acagaggagg acagagcaga cagcagagac catggagtct
14580 ccctcggccc ctccccacag atggtgcatc ccctggcaga ggctcctgct
cacaggtgaa 14640 gggaggacaa cctgggagag ggtgggagga gggagctggg
gtctcctggg taggacaggg 14700 ctgtgagacg gacagagggc tcctgttgga
gcctgaatag ggaagaggac atcagagagg 14760 gacaggagtc acaccagaaa
aatcaaattg aactggaatt ggaaaggggc aggaaaacct 14820 caagagttct
attttcctag ttaattgtca ctggccacta cgtttttaaa aatcataata 14880
actgcatcag atgacacttt aaataaaaac ataaccaggg catgaaacac tgtcctcatc
14940 cgcctaccgc ggacattgga aaataagccc caggctgtgg agggccctag
gaaccctcat 15000 gaactcatcc acaggaatct gcagcctgtc ccaggcactg
gggtgcaacc aagatc 15056 Mucin-TRE SEQ ID NO:15 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 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 .alpha.FP-TRE SEQ ID NO:16 gaattcttag
aaatatgggg gtaggggtgg tggtggtaat tctgttttca ccccataggt 60
gagataagca ttgggttaaa tgtgctttca cacacacatc acatttcata agaattaagg
120 aacagactat gggctggagg actttgagga tgtctgtctc ataacacttg
ggttgtatct 180 gttctatggg gcttgtttta agcttggcaa cttgcaacag
ggttcactga ctttctcccc 240 aagcccaagg tactgtcctc ttttcatatc
tgttttgggg cctctggggc ttgaatatct 300 gagaaaatat aaacatttca
ataatgttct gtggtgagat gagtatgaga gatgtgtcat 360 tcatttgtat
caatgaatga atgaggacaa ttagtgtata aatccttagt acaacaatct 420
gagggtaggg gtggtactat tcaatttcta tttataaaga tacttatttc tatttattta
480 tgcttgtgac aaatgttttg ttcgggacca caggaatcac aaagatgagt
ctttgaattt 540 aagaagttaa tggtccagga ataattacat agcttacaaa
tgactatgat ataccatcaa 600 acaagaggtt ccatgagaaa ataatctgaa
aggtttaata agttgtcaaa ggtgagaggg 660 ctcttctcta gctagagact
aatcagaaat acattcaggg ataattattt gaatagacct 720 taagggttgg
gtacattttg ttcaagcatt gatggagaag gagagtgaat atttgaaaac 780
attttcaact aaccaaccac ccaatccaac aaacaaaaaa tgaaaagaat ctcagaaaca
840 gtgagataag agaaggaatt ttctcacaac ccacacgtat agctcaactg
ctctgaagaa 900 gtatatatct aatatttaac actaacatca tgctaataat
gataataatt actgtcattt 960 tttaatgtct ataagtacca ggcatttaga
agatattatt ccatttatat atcaaaataa 1020 acttgagggg atagatcatt
ttcatgatat atgagaaaaa ttaaaaacag attgaattat 1080 ttgcctgtca
tacagctaat aattgaccat aagacaatta gatttaaatt agttttgaat 1140
ctttctaata ccaaagttca gtttactgtt ccatgttgct tctgagtggc ttcacagact
1200 tatgaaaaag taaacggaat cagaattaca tcaatgcaaa agcattgctg
tgaactctgt 1260 acttaggact aaactttgag caataacaca catagattga
ggattgtttg ctgttagcat 1320 acaaactctg gttcaaagct cctctttatt
gcttgtcttg gaaaatttgc tgttcttcat 1380 ggtttctctt ttcactgcta
tctatttttc tcaaccactc acatggctac aataactgtc 1440 tgcaagctta
tgattcccaa atatctatct ctagcctcaa tcttgttcca gaagataaaa 1500
agtagtattc aaatgcacat caacgtctcc acttggaggg cttaaagacg tttcaacata
1560 caaaccgggg agttttgcct ggaatgtttc ctaaaatgtg tcctgtagca
catagggtcc 1620 tcttgttcct taaaatctaa ttacttttag cccagtgctc
atcccaccta tggggagatg 1680 agagtgaaaa gggagcctga ttaataatta
cactaagtca ataggcatag agccaggact 1740 gtttgggtaa actggtcact
ttatcttaaa ctaaatatat ccaaaactga acatgtactt 1800 agttactaag
tctttgactt tatctcattc ataccactca gctttatcca ggccacttat 1860
ttgacagtat tattgcgaaa acttcctaac tggtctcctt atcatagtct tatccccttt
1920 tgaaacaaaa gagacagttt caaaatacaa atatgatttt tattagctcc
cttttgttgt 1980 ctataatagt cccagaagga gttataaact ccatttaaaa
agtctttgag atgtggccct 2040 tgccaacttt gccaggaatt cccaatatct
agtattttct actattaaac tttgtgcctc 2100 ttcaaaactg cattttctct
cattccctaa gtgtgcattg ttttccctta ccggttggtt 2160 tttccaccac
cttttacatt ttcctggaac actataccct ccctcttcat ttggcccacc 2220
tctaattttc tttcagatct ccatgaagat gttacttcct ccaggaagcc ttatctgacc
2280 cctccaaaga tgtcatgagt tcctcttttc attctactaa tcacagcatc
catcacacca 2340 tgttgtgatt actgatacta ttgtctgttt ctctgattag
gcagtaagct caacaagagc 2400 tacatggtgc ctgtctcttg ttgctgatta
ttcccatcca aaaacagtgc ctggaatgca 2460 gacttaacat tttattgaat
gaataaataa aaccccatct atcgagtgct actttgtgca 2520 agacccggtt
ctgaggcatt tatatttatt gatttattta attctcattt aaccatgaag 2580
gaggtactat cactatcctt attttatagt tgataaagat aaagcccaga gaaatgaatt
2640 aactcaccca aagtcatgta gctaagtgac agggcaaaaa ttcaaaccag
ttccccaact 2700 ttacgtgatt aatactgtgc tatactgcct ctctgatcat
atggcatgga atgcagacat 2760 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
[0410]
Sequence CWU 1
1
35 1 519 DNA Artificial Sequence IRES from encephelomycarditis
virus (EMCV) 1 gacgtcgact aattccggtt attttccacc atattgccgt
cttttggcaa tgtgagggcc 60 cggaaacctg gccctgtctt cttgacgagc
attcctaggg gtctttcccc tctcgccaaa 120 ggaatgcaag gtctgttgaa
tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 180 caaacaacgt
ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc 240
ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc
300 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag
cgtattcaac 360 aaggggctga aggatgccca gaaggtaccc cattgtatgg
gatctgatct ggggcctcgg 420 tgcacatgct ttacatgtgt ttagtcgagg
ttaaaaaacg tctaggcccc ccgaaccacg 480 gggacgtggt tttcctttga
aaaacacgat gtcgacgtc 519 2 188 DNA Artificial Sequence IRES from
vascular endothelial growth factor (VEGF) 2 acgtagtcga cagcgcagag
gcttggggca gccgagcggc agccaggccc cggcccgggc 60 ctcggttcca
gaagggagag gagcccgcca aggcgcgcaa gagagcgggc tgcctcgcag 120
tccgagccgg agagggagcg cgagccgcgc cggccccgga cggcctccga aaccatggtc
180 gacacgta 188 3 341 DNA Artificial Sequence 5' UTR region of HCV
3 gccagccccc tgatgggggc gacactccgc catgaatcac tcccctgtga ggaactactg
60 tcttcacgca gaaagcgtct agccatggcg ttagtatgag tgtcgtgcag
cctccaggac 120 cccccctccc gggagagcca tagtggtctg cggaaccggt
gagtacaccg gaattgccag 180 gacgaccggg tcctttcttg gattaacccg
ctcaatgcct ggagatttgg gcgtgccccc 240 gcaagactgc tagccgagta
gtgttgggtc gcgaaaggcc ttgtggtact gcctgatagg 300 gtgcttgcga
gtgccccggg aggtctcgta gaccgtgcac c 341 4 595 DNA Artificial
Sequence 5' UTR region of BiP 4 cccggggtca ctcctgctgg acctactccg
accccctagg ccgggagtga aggcgggact 60 tgtgcggtta ccagcggaaa
tgcctcgggg tcagaagtcg caggagagat agacagctgc 120 tgaaccaatg
ggaccagcgg atggggcgga tgttatctac cattggtgaa cgttagaaac 180
gaatagcagc caatgaatca gctggggggg cggagcagtg acgtttattg cggagggggc
240 cgcttcgaat cggcggcggc cagcttggtg gcctgggcca atgaacggcc
tccaacgagc 300 agggccttca ccaatcggcg gcctccacga cggggctggg
ggagggtata taagccgagt 360 aggcgacggt gaggtcgacg ccggccaaga
cagcacagac agattgacct attggggtgt 420 ttcgcgagtg tgagagggaa
