JP2003527128A - Osteocalcin promoter-directed adenovirus replication for therapy - Google Patents

Abstract

(57) Abstract: The present invention relates to prostate cancer cells, prostate stromal cells, perivascular cells, proliferative cancer-related osteoblasts and prostate cancer and related bone stromal cells that do not express PSA or androgen receptor (AR). The present invention relates to a replicable adenovirus vector specific to a cell, such as a cell, in which an osteocalcin transcription regulatory sequence functions, and a method for using such a virus is provided. These viruses contain an adenovirus gene under the control of osteocalcin transcriptional regulatory sequences. The gene can be, for example, a gene required for adenovirus replication. These viruses can also include at least one additional adenovirus gene under the control of at least one additional tissue-specific transcriptional regulatory sequence. As a result, viral replication can be restricted to target cells that exhibit osteocalcin gene expression. Such replicable adenovirus vectors are useful for treating prostate, brain, ovarian, thyroid, various tumors, osteosarcoma, ocular melanoma, lung or breast cancer.

Description

DETAILED DESCRIPTION OF THE INVENTION       [0001]Relationship with related applications   This application is related to co-pending U.S. Provisional Application No. 60 / 191,063, filed March 21, 2000.
Claims the benefit of 35 U.S.C. 119 (e).
The entire text of which is incorporated herein by reference without disclaimer.
And).       [0002]I. Introduction   The present invention relates to targeted therapy using a recombinant vector, particularly an adenovirus vector.
Related. In particular, the present invention relates to adenovirus vectors that conditionally replicate and
About their use. Such adenovirus vectors are tissue-specific and
And the adenoviral vector is capable of selectively replicating in a tumor-restricted manner.
Derived from its own presence and / or a heterologous gene product expressed from the vector
It can provide a therapeutic benefit. More specifically, the invention relates to metastatic cancer (previously
Prostate cancer, brain tumor, ovarian cancer, thyroid cancer, various tumors, osteosarcoma, ocular melanoma, lung cancer and
And breast cancer (including but not limited to)
Methods and compositions related to novel viral vectors that can be used.
In addition, the present invention relates to benign but severe, life-threatening conditions and diseases with calcification.
Disease (including but not limited to benign prostatic hyperplasia (BPH) and atherosclerosis)
) Find use in treatment. These uses of the present invention are primarily for the treatment of metastatic cancer.
In relation to, and then with respect to, treatment of other tissues. However,
There is one treatment modality common to all these uses. That is, osteoca
An adenovirus with a gene essential for replication under the control of a rucine transcription regulatory sequence
Is a systemic administration of       [0003]   Transcription regulatory sequences used with the adenovirus vectors of the invention are essential for replication.
Selective adenovirus gene expression in a tissue-specific and tumor-specific manner
Can be led to. Thus, transcriptional regulation used with viral vectors
Due to the tissue specificity of the sequence, the viral vector of the present invention is suitable for injection into target tissues, etc.
Not only when administered more directly, but also systemically by intravenous administration, oral administration, etc.
It is also an effective therapeutic agent when used. Because gene expression is restricted to specific cell types
Because it is localized.       [0004]II. Background of the Invention A. Cancer therapy   Bone carcinoma, a bone cancer that mainly affects teenagers and young adults,
Hit about 2100 people each year in the United States (Boring, C.C., Squires, T.S., Ton
g, T. and Montgomery, S., Cancer Statistics, 1994, CA Cancer J. Clin.,
44; 7-26, 1994). This malignancy accounts for about 5% of all childhood malignancies and
Accounts for 60% of childhood malignant bone tumors [Hudson, M., Jaffe, M.R., and Jaffe, N.,
Bone bone meat type: treatment strategies, outcomes and prognostic factors derived from 10 years of experience (Pedia
tric osteosarcoma: therapeutic strategies, results, and prognostic actor
s derived from a 10-year experience), J. Clin.Oncol., 8: 1988-1997, 1990
]. Thorough surgical resection of the primary tumor and aggressive adjuvant chemotherapy
Even so, the overall 2-year metastasis-free survival rate is only 66%. Patients with this disease
More than 30% of patients develop lung metastases within one year [Link, M.P., Goorin, A.M., Mixer, A
.W., Link, M.P., Goorin, A.M., Miser, A.W., Green, A.A., Pratt, C.H., Be
lasco, J.B., Pritchard, J., Malpas, J.S., Baker, A.R., Kirkpatrick, J.A.
, Ayala, A.O., Schuster, J.J., Abelson, H.T., Simone, J.V. and Vietti,
T.J., "Adjuvant for relapse-free survival in patients with osteosarcoma in the limbs.
The effect of adjuvant chemotherapy on relapse-fr
ee survival in patients with osteosarcoma of the extremeity), N. Engl. J
Med, 314: 1600-1602, 1991; Goorin, A.M., Perez-Atayde, A., Gebbhardt,
M. et al., High weekly doses of methotrexate and doxorubicin for bone and meat species: th
e Dunn-Farber Cancer Institute / The Children's Hospotal Study III (Weekly
high-dose methotrexate and doxorubicin: the Dunn-Farber Cancer Institute /
The Children's Hospotal-Study III), J. Clin. Oncol., 5: 1178-1184, 1987
]. Changes in adjuvant chemotherapy [Kane, M.J., "Advanced soft tissue and bone
Chemotherapy of advanced soft tissue and osteosarcoma,
 Semin. Oncol., 16: 297-304, 1989]
Survival rates have not changed significantly over the past decade.       [0005]   Prostate cancer often metastasizes to lymph nodes and bones and is the second leading cause of cancer death in North American men.
(Landis, S.H., Murray, T., Bolden, S. and Wingo,
P.A., Cancer Statistics 1998. CA Cancer J. Clin., 48: 6, 1998). Prostate cancer
Standard front-line treatment for metastases will eventually result in limited response to chemotherapy
Therefore, recurrence always occurs, but it is androgen deprivation therapy that slows the progression of the disease [K
antoff, P.W., Halabi, S., Conaway, M., Picus, J., Kirshner, J., Hars, V.
, Trump, D., Winer, E.P. and Vogelzang, N.J.
In the presence or absence of mitozantrone in men with
Thizon: Results of the Cancer and Leukocyte Group B 9182 Study "(Hydrocortizone with or
without mitoxantrone in men with hormone-refractory prostate cancer: res
ults of the Cancer and Leukemia Group B 9182 study), J. Clin.Oncol., 17
: 2506, 1999]. About 12 patients with androgen-independent progression
Died within months [Kantoff, P.W., Halabi, S., Conaway, M., Picus, J., Ki
rshner, J., Hars, V., Trump, D., Winer, E.P. and Vogelzang, N.J.
Presence or absence of mitozantrone in men with lemon-refractory prostate cancer
Hydrocortisone in the Presence: Results of the Cancer and Leukocyte Group B 9182 Study "(
Hydrocortizone with or without mitoxantrone in men with hormone-refracto
ry prostate cancer: results of the Cancer and Leukemia Group B 9182 stud
y), J. Clin. Oncol., 17: 2506, 1999]. Androgen refractory prostate cancer
Among the various treatment options for therapy are gene therapies that use: sand
In other words, tumor suppressors [Ko, S.C., Gotoh, A., Thalmann, G.N., Zhau, H.E.,
Johnston, D.A., Zhang, W.W., Kao, C. and Chung, L.W.K., "Androgen Non-
Using recombinant p53 adenovirus in a dependent metastatic human prostate cancer model
Child therapy '' (Molecular therapy with recombinant p53 adenovirus in an androge
n-independent, metastatic human prostate cancer model), Hum. Gene Ther.,
 7: 1683, 1996], immunomodulators [Sanda, M.G., Ayyagari, S.R., Jaffee,
E.M., Epstein, J.I., Clift, S.L., Cohen, L.K., Dranoff, G., Pardoll, D.M
., Mulligan, R.C. and Simons, J.W., "Synthesis for human prostate cancer gene therapy.
Demonstration of a rational strategy for human prostate
 cancer gene therapy), J. Urol., 151: 622, 1994], and a toxic gene [Gotoh
, A., Ko, S.C., Shirakawa, T., Cheon, J., Kao, C., Miyamoto, T., Gardner
, T.A., Ho, L.J., Cleutjens, C.B., Trapman, J., Graham, F.L. and Chung,
 L.W.K., "Prostate specific antigen pro for androgen-independent human prostate cancer.
Development of prostate-specific anti-motor
gen promoter-based gene therapy for androgen-independent human prostate
cancer), J. Urol., 160: 220, 1998; Eastham, J.A., Chen, S.H., Sehgel, L.
, Yang, G., Timme, T.L., Hall, S.J., Woo, S.L. and Thompson, T.C., "Previous
Prostate cancer gene therapy: simple perpesui in mouse and human prostate cancer models
Ruth thymidine kinase gene transfer followed by ganciclovir '' (Prostat
e cancer gene therapy: herpes simplex virus thymidine kinase gene transd
auction followed by ganciclovir in mouse and human prostate cancer models
), Hum. Gene Ther., 7: 515, 1996; Koeneman, K.S., Kao, C., Ko, S.C., Yang.
, L., Wada Y., Kallmes, D.A., Gillenwater, J.Y., Zhau, H.E., Chung, L.W.
K. and Gardner, T.A., "On Osteocalcin for Prostate Cancer Bone Metastasis.
Osteocalcin directed gene therapy for prostate canc
er born metastasis), World J. Urol., 18: 102, 2000; Gardner, T.A., Ko, S.
C., Kao, C., Shirakawa, S., Cheon, J. Gotoh, A., Wu, T.T., Sikes, R.A.,
Zhau, H.E., Cui, Q., Balian, G. and Chung, L.W.K., "
Use of stromal-epithelial interactions and genes for prostate cancer and osteosarcoma metastasis
Exploiting stromal-epithelial interaction for m
odel development and new strategies of gene therapy for prostate cancer
and osteosarcoma metastasis), Gene Ther. Mol. Biol., 2:41, 1998].
Treatment.       [0006]   For treatment with virulence genes, p53 and herpes simplex virus thymidine
Several therapeutic genes, including kinases, have been applied to cancer gene therapy with limited success
I only keep it. The main problem is the current viral vectors and delivery methods
Associated with inefficient gene transfer rates that can be achieved by This difficulty
Ability to propagate into viral vector, which is a target vehicle, and to infect other target cells
Can be overcome by giving them the ability to As a result, virus vector propagation
Thus, the desired recombinant gene construct can be transferred from a limited number of transduced cells to neighboring cells.
Will be delivered. Theoretically, if one cell in a tumor nodule is infected
The virus replicates in the cells to infect more nearby tumor cells.
Will produce a luz. This growth will continue until all tumor nodules are eradicated
U. However, virus replication needs to be controlled so that normal tissue is not compromised.
is there.       [0007]   Use of tissue-specific promoters to deliver therapeutic toxic genes to tumor cells
The concept of being expressed is well-recognized. This approach is
If mediated gene delivery results in infection of nearby normal cells,
The toxic effects of therapeutic genes on cells could be reduced. For example,
Use of albumin or α-fetoprotein to target patome cells [
Kuriyama, S., Yoshikawa, M, Ishizaka, S., Taujli, T., Ikenaka, K., Kagaw
a, T., Morita, N. and Mikoshiba, K.
Useful for gene therapy targeting hepatome using liver-specific promoter
A potential approach for gene therapy targeting hepatom
a using a liver-specific promoter on a retroviral vector), Cell Struct.
Punct., 16: 503-510, 1991], Bone morphology on the brain to target glioma cells
Use of metamorphic protein promoters [Shimizu, K. "Using brain-specific promoters"
Gene Therapy for Malignant Gliomas: Its Effectiveness and Basic Research "(Selective g
ene therapy of malignant glioma using brain-specific promoters; its effi
cacy and basic investigation), Nippon Rinsho 52: 3053-3058, 1994], black
Use of the tyrosinase promoter to kill tumor cells [Vile, R.G., Nelson, J.A.
., Castleden, S., Chong, H. and Hart, I.R., "Tissue-specific expression of the HSVtk gene.
`` Immune components are involved in systemic gene therapy for melanoma in mice using the present '' (Systemic ge
ne therapy of murine melanoma using tissue specific expression of the HS
Vkt gene involves an immune component), Cancer Res., 54: 6228-6234, 1994
], And use of the carcinoembryonic antigen promoter on gastric cancer cells [Tanaka, T., Kana
i, F., Okabe, S., Yoshida, Y., Wakimoto, H., Hamada, H., Shiratori, Y.,
Lan, K-H., Ishitobi, M. and Omata, M. "Human gastric cancer cells producing carcinoembryonic antigen.
Adenovirus-mediated prodrug gene therapy for vesicles in vitro "(Adenovirus-
mediated prodrug gene therapy for carcinoembryonic antigen-producing hum
an gastric carcinoma cells in vitro), Cancer Res., 46: 1341-1345, 1996]
Is included. The most studied therapeutic gene to date is simple perpesui
Lus TK gene. Herpes simplex virus TK is dividing the prodrug ACV,
Transforms into a phosphorylated form that is toxic to the cells of the cell [Moolten, F.L.
Tumor Chemosensitivity Conferred by the Herpes Thymidine Kinase Gene: General
Tumor chemosensitivity conferred by inserted
herpes thymidine kinase genes; paradigm for a prospective cancer control
 strategy), Cancer Res., 46: 5276-5281, 1986]. Essential for successful treatment
Confer cytotoxicity to nearby non-transduced cells by the `` bystander ''
Effect; delivering and expressing a suicide gene to any tumor cell in the body.
Instead, it can achieve effective killing of tumor cells. Recently, this appro
Has been shown to be effective in causing regression in many solid tumors. It
These solid tumors include metastatic colon cancer in rat liver [Chen, S.II., Chen, X.H.
L., Wang, Y., Kosal, K.E., Finegold, J.J., Rich, S.S. and Woo, S.L.C.,
`` In vivo combination gene therapy for liver metastasis of colon cancer ''
rapy for liver metastasis of colon carcinoma in vivo), Proc. Natl. Acad
. Sci. USA 92: 2577-2581, 1995], gastric cancer [Tanaka, T., Kanai, F., Okabe, S., Y
oshida, Y., Wakimoto, H., Hamada, H., Shiratori, Y., Lan, K-H., Ishitobi
, M. and Omata, M. "In vitro for human gastric cancer cells producing carcinoembryonic antigen"
Adenovirus-mediated prodrug g
ene therapy for carcinoembryonic antigen-producing human gastric carcino
ma cells in vitro), Cancer Res., 46: 1341-1345, 1996], and malignant mesothelioma [
Smythe, W.R., Hwang, B.S., Elshami, A.A., Amin, K.M., Eck, S., Davidson,
 B.L., Wilson, J.M., Kaiser, L.R. and Albelda, S.M.
Experimental human mesothelioma using adenovirus transfer of the midin kinase gene.
Treatment '' (Treatment of experimental human mesothelioma using adenovirus tra
nsfer of the herpes simplex thymidine kinase gene), Ann.Surg., 222: 78-8
6, 1995].       [0008]   For replicable vectors, normal tissue is compromised during gene therapy
Conditionally based on the assumption that virus replication must be controlled
Several adenoviruses that can be produced have been developed. For example, adenovirus
ONYX-015 replicates only in p53-deficient tumor cells (Heise, C.C. et al., Cancer Gene The
r. 6: 499-504 (1999), the entire disclosure of which is incorporated herein by reference).
. Thus, another recent therapy for the treatment of androgen-refractory prostate cancer is
Its replication is conditioned by tissue-specific promoters such as gland-specific antigen (PSA)
Gene therapy using a replicable adenovirus (Ad) [Rodrigez, R
., Schuur, E.R., Lim, H.Y., Henderson, G.A., Simons, J.W. and Henderson
, D.R., "Attenuated Replicatable Adenovirus for Prostate (ARCA) CN706: Prostate-Specific Antigen
Prostate attenuated replicati
on competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate
 specific antigen-positive prostate cancer cells), Cancer Res., 57: 2559
, 1997; Yu, D-.C., Sakamoto, G.T. and Henderson, D.R.
Of transcriptional regulatory sequences for adenosine 2 and attenuated replicable adenovirus for the treatment of prostate cancer
Lus their use in the construction of the calydon virus 764 '' (Ident
ification of the transcriptional regulatory sequences of human kallikrei
n 2 and their use in the construction of calydon virus 764, an attenuate
d replication competent adenovirus for prostate cancer therapy), Cancer
Res., 59: 1948, 1999; Yu, D-.C., Chen, Y., Seng, M., Dilley, J. and Her.
derson, D.R. "Addition of the adenovirus type 5 E3 region is due to the
To eliminate the prostate tumor xenograft. '' (The addition of adenovi
rus type 5 region E3 enables calydon virus 787 to eliminate distant pros
tate tumor xenografts), Cancer Res., 59: 4200, 1999].