gcgccgcggc ctgtatttct agacctgccc ttcgcctggt 480 tcgtggcgcc
ttgtgacccc gggcccctgc cgcctgcaag tcgaaattgc gctgtgctcc 540
tgtgctacgg cctgtggctg gactgcctgc tgctgcccaa ctggctggca agatg 595 5
575 DNA Artificial Sequence 5' UTR of PDGF 5 gtttgcacct ctccctgccc
gggtgctcga gctgccgttg caaagccaac tttggaaaaa 60 gttttttggg
ggagacttgg gccttgaggt gcccagctcc gcgctttccg attttggggg 120
ctttccagaa aatgttgcaa aaaagctaag ccggcgggca gaggaaaacg cctgtagccg
180 gcgagtgaag acgaaccatc gactgccgtg ttccttttcc tcttggaggt
tggagtcccc 240 tgggcgcccc cacaccccta gacgcctcgg ctggttcgcg
acgcagcccc ccggccgtgg 300 atgctgcact cgggctcggg atccgcccag
gtagccggcc tcggacccag gtcctgcgcc 360 caggtcctcc cctgcccccc
agcgacggag ccggggccgg gggcggcggc gccgggggca 420 tgcgggtgag
ccgcggctgc agaggcctga gcgcctgatc gccgcggacc tgagccgagc 480
ccacccccct ccccagcccc ccaccctggc cgcgggggcg gcgcgctcga tctacgcgtc
540 cggggccccg cggggccggg cccggagtcg gcatg 575 6 2240 DNA
Artificial Sequence Human uroplakin II 5' flanking region 6
tcgataggta cccactatag ggcacgcgtg gtcgacggcc cgggctggtc tggcaacttc
60 aagtgtgggc ctttcagacc ggcatcatca gtgttacggg gaagtcacta
ggaatgcaga 120 attgattgag cacggtggct cacacctgta atcccaacac
tctgggaggc caaggcaggt 180 ggatcacttg tggtcaggag tttgagacca
gcctggccaa catggtgaaa cctcatctct 240 actaaaaata caaaaattag
ctgggaatgg tggcacatgc ctataatccc agttactcag 300 gaggctgagg
caggagaatc atttgaacct gggaggcaga ggttgcagtg agccgagatc 360
acgccactgc actccagcct gggtgacaca gcgagactct gtctcaaaaa aaaaaaaatg
420 cagaatttca ggcttcaccc cagacccact gcatgactgc atgagaagct
gcatcttaac 480 aagatccctg gtaattcata cgcatattaa atttggagat
gcactggcgt aagaccctcc 540 tactctctgc ttaggcccat gagttcttcc
tttactgtca ttctccactc accccaaact 600 ttgagcctac ccttcccacc
ttggcggtaa ggacacaacc tccctcacat tcctaccagg 660 accctaagct
tccctgggac tgaggaagat agaatagttc gtggagcaaa cagatataca 720
gcaacagtct ctgtacagct ctcaggcttc tggaagttct acagcctctc ccgacaaagt
780 attccacttt ccacaagtaa ctctatgtgt ctgagtctca gtttccactt
ttctctctct 840 ctctctctct caactttctg agacagagtt tcacttagtc
gcccaggctg gagtgcaggg 900 gcacaatctc ggctcactgc aacctccacc
tcctgggttc aagtgtttct cctgtctcag 960 cctcccgagt agctgggatt
acaggcacac accaccgcgt tagtttttgt atttttggta 1020 gagatggtgt
ttcgccatat tggccaggct gatctcgaac tcctgacctc aggtgatccg 1080
cccacctcgg cctcccaaag tgctgggatt acaggcatga gccaccacgc ccggctgatc
1140 tcttttctat tttaatagag atcaaactct ctgtgttgcc taggctggtc
ttgaactcct 1200 ggcctcgagt gatcctccca ccttggcctc ccaaagtgtt
gagattacag gcatgagcca 1260 ctgtgcctgg cctcagttct actacaaaag
gaagccagta ccagctacca cccagggtgg 1320 ctgtagggct acaatggagc
acacagaacc cctacccagg gcccggaaga agccccgact 1380 cctctcccct
ccctctgccc agaactcctc cgcttctttc tgatgtagcc cagggccgga 1440
ggaggcagtc agggaagttc tgtctctttt tcatgttatc ttacgaggtc tcttttctcc
1500 attctcagtc caacaaatgg ttgctgccca aggctgactg tgcccacccc
caacccctgc 1560 tggccagggt caatgtctgt ctctctggtc tctccagaag
tcttccatgg ccaccttcgt 1620 ccccaccctc cagaggaatc tgaaaccgca
tgtgctccct ggcccccaca gcccctgcct 1680 ctcccagagc agcagtacct
aagcctcagt gcactccaag aattgaaacc ctcagtctgc 1740 tgcccctccc
caccagaatg tttctctccc attcttaccc actcaaggcc ctttcagtag 1800
ccccttggag tattctcttc ctacatatca gggcaacttc caaactcatc acccttctga
1860 ggggtggggg aaagaccccc accacatcgg gggagcagtc ctccaaggac
tggccagtct 1920 ccagatgccc gtgcacacag gaacactgcc ttatgcacgg
gagtcccaga agaaggggtg 1980 atttctttcc ccaccttagt tacaccatca
agacccagcc agggcatccc ccctcctggc 2040 ctgagggcca gctccccatc
ctgaaaaacc tgtctgctct ccccacccct ttgaggctat 2100 agggcccaag
gggcaggttg gactggattc ccctccagcc cctcccgccc ccaggacaaa 2160
atcagccacc ccaggggcag ggcctcactt gcctcaggaa ccccagcctg ccagcaccta
2220 ttccacctcc cagcccagca 2240 7 3592 DNA Artificial Sequence
Mouse uroplakin II 5' flanking region 7 ctcgaggatc tcggccctct
ttctgcatcc ttgtcctaaa tcattttcat atcttgctag 60 acctcagttt
gagagaaacg aaccttctca ttttcaagtt gaaaaaaaaa agaggttcaa 120
agtggctcac tcaaagttac aagccaacac tcaccactac gagtacaatg gccaccatta
180 gtgctggcat gccccaggag acaggcatgc atattattct agatgactgg
gaggcagagg 240 ggtggcctag tgaggtcaga ctgtggacag atcaggcaga
tgtgggttct gatcccaatt 300 cctcaggccg cagaactact gtggttcaag
aaggggacaa aaggactgca gtccggaaca 360 ggaggtccat ttgagagctg
actgagcaga agaggaaagt gaagaacttc tggggcaaga 420 gcttacccta
ctttacagct ttgttgtctt ctttactcca ggggcgtccc tggtactcag 480
taaatgtctg ttggcttgag gaacatatgt gtaaggagga aggagaggga acttgaggga
540 gttaagactc aagaatcaat caaggagagg acagcagaga agacagggtt
tgggagagag 600 actccagaca ttggccctgg ttcccttctt ggccactgtg
aaaccctcca gaggaactga 660 gtgctgtggc tttaaatgat ctcagcactg
tcagtgaagc gctctgctca aagagttatc 720 ctcttgctcc tgtgccgggg
cctccccctc ctctcagctc ccaaaccctt ctcagccact 780 gtgatggcat
aattagatgc gagagctcag accgtcaggt ctgctccagg aaccacccat 840
tttccccaac cccagagaaa ggtcctagtg gaaaagtggg ggccactgaa gggctgatgg
900 ggttctgtcc tttcccccat gctgggtgga cttaaagtct gcgatgtgtg
tagggggtag 960 aagacaacag aacctggggg ctccggctgg gagcaggagg
aactctcacc agacgatctc 1020 caaatttact gtgcaatgga cgatcaggaa
actggttcag atgtagcttc tgatacagtg 1080 ggtctgaggt aaaacccgaa
acttaatttc tttcaaaaat ttaaagttgc atttattatt 1140 ttatatgtgt
gcccatatgt gtgccacagt gtctatgtgg aggtcagagg gcaagttgtg 1200
ggcattggct ctctcctttc ataatgtggc ttctggggac caaaatgtca ggcatggtgg
1260 caagagcttt tacctgttga gccatctcat ggtttcgtaa aacttcctat
gacgcttaca 1320 ggtaacgcag agacacagac tcacatttgg agttagcaga
tgctgtattg gtgtaaacac 1380 tcatacacag acacacacac atactcatac
acacacacac acacttatca catgcacaca 1440 catactcgta tacacacaga
cacacacaca tgcactctca cattcacata ttcatacaca 1500 tccacacaca
cactcatcca cacacacaga cacacatact catccacaca cacacacaca 1560
catactcata cacacacaca gacacacata ctcatacaca cacacagaca cacacatata
1620 atcatacata cacagacaca ctcatacatg tgcacacaca cactcatcca
cacacacaca 1680 ctcatacaca cacacactca tacacacaca cactcataca
cacacacacg aggtttttct 1740 caggctgcct ttgggtggag actggaactg
atttctgttt ttcagctcct tggctttttg 1800 tccctttaga tgagatctcc
tcctcacttt acacacagaa agatcacaca cgagggagaa 1860 ctggcggtgc
ggaagagggc tacacggtag ggtgtcaggg tcaggagatc ttcctggcaa 1920
gtctcaaacc tccacatagc acagtgttta cgtgaggatt taggaggaat caggaagagg
1980 attggtttac tgcagagcag accatatagg tccactccta agccccattt
gaaattagaa 2040 gtgagacagt gtgggataaa aagagcagat ctctggtcac