Incorporated herein by reference). But these replicable adenoviruses
Can only replicate in cells that express PSA.       [0009]   Osteocalci, a non-collagenous Gla protein specifically produced in osteoblasts
(OC) is synthesized, secreted, and deposited during bone calcification [Price, P.A.
Vitamin K-dependent formation of A protein (osteocalcin) and its function "(Vit
amin-K dependent formation of bone GLA protein (osteocalcin) and its fun
ction), Vitam. Horm., 42: 65-108, 1985]. Recent studies have focused on OC immune system chemistry
Staining was performed on primary and chondroblastic osteosarcoma specimens and 7
Shows that 5 of 5 fibroblastic osteosarcomas were positive
[Park, Y.K., Yung, M.H., Kim, Y.W. and Park, H.R.
Osteocalcin expression: in situ hybridization and immune organization
`` Osteocalcin expression in primary bone tumors: in situ hybrid
ization and immunohistochemical study), J. Korean Med.Sci., 10: 268-273,
 1995]. In addition, OC activity was detected in a wide range of human tumors. This is
Clinical evidence that human tumors showed calcification characteristics in both primary and distant metastases
Consistent with observation. Osteocalcin expression is a disease associated with calcium deposition.
Tissues such as, but not limited to, benign prostatic hyperplasia (BPH), cancer and
Atherosclerosis, etc. (see published International Application No. WO 98/313376).
I want to. The entire disclosure of this application is incorporated herein by reference).       [0010]   The osteocalcin (OC) promoter is used in both rat and human osteosarcoma cells.
Has been shown to be very effective in directing reporter gene transcription in strains
(Ward, W. et al., J. Clin.Oncol. 1994, 12: 1849-1858; Ducy P. et al., Molecular
and Cellular Biology, 1995, 15: 1858-1869; see entire disclosure of these references.
Which is incorporated herein by reference). In the study of transgenic mice, males
The activity of the theocalcin promoter was also shown to be osteoblast-specific. OC
Motors are several species-specific and mutually overlapping regulatory elements.
Heinrichs, A.A., Banerjee, C., Bortell, R., Owen, T.A., S
tein, J.L., Stein, G.S. and Lian, J.B.
Two contiguous elements in the promoter that may confer species-specific regulation
Identification and characterization of two pr
oximal elements in the rat osteocalcin gene promoter that may confer spe
cies-specific regulation), J. Cell. Biochem., 53: 240, 1993]. "Osteo
Calcine box '' is homeobox MSX protein, AP1, AP2, NF-1,
Factors such as ruscor enhancer, c-AMP, and vitamin D and glucocorti
Contains sites for binding to the coid receptor [Heinrichs, A.A., Banerjee, C., Bo
rtell, R., Owen, T.A., Stein, J.L., Stein, G.S. and Lian, J.B.
・ Can provide species-specific regulation of osteocalcin gene promoter
Identification and Characterization of Two Neighboring Neighboring Elements ''
racterization of two proximal elements in the rat osteocalci n gene promoter that may confer species-specific regulation), J. Cell. B
iochem., 53: 240, 1993; Hoffmann, H.M., Beumer, T.L., Rahman, S., McCabe,
 L.R., Benerjee, C., Aslam, F., Tiro, J.A., Wijnen, A.F.V., Stein, J.L.,
 Stein, G.S. and Lian, J.B., "Bone tissue-specific transcription of the osteocalcin gene.
: Activator osteoblast-specific complex and suppressor binding to OC box
-The role of Hox protein '' (Bone tissue specific transcription of the osteoc
alcin gene: role of an activator osteoblast-specific complex, and suppre
ssor Hox proteins that bind the OC box), J. Cell Biochem., 61: 310, 1996;
 Towler, D.A., Rutledge, S.J. and Rodan, G.A., "Msx-2 / Hox 8.1: Rat
Transcriptional regulator of osteocalcin promoter '' (Msx-2 / Hox 8.1: a transcript
ional regulator of the rat osteocalcin promoter), Mol.Endocrinol., 8:14
84, 1994; Liu, M. and Freedman, L.P. "Vitamin D3 receptors and other non-receptor translocations.
Transcriptional synergism between the vitam
in D3 receptor and other nonreceptor transcription factors), Mol.Endocr
inol., 8: 1593, 1994; Sneddon, W.B., Bogado, C.E., Kiernan, M.S. and Dem.
ay, M.B., "Downstream from the vitamin D response element of the rat osteocalcin gene.
Are required for ligand-dependent transcriptional activity "(DNA sequences downstream
 from the vitamin D response element of the rat osteocalcin gene are req
uired for ligand-dependent transactivation), Mol.Endocrinol., 11: 210, 1
997; Sneddon, W.B. and Demay, M.B., `` 1,25 of the rat osteocalcin gene.
Of enhancers required for D-dihydroxyvitamin D3-dependent transcriptional activity
Ke '' (Characterization of an enhancer required for 1,25-dihydroxyvitamin
D3-dependent transactivation of the rat osteocalcin gene), J. Cell Bioch
em., 73: 400, 1999]. The osteoblast-specific cis-acting element OSE2 is
Binds to the transcriptional activator Osf2 / Cbfa1 [Ducy, P., Zhang, R., Ge
offroy, V., Ridall, A.L. and Karsenty, G., "Osf2 / Cbfa1: Osteoblast differentiation
Transcriptional activator '' (Osf2 / Cbfa1: a transcriptional activation of osteobl
ast differentiation), Cell, 89: 747,1997]. Li YP are human osteocalci
A sequence located in the first exon of an inducible gene has a differentiation-related
-Element (OSE). After deleting the area from -3 to +51,
Osteocalcin can be transcribed in UMR-106 cells and growing normal osteoblasts
Functioned. Site-directed mutagenesis of this region resulted in a 7 bp sequence (TGGCCCT) (+29
(+35) were critical for silencer function (Li YP et al.,
Nucleic Acids Res. December 25, 1995; 23 (24): 5064-72;
Incorporated herein by reference).       [0011]   The mouse OC promoter has an additional OSE1 cis-acting element but [Ducy,
 P. and Karsenty, G., "Two distinct osteoblast-specific cis-acting elements
Regulates mouse osteocalcin gene expression '' (Two distinct osteoblast
-specific cis-acting elements control expression of a mouse osteocalcin
gene), Mol. Cell. Biol., 15: 1858, 1995], a non-functional vitamin D-responsive element.
[Heinrichs, A.A., Banerjee, C., Bortell, R., Owen, T.A., Ste.
in, J.L., Stein, G.S. and Lian, J.B., "The Rat Osteocalcin Gene Map.
Two adjacent elements in the lomomotor that may confer species-specific regulation
Identification and characterization of two prox
imal elements in the rat osteocalcin gene promoter that may confer speci
es-specific regulation), J. Cell. Biochem., 53: 240, 1993]. Rat male
For the theeocalcin regulatory sequence, previous studies have shown that the intron sequence TTTCTTT (+11
8 to +124) is an osteoblast cell lineage that interacts with
Mediates transcriptional repression of osteocalcin-CAT fusion gene in human cells
Is shown. Further analysis of the intron sequence will require a second silencer within this region.
-The motif was identified. Two copies of the CCTCCT motif are rat osteocal
Located in the first intron of the syngene (+106 to +111 and +135 to +140)
They inhibited the transcription of the osteocalcin-CAT fusion gene in rat osteosarcoma cells.
(Kearns, A.E. et al., Endocrinology 1999 Sep: 140 (9): 412
0-6; the entire contents of this document are incorporated herein by reference). Rat Oste
The sequence located 3 'to the vitamin D response element (VDRE) in the ocarcin gene is
Activates osteocalcin-CAT (OC-CAT) fusion gene transcription by 1,25 (OH) 2D3
Contains the essential GGTTTGG motif (-420 to -414). Sneddon, W.B.
The VDR-dependent transcriptional activity of the steocalcin gene is not only VDRE (-456 to -442)
, -430 to -414 were also required. Interact with these arrays
Acting proteins promote transcription in a manner that is independent of both position and orientation
(Sheddon, W.B. et al., J. Cell Biochem. Jun. 1, 1999: 73 (3): 400
-7; the entire contents of this document are incorporated herein by reference).       [0012]   In the case of atherosclerosis, this calcification disease process surrounds the active blood vessels
Although osteocalcin expression is associated with the formation of atherosclerotic plaques,
Increase. Often, overexpression of OC in these tissues
With increased deposition. Jie et al., Calcified Tissue Intl. 59: 352-356 (1996) and J.
ie et al., Atherosclerosis 116: 117-123 (1995). Balica et al., Circulation 95: 1954-
See also 1960 (1997).       [0013]   US Patent No. 5,772,993, the entire contents of which are incorporated herein by reference.
And in a co-published publication, a recombination involving the TK gene under the control of the OC promoter
Adenoviruses, when supplemented with the prodrug ACV,
Intralesional injection was shown to be able to suppress bone meat growth in both models
(Cheon, J. et al., Cancer Gene Therapy, 1997; 4: 359-365 and Ko., S.C. et al., Canc.
er Research, 1996; 56: 4614-4619). Toxic inheritance driven by the OC promoter
Offspring, or herpes simplex virus thymidine kinase (HSV-TK),
And in human prostate cancer cells when injected intratumorally at the site of skeletal metastasis of prostate cancer
(Unpublished observations) [Koeneman, K.S., Kao, C., Ko, S.
C., Yang, L., Wada Y., Kallmes, D.A., Gillenwater, J.Y., Zhau, H.E., Chu
ng, L.W.K. and Gardner, T.A., "Osteocalcin for prostate cancer bone metastasis.
Osteocalcin directed gene therapy for prost
ate cancer born metastasis), World J. Urol., 18: 102, 2000]. In addition, arteries
For the treatment of sclerosis, an excess of OC around the formed atherosclerotic plaque
Expression and concomitant calcification is useful for Ad-OC-TK-mediated gene therapy. Depending on the case
Administration of this therapeutic agent in combination with administration of ACV
It provides gene therapy to regress and effective treatment of arteriosclerosis. Ad-OC-TK is bone meat
Animals are administered by intravenous route of administration for the treatment of seed lung metastases [Shirakawa, T., Ko, S.
C., Gardner, T.A., Cheon, J., Miyamoto, T., Gotoh, A., Chung, L.W.K.
And Kao, C. "Using toxic gene therapy based on the osteocalcin promoter for intravenous injection.
In vivo suppression of osteosarcoma pulm
onary metastasis with intravenous osteocalcin promoter-based toxic gene
therapy), Cancer Gene Ther., 5: 274, 1998], and the sera of treated patients.
Primary and metastatic prostate cancer despite adenovirus detection in
(Eg bone and lymph nodes) by intratumoral administration [Koeneman, K.S., Kao, C., Ko
, S.C., Yang, L., Wada Y., Kallmes, D.A., Gillenwater, J.Y., Zhau, H.E.,
 Chung, L.W.K. and Gardner, T.A., "Osteocal for prostate cancer bone metastasis.
Osteocalcin directed gene therapy for p
rostate cancer bone metastasis), World J. Urol., 18: 102, 2000] Safe administration
can do. In fact, the safety of Ad-OC-TK is that general-purpose promoters
Another Adenovirus Virulence Inheritance Where Cytomegalovirus (CMV) Is Used
Much better than the security of child Ad-CMV-TK. Ad-CMV-TK and prodrug
75% of animals died from an intravenous ganciclovir (GCV), but Ad-OC-
None of the animals treated with TK / GCV died from the treatment [Shirakawa, T. et al.
, Ko, S.C., Gardner, T.A., Cheon, J., Miyamoto, T., Gotoh, A., Chung, L.
W.K. and Kao, C. "Toxic genes based on the osteocalcin promoter for intravenous injection.
In vivo suppression of osteosarcoma lung metastasis using therapy ''
oma pulmonary metastasis with intravenous osteocalcin promoter-based tox
ic gene therapy), Cancer Gene Ther., 5: 274, 1998; Brand, K., Arnold, W.,
 Bartels, T., Lieber, A., Kay, M.A., Strauss, M. and Dorken, B., `` HSV-t
k / GCV Approach and Liver-Related Toxicity of Adenovirus Vectors "(Liver-asso
ciated toxicity of the HSV-tk / GCV approach and adenoviral vectors), Canc
er Gene Ther., 4: 9, 1997]. Despite the advantage of Ad-OC-TK over Ad-CMV-TK
Need for more therapeutically effective adenovirus vectors for cancer therapy
I have.       [0014]   Thus, in view of the above, androgen receptor and PS in prostate tumor cells
Novel tissue-specific and tumors for cancer treatment that can replicate regardless of A status
The development of limited adenovirus vectors represents a significant advance in the area of cancer treatment
Will taste. The present invention relates to osteocalcin promoter activity and E1a
Ad-OC-E1a, another conditionally replicable adenovirus based on the gene product
Is pointed. This Ad-OC-E1a adenovirus is osteocalcin promoter
Can replicate only in cells that support
Can be broken. Such cells include prostate cancer, brain tumor, ovarian cancer,
Cancerous cells of thyroid cancer, osteosarcoma, ocular melanoma, lung cancer, breast cancer, and the like. The present invention
Also includes, but is not limited to, benign prostate hyperplasia (BPH) and atherosclerosis
Find use in the treatment of diseases with no calcification.       [0015]III. Summary of the Invention   The present invention relates to metastatic cancer (prostate cancer, brain tumor, ovarian cancer, thyroid cancer, various tumors, bone tumors,
Treats flesh, ocular melanoma, lung cancer, and breast cancer)
Related to novel viral vectors that can be used as therapeutics for cancer
About things. In addition, the present invention relates to benign but severe and life-threatening conditions and stones.
Diseases associated with ash (including but not limited to benign prostatic hyperplasia (BPH) and atherosclerosis)
It finds use in (but not limited to) treatment. The present invention further provides the use of the above therapeutic composition.
About new ways.       [0016]   The present invention has been constructed using the osteocalcin transcription regulatory sequences described herein.
Adenovirus vectors integrate the expression of adenovirus genes essential for replication.
The fact that it can be selectively extracted in a tissue-specific and tumor-limited manner
Based on the division. The present invention further provides that such adenoviral vectors
Prostate cancer, brain tumor, ovarian cancer, thyroid cancer, osteosarcoma, ocular melanoma, lung cancer, breast cancer, and
Stones including but not limited to benign prostatic hyperplasia (BPH) and atherosclerosis
Partially due to the discovery that it can be used as a therapeutic to treat diseases associated with incineration
Is based on Thus, the osteotype used with the adenovirus vector
Due to the tissue specificity and tumor specificity of the calcine transcription regulatory sequence, the adenovirus
Of the adenovirus gene and / or heterologous nucleotide sequence essential for replication
Tumors by using osteocalcin transcriptional regulatory sequences to allow tissue-specific expression
It can be administered in a limited and tissue-specific manner. Such osteooka
Examples of rucine transcription regulatory sequences are osteoblast cell lines and diseased tissue with calcification (eg,
(Eg, benign prostatic hyperplasia (BPH) and atherosclerosis)
Osteocalcin promoter. Therefore, osteocalcin transcriptional tone
Adenovirus vectors constructed using essential genes under the control of knot sequences
Can be expressed effectively and specifically in targeted cells and tissues
, Which results in non-osteoblasts and non-cancerous or non-atherosclerotic cells
Side effects of virus expression can be minimized.       [0017]   Additionally, osteocalcin transcription regulation used with adenovirus vectors
Due to the tissue specificity of the sequence, the viral vector of the present invention can be directly applied by injection, etc.
If administered systemically by intravenous administration, oral administration, etc.
In any case, it is an effective therapeutic agent. Because gene expression is specific to osteoblasts and stones
Diseased tissue with ash (eg, benign prostatic hyperplasia (BPH), cancer and atherosclerosis, etc.
), And is localized.       [0018]   In one embodiment, the invention provides transcription control of an osteocalcin transcription regulatory sequence.
Adenovirus vectors containing adenoviruses with underlying essential genes
Offer. This osteocalcin transcription regulatory sequence is an osteocalcin transcription regulatory sequence.
Cells that function (eg, prostate cancer cells, prostate stromal cells, pericytes,
Refractory cancer-related osteoblasts and do not express PSA or androgen receptor (AR)
Mediate gene expression specific to prostate cancer and related bone stromal cells)
Can be. The osteocalcin transcription regulatory sequence, wherein the sequence is osteocalcin
Osteocalcin inheritance should be able to mediate gene expression specific to expressing cells
A promoter and / or enhancer or enhancer from a child
Sequence can be included. In one embodiment, osteocalcin transcription
The regulatory sequences include a promoter from the osteocalcin gene. One embodiment
In, the osteocalcin transcription control sequence is an element derived from the osteocalcin gene.
Includes sequences similar to enhancers or enhancers. In one embodiment
Indicates that the osteocalcin transcription regulatory sequence is a promoter derived from the osteocalcin gene.
-And enhancers from osteocalcin gene or similar to enhancers
Included sequence. In one embodiment, the osteocalcin transcription regulatory sequence is
Cells that allow osteocalcin transcription regulatory sequences to function (eg, osteocalcin
Transcriptionally active in expressing cells, etc.).       [0019]   In certain embodiments, the osteocalcin transcription regulatory sequence is the sequence shown in FIG.
Contains the 1,370 bp nucleotide sequence of No. 1. In certain embodiments, osteocal
Syn transcription regulatory sequences can be found in osteocalcin-producing cells (eg, PSA or andro).
Prostate cancer cells, prostate stromal cells, perivascular cells,
Cancer-related osteoblasts, and prostate cancer and related bone stromal cells, etc.)
A portion of SEQ ID NO: 1 capable of mediating cell-specific transcription. Another implementation
In some embodiments, the osteocalcin transcriptional regulatory sequence is a osteocalcin gene inversion.
A sequence from about -290 to about +30 based on the transcription start site (approximately 141
454 to about 454th nucleotide). In another embodiment, osteo
The calsin transcription regulatory sequence is based on the osteocalcin gene transcription start site.
A sequence from -250 to about +30 (nucleotides from about 1 to about 454 of SEQ ID NO: 1)
C). In another embodiment, the osteocalcin transcription regulatory sequence is non-male.
Transcription of the osteocalcin gene linked by the theocalcin promoter
About -236 to about -223 sequences and / or about -140 to about
-117 (from about position 191 to about position 204 of SEQ ID NO: 1, and / or
Or about nucleotides 286 to 310). Yet another implementation
In some embodiments, the osteocalcin transcription control sequence is about the first of each of SEQ ID NO: 1.
From about 1 to about 150, from about 1 to about 200, about 1
From the 250th, from the 1st to the 300th, from the 1st to the 350th
From about 1 to about 400, from about 1 to about 450, from about 1 to about 500
, From about 1 to about 550, from about 1 to about 600, about 1
From about 1 to about 700, from about 1 to about 750, about 1
From the 1st to the 900th, from the 1st to the 850th, from the 1st to the 900th
From about 1 to about 950, from about 1 to about 1000, from about 1 to about 10
Up to 50th, from about 1st to about 1100th, from about 1st to about 1150th, about 1st
From the eyes to the 1200th, from the 1st to the 1250th, from the 1st to the 1300th
, From about 1 to about 1350, and from about 1 to about 1370
Includes nucleotide sequence consisting of tide. In each embodiment, osteocalcin transcription regulation
A sequence is a transcription regulatory sequence, i.e., an element that allows the osteocalcin transcription regulatory sequence to function.
Vesicles (eg, prostate cancer cells that do not express PSA or the androgen receptor (AR),
Prostate stromal cells, perivascular cells, proliferative cancer-associated osteoblasts, and prostate cancer and
Transcription regulatory sequences capable of effecting transcription in associated bone stromal cells, etc.)
Is done.       [0020]   In some embodiments, the osteocalcin transcription regulatory sequence is human, mouse,
Sequence of rat or rat origin. In some embodiments, the mouse
Or rat osteocalcin transcriptional regulatory sequence is prostate-specific in humans
It is possible to mediate offspring expression.       [0021]   In some embodiments, under the control of an osteocalcin transcription regulatory sequence.
Adenovirus genes, such as those essential for viral replication, are cytotoxic
Directly or indirectly. In one embodiment, the adenovirus
Genes are early genes. In another embodiment, the early gene is E1A.
is there. In another embodiment, the early gene is E1B. Yet another implementation
In one embodiment, both E1A and E1B regulate transcription of the osteocalcin transcription regulatory sequence.
Below. In another embodiment, the adenovirus gene essential for replication is late.
Is a gene. In various embodiments, the late gene is L1, L2, L3, L4 or
Is L5.       [0022]   In another embodiment, the osteocalcin transcription regulatory sequence is under transcriptional control
An adenovirus vector containing an adenovirus gene contains at least one additional
At least one osteocalcin-specific transcription regulatory sequence under the transcriptional control of
Contains additional adenovirus genes. In one embodiment, the composition is
Of adenovirus. In one embodiment, the composition is pharmaceutically acceptable
Further comprising an excipient. In one embodiment, the at least one
Consists of a second osteocalcin-specific transcription regulatory sequence
Is an array. In one embodiment, the at least one additional osteo
The calcine transcription regulatory sequence is different from the sequence of the first osteocalcin transcription regulatory sequence.
You can have an array that In one embodiment, the at least one
Osteocalcin-specific transcription regulatory sequence consists of an osteocalcin transcription regulatory sequence.
Including.       [0023]   In other embodiments, the adenovirus vector contains a heterologous gene or a transgene.
The gene may further comprise a gene, wherein the heterologous gene or transgene is
It is under the transcriptional control of an osteocalcin transcription regulatory sequence. In one embodiment,
The heterologous gene is, for example, a luciferase reporter gene or β-galactosida.
Reporter genes, such as a reporter gene. In one embodiment
Is a gene that is conditionally required for cell survival. Several
In embodiments, the transgene is a cytotoxic gene.       [0024]   In another embodiment, a method for treating metastatic cancer in an individual is provided.
. In this method, the adenovirus gene regulates the transcription of the osteocalcin transcription regulatory sequence.
Administering to the individual an effective amount of the adenovirus vector under control,
Here, the above metastatic cancer is prostate cancer, brain tumor, ovarian cancer, thyroid cancer, osteosarcoma, ocular melanoma
, Lung cancer or breast cancer. In another embodiment, treating metastatic cancer in an individual
A method is provided for treating osteocalcin conversion of an adenovirus gene.
Administer an effective amount of an adenovirus vector under transcriptional control of a transcript regulatory sequence to an individual
Wherein said metastatic cancer is prostate cancer, wherein said prostate cancer cells,
Prostate stromal cells, perivascular cells, proliferative cancer-associated osteoblasts, and related bone stromal cells
The vesicle does not express PSA or the androgen receptor (AR).       [0025]   In one embodiment, the adenovirus gene is essential for virus replication
Is the gene. In one embodiment, the adenovirus gene is in the early stage.
It is a messenger. In one embodiment, the adenovirus gene is E1A.
In one embodiment, the adenovirus gene is E1B. One implementation
In one embodiment, the osteocalcin transcription regulatory sequence is derived from an osteocalcin gene.
Includes native enhancers or sequences similar to enhancers. In one embodiment
In the above, the osteocalcin transcription regulatory sequence is derived from the osteocalcin gene.
Includes promoter. In one embodiment, the osteocalcin transcription control
The knot sequence is a promoter derived from the osteocalcin gene and the osteocalcin gene.
Includes enhancers or sequences similar to enhancers from the gene. One implementation
In an embodiment, the adenovirus has at least one additional transcription regulatory sequence.
Includes additional adenovirus genes under transcriptional control. In one embodiment
In some embodiments, the second transcription control sequence comprises an osteocalcin transcription control sequence. One implementation
In a form, the additional adenovirus gene is a gene essential for viral replication.
I am a child. In one embodiment, the additional adenovirus gene is an early adenovirus gene.
Is a gene. In one embodiment, the additional adenovirus gene is E
1A. In one embodiment, the additional adenovirus gene is E1B.
is there. In one embodiment, the additional adenovirus gene is a late inherited gene.
I am a child. In various embodiments, the late gene is L1, L2, L3, L4 or L5.
It is possible.       [0026]   In another aspect, the invention relates to any of the adenoviral vectors described herein.
To provide a host cell transformed with       [0027]   In another embodiment, the present invention provides for the transcriptional control of an osteocalcin transcriptional regulatory sequence.
A composition comprising an adenovirus vector comprising an adenovirus gene
. In one embodiment, the composition further comprises a pharmaceutically acceptable excipient
.       [0028]   In another aspect, the invention includes an adenovirus vector described herein.
To provide a kit.       [0029]   In another embodiment, the osteocalcin transcription regulatory sequence is specific to a cell that functions.
Methods for propagating a specific adenovirus vector are provided. With such cells
For example, osteocalcin is expressed and PSA or androgen receptor is expressed.
Prostate cancer cells that do not express the body (AR), prostate stromal cells, perivascular cells, proliferative cancer
Osteoblasts and related bone stromal cells, and the like. This method is osteocal
Such cells in which a syn-transcription regulatory sequence is functioning can be prepared by any of the methods described herein.
Infecting the adenovirus with the adenovirus vector
The method is characterized in that the vector is propagated.       [0030]   In another aspect, a method is provided for altering the genotype of a target cell. this
The method describes cells that function osteocalcin transcriptional regulatory sequences herein.
Contacting with any adenovirus, whereby said adenovirus
Enter the cell. Such cells include, for example, PSA and
Indicates prostate cancer cells that do not express androgen receptor (AR), prostate stromal cells,
Includes pericytes, proliferative cancer-related osteoblasts and related bone stromal cells.       [0031]   In another embodiment, osteocalcin is expressed in a biological sample
A method of detecting cells, wherein the cells in a biological sample are described herein.
Contacting with an adenovirus vector and replicating the adenovirus vector
The method is provided that includes detecting (if occurring).       [0032]   In one embodiment, osteocalcin transcription regulation in a biological sample
Cells that allow the sequence to function (eg, do not express PSA or the androgen receptor (AR)).
Prostate cancer cells, prostate stromal cells, perivascular cells, proliferative cancer-related osteoblasts and
Associated bone stromal cells, etc.). This method is osteo
Osteocalcin transcription regulatory sequences in cells that function calcine transcription regulatory sequences
Osteocalcin transcription of biological samples under conditions appropriate for row-mediated gene expression
Contains essential adenovirus previously or late genes under the transcriptional control of regulatory sequences
Contacting with an adenovirus vector; and wherein the osteocalcin transcription regulatory sequence is
Confirming whether to mediate gene expression in the biological sample.
Osteocalcin transcription regulatory sequence-mediated gene expression
Indicates the presence of cells that make the sequence work. In one embodiment, the gene is a different gene.
Species (non-adenovirus) gene. In one embodiment, the heterologous genetic
The offspring is a reporter gene, and production of this reporter gene product is detected
Is done.       [0033]   In another embodiment, the method of imparting selective toxicity or cytotoxicity to target cells
A law is provided. This method is used for cells in which osteocalcin transcription regulatory sequences function.
(Eg, expressing osteocalcin and PSA or androgen receptor (AR)
Prostate cancer cells, prostate stromal cells, perivascular cells, proliferative cancer-related osteoblasts that do not express
Cells and associated bone stromal cells, etc.)
The adenovirus to enter the cell.
It is a way to sign.       [0034]   In yet another embodiment, the osteocalcin transcription regulatory sequence is under transcriptional control.
An adenovirus comprising a heterologous gene is provided. In one embodiment
, The heterologous gene is a reporter gene. In one embodiment, the difference
A seed gene is a gene that is conditionally required for cell survival. In one embodiment
In addition, cells that function osteocalcin transcription regulatory sequence in a sample (eg,
Expresses osteocalcin and expresses PSA or androgen receptor (AR)
Prostate cancer cells, prostate stromal cells, perivascular cells, proliferative cancer-related osteoblasts and
And related bone stromal cells, etc.). This method is
Regulation of osteocalcin transcription in cells functioning ocarcin transcription regulatory sequences
Biological samples are converted to osteocalcin under conditions appropriate for sequence-mediated gene expression.
Contacting with an adenovirus vector containing a gene under the transcriptional control of a transcript regulatory sequence
And osteocalcin transcriptional regulatory sequences regulate gene expression in the biological sample.
Osteocalcin transcription regulatory sequence medium
Transgene expression indicates the presence of cells expressing osteocalcin.       [0035]   As described in more detail herein, osteocalcin transcription regulatory sequences are numerous.
Can be configured. These configurations include, for example, one
OC promoter; one OC enhancer or a sequence similar to the OC enhancer; 1
1 OC silencer; 1 OC promoter and 1 OC enhancer or OC
Sequence similar to enhancer; one OC promoter and one non-OC (heterologous) enhancer
Hansers; one non-OC (heterologous) promoter and one OC enhancer or OC enhancer
Sequence similar to enhancer; one non-OC promoter and multiple copies of enhancer
And multimers described above, but is not limited thereto. Oste
The activity of the ocarcin transcription regulatory sequence is measured, and as a result, certain cells
Methods for determining whether a calcine transcription regulatory sequence is functional are described herein.
Have been. Osteocalcin transcriptional regulatory sequence promoter and enhancer
Or a sequence similar to the OC enhancer can provide the desired OC cell-specific transcriptional activity
As long as they are located in any orientation, and / or
It may be at any distance and may be in the form of a multimer of the above composition. Transcription activity
Is known in the art and is measured by a number of methods detailed later in this specification.
In general, this is under the control of the osteocalcin transcription regulatory sequence.
Coding sequence mRNA or (ie, operably linked thereto)
It is measured by detecting and / or quantifying the protein product. Discussed herein
As such, osteocalcin transcription regulatory sequences have different lengths and different sequence configurations.
Can be taken.       [0036]   By the terms "transcriptional activation" or "increased transcription", transcription is carried out in the target cell (
That is, cells that function osteocalcin transcription regulatory sequence, such as osteocalcin
Prostate that does not express calcin and does not express PSA or androgen receptor (AR)
Cancer cells, prostate stromal cells, perivascular cells, proliferative cancer-related osteoblasts and related
Bone stromal cells, etc.) at least about 20 times, preferably at least about
Also about 50 times, more preferably at least about 100 times, even more preferably at least
About 200 times, even more preferably at least about 400 to about 500 times, even more preferably
Preferably it is intended to increase by at least about 1000 times. The base level is
Generally, the level of activity (if present) in non-osteocalcin producing cells is increased.
Osteocalcin transcription tested in bell or osteocalcin producing cells
The level of activity (if any) of the reporter construct lacking regulatory sequences
. Optionally, use a transcription terminator or transcription "silencer"
Placed upstream of the syn transcript regulatory sequence, thereby
Undesirable read-through of code segments under transcriptional control
It is possible to prevent transcription. In some cases, osteocalci
Endogenous promoter of a coding segment to be placed under the transcriptional control of a transcription regulatory sequence
Can be deleted.       [0037]   Another embodiment of the present invention is directed to mammalian cells expressing osteocalcin.
Is an adenovirus that replicates specifically.       [0038]IV. Brief description of drawings   The following drawings form part of the present specification and further illustrate certain aspects of the present invention.
Included to illustrate. The invention features one or more of these drawings.
Better understanding by reference to the detailed description of certain embodiments
Can be. (See end of this specification for a brief description of the drawings.)       [0039]V. Definition   For convenience, reference to the specification, examples, and appended claims are made.
The meanings of certain terms and phrases are set forth below.       [0040]   The term “tissue-specific” refers to a gene in which genes essential for viral replication can function.
The transcriptional regulatory sequence linked thereto is used to promote replication in the tissue.
It means functioning in the organization.       [0041]   The term "transcription regulatory sequence" is used according to its art-recognized meaning.
Have been. The term refers to a gene linked by its sequence in a particular cell.
Can cause up-regulation or down-regulation
It means any DNA sequence that can be used. In one embodiment of the present invention,
The native transcription regulatory sequence is completely deleted from the vector and
Will be replaced. Transcriptional regulatory sequences are located near the coding region of genes essential for replication.
Or away from it. Therefore, in the case of a promoter
The promoter will generally be located near the coding region. But Enha
If the sequence is similar to an enhancer or enhancer, the enhancer or enhancer
As there is a DNA sequence intervening between the coding region and a sequence similar to sir,
Enhancers or enhancer-like sequences may be slightly distant from the coding region
May be found at the time. In some cases, the natural transcriptional regulatory sequences are
Remains above, but is non-functional for transcription of genes essential for replication. A place
In that case, the native transcriptional regulatory sequence remains on the vector and is essential for replication.
A tissue-specific, tumor-restricted transcriptional regulatory element to which the gene is operably linked
Augmented by placing rows.       [0042]   The term "adenoviral vector" or "adenoviral vector"
Is used interchangeably, but is a term well understood in the art.
In general, a polynucleotide containing all or part of the adenovirus genome (this book)
(As defined in the specification). For the purposes of the present invention, adenovirus vectors are
Osteocalcin transcription regulatory sequence operably linked to the oligonucleotide
No. Is the operably linked polynucleotide adenoviral?