atttttaaag ggatatgagg 2100 gtcctgtgcc tttaagcctt cccatctccc
tccaatcccc cctcaccttc cccaccctaa 2160 ccctccccag gtttctggag
gagcagagtt gcgtcttctc cctgccctgc cgagctgctc 2220 actggctgct
ctagaggctg tgctttgcgg tctccatgga aaccattagt tgctaagcaa 2280
ctggagcatc atctgtgctg agctcaggtc ctatcgagtt cacctagctg agacacccac
2340 gcccctgcag ccactttgca gtgacaagcc tgagtctcag gttctgcatc
tataaaaacg 2400 agtagccttt caggagggca tgcagagccc cctggccagc
gtctagagga gaggtgactg 2460 agtggggcca tgtcactcgt ccatggctgg
agaacctcca tcagtctccc agttagcctg 2520 gggcaggaga gaaccagagg
agctgtggct gctgattgga tgatttacgt acccaatctg 2580 ttgtcccagg
catcgaaccc cagagcgacc tgcacacatg ccaccgctgc cccgccctcc 2640
acctcctctg ctcctggtta caggattgtt ttgtcttgaa gggttttgtt gttgctactt
2700 tttgctttgt tttttctttt ttaacataag gtttctctgt gtagccctag
ctgtcctgga 2760 actcactctg tagaccaggc tggcctcaaa ctcagaaatc
caccttcctc ccaagtgctg 2820 ggattaaagg cattcgcacc atcgcccagc
ccccggtctt gtttcctaag gttttcctgc 2880 tttactcgct acccgttgca
caaccgcttg ctgtccaagt ctgtttgtat ctactccacc 2940 gcccactagc
cttgctggac tggacctacg tttacctgga agccttcact aacttccctt 3000
gtctccacct tctggagaaa tctgaaggct cacactgata ccctccgctt ctcccagagt
3060 cgcagtttct taggcctcag ttaaatacca gaattggatc tcaggctctg
ctatccccac 3120 cctacctaac caaccccctc ctctcccatc cttactagcc
aaagcccttt caacccttgg 3180 ggcttttcct acacctacac accagggcaa
ttttagaact catggctctc ctagaaaacg 3240 cctacctcct tggagactga
ccctctacag tccaggaggc agacactcag acagaggaac 3300 tctgtccttc
agtcgcggga gttccagaaa gagccatact cccctgcaga gctaactaag 3360
ctgccaggac ccagccagag catccccctt tagccgaggg ccagctcccc agaatgaaaa
3420 acctgtctgg ggcccctccc tgaggctaca gtcgccaagg ggcaagttgg
actggattcc 3480 cagcagcccc tcccactccg agacaaaatc agctaccctg
gggcaggcct cattggcccc 3540 aggaaacccc agcctgtcag cacctgttcc
aggatccagt cccagcgcag ta 3592 8 822 DNA Artificial Sequence APF-TRE
8 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 9 451 DNA
Artificial Sequence Probasin-TRE 9 aag ctt cca caa gtg cat tta gcc
tct cca gta ttg ctg atg aat cca 48 cag ttc agg ttc aat ggc gtt caa
aac ttg atc aaa aat gac cag act 96 tta tat tta cac caa cat cta tct
gat tgg agg aat gga taa tag tca 144 tca tgt tta aac atc tac cat tcc
agt taa gaa aat atg ata gca tct 192 tgt tct tag tct ttt tct taa tag
gga cat aaa gcc cac aaa taa aaa 240 tat gcc tga aga atg gga cag gca
ttg ggc att gtc cat gcc tag taa 288 agt act cca aga acc tat ttg tat
act aga tga cac aat gtc aat gtc 336 tgt gta caa ctg cca act ggg atg
caa gac act gcc cat gcc aat cat 384 cct gaa aag cag cta taa aaa gca
gga agc tac tct gca cct tgt cag 432 tag gtc cag ata cct aca g 451
10 546 DNA Artificial Sequence Tyrosinase-TRE 10 ccggttgaaa
atgataagtt gaattctgtc ttcgagaaca tagaaaagaa ttatgaaatg 60
ccaacatgtg gttacaagta atgcagaccc aaggctcccc agggacaaga agtcttgtgt
120 taactctttg tggctctgaa agaaagagag agagaaaaga ttaagcctcc
ttgtggagat 180 catgtgatga cttcctgatt ccagccagag cgagcatttc
catggaaact tctcttcctc 240 ttcactcgag attactaacc ttattgttaa
tattctaacc ataagaatta aactattaat 300 ggtgaataga gtttttcact
ttaacatagg cctatcccac tggtgggata cgagccaatt 360 cgaaagaaaa
agtcagtcat gtgcttttca gaggatgaaa gcttaagata aagactaaaa 420
gtgtttgatg ctggaggtgg gagtggtatt atataggtct cagccaagac atgtgataat
480 cactgtagta gtagctggaa agagaaatct gtgactccaa ttagccagtt
cctgcagacc 540 ttgtga 546 11 12047 DNA Artificial Sequence Human
glandular kallikrein-TRE 11 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 ggtatgaagc cctgcctgca ttagctcttt tccctaatgc
tctcactact gccccaccct 3240 caccctgaca gggcaaacag acaacctaca
gaatgggagg aaatttttgc aatctattca 3300 tctgacaaag gtcaagaata
tccagaatct acaaggaact taagcaaatt tttacttttt 3360 aataatagcc
actctgactg gcgtgaaatg gtatctcatt gtggttttca tttgaatttc 3420
tctgatgatc agtgacgatg agcatttttt catatttgtt ggctgcttgt acgtcttttg
3480 agaagtgtct cttcatgcct tttggccact ttaatgggat tattttttgc
tttttagttt 3540 aagttcctta tagattctgg atattagact tcttattgga
tgcatagttt gtgaatactc 3600 tcttccattc tgtaggttgt ctgtttactc
tattgatggc ttcttttgct gtgccgaagc 3660 atcttagttt aattagaaac
cacctgccaa tttttgtttt tgttgcaatt gcttttgggg 3720 acttagtcat
aaactctttg ccaaggtctg ggtcaagaag agtatttcct aggttttctt 3780
ctagaatttt gaaagtctga atgtaaacat ttgcattttt aatgcatctt gagttagttt
3840 ttgtatatgt gaaaggtcta ctctcatttt ctttccctct ttctttcttt
ctttcttttc 3900 tttctttctt tctttctttc tttctttctt tctttctttc
tttctttttg tccttctttc 3960 tttctttctt tctctttctt tctctctttc
tttttttttt ttgatggagt attgctctgt 4020 tgcccaggct gcagtgcagc
ggcacgatct cggctcactg caacctctgc ctcctgggtt 4080 caactgattc
tcctgcatca gccttccaag tagctgggat tataggcgcc cgccaccacg 4140
cccgactaat ttttgtattt ttagtagaga cggggttgtg ccatgttggc
caggctggtt 4200 tgaaactcct gacctcaaac gatctgcctg ccttggcctc
ccaaagtgct gggattacag 4260 gtgtgagcca ctgtgcccag ccaagaatgt
cattttctaa gaggtccaag aacctcaaga 4320 tattttggga ccttgagaag
agaggaattc atacaggtat tacaagcaca gcctaatggc 4380 aaatctttgg
catggcttgg cttcaagact ttaggctctt aaaagtcgaa tccaaaaatt 4440
tttataaaag ctccagctaa gctaccttaa aaggggcctg tatggctgat cactcttctt
4500 gctatacttt acacaaataa acaggccaaa tataatgagg ccaaaattta
ttttgcaaat 4560 aaattggtcc tgctatgatt tactcttggt aagaacaggg
aaaatagaga aaaatttaga 4620 ttgcatctga cctttttttc tgaattttta
tatgtgccta caatttgagc taaatcctga 4680 attattttct ggttgcaaaa
actctctaaa gaagaacttg gttttcattg tcttcgtgac 4740 acatttatct
ggctctttac tagaacagct ttcttgtttt tggtgttcta gcttgtgtgc 4800
cttacagttc tactcttcaa attattgtta tgtgtatctc atagttttcc ttcttttgag
4860 aaaactgaag ccatggtatt ctgaggacta gagatgactc aacagagctg
gtgaatctcc 4920 tcatatgcaa tccactgggc tcgatctgct tcaaattgct
gatgcactgc tgctaaagct 4980 atacatttaa aaccctcact aaaggatcag
ggaccatcat ggaagaggag gaaacatgaa 5040 attgtaagag ccagattcgg
ggggtagagt gtggaggtca gagcaactcc accttgaata 5100 agaaggtaaa
gcaacctatc ctgaaagcta acctgccatg gtggcttctg attaacctct 5160
gttctaggaa gactgacagt ttgggtctgt gtcattgccc aaatctcatg ttaaattgta
5220 atccccagtg ttcggaggtg ggacttggtg gtaggtgatt cggtcatggg
agtagatttt 5280 cttctttgtg gtgttacagt gatagtgagt gagttctcgt
gagatctggt catttaaaag 5340 tgtgtggccc ctcccctccc tctcttggtc
ctcctactgc catgtaagat acctgctcct 5400 gctttgcctt ctaccataag
taaaagcccc ctgaggcctc cccagaagca gatgccacca 5460 tgcttcctgt