Or a heterologous polynucleotide. Construction of Adenoviral Vector of the Invention
An object can take any of several forms. These forms include naked
DNA, DNA wrapped in adenovirus coat, DNA wrapped in liposome,
DNA complexed with lysine, DNA complexed with synthetic polycationic molecules,
DNA conjugated to spherin, immunologically "masks" the molecule, and
DNA conjugated with a compound such as PEG to increase the half-life
Includes, but is not limited to, DNA conjugated to
Not determined. Preferably, the polynucleotide is DNA. Used in this specification
The term "DNA" refers to any of the bases A, T, C, and G, as well as their analogs.
Or modified forms of these bases, such as methylated nucleotides
Etc., internucleotide modification (uncharged bond and thioate etc.), sugar analog
Use, and modified and / or alternative backbone structures (such as polyamides)
Including. For the purposes of the present invention, adenovirus vectors are used to target cells, such as tumor cells.
Can be replicated in.       [0043]   As used herein, the term “polynucleotide” or “nucleic acid” refers to any length
Nucleotide (either ribonucleotide or deoxyribonucleotide)
May be used). Therefore, the term is not limiting.
However, single-stranded, double-stranded or multi-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA
Hybrid, or purine and pyrimidine bases or other natural, chemical
Or a biochemically modified, non-natural or derivatized nucleotid
Includes polymers containing a base. The backbone of the polynucleotide is composed of sugar and phosphate groups (reference
(Typically found in RNA or DNA), or modified or substituted
Sugar or phosphate groups. Alternatively, the backbone of the polynucleotide is
Can comprise a polymer consisting of synthetic subunits, such as suformamidate,
Thus oligodeoxynucleoside phosphoramidate (P--NH_Two) Or mixed
It can be a suformamidate-phosphodiester oligomer. Peyrottes
Et al., (1996) Nucleic Acids Res. 24: 1841-8; Chaturvedi et al., (1996) Nucleic Acid.
s Res. 24: 2318-23; Schultz et al., (1996) Nucleic Acids Res. 24: 2966-73. Hos
Phosphorothiate linkages can be used in place of the homodiester linkages. Brau
n et al., (1998) J. Immunol. 141: 2084-9; Latimer et al., (1995) Mol. Immunol. 32: 10
57-1064. Furthermore, a complementary strand is synthesized from the chemically synthesized single-stranded polynucleotide product.
By synthesizing and annealing the two strands under appropriate conditions, or
By synthesizing a new complementary strand using merase with appropriate primers
And a double-stranded polynucleotide can be obtained.       [0044]   The following are non-limiting examples of polynucleotides: genes or gene fragments,
Exon, intron, mRNA, tRNA, rRNA, ribozyme, cDNA, recombinant polynucleotide
Otide, branched polynucleotides, plasmids, vectors, any sequence isolated
DNA, isolated RNA of any sequence, nucleic acid probes, and primers. Polinu
Nucleotides are modified nucleosides such as methylated nucleotides and nucleotide analogs.
Other sugars and linking groups such as tide, uracil, fluororibose and thioate,
And nucleotide branches. Nucleotide sequence is non-nucleotide
It may be interrupted by a tide component. The polynucleotide is combined with the labeling component.
Can be further modified after polymerization, such as by forming
You. Other types of modifications included in this definition include caps, naturally occurring nucleo
Substitution of one or more analogs of the tide and the polynucleotide for the protein
Bound to the substrate, metal ions, labeling components, other polynucleotides or solid supports.
It is the introduction of the means for.       [0045]   A polynucleotide or polynucleotide region is a specified percentage of another sequence
Having a "sequence identity" at a rate (eg, 80%, 85%, 90%, 95%, 98% or 99%)
Indicates that the above percentages of bases are identical when the two sequences are aligned and compared.
Means This alignment and percent homology or sequence identity
Software programs known in the art, for example, Current Protocols in
 Molecular Biology (Ausubel et al., Eds., 1987) Addendum 30, Section 7.7.18, Table 7.7.
It can be determined using the program described in 1. Preferred Arai
ALIGN Plus (Scientific and Educational Software, Penns
ylvania).       [0046]   As used herein, “cells that function osteocalcin transcription regulatory sequence”, male
In cells where the function of the theeocalcin transcription regulatory sequence is “well conserved”, or
`` Cells in which the ocarcin transcription regulatory sequence is functioning ''
If the sequence is operably linked to a reporter gene, for example,
The reporter when not operably linked to an ocarcin transcription regulatory sequence
Preferably, the expression of the reporter gene is at least about 20-fold greater than the expression of the gene.
Or at least about 50 times, more preferably at least about 100 times, even more preferably
Or at least about 200 times, even more preferably at least about 400 to 500 times,
Even more preferred are cells that increase at least about 1000-fold. Absolutely relative
Methods for measuring expression levels, whether in pairs, are known in the art and are described herein.
Has been described.       [0047]   The term "under transcriptional control" is well understood in the art and refers to polynucleotides.
Transcription of the peptide sequence (usually a DNA sequence) contributes to or promotes transcription.
The polynucleotide sequence can function as a translating element, ie, a transcriptional regulatory sequence
Depends on whether they are connected in a shape. As described below
"Operably linked" means that the above-mentioned element, ie, a transcription regulatory sequence,
It refers to a parallel arrangement in which they function.       [0048]   As used herein, the terms "cytotoxic" or "cytotoxic" are used in the art.
One or two of the normal biochemical or biological functions of a cell
A condition in which one or more are abnormally compromised (ie, suppressed or enhanced).
These activities include, but are not limited to, metabolism, cell replication, DNA replication, transcription
, Translation, and incorporation of molecules. “Cytotoxicity” refers to cell death and / or
Includes cell lysis. Dye exclusion method,^ThreeH-thymidine incorporation and plaque uptake
Assays that show cytotoxicity, such as Sasay, are known in the art. As used herein
The term “selective cytotoxicity” refers to the function of the osteocalcin transcription regulatory sequence.
Of the cytotoxicity conferred by the adenovirus of the invention on uninfected cells
As a result, the adenovirus of the present invention can
It refers to the cytotoxicity conferred by a viral vector. Such cell wounds
The harm may be, for example, a plaque assay, a reduction in the size of target cells, including tumors, or
Stabilization, or markers characteristic of tumor cells or tissue-specific markers (eg,
For example, by lowering or stabilizing serum levels of cancer markers such as prostate specific antigen)
Can be measured.       [0049]   The terms "replication" and "proliferation" are used interchangeably and are used in the present invention.
Refers to the ability of an adenovirus vector to propagate or propagate. This term is
Well understood in the technical field. For the purposes of the present invention, replication is adenoviral
Involves the production of protein and is generally directed to the reproduction of adenovirus
ing. Replication is a standard assay in the art and is described herein.
(Burst assay or plaque assay, etc.)
Wear. "Replication" and "propagation" are direct or indirect
Includes any activity involved in These include, but are not limited to,
Expression of the viral gene; production of viral proteins, nucleic acids or other components; virus
Packaging of the components into complete virus; and cell lysis.       [0050]   The term "heterologous" refers to a DNA sequence not found in the natural vector genome
Means With respect to the expression "heterologous transcription control sequence", "heterologous"
The knot sequence is naturally linked to the DNA sequence of a gene essential for vector replication.
Indicates no.       [0051]   “Heterologous gene” or “transgene” is present in wild-type adenovirus
Not any gene. Preferably, the transgene is also an adenovirus
Not expressed in the target cell prior to introduction by the transfer vector, or
Not present in the cell. Examples of preferred transgenes are shown below.       [0052]   The term "promoter" is used according to its art-recognized meaning.
Have been used. It is usually located upstream of the coding sequence or operon of the gene.
DNA region that binds RNA polymerase and directs the enzyme to the correct transcription start site
Means the area.       [0053]   The term "enhancer" is used according to its art-recognized meaning.
Have been. It is a variant found in eukaryotes and certain eukaryotic viruses.
Rows, located several kilobases from the gene being tested
In some cases (either direction), a sequence that can increase transcription from the gene
It means a column. These sequences are usually located on the 5 'side (upstream) of the gene of interest.
Acts as an enhancer when placed. But some enhancers
Is active when placed on the 3 'side (downstream) of the gene. This enhancer
It may be a sequence similar to an enhancer.       [0054]   The term "silencer" used in its art-recognized meaning,
, A sequence found in eukaryotic viruses and eukaryotes,
Reduces transcription of the gene when placed within a few kilobases, ie silence
Means an array that can be       [0055]   A "heterologous" promoter or enhancer is defined as the osteocalcin gene 5 '.
A promoter not associated with or derived from a flanking sequence
Or enhancer. Examples of heterologous promoters include α-fetopro
Thein, PSA, DF3, tyrosinase, CEA, detergent protein and ErbB2
Motor. Examples of heterologous enhancers include α-fetoprotein
, PSA, DF3, tyrosinase, CEA, detergent protein, ErbB2 and SV40
Hansers.       [0056]   An "endogenous" promoter, enhancer or transcription regulatory sequence
Or derived from adenovirus.       [0057]   The phrase “operably linked” refers to multiple functionally related
Regarding the arrangement direction of nucleotide elements. The transcriptional regulatory sequence is
If the sequence promotes transcription of the coding sequence, it can function in the coding segment.
Are linked. The term “operably linked” refers to a plurality of linked DNA sequences.
The rows are generally contiguous and link the two protein coding regions
Means that they have to be contiguous and have the same reading frame
. However, enhancers generally take advantage of a few kilobases away from the promoter.
Work, and the intron sequences can be of various lengths,
Elements may be functionally linked but not contiguous
.       [0058]   The term "host cell" can be a receptor for any of the vectors of the invention.
Or individual cells or cell cultures that have received or received the vector. Host details
The vesicle contains the progeny of one host cell, and the progeny is natural, incidental, or
Deliberate mutations and / or changes are not necessarily completely identical to the original parent cell (
In the form or in the total DNA complement). Host details
Vesicles can be traversed in vivo or in vitro by the adenovirus vector of the invention.
Includes transfected or infected cells.       [0059]   A “target cell” is any cell in which an osteocalcin transcription regulatory sequence functions.
You. Preferably, the target cell is a mammalian cell in which the osteocalcin transcription regulatory sequence functions.
Cells, e.g., cells expressing osteocalcin, preferably endogenously osteocalcin
Mammalian cells expressing ocarcin, more preferably human cells, and even more preferably
Preferably, a human cell capable of functioning the osteocalcin transcription regulatory sequence,
Or cells that do not express the androgen receptor (AR).       [0060]   As used herein, “neoplastic cell”, “tumorigenesis”, “tumor”, “tumor cell”, “
The terms "cancer" and "cancer cell" indicate relatively autonomous growth and, as a result,
Cells exhibiting an abnormal growth phenotype characterized by a severe decrease in cell growth control
As expected. Neoplastic cells can be benign or malignant.       [0061]   "Biological sample" encompasses various types of samples obtained from an individual, and
It can be used for cleavage or monitoring assays. Definition of this term
Are solid tumor samples such as blood and other liquid samples of biological origin, biopsy specimens, etc.
Or tumor cultures or cells derived therefrom and progeny thereof. this
The definition of terms also includes post-acquisition treatment with reagents, solubilization, or
Samples that have undergone some manipulation due to enrichment of proteins, polynucleotides, etc.)
Is also included. The term "biological sample" encompasses clinical samples, and
Cells in culture, cell supernatants, cell lysates, serum, plasma, body fluids and tissue samples
Including.       [0062]   An "individual" is a vertebrate, preferably a mammal, more preferably a human. Feeding
Animals include, but are not limited to, livestock, sports / hunting animals and pets.
Not limited.       [0063]   An "effective amount" is an amount sufficient to effect beneficial or desired clinical results. Yes
The effective dose can be administered once or in several divided doses. For the purposes of the present invention
What is the effective amount of adenovirus vector?
The amount is sufficient to cause the compound to reverse, reverse, slow or delay.       [0064]   As used herein, the term “treatment” is used to refer to a treatment that achieves a beneficial or desired clinical outcome.
Approach. For the purposes of the present invention, beneficial or desired clinical outcomes include:
In a non-limiting manner, symptoms are reduced, disease spread is reduced, and
That is, the condition does not worsen), the spread of the disease (ie, metastasis),
Slowing or slowing, amelioration or alleviation of pathology, and (partially or wholly)
Includes remissions, whether available or not. “Treatment” also refers to receiving treatment.
It also means prolonging survival compared to expected survival if not.       [0065]   “Palliating” of a disease refers to the absence of administration of the adenoviral vector of the present invention.
Spread and / or undesired clinical symptoms as compared to
Meaning that the reality is less and / or the course of progress is slower or longer
To taste.       [0066]   Various combinations of transcription control sequences can be included in the vector. One or two of them
One or more can be a heterologous sequence. Furthermore, one or more of them may
May have. One or more of the transcription regulatory sequences can be inducible. For example, one
Using transcriptional regulatory sequences to direct replication by two or more genes essential for replication
Would be possible. For example, the product of one gene among multiple genes
This is the case that leads to the transcription of One example is the E1a coding sequence (E1a promoter
Operably linked to a cassette containing the entire E1b gene
Heterologous promoter. In this case, only one heterologous transcription regulatory sequence is required.
Will be done. However, if multiple genes are individually controlled,
If it is desired that two or more of the genes control replication, two or more transcriptional regulators
Arrays can be used.       [0067]   The term "gene essential for replication" refers to the replication of a viral vector in a target cell.
A gene sequence whose transcription is required to perform       [0068]   Thus, the vectors of the present invention comprise two or more heterologous transcriptional regulatory sequences,
Combinations of transcriptional regulatory sequences, one or more of which are not tissue-specific or tumor-specific
Including. For example, one transcription regulatory sequence is a basal level constitutive transcription regulatory sequence.
Can be For example, a tissue-specific enhancer or promoter
Can be combined with Bell constitutive promoters. In another example, the pair
Combining a tissue-specific enhancer or promoter with an inducible promoter
be able to.       [0069]VI. Detailed Description of the Invention   The present invention relates to methods and compositions for adenovirus cell therapy. In particular,
The compositions of the present invention provide for the regulation of the adenovirus early genes required for viral replication.
Using osteocalcin transcriptional regulatory sequences to direct viral replication
Includes ills vector. The method of the present invention relates to metastatic cancer (prostate cancer, brain tumor, ovarian cancer).
, Thyroid cancer, various tumors, osteosarcoma, ocular melanoma, lung cancer and breast cancer
Adenovirus required for viral replication to treat (but not limited to)
Osteocalcin transcriptional regulatory sequences direct viral replication by regulating early genes
The use of the adenovirus vector used. Furthermore, the present invention is not benign.
Severe but life-threatening conditions and calcifications (benign prostate hyperplasia (BPH)
Also find use in the treatment of (including but not limited to atherosclerosis)
.       [0070]   Three types of evidence suggest that we have promising new therapies for prostate cancer skeletal metastases
Prompted the development of Ad-OC-E1a. First, osteocalcin (OC) protein is pre-human
It was found to be uniformly and highly expressed in skeletal metastases of prostate cancer. Ad-O
Targeting both prostate and proliferative cancer-associated osteoblasts using C-E1a
This form of therapy not only suppresses the growth of prostate cancer cells,
Prostate tumor-bone stroma by direct targeting and destruction of proliferating osteoblasts
It will also hinder the interaction. Second, the ad promoter regulated by the OC promoter
Virus replication is associated with prostate cancer and non-PSA or androgen receptor (AR) expression.
And may be much more effective on bone stromal cells associated therewith. did
Thus, it was shown to express OC and support tumor epithelial growth and progression
Prostate stromal cells [Koeneman, K.S., Kao, C., Ko, S.C., Yang, L., Wada Y., Ka
llmes, D.A., Gillenwater, J.Y., Zhau, H.E., Chung, L.W.K. and Gardner,
T.A., "Geotherapy guided by osteocalcin for prostate cancer bone metastases.
Act '' (Osteocalcin directed gene therapy for prostate cancer born metastas
is), World J. Urol., 18: 102, 2000; Gardner, T.A., Ko, S.C., Kao, C., Shi.
rakawa, S., Cheon, J. Gotoh, A., Wu, T.T., Sikes, R.A., Zhau, H.E., Cui,
 Q., Balian, G. and Chung, L.W.K., "Stromal-epithelial interactions for model development.
In the use of gene therapy and gene therapy for prostate cancer and osteosarcoma metastasis
Exploiting stromal-epithelial interaction for model development a
nd new strategies of gene therapy for prostate cancer and osteosarcoma m
etastasis), Gene Ther. Mol. Biol., 2:41, 1998; Koeneman, K.S., Yeung, F.
And Chung, L.W.K. "Bone mimetic properties of prostate cancer cells: prostate cancer conversion in the bone environment.
Hypothesis Supporting Transfer and Proliferation Preferences "(Osteomimetic properties of prostate c
ancer cells: a hypothesis supporting the predilection of prostate cancer
 metastasis and growth in the bone environment), Prostate, 39: 246, 1999]
And perivascular cells [Doherty, M.J., Ashton, B.A., Walsh, S., Beresford, J.
N., Grant, M.E. and Ganfield, A.E., "Perivascular cells are used in vitro and in vi.
Indicate bone formation potential in vo '' (Vascular pericytes express osteogenic p
otential in vivo and in vitro), J. Bone Miner. Res., 13: 828, 1998].
It is possible to direct adenovirus replication using the OC promoter. Third
The results of this study show that Ad-OC-E1a was previously present in humans when administered intravenously.
It was shown for the first time to induce regression of prostate cancer bone xenografts. This study is based on Ad-O
C-E1a is an attractive intravenous Ad vector for treating human prostate cancer skeletal metastases
It was proved that.       [0071]   Preferred vectors of the present invention are adenovirus vectors. One preferred
In embodiments, the adenovirus vector is a human adenovirus. Ad
There are many different types of adenovirus, such as 2, Ad5 and Ad40, which are trivial.
To varying degrees of significance. Ad5 and Ad40 in particular have affinity for host cells
And the nature of the disease induced by the virus
You.       [0072]   In another embodiment, the adenovirus used in the compositions and methods of the invention
The vector is canine adenovirus type 1 or canine adenovirus type 2. Limited
By way of example, and not by way of illustration, canine adenoviruses that can be used in the present invention are international patents.
Nos. WO 91/11525 and WO 94/26914 (the entire contents of each application being referenced
(Incorporated herein).       [0073]   In another embodiment, the adenovirus used in the compositions and methods of the invention
The vector is a bovine adenovirus. By way of example but not limitation,
One example of a denovirus is described in International Patent Application No. WO 96/16048 (see the entire contents of this application).
, Incorporated herein by reference).       [0074]   In another embodiment, the adenovirus used in the compositions and methods of the invention
The vector is a sheep adenovirus. As an example and not as a limitation,
One example of a sheep adenovirus suitable for use in Ming is described in US Pat. No. 6,020,172 (
OAV2 as described in the entire contents of this patent, which is incorporated herein by reference).
87.       [0075]   For the purpose of the present invention, Ad5 is exemplified. The following description is based on adenovirus
Brief about vectors in general, and especially for replicable adenovirus vectors
It is a simple explanation.       [0076]A. Adenovirus-based vectors   Adenovirus has a dense protein capsid and a large (36 kb) line
It is a large non-enveloped virus consisting of a stranded double-stranded genome. Adenovirus
Is a receptor-mediated endothelium that infects both dividing and non-dividing cells.
Incorporation into the cell enters the cell, after which internalization
Occur. After encapsulation, the adenovirus genome alters viral replication and host cell metabolism.
Many different gene products involved in packaging of variant and progeny virions
Is expressed. Three adenovirus gene products are essential for viral genome replication
There are: (1) end-binding proteins that prime DNA replication, (2) viruses
DNA polymerase and (3) a DNA binding protein (Tamanoi and Stillman,
 1983, Immunol. 109: 75-87). In addition, subsequent rounds of reinfection are possible.
(Stillman et al., 1981, Cell, 23: 497-508), and adenovirus
To complete the self-assembly of the capsid by processing the structural proteins (B
hatti and Weber, 1979, Virology, 96: 478-485), adenovirus 23 kDa L3
Processing of the terminal binding protein by a Rotase is required.       [0077]   Packaging of the developing adenovirus particles occurs in the nucleus and is cis-acting
Requires both sex DNA elements and trans-acting viral factors. the latter
Is generally understood to be some viral structural polypeptide. Adeno
Packaging of adenovirus DNA sequences into viral capsids
The genome needs to have a functional adenovirus envelope signal, and these
The signals are located at the left and right ends of the linear viral genome (Hearing et al., 1987,
J. Virol. 61: 2555-2558). In addition, packaging arrays are not
Must be present near the end of the irs genome (Hearing et al., 1987, J. V.
irol. 61: 2555-2558; Grable and Hearing, 1992, J. Virol., 66: 723-731). E
1a enhancer, viral origin of replication and envelope signal are adenovirus genome
It constitutes a double inverted terminal repeat (ITR) sequence located at both ends of the DNA. The replication origin is
It is disjointly defined by a series of conserved nucleotide sequences in the ITR. This
These sequences must be at the ends of the genome to function as replication priming elements.
(Challberg and Kelly, 1989, Biochem., 58: 671-
717; Tamanoi and Stillman, 1983, Immunol. 109: 75-87).
As several groups have shown, ITRs can be expressed in the presence of complementary adenovirus function.
Is sufficient to confer replication on the heterologous DNA. Surround the terminal ITRs and their sides
Adenovirus "mi" consisting of short linear DNA fragments (in some cases, non-viral DNA)
`` Chromosomes '' are low levels in vivo in the presence of infectious wild-type adenovirus,
Or replicate at low levels in vitro in extracts prepared from infected cells
(Eg, Hay et al., 1984, J. Mol. Biol. 175: 493-510; Tamanoi et al.
And Stillman, 1983, Immunol. 109: 75-87).       [0078]   High expression of foreign genes in "replication defective" adenovirus (E1 region deleted)
Has been used for many years in laboratories. And various published reports are
Several different approaches often used in constructing these vectors
(Vernon et al., 1991, J. Gen. Virol., 72: 1243-1251; Wilkinson et al.
And Akrigg, 1992, Nuc. Acids Res., 20: 2233-2239; Eliot et al., 1990, J. Gen.
Virol., 71: 2425-2431; Johnson, 1991; Prevec et al., 1990, J. Infect. Dis., 16
1: 27-30; Haj-Ahmad and Graham, 1986, J. Virol., 57: 267-274; Lucito and
And Schneider, 1992, J. Virol., 66: 983-991; review by Graham and Prevec, 1992,
 Butterworth-Heinemann, 363-393). Generally, replication defective virus is required E1
For some or all of the in vitro direct ligation to the viral genome or intracellular in v
ivo by homologous recombination
(The methods are described in Berkner, 1992, Curr. Topics Micro. Immunol., 158
: 39-66). All of these methods transduce the E1 gene product.
Adenovirus vector that replicates in complementing cell lines such as 293 cells
Bring. Instead of the non-essential region E3 (eg, Haj-Ahmad and Graham, 1986, J
Virol., 57: 267-274), or between the right ITR and region E4 (Saito, 1985, J. Virol.
, 54: 711-719) Replicable adenovirus with inserted heterologous gene of interest.
Lus vectors have also been described. Replication defective and replicable viruses
In both cases, the heterologous gene of interest is the one in the recombinant adenovirus genome.
Incorporation into viral particles by packaging.       [0079]   The E1A gene is linked to any of the other viral genes immediately after viral infection (0-2 hours).
Is also expressed earlier. E1A protein is a trans-acting positive-acting transcriptional regulator
Acts as a nodal factor, and serves as a control for other early viral genes E1B, E2, E3, E4,
Required for expression of major late genes proximal to the lomotor. I'm also concerned with the nomenclature
However, the promoter proximal gene driven by the major late promoter is Ad5
It is expressed early after infection. Flint (1982) Biochem. Biophys. Acta 651: 1
75-208; Flint (1986) Advances Virus Research 31: 169-228; Grand (1987) B
iochem. J. 241: 25-38. Viral infection progresses in the absence of a functional E1A gene
Absent. This is because gene products required for viral DNA replication are not produced.
You. Nevins (1989) Adv. Virus Res. 31: 35-81. Ad5 E1A transcription initiation site
At nucleotide 498 of the lus genome, and the ATG start site of the E1A protein is
At nucleotide 560 of the genome.       [0080]   E1B protein functions in trans and transports late mRNA from the nucleus to the cytoplasm
Needed to work. Deficiency in E1B expression results in poor expression of late viral proteins
And an inability to block host cell protein synthesis. E1B promoter is Ad4
Has been used as an element to clarify the host range difference between 0 and Ad5: clinical
Ad40 is an enteric virus, while Ad5 causes acute conjunctivitis. Bailey et al.
(1993) Virology 193: 631; Bailey et al. (1994) Virology 202: 695-706. E1B Tampa
The virus overcomes the limitations imposed on viral replication by the host cell cycle
Also required for viruses to reduce the apoptotic effect of E1A
is there. Goodrum et al. (1997) J. Virology 71: 548-561. Ad5 E1B promoter is Sp1
And a single high affinity recognition site for the TATA box.       [0081]   The E2 region of adenovirus is a 72-kDa DNA binding protein, 80-kDa precursor terminal tag.
Adenovirus genome replication, including protein and viral DNA polymerase
Encodes a protein associated with The E2 region of Ad5 is called early E2 and late E2
Right from the two promoters located at 76.0 and 72.0 map units, respectively.
It is transferred in the direction. The late E2 promoter is transiently active late in infection and
And independent of the E1A transactivator protein, but
Is critical in the early stages of viral replication.       [0082]   The E2 early promoter, located at 27050-27150 of Ad5, has a major transcription start site and
Small transcription initiation site (the latter accounts for about 5% of E2 transcripts), two non-canonical TATA boxes
And two A2 transcription factor binding sites and one ATF transcription factor binding site. E2
For a detailed review of promoter structures, see Swaminathan et al., Curr. Topics in M.
icro. and Imm. (1995) 199 Part 3: 177-194.       [0083]   The E2 late promoter is the promoter of the gene encoded by the counterstrand.
It overlaps with the coding sequence, so that genetic manipulation is not possible. But,
The E2 early promoter contains a 33 kDa protein-encoding sequence
The base pairs only overlap. Notably, the SpeI restriction site (Ad5
, Position 27082) is part of the stop codon of the 33 kDa protein and has a major E2
Transcription factor binding site E2F upstream from the initial transcription initiation site and TATA binding protein site
And is conveniently separated from ATF. Therefore, Osteo with SpeI end
Insertion of the calcine transcriptional regulatory sequence into the SpeI restriction site of the first strand is responsible for the endogenous E2 of Ad5.
Disruption of the early promoter, resulting in osteocalcin
Must allow expression restricted by the protein.       [0084]   The E4 gene produces multiple transcripts. The E4 region is responsible for the replication of viral genomic DNA.
Two polypep involved in stimulating and stimulating late gene expression
Code the tide. Open reading frame (ORF) 3 and 6 proteins
Both products are 55-kDa protein from E1B and heterodimers E2F-1 and DP
These functions can be achieved by binding to -1. ORF 6 Tampa
Proteins require interaction with 55-kDa protein from E1B to become active
However, the ORF3 protein, on the other hand, does not need it. Machines from ORF 3 and ORF 6
In the absence of functional proteins, 10^-6Plaque with less efficiency
Generated. Cells that make osteocalcin transcription regulatory sequences function for viral replication
(Or osteocalcin-producing cells) by deleting ORFs 1-3 of E4
Makes viral DNA replication and late gene synthesis dependent on the E4 ORF6 protein
Can be. Such a vector is transformed into E1B by the osteocalcin transcription regulatory sequence.
By combining with sequences whose regions are regulated, both E1B and E4 functions
Can obtain viruses that depend on osteocalcin transcriptional regulatory sequences leading to E1B
it can.       [0085]   The major late genes involved in the present invention are L1, L2, L3, L4 and L5, which are Ad5
Encodes a viral virion protein. These genes (typically
All encoding structural proteins) are probably required for adenovirus replication
Is done. All of these late genes are major, located from about +5986 to about +6048 of Ad5
Under the control of the late promoter (MLP).       [0086]   In some embodiments, the osteocalcin transcription regulatory sequence functions
Replication ability can be achieved preferentially in target cells (osteocalcin-expressing cells, etc.)
Thus, the osteocalcin transcription regulatory sequence is associated with an adenovirus gene essential for growth.
Used for Preferably, the gene is an early gene such as E1A, E1B, E2 or E4.
I am a child. (As noted above, E3 is not essential for viral replication.)
Preferably, the early genes under the control of the osteocalcin transcription regulatory sequence are E1A and
And / or E1B. Combining two or more early genes into one osteocalcin transcription regulatory
Can be under column control. Example 1 provides a more detailed description of such a construct
I will provide a.       [0087]   In other embodiments, the cells that function osteocalcin transcriptional regulatory sequences (
Selective cytotoxicity due to preferential replication ability in osteocalcin expressing cells
And / or in addition to conferring cytolytic activity, the adenovirus of the invention
The vector contains a heterologous gene (trans) under the control of osteocalcin transcription regulatory sequences.
Gene). In this way, various genetic capabilities can be
The steocalcin transcription regulatory sequence can be introduced into target cells that function. So
Such cells include osteocalcin-expressing cells, particularly prostate cancer and cerebral tumors.
Tumors, ovarian cancer, thyroid cancer, various tumors, osteosarcoma, ocular melanoma, lung cancer or breast cancer
Vesicles and prostate cancer cells that do not express PSA or androgen receptor (AR),
Prostate stromal cells, perivascular cells, proliferative cancer-related osteoblasts and related bone stromal cells
And the like. For example, in certain cases, osteocalcin production targets
Due to the relatively refractory nature of the cells or special aggression, etc.
It may be desirable to increase the degree and / or speed. This is the above cell
Cytotoxic activity by specific replication can be assessed, for example, by HSV-tk and / or cytosine
Aminase (cd) [These cells express 5-fluorocytosine (5-FC)
Enables metabolism to Olouracil (5-FU)] in combination with cell-specific expression
Could be achieved by: These types of heterologous genes or trans
The use of genes can also provide a bystander effect.       [0088]   Other desirable transgenes that can be introduced by adenovirus vectors
May include cytotoxic proteins such as diphtheria toxin, ricin, or the A-chain of abrin.
Gene encoding protein [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. (1
992) 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) Mo. Cell. Biol. 9: 415], Apot.
Antisense transcript, a gene encoding a factor capable of initiating lysis
Or, it is a ribozyme-encoding sequence, especially for structural proteins.
Those directed to mRNAs encoding proteins or transcription factors essential for propagation,
A gene encoding a viral or other pathogenic protein, said gene comprising
That grows in cells, creatase (eg, RNase A) or protease (eg,
Ausin, papain, proteinase K, carboxypeptidase, etc.)
A gene encoding a genetically engineered cytoplasmic variant, or a Fas gene, etc.
Is included. Other genes of interest include IL-1, -2, -6
, -12, GM-CSF, G-CSF, M-CFS, IFN-α, -β, -γ, TNF-α, -β, NGF, etc.
It includes intrakines, antigens, transmembrane proteins, and the like. Positive effe in the early stages
Gene can be used to subsequently produce cytotoxic activity by replication.
Will be. In another embodiment, the adenovirus vector regulates heterologous transcription.
Any of the other genes essential for replication under sequence control (eg, E2 or
Are provided with E4, etc.).       [0089]   With respect to the packaging capacity of the adenovirus vector of the present invention,
If no virus sequence was deleted, the adenovirus vector would have
Package with inserted sequence up to about 5% of the program size, ie, up to about 1.8 kb
Can be performed. If non-essential sequences have been deleted from the viral genome, then 4.
6 kb insert can be accommodated (ie, a total of about 1.8 kb and 4.6 kb)
About 6.4 kb). Examples of non-essential adenovirus sequences that can be deleted include (E4
 E3 and E4 (as long as ORF6 is maintained).       [0090]   Any of the adenovirus vectors described herein can be replaced with a naked polynucleotide.
Used in a variety of forms, including but not limited to otide (usually DNA) constructs
be able to. Alternatively, adenovirus vectors can be used to facilitate cell entry.
Complex with other substances (cationic liposomes or other compounds such as polylysine, etc.)
Integrated polynucleotide construct; packaging into infectious adenovirus particles
Polynucleotide construct (which makes adenovirus vectors more immunogenic)
A polynucleate complexed with an agent that enhances or reduces the immune response
Reotide constructs; or agents that facilitate in vivo transfection (DO
TMA, DOTAP^TMAnd polyamines).
Can be achieved.       [0091]   Adenovirus vectors can be delivered to target cells in a variety of ways. So
These methods include, but are not limited to, liposomes, one of those well known in the art.
General transfection methods such as calcium phosphate precipitation, electroporation
Injection, direct injection, and intravenous infusion. Delivery means
Certain adenovirus vectors (including their morphology), as well as target cell types and
And where they are present (ie, whether the cells are in vitro or in vivo).
Depends on       [0092]   When used in the form of a packaged adenovirus, it must be
About 10 adenovirus vectors using the carrier^Four About 10 from PFU¹⁴ PF
It can be administered in U doses. Multiplicity of infection is generally about 0.001 PFU to 100 PFU
Will be within the range. Polynucleotide constructs (ie, packaged as a virus)
If not administered, approximately 0.01 μg to 1000 μg of adeno
A viral vector can be administered. Adenovirus vectors are intended
One or several doses, depending on the intended use and the host's potential for immune response.