acagcctgca gaaccatcag ccaattaaac ctcttttctg tataaattac 5520
cagtcttgag tatctcttta cagcagtgtg agaacggact aatacaaggg tctccaaaat
5580 tccaagttta tgtattcttt cttgccaaat agcaggtatt taccataaat
cctgtcctta 5640 ggtcaaacaa ccttgatggc atcgtacttc aattgtctta
cacattcctt ctgaatgact 5700 cctcccctat ggcatataag ccctgggtct
tgggggataa tggcagaggg gtccaccatc 5760 ttgtctggct gccacctgag
acacggacat ggcttctgtt ggtaagtctc tattaaatgt 5820 ttctttctaa
gaaactggat ttgtcagctt gtttctttgg cctctcagct tcctcagact 5880
ttggggtagg ttgcacaacc ctgcccacca cgaaacaaat gtttaatatg ataaatatgg
5940 atagatataa tccacataaa taaaagctct tggagggccc tcaataattg
ttaagagtgt 6000 aaatgtgtcc aaagatggaa aatgtttgag aactactgtc
ccagagattt tcctgagttc 6060 tagagtgtgg gaatatagaa cctggagctt
ggcttcttca gcctagaatc aggagtatgg 6120 ggctgaagtc tgaagcttgg
cttcagcagt ttggggttgg cttccggagc acatatttga 6180 catgttgcga
ctgtgatttg gggtttggta tttgctctga atcctaatgt ctgtccttga 6240
ggcatctaga atctgaaatc tgtggtcaga attctattat cttgagtagg acatctccag
6300 tcctggttct gccttctagg gctggagtct gtagtcagtg acccggtctg
gcatttcaac 6360 ttcatataca gtgggctatc ttttggtcca tgtttcaacc
aaacaaccga ataaaccatt 6420 agaacctttc cccacttccc tagctgcaat
gttaaaccta ggatttctgt ttaataggtt 6480 catatgaata atttcagcct
gatccaactt tacattcctt ctaccgttat tctacaccca 6540 ccttaaaaat
gcattcccaa tatattccct ggattctacc tatatatggt aatcctggct 6600
ttgccagttt ctagtgcatt aacatacctg atttacattc ttttacttta aagtggaaat
6660 aagagtccct ctgcagagtt caggagttct caagatggcc cttacttctg
acatcaattg 6720 agatttcaag ggagtcgcca agatcatcct caggttcagt
gattgctggt agccctcata 6780 taactcaatg aaagctgtta tgctcatggc
tatggtttat tacagcaaaa gaatagagat 6840 gaaaatctag caagggaaga
gttgcatggg gcaaagacaa ggagagctcc aagtgcagag 6900 attcctgttg
ttttctccca gtggtgtcat ggaaagcagt atcttctcca tacaatgatg 6960
tgtgataata ttcagtgtat tgccaatcag ggaactcaac tgagccttga ttatattgga
7020 gcttggttgc acagacatgt cgaccacctt catggctgaa ctttagtact
tagcccctcc 7080 agacgtctac agctgatagg ctgtaaccca acattgtcac
cataaatcac attgttagac 7140 tatccagtgt ggcccaagct cccgtgtaaa
cacaggcact ctaaacaggc aggatatttc 7200 aaaagcttag agatgacctc
ccaggagctg aatgcaaaga cctggcctct ttgggcaagg 7260 agaatccttt
accgcacact ctccttcaca gggttattgt gaggatcaaa tgtggtcatg 7320
tgtgtgagac accagcacat gtctggctgt ggagagtgac ttctatgtgt gctaacattg
7380 ctgagtgcta agaaagtatt aggcatggct ttcagcactc acagatgctc
atctaatcct 7440 cacaacatgg ctacagggtg ggcactacta gcctcatttg
acagaggaaa ggactgtgga 7500 taagaagggg gtgaccaata ggtcagagtc
attctggatg caaggggctc cagaggacca 7560 tgattagaca ttgtctgcag
agaaattatg gctggatgtc tctgccccgg aaagggggat 7620 gcactttcct
tgacccccta tctcagatct tgactttgag gttatctcag acttcctcta 7680
tgataccagg agcccatcat aatctctctg tgtcctctcc ccttcctcag tcttactgcc
7740 cactcttccc agctccatct 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 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 12 67 DNA
Artificial Sequence HRE-TRE 12 ccccgaggca gtgcatgagg ctcagggcgt
gcgtgagtcg cagcgagacc ccggggtgca 60 ggccgga 67 13 5835 DNA
Artificial Sequence PSA-TRE 13 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 aaaaaaaaaa
gagatgacct ctcaggctct 720 gaggggaaac gcctgaggtc tttgagcaag
gtcagtcctc tgttgcacag tctccctcac 780 agggtcattg tgacgatcaa
atgtggtcac gtgtatgagg caccagcaca tgcctggctc 840 tggggagtgc
cgtgtaagtg tatgcttgca ctgctgaatg gctgggatgt gtcagggatt 900
atcttcagca cttacagatg ctcatctcat cctcacagca tcactatggg atgggtatta
960 ctggcctcat ttgatggaga aagtggctgt ggctcagaaa ggggggacca
ctagaccagg 1020 gacactctgg atgctgggga ctccagagac catgaccact
caccaactgc agagaaatta 1080 attgtggcct gatgtccctg tcctggagag
ggtggaggtg gaccttcact aacctcctac 1140 cttgaccctc tcttttaggg
ctctttctga cctccaccat ggtactagga ccccattgta 1200 ttctgtaccc
tcttgactct atgaccccca ccgcccactg catccagctg ggtcccctcc 1260
tatctctatt cccagctggc cagtgcagtc tcagtgccca cctgtttgtc agtaactctg
1320 aaggggctga cattttactg acttgcaaac aaataagcta actttccaga
gttttgtgaa 1380 tgctggcaga gtccatgaga ctcctgagtc agaggcaaag
gcttttactg ctcacagctt 1440 agcagacagc atgaggttca tgttcacatt
agtacacctt gcccccccca aatcttgtag 1500 ggtgaccaga gcagtctagg
tggatgctgt gcagaagggg tttgtgccac tggtgagaaa 1560 cctgagatta
ggaatcctca atcttatact gggacaactt gcaaacctgc tcagcctttg 1620
tctctgatga agatattatc ttcatgatct tggattgaaa acagacctac tctggaggaa
1680 catattgtat cgattgtcct tgacagtaaa caaatctgtt gtaagagaca
ttatctttat 1740 tatctaggac agtaagcaag cctggatctg agagagatat
catcttgcaa ggatgcctgc 1800 tttacaaaca tccttgaaac aacaatccag
aaaaaaaaag gtgttactgt ctttgctcag 1860 aagacacaca gatacgtgac
agaaccatgg agaattgcct cccaacgctg ttcagccaga 1920 gccttccacc
ctttctgcag gacagtctca acgttccacc attaaatact tcttctatca 1980
catcccgctt ctttatgcct aaccaaggtt ctaggtcccg atcgactgtg tctggcagca
2040 ctccactgcc aaacccagaa taaggcagcg ctcaggatcc cgaaggggca
tggctgggga 2100 tcagaacttc tgggtttgag tgaggagtgg gtccaccctc
ttgaatttca aaggaggaag 2160 aggctggatg tgaaggtact gggggaggga
aagtgtcagt tccgaactct taggtcaatg 2220 agggaggaga ctggtaaggt
cccagctccc gaggtactga tgtgggaatg gcctaagaat 2280 ctcatatcct
caggaagaag gtgctggaat cctgaggggt agagttctgg gtatatttgt 2340
ggcttaaggc tctttggccc ctgaaggcag aggctggaac cattaggtcc agggtttggg
2400 gtgatagtaa tgggatctct tgattcctca agagtctgag gatcgagggt
tgcccattct 2460 tccatcttgc cacctaatcc ttactccact tgagggtatc
accagccctt ctagctccat 2520 gaaggtcccc tgggcaagca caatctgagc
atgaaagatg ccccagaggc cttgggtgtc 2580 atccactcat catccagcat
cacactctga gggtgtggcc agcaccatga cgtcatgttg 2640 ctgtgactat
ccctgcagcg tgcctctcca gccacctgcc aaccgtagag ctgcccatcc 2700
tcctctggtg ggagtggcct gcatggtgcc aggctgaggc ctagtgtcag acagggagcc
2760 tggaatcata gggatccagg actcaaaagt gctagagaat ggccatatgt
caccatccat 2820 gaaatctcaa gggcttctgg gtggagggca cagggacctg
aacttatggt ttcccaagtc 2880 tattgctctc ccaagtgagt ctcccagata
cgaggcactg tgccagcatc agccttatct 2940 ccaccacatc ttgtaaaagg
actacccagg gccctgatga acaccatggt gtgtacagga 3000 gtagggggtg
gaggcacgga ctcctgtgag gtcacagcca agggagcatc atcatgggtg 3060
gggaggaggc aatggacagg cttgagaacg gggatgtggt tgtatttggt tttctttggt
3120 tagataaagt gctgggtata ggattgagag tggagtatga agaccagtta
ggatggagga 3180 tcagattgga gttgggttag ataaagtgct gggtatagga
ttgagagtgg agtatgaaga 3240 ccagttagga tggaggatca gattggagtt
gggttagaga tggggtaaaa ttgtgctccg 3300 gatgagtttg ggattgacac