Can be given. Alternatively, they can be administered as multiple simultaneous injections. Immunity
If a response is not desired, the species should be used to allow multiple doses without a strong immune response.
The use of various immunosuppressants can reduce the immune response.       [0093]   The present invention also provides a composition comprising an adenovirus vector described herein.
(Including pharmaceutical compositions). Such compositions can be used, for example, in an individual.
Useful for in vivo administration when measuring the degree of transduction and / or the effect of cell killing.
It is for. Preferably, these compositions further comprise a pharmaceutically acceptable excipient
. An effective amount of the adenovirus vector of the present invention is contained in a pharmaceutically acceptable excipient.
These compositions can be in unit dosage form, sterile parenteral solution or suspension, sterile
Non-parenteral solution or oral solution or suspension, oil-in-water or water-in-oil emulsion
It is suitable for systemic administration to individuals in the form of Parenteral or non-parenteral drug delivery
Formulations are known in the art and include those described in Remington's Pharmaceutical Scie
nces, 18th Edition, Mack Publishing (1990). The composition may also be freeze-thawed.
The adenovirus vector of the invention in unraveled and / or reconstituted form (additional
(Including those packaged as viruses).       [0094]   The present invention also includes a kit containing the adenovirus vector of the present invention. This
These kits can be used for diagnostic and / or monitoring purposes, preferably
Used for monitoring. Methods using these kits are available in clinical and experimental laboratories.
, A practitioner, or an individual. Kit embodied by the present invention
Regulates osteocalcin transcription in appropriate biological samples, such as biopsy specimens
Detecting the presence of cells that function the sequence (osteocalcin-producing cells, etc.)
Is possible.       [0095]   The kits of the present invention can be suitably packaged, as described in
Includes viral vectors. This kit may optionally include buffers, development reagents, and labels
Including reactive surfaces, detection means, control samples, instructions, and interpretive information.
Provide additional components useful for processing, including but not limited to
it can.       [0096]VII. Method of using the adenovirus vector of the present invention   The subject vectors can be used for a wide variety of purposes, and
It depends on the result. Therefore, the present invention provides the above-described adenovirus vector
The method of use is included.       [0097]   In one embodiment, the cells that function the osteocalcin transcription regulatory sequence (
Confer selective cytotoxicity in cells expressing, for example, osteocalcin
A method comprising contacting said cells with an adenovirus vector described herein.
A method is provided that includes touching. Cytotoxicity, dye exclusion, H-thymidine
Measured using standard assays in the industry, such as uptake and / or lysis.
Can be determined.       [0098]   In another embodiment, a cell that allows the osteocalcin transcription regulatory sequence to function (eg,
For example, adenovirus propagation method specific to osteocalcin-expressing cells)
Provided. These methods involve infecting cells with an adenovirus vector.
And the adenovirus multiplies by doing so.       [0099]   Another embodiment is to allow osteocalcin transcription regulatory sequences to function in a mixture of cells.
Cells that kill germ cells (eg, cells that express osteocalcin).
A method as described above comprising infecting a mixture of vesicles with an adenovirus vector of the invention
Is provided. A mixture of cells usually produces normal cells and osteocalcin
A mixture with cancer cells, which may be an in vivo mixture or an in vitro mixture.
Is also good.       [0100]   The invention also provides for osteocalcin transcription regulatory sequences in biological samples.
Includes a method for detecting cells to be activated (eg, cells expressing osteocalcin)
. These methods (in both experimental and clinical situations) can
Particularly useful for monitoring clinical and / or physiological symptoms of mammals)
It is. In one method, cells from a biological sample are transformed into an adenovirus vector.
Contact and detect replication of the adenovirus vector. Or, alternatively,
If the sample is reporter gene is under the control of osteocalcin transcription regulatory sequence
It can be contacted with an adenovirus. If the reporter gene is expressed,
Cells that function osteocalcin transcription regulatory sequences (eg, osteocalcin production)
Cells) are present. Reporter remains for use in the method of the invention
Non-limiting examples of genes include luciferase and β-galactosidase.
I can do it.       [0101]   Activation or reversal of transcription found in such osteocalcin producing cells
Increased transcription is defined as the function of the target cell (ie, osteocalcin transcription regulatory sequence
Cells such as prostate cancer cells, prostate stromal cells, perivascular cells, proliferative cancer-related bone
Blasts and osteocalcin are expressed but PSA or androgen receptor
(AR) does not express related bone stromal cells)
20-fold, more preferably at least about 50-fold, more preferably at least about 100
Times, even more preferably at least about 200 times, even more preferably at least
Also increases by about 400 to about 500 times, even more preferably at least about 1000 times more
Transfer. In some cases, increased transcription is
About 2 times, about 5 times, or about 10 times above the basal level in the body.       [0102]   Alternatively, the gene conditionally required for cell survival is osteocalcin.
Adenoviruses that are under the control of transcriptional regulatory sequences can be constructed.
This gene may for example code for antibiotic resistance. This Ade
Virus into a biological sample and then at certain time intervals,
And treat it with antibiotics. If viable cells expressing antibiotic resistance are present,
This indicates that there are cells in which the theocalcin transcription regulatory sequence functions. Suitable
A biological sample may contain osteocalcin-producing cells.
Something that seems to exist or exists. Usually, in mammals,
Suitable clinical samples include cancer cells that produce osteocalcin therein (eg,
Prostate cancer cells). Such cells are, for example,
It can be obtained by needle biopsy or any other suitable surgical procedure. Contact
The cells to be tested can be assay conditions (eg, selective enrichment and / or solubilization).
Can be treated to facilitate. In these methods, osteocalcic
Producer cells can be detected using in vitro assays to detect proliferation, and
Standard in the industry. Examples of such standard assays include, without limitation,
There is no, but a burst assay (measures virus production) and
And plaque assay (measuring infectious particles per cell). Also multiply
Can also be detected by measuring specific adenovirus DNA replication,
Is also a standard assay.       [0103]   The present invention also provides for contacting a target cell with an adenovirus vector described herein.
A method of altering the genotype of said target cell, said method comprising:
The above-described method is provided, wherein the sub-vector enters the cell.       [0104]   The invention further relates to a tumor cell (preferably a tumor cell expressing osteocalcin).
), Wherein the tumor cells and the non-tumor cells are treated with the adenovirus of the present invention.
The adenovirus vector into tumor cells upon contact with
Providing the above method, wherein the method comprises displaying selective cytotoxicity to cells.
You. Tumor cell growth can be assessed by any means known in the art,
For example, but not limited to, measuring the size of a tumor,^ThreeH-thymidine
Using an uptake assay to determine if the tumor cells are growing, or
Counting tumor cells. To “suppress” the growth of tumor cells means:
Means any one or all of the following conditions: slowing, delaying tumor growth, and
Stopping, as well as tumor shrinkage. "Inhibiting" tumor growth is defined herein as
Not contacted with the adenovirus vector described in the
(Not transfected with the Lus vector).
The growth state.       [0105]   The present invention also provides a method of reducing the level of a tumor cell marker in an individual.
And administering the adenovirus vector of the present invention to the individual.
Wherein the adenovirus vector is a tumor cell marker
-Selectively cytotoxic to producer cells. Limited tumor cell markers
Although not specified, includes PSA, hK2 and carcinoembryonic antigen. Tumor cells
The method of measuring the marker level is known to those of skill in the art and is not limited to
Immunological assays using antibodies specific for tumor cell markers (eg, enzyme
Epidemiological Assay (ELISA)). In general, test biological samples
Suitable assays (e.g.,
ELISA).       [0106]   The present invention also provides that an effective amount of an adenoviral vector described herein is administered to an individual.
Provided a method of treatment. Treatment with adenovirus vectors is promising
Adenocarcinoma, brain cancer, ovarian cancer, thyroid cancer, various tumors, osteosarcoma, ocular melanoma, lung cancer or
In individuals suffering from metastatic cancer, such as, but not limited to, breast cancer
Things. In addition, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, various tumors, osteosarcoma
Metastatic cancers such as, but not limited to, ocular melanoma, lung cancer and breast cancer
In individuals considered to be at risk of developing a disease associated with
, Such individuals include prostate cancer, brain cancer, ovarian cancer, thyroid cancer, various tumors,
Involvement of, but not limited to, osteosarcoma, ocular melanoma, lung cancer or breast cancer
Individuals undergoing resection for metastatic cancer-related disease and family history of those diseases
Certain individuals are mentioned. Adaptation of administering the adenovirus vector of the invention
Sex determination is particularly based on evaluable clinical parameters such as serologic indicators and tissue
Determined by biopsy histological experiments. Generally, in a pharmaceutically acceptable excipient
A pharmaceutical composition comprising the adenovirus vector is administered. The pharmaceutical composition is described above.
ing.       [0107]   The dose of the adenovirus vector will depend on the route of administration, the condition of the individual, and the invasiveness of the disease.
To the extent, the particular osteocalcin transcription regulatory sequence used, and the particular vector construction
(Ie, the adenovirus gene regulates osteocalcin transcriptional regulatory sequences)
Depending on a number of factors, such as the one below.       [0108]   When administered as an encapsulated adenovirus, approximately 10^FourPFU ~ 10¹⁴ PFU
, Preferably about 10^FourPFU ~ 10¹² PFU, more preferably about 10^FourPFU ~ 10^TenPFU
It is. The polynucleotide construct (ie, not encapsulated as a virus
) Can be administered at about 0.01 μg to about 100 μg, preferably
Preferably, it is 0.1 μg to about 500 μg, more preferably about 0.5 μg to about 200 μg. 2 or more
The above adenovirus vectors may be administered simultaneously or sequentially. Administration is typical
Typically, this is done at regular intervals while monitoring all responses. Administration
Can be performed, for example, intratumorally, intravenously or intraperitoneally.       [0109]   The adenoviral vectors of the present invention can be used alone or in other forms to enhance a desired purpose.
(Eg, chemotherapeutic agents).       [0110]   Thus, according to the present invention, when expressed, the growth of target tissue or tumor cells
Substances that can cause suppression, inhibition or destruction also include heterologous genes or transgenes.
Negative selectable marker provided as a gene, ie chemotherapeutic agent or interaction
Inhibits, blocks or destroys target cell growth in combination with an interaction agent
Substance.       [0111]   That is, when the negative selectable marker is introduced into the cell,
Administered to the host. The interacting agent interacts with the negative selectable marker to
Inhibits, suppresses, or destroys the growth of       [0112]   Negative selectable markers that can be used in the methods of the invention include, but are not limited to:
But not thymidine kinase and cytosine deaminase. One
In embodiments, the negative selectable marker is herpes simplex virus thymidine quina.
, Cytomegalovirus thymidine kinase and varicella-zoster virus
A viral thymidine kinase selected from the group consisting of
You. When using viral thymidine kinase, the interacting or chemotherapeutic agent
, Preferably nucleoside analogues, such as ganciclovir, acyclovir
And 1-2-deoxy-2-fluoro-β-D-arabinofuranosyl-5-iodouracil
(FIAU). Such interacting agents include
It is efficiently used as a substrate by viral thymidine kinase, and
An interacting agent such as the DNA of a tumor cell that expresses viral thymidine kinase
Lethal incorporation into the cell, causing its target cells to be killed.       [0113]   When cytosine deaminase is a negative selectable marker, a preferred interactant is
5-fluorocytosine. Cytosine deaminase converts 5-fluorocytosine to 5-
Converts to fluorouracil, which shows high cytotoxicity. Therefore,
The target cells expressing the endoaminase gene can be obtained by converting 5-fluorocytosine to 5-fluorocytosine.
Converted to uracil and killed.       [0114]   The interacting agent is administered in an amount effective to inhibit, arrest or destroy the growth of the target cell.
Given. For example, the interacting agent may affect the patient's weight and overall toxicity to the patient.
Is administered in an amount based on Interaction agents are preferably administered, e.g.
It is administered systemically by oral, intraperitoneal or intramuscular administration.       [0115]   The vector of the present invention induces a negative selection marker, and is used for tissue or tumor.
When administered in vivo to a patient, it has a "bystander effect", i.e.,
Cells that have not been transduced with the nucleic acid sequence encoding the
Administration of the agent may have the effect of causing killing. Scope of the invention
Is not limited for any theoretical reason, but is not
Vesicles act extracellularly on interacting agents or are taken up by neighboring non-target cells
May produce a diffusible form of a negative selectable marker, and
It becomes more susceptible to the action of an agent. In addition, one of the negative selectable marker and interactor
Alternatively, both can be transmitted between target cells.       [0116]   In one embodiment, results in inhibition, arrest or destruction of tumor cell growth
The substance is a cytokine. In one embodiment, the cytokine is
Leukin. Other cytokines that can be used include interferon and
Colony stimulating factors (eg, GM-CSF). As an interleukin,
Without limitation, interleukin-1, interleukin-1β and
Interleukin-2 to 15. In one embodiment, the interleak
Is interleukin-2.       [0117]   In one preferred embodiment of the invention, the target tissue is a metastatic cancer tissue, e.g.
For example, but not limited to, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, various tumors
Tumor, osteosarcoma, ocular melanoma, lung cancer, breast cancer. The virus can be found in tissues or
Are dispersed throughout the tumor mass. In another preferred embodiment, the target set
Weave is a cell that allows the osteocalcin transcription regulatory sequence to function, e.g.,
But not prostate cancer cells, prostate stromal cells, perivascular cells, proliferative cancer-related bone
Blasts and prostate cancer and non-PSA or androgen receptor (AR)
And related bone stromal cells. The virus spreads throughout a tissue or tumor mass
Distributed throughout.       [0118]VIII. Other embodiments of the present invention   In another embodiment, the invention further comprises a prostate, brain, ovarian, thyroid cancer
, Various tumors, osteosarcoma, lung cancer, breast cancer and diseases involving calcification (eg, limited
But not for the treatment of benign prostate hyperplasia (BPH) and atherosclerosis)
Use of the adenovirus compositions and methods of the present invention in conjunction with gene therapy
Including. A tissue-specific and tumor-specific promoter, such as shown in SEQ ID NO: 1
Osteocalcin promoter sequence or any other tissue-specific
Used to drive tissue-specific and tumor-specific expression of therapeutic molecules
And introduced into cancer cells. This method uses a tissue-specific promoter (eg, a sequence
Osteocalcin promoter sequence shown in No. 1)
Adenovirus vector constructed in
Another tissue-specific promoter operably linked to a nucleic acid encoding a therapeutic molecule
Cancer (including but not limited to prostate cancer, brain cancer, ovary)
Cancer, thyroid cancer, various tumors, osteosarcoma, lung cancer, and breast cancer) cells and stones
Diseases associated with incineration (eg, but not limited to benign prostatic hyperplasia (BPH)
And arteriosclerosis).       [0119]   Specifically, it is operably linked to a heterologous reporter gene (eg, LacZ).
Expression vector containing osteocalcin transcription regulatory sequence and its transcriptionally active fragment,
And host cells and transgenic animals containing such vectors are provided.
Provided. The invention also relates to prostate-related disorders and diseases associated with calcification (eg,
Jaw of benign prostate hyperplasia (BPH), cancer and atherosclerosis
Such a method for screening candidate molecules for nysts and antagonists
The present invention provides methods for using various vectors, cells and animals. Also this screening
Methods of using molecules and compounds identified by the assay for therapeutic treatment are also provided.
Provided.       [0120]   For example, but not limited to, compositions containing a reporter gene may be
It is operably linked to an ocarcin transcription regulatory sequence. Osteocalcin Promo
Tissue-specific promoters, such as reporter-driven reporter genes,
Expressed as a transgene. Transgenic animals as described above and such
Cells from the prostate of the transgenic animal have prostate-related damage and calcification
Compounds for Candidates Useful for Modulating Diseases Associated with
Can be used to Without being bound by any particular theory,
The compound may be a trans-acting factor (eg, a transcription factor), a cis-acting element (eg,
E.g. promoters and enhancers), and prostate related disorders and stones
The role of post-transcriptional, translational and post-translational compounds of all classes in diseases involving incineration
It seems to interfere with Noh. For that reason, they are such cancers and disorders
Is a promising candidate for the treatment of       [0121]   In one embodiment, the invention relates to prostate cancer, brain cancer, ovarian cancer, thyroid cancer, species
Various tumors, osteosarcoma, lung cancer, breast cancer and diseases associated with calcification (eg, but not limited to
But not the characteristics of genes in cells of benign prostate hyperplasia (BPH) and atherosclerosis)
High-throughput screening method for compounds that modulate constant expression
Offer. In this aspect of the invention, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, various
Tumors, osteosarcoma, lung cancer, breast cancer and diseases associated with calcifications (eg, but not limited to
Cells from benign prostate hyperplasia (BPH) and atherosclerosis)
Removed from the transgenic animal and cultured in vitro. Reporter gene expression
Used to monitor the activity of osteocalcin-specific genes. One
In certain embodiments, LacZ is a reporter gene. In another specific embodiment,
Luciferase is a reporter gene. Compound identified by this method
Further includes prostate cancer, brain cancer, ovarian cancer, thyroid cancer, various tumors, osteosarcoma, lung cancer,
Diseases associated with breast cancer and calcification in normal animals (eg, but not limited to
But their effects on benign prostatic hyperplasia (BPH) and atherosclerosis)
Can be tested.       [0122]   In another embodiment, the transgenic animal model of the invention comprises prostate cancer
Associated with brain cancer, ovarian cancer, thyroid cancer, various tumors, osteosarcoma, lung cancer, breast cancer and calcification
Diseases such as, but not limited to, benign prostatic hyperplasia (BPH) and
In vivo studies to determine the mechanism of action of candidate drugs on pulse sclerosis
Can be used for cleaning.       [0123]   Specifically, prostate cancer, brain cancer, ovarian cancer, thyroid cancer, various tumors, osteosarcoma
, Lung cancer, breast cancer and diseases associated with calcification (eg, but not limited to benign
The effect of the drug on prostate hyperplasia (BPH) and atherosclerosis can be assayed.       [0124]A. Polynucleotides and nucleic acids of the invention   The present invention relates to a 5 'regulatory region of an osteocalcin transcription regulatory sequence and its transcriptional activity.