tgtggaggtg gtttgggatg gcatggcttt gggatggaaa 3360 tagatttgtt
ttgatgttgg ctcagacatc cttggggatt gaactgggga tgaagctggg 3420
tttgattttg gaggtagaag acgtggaagt agctgtcaga tttgacagtg gccatgagtt
3480 ttgtttgatg gggaatcaaa caatggggga agacataagg gttggcttgt
taggttaagt 3540 tgcgttgggt tgatggggtc ggggctgtgt ataatgcagt
tggattggtt tgtattaaat 3600 tgggttgggt caggttttgg ttgaggatga
gttgaggata tgcttgggga caccggatcc 3660 atgaggttct cactggagtg
gagacaaact tcctttccag gatgaatcca gggaagcctt 3720 aattcacgtg
taggggaggt caggccactg gctaagtata tccttccact ccagctctaa 3780
gatggtctta aattgtgatt atctatatcc acttctgtct ccctcactgt gcttggagtt
3840 tacctgatca ctcaactaga aacaggggaa gattttatca aattcttttt
tttttttttt 3900 tttttttgag acagagtctc actctgttgc ccaggctgga
gtgcagtggc gcagtctcgg 3960 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 14
15056 DNA Artificial Sequence CEA TRE 14 aagcttttta gtgctttaga
cagtgagctg gtctgtctaa cccaagtgac ctgggctcca 60 tactcagccc
cagaagtgaa gggtgaagct gggtggagcc aaaccaggca agcctaccct 120
cagggctccc agtggcctga gaaccattgg acccaggacc cattacttct agggtaagga
180 aggtacaaac accagatcca accatggtct ggggggacag ctgtcaaatg
cctaaaaata 240 tacctgggag aggagcaggc aaactatcac tgccccaggt
tctctgaaca gaaacagagg 300 ggcaacccaa agtccaaatc caggtgagca
ggtgcaccaa atgcccagag atatgacgag 360 gcaagaagtg aaggaaccac
ccctgcatca aatgttttgc atgggaagga gaagggggtt 420 gctcatgttc
ccaatccagg agaatgcatt tgggatctgc cttcttctca ctccttggtt 480
agcaagacta agcaaccagg actctggatt tggggaaaga cgtttatttg tggaggccag
540 tgatgacaat cccacgaggg cctaggtgaa gagggcagga aggctcgaga
cactggggac 600 tgagtgaaaa ccacacccat gatctgcacc acccatggat
gctccttcat tgctcacctt 660 tctgttgata tcagatggcc ccattttctg
taccttcaca gaaggacaca ggctagggtc 720 tgtgcatggc cttcatcccc
ggggccatgt gaggacagca ggtgggaaag atcatgggtc 780 ctcctgggtc
ctgcagggcc agaacattca tcacccatac tgacctccta gatgggaatg 840
gcttccctgg ggctgggcca acggggcctg ggcaggggag aaaggacgtc aggggacagg
900 gaggaagggt catcgagacc cagcctggaa ggttcttgtc tctgaccatc
caggatttac 960 ttccctgcat ctacctttgg tcattttccc tcagcaatga
ccagctctgc ttcctgatct 1020 cagcctccca ccctggacac agcaccccag
tccctggccc ggctgcatcc
acccaatacc 1080 ctgataaccc aggacccatt acttctaggg taaggagggt
ccaggagaca gaagctgagg 1140 aaaggtctga agaagtcaca tctgtcctgg
ccagagggga aaaaccatca gatgctgaac 1200 caggagaatg ttgacccagg
aaagggaccg aggacccaag aaaggagtca gaccaccagg 1260 gtttgcctga
gaggaaggat caaggccccg agggaaagca gggctggctg catgtgcagg 1320
acactggtgg ggcatatgtg tcttagattc tccctgaatt cagtgtccct gccatggcca
1380 gactctctac tcaggcctgg acatgctgaa ataggacaat ggccttgtcc
tctctcccca 1440 ccatttggca agagacataa aggacattcc aggacatgcc
ttcctgggag gtccaggttc 1500 tctgtctcac acctcaggga ctgtagttac
tgcatcagcc atggtaggtg ctgatctcac 1560 ccagcctgtc caggcccttc
cactctccac tttgtgacca tgtccaggac cacccctcag 1620 atcctgagcc
tgcaaatacc cccttgctgg gtgggtggat tcagtaaaca gtgagctcct 1680
atccagcccc cagagccacc tctgtcacct tcctgctggg catcatccca ccttcacaag
1740 cactaaagag catggggaga cctggctagc tgggtttctg catcacaaag
aaaataatcc 1800 cccaggttcg gattcccagg gctctgtatg tggagctgac
agacctgagg ccaggagata 1860 gcagaggtca gccctaggga gggtgggtca
tccacccagg ggacaggggt gcaccagcct 1920 tgctactgaa agggcctccc
caggacagcg ccatcagccc tgcctgagag ctttgctaaa 1980 cagcagtcag
aggaggccat ggcagtggct gagctcctgc tccaggcccc aacagaccag 2040
accaacagca caatgcagtc cttccccaac gtcacaggtc accaaaggga aactgaggtg
2100 ctacctaacc ttagagccat caggggagat aacagcccaa tttcccaaac
aggccagttt 2160 caatcccatg acaatgacct ctctgctctc attcttccca
aaataggacg ctgattctcc 2220 cccaccatgg atttctccct tgtcccggga
gccttttctg ccccctatga tctgggcact 2280 cctgacacac acctcctctc
tggtgacata tcagggtccc tcactgtcaa gcagtccaga 2340 aaggacagaa
ccttggacag cgcccatctc agcttcaccc ttcctccttc acagggttca 2400
gggcaaagaa taaatggcag aggccagtga gcccagagat ggtgacaggc agtgacccag
2460 gggcagatgc ctggagcagg agctggcggg gccacaggga gaaggtgatg
caggaaggga 2520 aacccagaaa tgggcaggaa aggaggacac aggctctgtg
gggctgcagc ccagggttgg 2580 actatgagtg tgaagccatc tcagcaagta
aggccaggtc ccatgaacaa gagtgggagc 2640 acgtggcttc ctgctctgta
tatggggtgg gggattccat gccccataga accagatggc 2700 cggggttcag
atggagaagg agcaggacag gggatcccca ggataggagg accccagtgt 2760
ccccacccag gcaggtgact gatgaatggg catgcagggt cctcctgggc tgggctctcc
2820 ctttgtccct caggattcct tgaaggaaca tccggaagcc gaccacatct
acctggtggg 2880 ttctggggag tccatgtaaa gccaggagct tgtgttgcta
ggaggggtca tggcatgtgc 2940 tgggggcacc aaagagagaa acctgagggc
aggcaggacc tggtctgagg aggcatggga 3000 gcccagatgg ggagatggat
gtcaggaaag gctgccccat cagggagggt gatagcaatg 3060 gggggtctgt
gggagtgggc acgtgggatt ccctgggctc tgccaagttc cctcccatag 3120
tcacaacctg gggacactgc ccatgaaggg gcgcctttgc ccagccagat gctgctggtt
3180 ctgcccatcc actaccctct ctgctccagc cactctgggt ctttctccag
atgccctgga 3240 cagccctggc ctgggcctgt cccctgagag gtgttgggag
aagctgagtc tctggggaca 3300 ctctcatcag agtctgaaag gcacatcagg
aaacatccct ggtctccagg actaggcaat 3360 gaggaaaggg ccccagctcc
tccctttgcc actgagaggg tcgaccctgg gtggccacag 3420 tgacttctgc
gtctgtccca gtcaccctga aaccacaaca aaaccccagc cccagaccct 3480
gcaggtacaa tacatgtggg gacagtctgt acccagggga agccagttct ctcttcctag
3540 gagaccgggc ctcagggctg tgcccggggc aggcgggggc agcacgtgcc
tgtccttgag 3600 aactcgggac cttaagggtc tctgctctgt gaggcacagc
aaggatcctt ctgtccagag 3660 atgaaagcag ctcctgcccc tcctctgacc
tcttcctcct tcccaaatct caaccaacaa 3720 ataggtgttt caaatctcat
catcaaatct tcatccatcc acatgagaaa gcttaaaacc 3780 caatggattg
acaacatcaa gagttggaac aagtggacat ggagatgtta cttgtggaaa 3840
tttagatgtg ttcagctatc gggcaggaga atctgtgtca aattccagca tggttcagaa
3900 gaatcaaaaa gtgtcacagt ccaaatgtgc aacagtgcag gggataaaac
tgtggtgcat 3960 tcaaactgag ggatattttg gaacatgaga aaggaaggga
ttgctgctgc acagaacatg 4020 gatgatctca cacatagagt tgaaagaaag
gagtcaatcg cagaatagaa aatgatcact 4080 aattccacct ctataaagtt
tccaagagga aaacccaatt ctgctgctag agatcagaat 4140 ggaggtgacc
tgtgccttgc aatggctgtg agggtcacgg gagtgtcact tagtgcaggc 4200
aatgtgccgt atcttaatct gggcagggct ttcatgagca cataggaatg cagacattac
4260 tgctgtgttc attttacttc accggaaaag aagaataaaa tcagccgggc
gcggtggctc 4320 acgcctgtaa tcccagcact ttagaaggct gaggtgggca
gattacttga ggtcaggagt 4380 tcaagaccac cctggccaat atggtgaaac
cccggctcta ctaaaaatac aaaaattagc 4440 tgggcatggt ggtgcgcgcc
tgtaatccca gctactcggg aggctgaggc tggacaattg 4500 cttggaccca
ggaagcagag gttgcagtga gccaagattg tgccactgca ctccagcttg 4560
ggcaacagag ccagactctg taaaaaaaaa aaaaaaaaaa aaaaaaagaa agaaagaaaa
4620 agaaaagaaa gtataaaatc tctttgggtt aacaaaaaaa gatccacaaa
acaaacacca 4680 gctcttatca aacttacaca actctgccag agaacaggaa
acacaaatac tcattaactc 4740 acttttgtgg caataaaacc ttcatgtcaa
aaggagacca ggacacaatg aggaagtaaa 4800 actgcaggcc ctacttgggt
gcagagaggg aaaatccaca aataaaacat taccagaagg 4860 agctaagatt
tactgcattg agttcattcc ccaggtatgc aaggtgattt taacacctga 4920
aaatcaatca ttgcctttac tacatagaca gattagctag aaaaaaatta caactagcag
4980 aacagaagca atttggcctt cctaaaattc cacatcatat catcatgatg
gagacagtgc 5040 agacgccaat gacaataaaa agagggacct ccgtcacccg
gtaaacatgt ccacacagct 5100 ccagcaagca cccgtcttcc cagtgaatca
ctgtaacctc ccctttaatc agccccaggc 5160 aaggctgcct gcgatggcca
cacaggctcc aacccgtggg cctcaacctc ccgcagaggc 5220 tctcctttgg
ccaccccatg gggagagcat gaggacaggg cagagccctc tgatgcccac 5280
acatggcagg agctgacgcc agagccatgg gggctggaga gcagagctgc tggggtcaga
5340 gcttcctgag gacacccagg cctaagggaa ggcagctccc tggatggggg
caaccaggct 5400 ccgggctcca acctcagagc ccgcatggga ggagccagca
ctctaggcct ttcctagggt 5460 gactctgagg ggaccctgac acgacaggat
cgctgaatgc acccgagatg aaggggccac 5520 cacgggaccc tgctctcgtg
gcagatcagg agagagtggg acaccatgcc aggcccccat 5580 ggcatggctg
cgactgaccc aggccactcc cctgcatgca tcagcctcgg taagtcacat 5640
gaccaagccc aggaccaatg tggaaggaag gaaacagcat cccctttagt gatggaaccc
5700 aaggtcagtg caaagagagg ccatgagcag ttaggaaggg tggtccaacc
tacagcacaa 5760 accatcatct atcataagta gaagccctgc tccatgaccc
ctgcatttaa ataaacgttt 5820 gttaaatgag tcaaattccc tcaccatgag
agctcacctg tgtgtaggcc catcacacac 5880 acaaacacac acacacacac
acacacacac acacacacac acagggaaag tgcaggatcc 5940 tggacagcac
caggcaggct tcacaggcag agcaaacagc gtgaatgacc catgcagtgc 6000
cctgggcccc atcagctcag agaccctgtg agggctgaga tggggctagg caggggagag
6060 acttagagag ggtggggcct ccagggaggg ggctgcaggg agctgggtac
tgccctccag 6120 ggagggggct gcagggagct gggtactgcc ctccagggag
ggggctgcag ggagctgggt 6180 actgccctcc agggaggggg ctgcagggag
ctgggtactg ccctccaggg agggggctgc 6240 agggagctgg gtactgccct
ccagggaggc aggagcactg ttcccaacag agagcacatc 6300 ttcctgcagc
agctgcacag acacaggagc ccccatgact gccctgggcc agggtgtgga 6360
ttccaaattt cgtgccccat tgggtgggac ggaggttgac cgtgacatcc aaggggcatc
6420 tgtgattcca aacttaaact actgtgccta caaaatagga aataacccta
ctttttctac 6480 tatctcaaat tccctaagca caagctagca ccctttaaat
caggaagttc agtcactcct 6540 ggggtcctcc catgccccca gtctgacttg
caggtgcaca gggtggctga catctgtcct 6600 tgctcctcct cttggctcaa
ctgccgcccc tcctgggggt gactgatggt caggacaagg 6660 gatcctagag
ctggccccat gattgacagg aaggcaggac ttggcctcca ttctgaagac 6720
taggggtgtc aagagagctg ggcatcccac agagctgcac aagatgacgc ggacagaggg
6780 tgacacaggg ctcagggctt cagacgggtc gggaggctca gctgagagtt
cagggacaga 6840 cctgaggagc ctcagtggga aaagaagcac tgaagtggga
agttctggaa tgttctggac 6900 aagcctgagt gctctaagga aatgctccca
ccccgatgta gcctgcagca ctggacggtc 6960 tgtgtacctc cccgctgccc
atcctctcac agcccccgcc tctagggaca caactcctgc 7020 cctaacatgc
atctttcctg tctcattcca cacaaaaggg cctctggggt ccctgttctg 7080
cattgcaagg agtggaggtc acgttcccac agaccaccca gcaacagggt cctatggagg
7140 tgcggtcagg aggatcacac gtccccccat gcccagggga ctgactctgg
gggtgatgga 7200 ttggcctgga ggccactggt cccctctgtc cctgagggga
atctgcaccc tggaggctgc 7260 cacatccctc ctgattcttt cagctgaggg
cccttcttga aatcccaggg aggactcaac 7320 ccccactggg aaaggcccag
tgtggacggt tccacagcag cccagctaag gcccttggac 7380 acagatcctg
agtgagagaa cctttaggga cacaggtgca cggccatgtc cccagtgccc 7440
acacagagca ggggcatctg gaccctgagt gtgtagctcc cgcgactgaa cccagccctt
7500 ccccaatgac gtgacccctg gggtggctcc aggtctccag tccatgccac
caaaatctcc 7560 agattgaggg tcctcccttg agtccctgat gcctgtccag
gagctgcccc ctgagcaaat 7620 ctagagtgca gagggctggg attgtggcag
taaaagcagc cacatttgtc tcaggaagga 7680 aagggaggac atgagctcca
ggaagggcga tggcgtcctc tagtgggcgc ctcctgttaa 7740 tgagcaaaaa
ggggccagga gagttgagag atcagggctg gccttggact aaggctcaga 7800
tggagaggac tgaggtgcaa agagggggct gaagtagggg agtggtcggg agagatggga
7860 ggagcaggta aggggaagcc ccagggaggc cgggggaggg tacagcagag
ctctccactc 7920 ctcagcattg acatttgggg tggtcgtgct agtggggttc
tgtaagttgt agggtgttca 7980 gcaccatctg gggactctac ccactaaatg
ccagcaggac tccctcccca agctctaaca 8040 accaacaatg tctccagact
ttccaaatgt cccctggaga gcaaaattgc ttctggcaga 8100 atcactgatc
tacgtcagtc tctaaaagtg actcatcagc gaaatccttc acctcttggg 8160
agaagaatca caagtgtgag aggggtagaa actgcagact tcaaaatctt tccaaaagag
8220 ttttacttaa tcagcagttt gatgtcccag gagaagatac atttagagtg
tttagagttg 8280 atgccacatg gctgcctgta cctcacagca ggagcagagt
gggttttcca agggcctgta 8340 accacaactg gaatgacact cactgggtta
cattacaaag tggaatgtgg ggaattctgt 8400 agactttggg aagggaaatg
tatgacgtga gcccacagcc taaggcagtg gacagtccac 8460 tttgaggctc
tcaccatcta ggagacatct cagccatgaa catagccaca tctgtcatta 8520
gaaaacatgt tttattaaga ggaaaaatct aggctagaag tgctttatgc tcttttttct
8580 ctttatgttc aaattcatat acttttagat cattccttaa agaagaatct
atccccctaa 8640 gtaaatgtta tcactgactg gatagtgttg gtgtctcact
cccaacccct gtgtggtgac 8700 agtgccctgc ttccccagcc ctgggccctc
tctgattcct gagagctttg ggtgctcctt 8760 cattaggagg aagagaggaa
gggtgttttt aatattctca ccattcaccc atccacctct 8820 tagacactgg
gaagaatcag ttgcccactc ttggatttga tcctcgaatt aatgacctct 8880
atttctgtcc cttgtccatt tcaacaatgt gacaggccta agaggtgcct tctccatgtg
8940 atttttgagg agaaggttct caagataagt tttctcacac ctctttgaat
tacctccacc 9000 tgtgtcccca tcaccattac cagcagcatt tggacccttt
ttctgttagt cagatgcttt 9060 ccacctcttg agggtgtata ctgtatgctc
tctacacagg aatatgcaga ggaaatagaa 9120 aaagggaaat cgcattacta
ttcagagaga agaagacctt tatgtgaatg aatgagagtc 9180 taaaatccta
agagagccca tataaaatta ttaccagtgc taaaactaca aaagttacac 9240
taacagtaaa ctagaataat aaaacatgca tcacagttgc tggtaaagct aaatcagata
9300 tttttttctt agaaaaagca ttccatgtgt gttgcagtga tgacaggagt
gcccttcagt 9360 caatatgctg cctgtaattt ttgttccctg gcagaatgta
ttgtcttttc tccctttaaa 9420 tcttaaatgc aaaactaaag gcagctcctg
ggccccctcc ccaaagtcag ctgcctgcaa 9480 ccagccccac gaagagcaga
ggcctgagct tccctggtca aaataggggg ctagggagct 9540 taaccttgct
cgataaagct gtgttcccag aatgtcgctc ctgttcccag gggcaccagc 9600