Includes polynucleotide sequences including fragments. In particular, the present invention relates to SEQ ID NO: 1.
Polynucleotide containing osteocalcin promoter sequence and its transcriptionally active fragment
Provide Otide.       [0125]   The present invention further relates to a tissue-specific promoter (eg, the osteo-
Probes, primers and fragments of the calcine promoter sequence). 1
In one embodiment, the tissue-specific promoter (eg, osteocalcin regulation)
Sequence) of at least 8 nucleotides (ie, a hybridizable portion)
A purified nucleic acid comprising: In another embodiment, the nucleic acid is
A tissue-specific promoter (eg, the osteocalcin promoter shown in SEQ ID NO: 1)
Sequence) at least 20 (adjacent) nucleotides, 25 nucleotides, 50 nucleotides
Nucleotide, 100 nucleotides, 200 nucleotides or 500 nucleotides.
You. Using sequences known to those skilled in the art, these sequences can be isolated (in isolated form or generated).
(Contained in the current vector). These methods include, for example,
, In vitro recombinant DNA methods, synthetic methods, and in vivo genetic recombination. An example
See, for example, Sambrook et al., 1989 (supra) and Ausabel et al., 1989 (supra).
Please refer to the techniques that are available. In addition, “Oligonucleotide Synthesis”
Otide Synthesis) ”, 1984, edited by Gait M.J., IRL Press, Oxford (this is incorporated by reference.
, Incorporated herein in its entirety).       [0126]   In another embodiment, the nucleic acid is 20, 25, 35, 200 or 500 nucleotides in length.
It is smaller than Ochide. Nucleic acids can be single-stranded or double-stranded
. The invention also encompasses nucleic acids that are hybridizable or complementary to the above sequences.
I do. In certain embodiments, a tissue-specific promoter (eg, as set forth in SEQ ID NO: 1)
Osteocalcin promoter sequence) of at least 10, 20, 25, 50, 100, 200,
Nucleic acids are provided that comprise a sequence complementary to 500 nucleotides or the entire regulatory region.       [0127]   The tissue-specific promoter provided by the present invention (for example,
Probes, primers and fragments of the steocalcin promoter sequence
Can be used by a research institution. They are molecular weight markers in Southern gels.
-To identify chromosomes or map the location of related genes
As a chromosome marker or tag (if appropriately labeled) for
To compare potential DNA sequences to identify potential hereditary disorders;
A probe for finding new related DNA sequences by bridging
To derive PCR primers for gene fingerprinting
As a source of information; and in the process of finding other novel polynucleotides.
It can be used as a probe to "subtract-out" a known sequence. Up
The manner of carrying out the uses mentioned above is known to those skilled in the art. Disclosure such a method
References include, but are not limited to, "Molecular Cloning: A Lab
oratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Sambrook, J.
, E.F.Fritsch and T. Maniatis, eds., 1989 and Methods in Enzymology: Gu
ide to Molecular Cloning Techniques, '' Academic Press, Berger, S.L.
And A.R. Kimmel, 1987.       [0128]   The nucleotide sequence of the present invention also comprises the osteocalcin promoter set forth in SEQ ID NO: 1.
At least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98
%, A nucleotide sequence having 99% or more nucleotide sequence identity,
And / or transcriptionally active fragments thereof.       [0129]   Optimal for determining% identity between two amino acid sequences or between two nucleic acids
Align those sequences for comparison purposes (eg, the first amino acid
Or for nucleic acid sequences, optimal alignment with the second amino acid or nucleic acid sequence
Can introduce a gap). Next, the corresponding amino acid position or
Compare the amino acid residue or nucleotide at the position of the leotide. First array
The position of the same amino acid residue or nucleotide as the corresponding position in the second sequence
When occupied by a tide, the molecules are said to be identical at that position.
The% identity between two sequences is the number of identical positions common to the sequences.
Function (ie,% identity = number of duplicate locations that are identical / total number of locations × 100)
). In one embodiment, the two sequences are the same length.       [0130]   The determination of% identity between two sequences can also be achieved using a mathematical algorithm.
it can. One preferred non-linear algorithm of the mathematical algorithm used to compare two sequences
Non-limiting examples are described in Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:
The algorithm of 2264-2268 is described in Karlin and Altschul (1993) Proc.
 Sci. USA 90: 5873-5877 with modifications. Such al
The algorithm is described in NBLAST and XB in Altschul et al., (1990) J. Mol. Biol. 215: 403-410.
Built into the LAST program. BLAST nucleotide search using NBLAST program
, A score of 100 and a word length of 12 yields a sequence homologous to the nucleic acid molecule of the present invention.
A nucleotide sequence can be obtained. XBLAST program for BLAST protein search
, Score = 50, wordlength = 3, homologous to the protein molecule of the present invention.
An amino acid sequence can be obtained. Obtain gapped alignments for comparison purposes
For this, see Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402.
Gapped BLAST can be used. Alternatively, PSI-Blast
Can be used to perform an iterated search to detect distance relationships between molecules.
And can be (ibid.). Use BLAST, Gapped BLAST and PSI-Blast programs
Default parameters for each program (for example, XBLAST and
NBLAST) can be used (see http://www.ncbi.nlm.nih.gov). Array
Another preferred, non-limiting example of a mathematical algorithm used to compare one another is My
ers and Miller, (1988) CABIOS 4: 11-17 algorithm. Such a
The algorithm uses the ALIGN program (version 2.0) (GCG sequence alignment software).
Software package). AL for comparing amino acid sequences
When utilizing the IGN program, PAM120 weight residue table, 1
A gap length penalty of 2 and a gap penalty of 4 can be used. Another fruit
In an embodiment, the alignment uses the NA-MULTIPLE-ALIGNMENT 1.0 program.
Gap Weight of 5 and Gap Length of 1
 Weight).       [0131]   The% identity between the two sequences can be determined in a manner similar to that described above (gap
May or may not be attached). By calculating% identity
Typically only counts exact matches.       [0132]   The invention also includes:   (A) the above-described tissue-specific promoter (for example, osteocalcin represented by SEQ ID NO: 1)
Syn promoter sequence) and / or its complement (ie, antisense)
A DNA vector containing any of the above;   (B) operably linked to a heterologous gene (eg, a reporter gene)
The tissue-specific promoter (for example, osteocalcin promoter shown in SEQ ID NO: 1)
A DNA expression vector comprising any of the following:   (C) a host cell engineered by genetic engineering, which can function for a heterologous gene;
The tissue-specific promoter (for example, the osteotype
Any of the tissue-specific promoters (eg,
Osteocalcin promoter element) in the host cell.
Such a host cell, wherein the host cell comprises to direct expression of the offspring.       [0133]   Also, various transcriptionally active fragments of this regulatory region are included in the scope of the present invention.
The tissue-specific promoter according to the present invention (for example, osteocalcin
Transcriptional activity (transcriptionally active) or transcriptionally functional (transcriptionally
A fragment) is a recombinant polypeptide or recombinant polypeptide in a recombinant host cell.
Such a polynucleotide, which is functional as a regulatory region for expressing a recombinant polynucleotide,
Refers to a polynucleotide containing a fragment of a nucleotide. For the purposes of the present invention, the core
The acid or polynucleotide is a recombinant polypeptide or a polynucleotide.
It can be said that “transcriptional activity” is a regulatory region for expressing
The oligonucleotide contains a nucleotide sequence containing transcriptional information, and
The nucleotide sequence encoding the polypeptide of interest or the polynucleotide of interest.
This is the case where it is operably linked to the oligonucleotide.       [0134]   In particular, the tissue-specific promoter of the present invention (for example, osteoca shown in SEQ ID NO: 1)
A transcriptionally active fragment of a rucine regulatory sequence may be a tissue-specific promoter (eg, SEQ ID NO:
Prostate cancer cells operably linked to the osteocalcin regulatory sequence shown in 1)
When transfected into a strain, a heterologous gene (eg, a reporter gene)
Includes fragments long enough to promote transcription. Typically, this regulatory region is
Located 5 'of the coding sequence and operably linked to the coding gene.
It is. The term "operably linked" as used herein,
Placing regulatory sequences 5 'of the reporter gene (upstream) and required for transcription initiation
Trans-acting factors such as transcription factors, polymerase subunits and
Sesary protein) is assembled in this region to form the RNA
Refers to enabling the initiation of remelase-dependent transcription.       [0135]   In one embodiment, selected to serve as a tissue-specific transcriptional regulatory sequence
The polynucleotide sequence is the nucleotide sequence of the osteocalcin regulatory sequence shown in SEQ ID NO: 1.
In addition to the reotide sequence, it can further include other nucleotide sequences. That
Such tissue-specific transcription regulatory sequences include, but are not limited to, α
-Fetoprotein, PSA, DF3, Tyrosinase, CEA, Surfactant protein
Quality and the ErbB2 promoter. In a particularly preferred embodiment
However, additional tissue-specific transcription regulatory sequences include, but are not limited to, for example,
However, PSA promoter, prostate specific enhancer (PSE), superPSE promoter
No. 5,998,205, the entire contents of which are incorporated herein by reference.
Modified artificial α-fetoprotein promoter sequence described in
allebbeck et al. (Hallenbeck et al., 1999, Human Gene Therapy 10: 1721-1733; see
The entire contents of which are incorporated herein by reference).
-Fetoprotein promoter sequence, and Nakabayashi et al. (Mol. Cell. Biol.
 1991 11 (12): 5885-93; the entire contents of which are incorporated herein by reference).
Mention may be made of the modified artificial α-fetoprotein promoter described.
it can.       [0136]   In yet another embodiment, the osteocalcin promoter set forth in SEQ ID NO: 1
-Multiple copies of the sequence or a fragment thereof can be linked together. For example
The osteocalcin promoter sequence shown in SEQ ID NO: 1 or a fragment thereof
Locomotor sequence, or another fragment thereof, with a head-to-tail, head-to-head or tail-
Can be connected in the direction of the tail. Thus, in another embodiment, as an example (but not limited)
Prostate cell-specific enhancer is a tissue-specific promoter.
(For example, the osteocalcin promoter sequence shown in SEQ ID NO: 1) or its
And a tissue-specific osteocalcin shown in SEQ ID NO: 1
Can be used to promote transcription from constructs containing the promoter sequence.       [0137]   The scope of the present invention also includes the osteocalcin promoter sequence shown in SEQ ID NO: 1.
Also included are variants of a row that do not substantially affect its transcriptional activity. That's it
Such modifications include adducts, deletions and substitutions. In addition, the string
Complementation of the osteocalcin promoter sequence shown in SEQ ID NO: 1 under gentle conditions
Genes that selectively hybridize to the body and are essential for adenovirus replication
Any nucleotide sequence capable of activating the expression of is encompassed by the present invention. Typical
Moderately stringent hybridization conditions are as follows:
: Pre-hybridization of the filter containing DNA for 8 hours to overnight
6 × SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP, 0.02%
Run in a buffer consisting of 2% Ficoll, 0.02% BSA and 500 μg / ml denatured salmon sperm DNA.
U. The filters were filtered at 65 ° C. for 48 hours at 100 μg / ml denatured salmon sperm DNA and
5-20 × 10⁶cpm³²In a prehybridization mixture containing a P-labeled probe
To hybridize. Washing of filters was carried out at 37 ° C. for 1 hour with 2 ×
Performed in a solution containing SSC, 0.01% PVP, 0.01% Ficoll and 0.01% BSA. continue
After washing in 0.1 × SSC at 50 ° C. for 45 minutes, autoradiography was performed.
You. Alternatively, representative conditions for high stringency are as follows:
For example, hybridization to filter-bound DNA is performed using 0.5M NaHPO._Four, 7%
Performed in sodium dodecyl sulfate (SDS), 1 mM EDTA at 65 ° C, 0.1 x SSC / 0.1
Wash at 68 ° C. in% SDS (Ausubel F.M. et al., Eds., 1989, Current Protocols in
Molecular Biology, Volume I, Green Publishing Associates, Inc., and John
 Wiley & sons, Inc., New York, page 2.10.3). Other high stringency available
Sea conditions are well known in the art. Generally, pros of 14-70 nucleotides in length
For tubes, the melting temperature (TM) is calculated using the following equation: Tm (° C.) = 81.5 + 16.6
(log [monovalent cation (mol)]) + 0.41 (% G + C) − (500 / N) (where N is the length of the probe
Is). When hybridization is performed in a solution containing formamide
The melting temperature is calculated using the following formula: Tm (° C.) = 81.5 + 16.6 (log [monovalent cation
(Mol)] + 0.41 (% G + C) − (0.61% formamide) − (500 / N) (where N is a probe
Length). Generally, hybridization is about 20-25 ° C. below Tm
Temperature (for DNA-DNA hybrids) or 10-15 ° C below Tm (RNA-D
In the case of NA hybrid).       [0138]   A tissue-specific promoter such as the osteocalcin promoter sequence shown in SEQ ID NO: 1.
The motor, or a transcriptionally functional fragment thereof, is preferably derived from a mammalian organism
I do. In one embodiment, the osteocalcin promoter sequence is human,
It is derived from mouse or rat. In another embodiment, osteocalcin pro
The motor sequence is that shown in SEQ ID NO: 1 or a transcriptionally functional fragment thereof.
You. Various screening methods based on nucleic acid hybridization
To isolate the gene sequence from the product. The isolated as disclosed herein
The polynucleotide sequence or a fragment thereof is labeled, and
CDNA line constructed from mRNA obtained from various cells or tissues (eg, prostate tissue)
Can be used to screen a library. Hybridization used
The conditions for the cDNA library production are different from the type of organism from which the labeled sequence is derived.
If derived from a product, it should be of lower stringency. Low
Stringency conditions are known to those of skill in the art, and libraries and
It depends on the specific species from which it is derived. Guidance on such conditions includes:
For example, Sambrook et al., 1989, Molecular Cloning, A Laboratory Manual, 2nd edition,
 Cold Spring Harbor Press, N.Y. and Ausabel et al., 1989, Current Protocols
in Molecular Biology, Green Publishing Associates and Wiley Interscience
, N.Y., each of which is incorporated herein by reference in its entirety.
Can be done).       [0139]   In addition, mammalian osteocalcin transcription regulatory sequence homologs can be found, for example, in bovine
Or from another non-human nucleic acid, the male of SEQ ID NO: 1 disclosed herein.
Two kinds of designed based on the nucleotide sequence of the theeocalcin promoter sequence
By performing polymerase chain reaction (PCR) amplification using a primer pool
Can be isolated. For example, osteocalcin can be expressed as a template for this reaction.
MRNA prepared from a known bovine or other non-human cell line or tissue
Can be used. About such conditions
For guidance, see, for example, Innis et al. (Eds.), 1995, PCR Strategies, Academic Pres.
s Inc., San Diego; and Erlich (eds.), 1992, PCR Technology, Oxford Univ.
ersity Press, New York (each of which is incorporated by reference in its entirety)
Incorporated herein).       [0140]   5 'of a tissue-specific promoter (e.g., osteocalcin promoter sequence)
The promoter sequence in the non-coding region may be as shown in SEQ ID NO: 1.
Yes, digestion with exonuclease III or appropriate restriction endonuclease
Nested 5 'and / or 3' deletions may be constructed using conventional techniques such as
And may be further defined by: The resulting deletion fragment is used as a promoter reporter vector.
Into the cell, for example according to Coles et al. (Hum. Mol. Genet., 7: 791-800, 1998).