ctggagggtg gtgagcctca ctggtggcct gatgcttacc ttgtgccctc acaccagtgg
9660 tcactggaac cttgaacact tggctgtcgc ccggatctgc agatgtcaag
aacttctgga 9720 agtcaaatta ctgcccactt ctccagggca gatacctgtg
aacatccaaa accatgccac 9780 agaaccctgc ctggggtcta caacacatat
ggactgtgag caccaagtcc agccctgaat 9840 ctgtgaccac ctgccaagat
gcccctaact gggatccacc aatcactgca catggcaggc 9900 agcgaggctt
ggaggtgctt cgccacaagg cagccccaat ttgctgggag tttcttggca 9960
cctggtagtg gtgaggagcc ttgggaccct caggattact ccccttaagc atagtgggga
10020 cccttctgca tccccagcag gtgccccgct cttcagagcc tctctctctg
aggtttaccc 10080 agacccctgc accaatgaga ccatgctgaa gcctcagaga
gagagatgga gctttgacca 10140 ggagccgctc ttccttgagg gccagggcag
ggaaagcagg aggcagcacc aggagtggga 10200 acaccagtgt ctaagcccct
gatgagaaca gggtggtctc tcccatatgc ccataccagg 10260 cctgtgaaca
gaatcctcct tctgcagtga caatgtctga gaggacgaca tgtttcccag 10320
cctaacgtgc agccatgccc atctacccac tgcctactgc aggacagcac caacccagga
10380 gctgggaagc tgggagaaga catggaatac ccatggcttc tcaccttcct
ccagtccagt 10440 gggcaccatt tatgcctagg acacccacct gccggcccca
ggctcttaag agttaggtca 10500 cctaggtgcc tctgggaggc cgaggcagga
gaattgcttg aacccgggag gcagaggttg 10560 cagtgagccg agatcacacc
actgcactcc agcctgggtg acagaatgag actctgtctc 10620 aaaaaaaaag
agaaagatag catcagtggc taccaagggc taggggcagg ggaaggtgga 10680
gagttaatga ttaatagtat gaagtttcta tgtgagatga tgaaaatgtt ctggaaaaaa
10740 aaatatagtg gtgaggatgt agaatattgt gaatataatt aacggcattt
aattgtacac 10800 ttaacatgat taatgtggca tattttatct tatgtatttg
actacatcca agaaacactg 10860 ggagagggaa agcccaccat gtaaaataca
cccaccctaa tcagatagtc ctcattgtac 10920 ccaggtacag gcccctcatg
acctgcacag gaataactaa ggatttaagg acatgaggct 10980 tcccagccaa
ctgcaggtgc acaacataaa tgtatctgca aacagactga gagtaaagct 11040
gggggcacaa acctcagcac tgccaggaca cacacccttc tcgtggattc tgactttatc
11100 tgacccggcc cactgtccag atcttgttgt gggattggga caagggaggt
cataaagcct 11160 gtccccaggg cactctgtgt gagcacacga gacctcccca
cccccccacc gttaggtctc 11220 cacacataga tctgaccatt aggcattgtg
aggaggactc tagcgcgggc tcagggatca 11280 caccagagaa tcaggtacag
agaggaagac ggggctcgag gagctgatgg atgacacaga 11340 gcagggttcc
tgcagtccac aggtccagct caccctggtg taggtgcccc atccccctga 11400
tccaggcatc cctgacacag ctccctcccg gagcctcctc ccaggtgaca catcagggtc
11460 cctcactcaa gctgtccaga gagggcagca ccttggacag cgcccacccc
acttcactct 11520 tcctccctca cagggctcag ggctcagggc tcaagtctca
gaacaaatgg cagaggccag 11580 tgagcccaga gatggtgaca gggcaatgat
ccaggggcag ctgcctgaaa cgggagcagg 11640 tgaagccaca gatgggagaa
gatggttcag gaagaaaaat ccaggaatgg gcaggagagg 11700 agaggaggac
acaggctctg tggggctgca gcccaggatg ggactaagtg tgaagacatc 11760
tcagcaggtg aggccaggtc ccatgaacag agaagcagct cccacctccc ctgatgcacg
11820 gacacacaga gtgtgtggtg ctgtgccccc agagtcgggc tctcctgttc
tggtccccag 11880 ggagtgagaa gtgaggttga cttgtccctg ctcctctctg
ctaccccaac attcaccttc 11940 tcctcatgcc cctctctctc aaatatgatt
tggatctatg tccccgccca aatctcatgt 12000 caaattgtaa accccaatgt
tggaggtggg gccttgtgag aagtgattgg ataatgcggg 12060 tggattttct
gctttgatgc tgtttctgtg atagagatct cacatgatct ggttgtttaa 12120
aagtgtgtag cacctctccc ctctctctct ctctctctta ctcatgctct gccatgtaag
12180 acgttcctgt ttccccttca ccgtccagaa tgattgtaag ttttctgagg
cctccccagg 12240 agcagaagcc actatgcttc ctgtacaact gcagaatgat
gagcgaatta aacctctttt 12300 ctttataaat tacccagtct caggtatttc
tttatagcaa tgcgaggaca gactaataca 12360 atcttctact cccagatccc
cgcacacgct tagccccaga catcactgcc cctgggagca 12420 tgcacagcgc
agcctcctgc cgacaaaagc aaagtcacaa aaggtgacaa aaatctgcat 12480
ttggggacat ctgattgtga aagagggagg acagtacact tgtagccaca gagactgggg
12540 ctcaccgagc tgaaacctgg tagcactttg gcataacatg tgcatgaccc
gtgttcaatg 12600 tctagagatc agtgttgagt aaaacagcct ggtctggggc
cgctgctgtc cccacttccc 12660 tcctgtccac cagagggcgg cagagttcct
cccaccctgg agcctcccca ggggctgctg 12720 acctccctca gccgggccca
cagcccagca gggtccaccc tcacccgggt cacctcggcc 12780 cacgtcctcc
tcgccctccg agctcctcac acggactctg tcagctcctc cctgcagcct 12840
atcggccgcc cacctgaggc ttgtcggccg cccacttgag gcctgtcggc tgccctctgc
12900 aggcagctcc tgtcccctac accccctcct tccccgggct cagctgaaag
ggcgtctccc 12960 agggcagctc cctgtgatct ccaggacagc tcagtctctc
acaggctccg acgcccccta 13020 tgctgtcacc tcacagccct gtcattacca
ttaactcctc agtcccatga agttcactga 13080 gcgcctgtct cccggttaca
ggaaaactct gtgacaggga ccacgtctgt cctgctctct 13140 gtggaatccc
agggcccagc ccagtgcctg acacggaaca gatgctccat aaatactggt 13200
taaatgtgtg ggagatctct aaaaagaagc atatcacctc cgtgtggccc ccagcagtca
13260 gagtctgttc catgtggaca caggggcact ggcaccagca tgggaggagg
ccagcaagtg 13320 cccgcggctg ccccaggaat gaggcctcaa cccccagagc
ttcagaaggg aggacagagg 13380 cctgcaggga atagatcctc cggcctgacc
ctgcagccta atccagagtt cagggtcagc 13440 tcacaccacg tcgaccctgg
tcagcatccc tagggcagtt ccagacaagg ccggaggtct 13500 cctcttgccc
tccagggggt gacattgcac acagacatca ctcaggaaac ggattcccct 13560
ggacaggaac ctggctttgc taaggaagtg gaggtggagc ctggtttcca tcccttgctc
13620 caacagaccc ttctgatctc tcccacatac ctgctctgtt cctttctggg
tcctatgagg 13680 accctgttct gccaggggtc cctgtgcaac tccagactcc
ctcctggtac caccatgggg 13740 aaggtggggt gatcacagga cagtcagcct
cgcagagaca gagaccaccc aggactgtca 13800 gggagaacat ggacaggccc
tgagccgcag ctcagccaac agacacggag agggagggtc 13860 cccctggagc
cttccccaag gacagcagag cccagagtca cccacctccc tccaccacag 13920
tcctctcttt ccaggacaca caagacacct ccccctccac atgcaggatc tggggactcc
13980 tgagacctct gggcctgggt ctccatccct gggtcagtgg cggggttggt
ggtactggag 14040 acagagggct ggtccctccc cagccaccac ccagtgagcc
tttttctagc ccccagagcc 14100 acctctgtca ccttcctgtt gggcatcatc
ccaccttccc agagccctgg agagcatggg 14160 gagacccggg accctgctgg
gtttctctgt cacaaaggaa aataatcccc ctggtgtgac 14220 agacccaagg
acagaacaca gcagaggtca gcactgggga agacaggttg tcctcccagg 14280
ggatgggggt ccatccacct tgccgaaaag atttgtctga ggaactgaaa atagaaggga
14340 aaaaagagga gggacaaaag aggcagaaat gagaggggag gggacagagg
acacctgaat 14400 aaagaccaca cccatgaccc acgtgatgct gagaagtact
cctgccctag gaagagactc 14460 agggcagagg gaggaaggac agcagaccag
acagtcacag cagccttgac aaaacgttcc 14520 tggaactcaa gctcttctcc
acagaggagg acagagcaga cagcagagac catggagtct 14580 ccctcggccc
ctccccacag atggtgcatc ccctggcaga ggctcctgct cacaggtgaa 14640
gggaggacaa cctgggagag ggtgggagga gggagctggg gtctcctggg taggacaggg
14700 ctgtgagacg gacagagggc tcctgttgga gcctgaatag ggaagaggac
atcagagagg 14760 gacaggagtc acaccagaaa aatcaaattg aactggaatt
ggaaaggggc aggaaaacct 14820 caagagttct attttcctag ttaattgtca
ctggccacta cgtttttaaa aatcataata 14880 actgcatcag atgacacttt
aaataaaaac ataaccaggg catgaaacac tgtcctcatc 14940 cgcctaccgc
ggacattgga aaataagccc caggctgtgg agggccctgg gaaccctcat 15000
gaactcatcc acaggaatct gcagcctgtc ccaggcactg gggtgcaacc aagatc 15056
15 858 DNA Artificial Sequence Mucin-TRE 15 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 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 16 5224 DNA Artificial Sequence AlphaFP-TRE
16 gaattcttag aaatatgggg gtaggggtgg tggtggtaat tctgttttca
ccccataggt 60 gagataagca ttgggttaaa tgtgctttca
cacacacatc acatttcata agaattaagg 120 aacagactat gggctggagg
actttgagga tgtctgtctc ataacacttg ggttgtatct 180 gttctatggg
gcttgtttta agcttggcaa cttgcaacag ggttcactga ctttctcccc 240
aagcccaagg tactgtcctc ttttcatatc tgttttgggg cctctggggc ttgaatatct
300 gagaaaatat aaacatttca ataatgttct gtggtgagat gagtatgaga
gatgtgtcat 360 tcatttgtat caatgaatga atgaggacaa ttagtgtata
aatccttagt acaacaatct 420 gagggtaggg gtggtactat tcaatttcta
tttataaaga tacttatttc tatttattta 480 tgcttgtgac aaatgttttg
ttcgggacca caggaatcac aaagatgagt ctttgaattt 540 aagaagttaa
tggtccagga ataattacat agcttacaaa tgactatgat ataccatcaa 600
acaagaggtt ccatgagaaa ataatctgaa aggtttaata agttgtcaaa ggtgagaggg
660 ctcttctcta gctagagact aatcagaaat acattcaggg ataattattt
gaatagacct 720 taagggttgg gtacattttg ttcaagcatt gatggagaag
gagagtgaat atttgaaaac 780 attttcaact aaccaaccac ccaatccaac
aaacaaaaaa tgaaaagaat ctcagaaaca 840 gtgagataag agaaggaatt
ttctcacaac ccacacgtat agctcaactg ctctgaagaa 900 gtatatatct
aatatttaac actaacatca tgctaataat gataataatt actgtcattt 960
tttaatgtct ataagtacca ggcatttaga agatattatt ccatttatat atcaaaataa
1020 acttgagggg atagatcatt ttcatgatat atgagaaaaa ttaaaaacag
attgaattat 1080 ttgcctgtca tacagctaat aattgaccat aagacaatta
gatttaaatt agttttgaat 1140 ctttctaata ccaaagttca gtttactgtt
ccatgttgct tctgagtggc ttcacagact 1200 tatgaaaaag taaacggaat
cagaattaca tcaatgcaaa agcattgctg tgaactctgt 1260 acttaggact
aaactttgag caataacaca catagattga ggattgtttg ctgttagcat 1320
acaaactctg gttcaaagct cctctttatt gcttgtcttg gaaaatttgc tgttcttcat
1380 ggtttctctt ttcactgcta tctatttttc tcaaccactc acatggctac
aataactgtc 1440 tgcaagctta tgattcccaa atatctatct ctagcctcaa
tcttgttcca gaagataaaa 1500 agtagtattc aaatgcacat caacgtctcc
acttggaggg cttaaagacg tttcaacata 1560 caaaccgggg agttttgcct
ggaatgtttc ctaaaatgtg tcctgtagca catagggtcc 1620 tcttgttcct
taaaatctaa ttacttttag cccagtgctc atcccaccta tggggagatg 1680
agagtgaaaa gggagcctga ttaataatta cactaagtca ataggcatag agccaggact
1740 gtttgggtaa actggtcact ttatcttaaa ctaaatatat ccaaaactga
acatgtactt 1800 agttactaag tctttgactt tatctcattc ataccactca
gctttatcca ggccacttat 1860 ttgacagtat tattgcgaaa acttcctaac
tggtctcctt atcatagtct tatccccttt 1920 tgaaacaaaa gagacagttt
caaaatacaa atatgatttt tattagctcc cttttgttgt 1980 ctataatagt
cccagaagga gttataaact ccatttaaaa agtctttgag atgtggccct 2040
tgccaacttt gccaggaatt cccaatatct agtattttct actattaaac tttgtgcctc
2100 ttcaaaactg cattttctct cattccctaa gtgtgcattg ttttccctta
ccggttggtt 2160 tttccaccac cttttacatt ttcctggaac actataccct
ccctcttcat ttggcccacc 2220 tctaattttc tttcagatct ccatgaagat
gttacttcct ccaggaagcc ttatctgacc 2280 cctccaaaga tgtcatgagt
tcctcttttc attctactaa tcacagcatc catcacacca 2340 tgttgtgatt
actgatacta ttgtctgttt ctctgattag gcagtaagct caacaagagc 2400
tacatggtgc ctgtctcttg ttgctgatta ttcccatcca aaaacagtgc ctggaatgca
2460 gacttaacat tttattgaat gaataaataa aaccccatct atcgagtgct
actttgtgca 2520 agacccggtt ctgaggcatt tatatttatt gatttattta
attctcattt aaccatgaag 2580 gaggtactat cactatcctt attttatagt
tgataaagat aaagcccaga gaaatgaatt 2640 aactcaccca aagtcatgta
gctaagtgac agggcaaaaa ttcaaaccag ttccccaact 2700 ttacgtgatt
aatactgtgc tatactgcct ctctgatcat atggcatgga atgcagacat 2760
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 17 307
DNA Artificial Sequence Nucleotide sequence for ADP 17 gatgaccggc
tcaaccatcg cgcccacaac ggactatcgc aacaccactg ctaccggact 60
aacatctgcc ctaaatttac cccaagttca tgcctttgtc aatgactggg cgagcttgga
120 catgtggtgg ttttccatag cgcttatgtt tgtttgcctt attattatgt
ggcttatttg 180 ttgcctaaag cgcagacgcg ccagaccccc catctatagg
cctatcattg tgctcaaccc 240 acacaatgaa aaaattcata gattggacgg
tctgaaacca tgttctcttc ttttacagta 300 tgattaa 307 18 101 PRT
Artificial Sequence Amino acid sequence for ADP 18 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 19 28 DNA
Artificial Sequence PCR EMCV IRES (PCR primer 96.74.2) 19
gacgtcgact aattccggtt attttcca 28 20 28 DNA Artificial Sequence PCR
EMCV IRES (PCR primer 96.74.1) 20 gacgtcgaca tcgtgttttt caaaggaa 28
21 25 DNA Artificial Sequence Ad5 sequence to 1314 to 1338 (PCR
primer 96.74.3) 21 cctgagacgc ccgacatcac ctgtg 25 22 30 DNA
Artificial Sequence Antisense of Ad5 sequence 1572 to 1586 (PCR
primer 96.74.6) 22 gtcgaccatt cagcaaacaa aggcgttaac 30 23 30 DNA
Artificial Sequence Ad5 sequence 1714 to 1728 (PCR primer 96.74.4)
23 tgctgaatgg tcgacatgga ggcttgggag 30 24 26 DNA Artificial
Sequence Antisense of Ad5 sequence 2070 to 2094 (PCR primer
96.74.5) 24 cacaaaccgc tctccacaga tgcatg 26 25 29 DNA Artificial
Sequence Human UPII (PCR primer 127.2.1) 25 aggaccggtc actatagggc
acgcgtggt 29 26 31 DNA Artificial Sequence Human UPII (PCR primer
127.2.2) 26 aggaccggtg ggatgctggg ctgggaggtg g 31 27 29 DNA
Artificial Sequence PCR primer 100.113.1 27 aggggtaccc actatagggc
acgcgtggt 29 28 32 DNA Artificial Sequence PCR primer 100.113.2 28
acccaagctt gggatgctgg gctgggaggt gg 32 29 30 DNA Artificial
Sequence PCR primer 127.50.1 29 aggaccggtc aggcttcacc ccagacccac 30
30 24 DNA Artificial Sequence PCR primer 31.166.1 30 tgcgccggtg
tacacaggaa gtga 24 31 21 DNA Artificial Sequence PCR primer 32.32.1
31 gagtttgtgc catcggtcta c 21 32 21 DNA Artificial Sequence PCR
primer 32.32.2 32 aatcaatcct tagtcctcct g 21 33 25 DNA Artificial
Sequence PCR primer 51.176 33 gcagaaaaat cttccaaaca ctccc 25 34 27
DNA Artificial Sequence PCR primer 99.120.1 34 acgtacaccg
gtcgttacat aacttac 27 35 26 DNA Artificial Sequence PCR primer
99.120.2 35 ctagcaaccg gtcggttcac taaacg 26
* * * * *