As described, the deletion reduces or abolishes promoter activity.
Can be determined. In this way, the boundaries of the promoter
Can be defined. If desired, individual controls within the promoter
Nodal sites are identified using site-directed mutagenesis or linker scanning,
Take the potential transcription factor binding sites within the promoter individually or in combination.
May be removed. The effect of these mutations on transcription levels is
Checked by inserting the mutation into the cloning site of the reporter vector
Can be These types of assays are known to those skilled in the art (WO 97/173
59, U.S. Patent No.5,374,544, European Patent No.582796, U.S. Patent No.5,698,389, U.S.A.
(US Pat. No. 5,643,746, US Pat. No. 5,502,176 and US Pat. No. 5,266,488)
.       [0141]   Tissue-specific promoters, such as the osteocalcin promoter sequence,
No. 1 or a transcriptionally functional fragment thereof.
Helps identify tissue-specific promoters such as the calcine promoter sequence
The fragments and probes described herein include those shown in SEQ ID NO: 1 and
And fragments thereof, and can be made by recombinant DNA methods using techniques known in the art.
Can be made. These sequences can be isolated in isolated form or using methods known to those of skill in the art.
Can be constructed by being included in an expression vector. These methods include, for example, in vit
ro recombinant DNA methods, synthetic methods and in vivo genetic recombination. For example, Samb
See the techniques described in rook et al., 1989, supra, and Ausabel et al., 1989, supra.
I want to be illuminated. Also, Oligonucleotide Synthesis, 1984, edited by Gait M.J., IRL
Press, Oxford, which is incorporated herein by reference in its entirety.
See also the techniques used.       [0142]   Variants of the above regulatory sequences can be obtained by various chemical and enzymatic methods known to those skilled in the art.
It can be manufactured using a method. For example, the region of the sequence defined by the restriction site may be deleted.
Can be made. Defined formats using oligonucleotide-directed mutagenesis
Modify the sequence and / or introduce a restriction site in a specific region within the sequence
be able to. In addition, deletion mutants can be Bal31, ExoIII or S1 nuclei.
It can be prepared using a DNA nuclease such as zeolites. Insert the DNA with nuclease
Increasing the incubation time may create larger deletions in the regulatory sequences.
(See, eg, Ausubel et al., 1989, supra).       [0143]   The modified sequences will have their ability to direct expression of the heterologous coding sequence in a suitable host.
Evaluate power. Any that retains the ability to direct the expression of a coding sequence
The modified regulatory sequence may be incorporated into a recombinant expression vector for further use.
Is within the scope of the present invention.       [0144]IX. Analysis of osteocalcin-specific promoter activity   A tissue-specific promoter such as the osteocalcin promoter sequence shown in SEQ ID NO: 1.
The motor exhibits selective tissue and cell type specificity. That is, it
, Prostate cancer cells, prostate stromal cells, perivascular cells, proliferative cancer-related osteoblasts, and
And related bone stromal cells that do not express PSA or androgen receptor (AR)
To induce gene expression. Therefore, the osteocal according to the present invention shown in SEQ ID NO: 1
Prostate cancer cells, prostate
Stromal cells, perivascular cells, proliferative cancer-associated osteoblasts, and PSA or andro
Expression of heterologous coding sequences in related bone stromal cells that do not express the gen receptor (AR)
Can be induced. In one embodiment, the invention relates to a set of target genes.
A tissue-specific promoter (eg, SEQ ID NO: 1) to achieve tissue-specific expression
Osteocalcin promoter sequence shown). Shown in SEQ ID NO: 1
The activity of tissue-specific promoters such as the osteocalcin promoter sequence and
Specificity depends on the tissue-specific promoter in various types of cells and tissues.
(Eg, osteocalcin promoter sequence shown in SEQ ID NO: 1)
Monitoring the expression level of the detectable polynucleotide linked by
More can be evaluated. As described below, this detectable polynucleotide and
Is a polynucleotide that specifically hybridizes with a given oligonucleotide probe.
Polynucleotides which may be leotide or encode a detectable protein
It may be leotide.       [0145]X. Osteocalcin transcription regulatory sequence driven reporter construct   The osteocalcin transcription regulatory sequence according to the present invention is advantageously used to provide the desired host cells.
Used to express the coding sequence or reporter gene in the vesicle or host organism.
It can be part of a usable recombinant expression vector. Osteocal of the present invention
Syn transcription regulatory sequences and their transcriptionally active fragments direct the expression of heterologous coding sequences
Can be used for In particular, the present invention provides a mammalian osteocalcin transcription regulatory sequence.
Include. According to the present invention, the transcriptionally active fragment of the osteocalcin transcription regulatory sequence,
Promotes transcription of a reporter coding sequence to which the fragment is operably linked
A fragment of a region that is long enough to       [0146]   Various reporter gene sequences known to those of skill in the art are available, for example,
However, fluorescent proteins such as green fluorescent protein (GFP), enzymes (
CAT, β-galactosidase, luciferase) or antigenic markers
Genes to be loaded. For convenience, the screening assays of the present invention include
Enzymatic reporter analyzed by colorimetric or fluorometric assay
And light emitting reporters are preferred.       [0147]   In one embodiment, for example, bioluminescent, chemiluminescent or fluorescent
Can be used as the light-emitting reporter in the present invention. Substrates and
Types of light-emitting reporters that do not require cofactors include, but are not limited to:
Not available, but the wild-type green fluorescent protein (GFP) of O jellyfish (Victoria aequoria)
) (Chalfie et al., 1994, Science 263: 802-805) and modified GFP (Heim et al., 1995,
 Nature 373: 663-4; PCT Publication WO96 / 23810). This type of reporter
-When the gene is transcribed and translated, the fluorescent protein accumulates in the test cells,
It is analyzed by a fluorescence analyzer or flow cytometer, for example, as is known in the art.
Methods (e.g., Lackowicz, 1983, Principles of Fluorescence Spectroscopy,
Plenum Press, New York).       [0148]   Another type of reporter gene that can be used requires a cofactor to emit light
For example, but not limited to, Renilla mushroom (Renilla
) Luciferase. Other sources of luciferase are publicly available in the industry.
Knowledge, but not limited to, of Vibrio harveyi
Bacterial luciferase (luxAB gene product) (Karp, 1989, Biochim. Biophys. A
cta 1007: 84-90; Stewart et al., 1992, J. Gen. Microbiol., 138: 1289-1300) and
Luciferase from Photinus pyralis (De Wet et al., 1987, Mol. C)
ell. Biol. 7: 725-737), which can be assayed for light generation.
Good (Miyamoto et al., 1987, J. Bacteriol. 169: 247-253; Loessner et al., 1996, Env
iron, Microbiol.62: 1133-1140; and Schultz & Yarus, 1990, J. Bacteriol
172: 595-602).       [0149]   Reporter genes that can be analyzed using colorimetric analysis are limited.
But not β-galactosidase (Nolan et al., 1988, Proc. Natl. Acad. Sci. USA
85: 2603-07), β-glucuronidase (Roberts et al., 1989, Curr. Genet.Fifteen: 177
-180), luciferase (Miyamoto et al., 1987, J. Bacteriol. 169: 247-253)
Or β-lactamase. In one embodiment, the reporter genetics
The nucleotide sequence is a nucleotide sequence encoding β-galactosidase, a product of the LacZ gene.
Includes code array. Because this enzyme is very stable and has a wide range of specificities,
It allows the use of different histochemical, chromogenic or fluorescent substrates.
Examples of such substrates include, but are not limited to, 5-bromo-4-chloro-
3-Indoyl-β-D-galactoside (X-gal), lactose 2,3,5-triphenyl-2H
-Tetrazolium (lactose-tetrazolium) and fluorescein galacto
Pyranoside (see Nolan et al., 1988, supra).       [0150]   In another embodiment, the product of the E. coli β-glucuronidase gene (GUS) is liposome
(Roberts et al., 1989, Curr. Genet.Fifteen: 177-180)
. GUS activity is determined by X-glucuronide (Xgluc) and 4-methylumbelliferyl glu
It can be detected by various histochemical and fluorescent forming substrates such as clonides.       [0151]   Reporter gene sequences as described above, which provide a simple colorimetric response
In addition, other reporter gene sequences, such as a selectable reporter gene sequence,
Can be used for routines. For example, chloramphenicol acetyl transferase
(CAT) coding sequence is available and is dependent on the osteocalcin transcription regulatory sequence.
Causes the proliferation of cells that are resistant to chloramphenicol. Use and selection of CAT
The advantages of possible reporter genes are known to those skilled in the art (Eikmanns et al., 1991, Ge
ne 102: 93-98). Other selectable reporter gene sequences are also available and limited
But not zeocin resistance (Hegedus et al., 1998, gene 207: 241-249).
Or kanamycin resistance (Friedrich & Soriano, 1991, Genes. Dev. 5: 1513-1523
)).       [0152]   Other reporter genes, such as toxic gene products, potentially toxic genes
Gene products as well as antiproliferative or antiproliferative gene products can also be used. another
In embodiments, the detectable reporter polynucleotide is a given oligonucleotide.
Even for polynucleotides that specifically hybridize with nucleotide probes
Or a polynucleotide encoding a detectable protein.
No. This type of assay is known to those skilled in the art (US Pat. No. 5,502,176).
And U.S. Patent No. 5,266,488).       [0153]   The osteocalcin transcription regulatory sequence driven reporter construct is a standard recombinant DN
A [Methods in Enzymology, 1987, Vol. 154, Aca
demic Press; Sambrook et al., 1989, Molecular Cloning-A Laboratory Manual,
Second Edition, Cold Spring Harbor Press, New York; and Ausubel et al., Current Pro.
tocols in Molecular Biology, Greene Publishing Associates and Wiley Inte
rscience, New York (each of which is incorporated herein by reference in its entirety.
See).       [0154]   Assays for promoter activity are known to those of skill in the art (eg, Sambrook
Et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory
, Cold Spring Harbor, NY, 1989). As an example of a typical method that can be used
Preserves sequences derived from the reporter gene and osteocalcin transcription regulatory sequence.
And a recombinant vector having the same. Briefly, reporter genes (eg,
Green fluorescent protein, luciferase, β-galactosidase or chloram
Phenicol acetyltransferase) is expressed in biologically active polynucleotides.
Detected when placed under control of a nucleotide fragment. Osteocalcin gene
The genomic sequence located upstream of exon 1 of any suitable promoter
It can also be cloned into a porter vector. For example, manipulating many commercially available vectors
Osteocalcin conversion of the present invention to produce and express in a mammalian host cell.
Photoregulatory sequences can be inserted. Non-limiting of such vectors
Examples are pSAPBasic, pSEAP-Enhancer, pβgal-Basic, pβgal-Enhancer or
pEGFP-1 Promoter Reporter vector (Clontech, Palo Alto, CA) or pGL2
-basic or pGL3-basic promoterless luciferase reporter gene
Vector (Promega, Madison, WI). These promoter reporters
Each of the proteins contains a readily assayable protein (eg, a secreted alkaline host).
Phatase, green fluorescent protein, luciferase or β-galactosidase
) Contains a multiple cloning site upstream of the reporter gene
It is. The osteocalcin transcription regulatory sequence of the osteocalcin gene is
Inserted in both orientations into the cloning site upstream of the
Introduced into cells. Assay and clone reporter protein levels
Compare to the level obtained in the vector without insert at the site. Inn
The expression level in the vector containing the salt is increased compared to the control vector.
Indicates that a promoter or a functional fragment thereof is present in the insert.
Be inspired.       [0155]   An expression vector containing the osteocalcin transcription regulatory sequence may further comprise a selection marker.
May be included. Many selection systems are available and limited
Although not required, herpes simplex virus thymidine kinase (Wigler et al., 19
77, Cell 11: 223), hypoxanthine-guanine phosphoribosyl transferase
(Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026)
And adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:81
7) genes, each of which is tk⁻, Hgprt⁻Or aprt⁻The cells
Can be used in In addition, anti-metabolite resistance is dhfr (resistance to methotrexate).
(Wigler et al., 1980, Proc. Natl. Acad. Sci. USA 77: 3567; O'H.
are et al., 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpr (mycophenolic acid)
(Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. U
SA 78: 2072); neo (conferring resistance to aminoglycoside G-418) (Colberr
e-Garapin et al., 1981, J. Mol. Biol. 150: 1); and hygro (hygromycin).
(Stererre et al., 1984, Gene 30: 147)
Can be used as a basis for selection. Yet another selectable gene is trpB (fine
HisD (allows cells to use indole instead of tryptophan);
Histidine can be used instead of histidine) (Hartman &
Mulligan, 1988, Proc. Natl. Acad. Sci. USA 85: 8047); ODC (ornithine
Carboxylase) (2- (di-ortinine decarboxylase inhibitor)
(Confers resistance to fluoromethyl) -DL-ornithine (DFMO)) (McConlogu
e L., 1987, Current Communications in Molecular Biology, Cold Spring Har
bor Laboratory) and glutamine synthetase (Bebbington et al., 1992, Bi
otech 10: 169).       [0156]XI. Characterization of the transcriptional activity regulatory fragment of the osteocalcin transcription regulatory sequence   Fusion constructs containing osteocalcin transcriptional regulatory sequences or fragments thereof will
Can be assayed for For promoter analysis of osteocalcin transcriptional regulatory sequences
The first step in the process is to initiate transcription of the target osteocalcin transcriptional regulatory sequence
The point (+1 position) is determined by standard methods (Sambrook et al., 1989, Molecular Cloning: A Lab
oratory Manual, Cold Spring Harbor, Cold Spring Harbor Press)
Must be determined using a primer extension assay and / or an RNAase protection assay
I have to. The DNA sequence upstream of the +1 position is a gene involved in gene regulation.
It is generally considered to be the motor domain. However, the downstream sequence (intro
(Including sequences within the sequence) may also be involved in gene regulation. promoter
In conducting an activity test, a region of -3 kb to +3 kb (+1 is the transcription initiation
Dot) can be cloned upstream of the reporter gene coding region. Previous
To facilitate the identification of the regions involved in gonad-specific expression, 5 '
And / or two or more alternative reporter gene constructs containing 3 'truncated variants.
It may be produced. The type of the reporter gene is selected depending on the use.       [0157]   In one preferred embodiment, a GFP reporter gene construct is used. Previous
In studies of gonad-specific gene promoters, green fluorescent protein was used as a reporter.
It is particularly useful to use quality (GFP). Of using GFP as a reporter
The main advantage is that GFP does not require a substrate, and in newly isolated prostate cells
Lies in the fact that it can be detected.       [0158]   In another embodiment of the invention, a LacZ reporter construct is used. LacZ gene
The product β-galactosidase is very stable and has a wide range of properties.
, Various histochemical, chromogenic or fluorescent substrates, such as, but not limited to
But not 5-bromo-4-chloro-3-indoyl-β-D-galactoside (X-gal),
Lactose 2,3,5-triphenyl-2H-tetrazolium (lactose-tetrazolium
) And fluorescein galactopyranoside are available (
Nolan et al., 1988, 1988, supra).       [0159]   In the case of promoter analysis in transgenic mice,
GFP whose expression in the cell is optimized is preferred. Promoterless cloning
The vector pEGFP1 (Clontech, Palo Alto, CA) has a long wavelength shift mutation of wild-type GFP.
Body (to provide brighter fluorescence and higher expression in mammalian cells)
(Cormack et al., 1996, Gene 173: 33; Haas et al.,
1996, Curr. Biol. 6: 315). Furthermore, the maximum excitation peak of this enhanced GFP (EGFP)
Is at 488 nm and has fluorescein isothiocyanate (FITC) optics (450-50
GFP fluorescence using commonly used filter sets (illuminated at 0 nm)
Can be visualized. pEGFP1 is a promoter component in transgenic mice.
Has been found to be useful as a reporter vector for analysis (Okabe et al., 199).
7, FEBS Lett. 407: 313). In another embodiment, the osteocalcin transcription regulatory sequence
Contains a transgene upstream of the LacZ or luciferase reporter gene.
For example, transgenic mice are used.       [0160]   Putative promoter fragments can be obtained using methods known in the art for cloning.
(Usually a parent file containing 8 to 10 kb of genomic DNA including the promoter region).
Prepared from a large clone). However, the feasibility of this method is within the regulatory fragment
Depends on the availability of appropriate restriction endonuclease sites. One
In a preferred embodiment, the required promoter fragment is a polymerase chain.
Restriction endonuclease by strand reaction (PCR; Saiki et al., 1988, Science 239: 487)
Amplification using oligonucleotide primers containing appropriate sites for cleavage
Is done. The sequence required for restriction cleavage is adjacent to the regulatory fragment to be amplified
Included at the 5 'end of the forward and reverse primers. PCR amplification
Afterwards, restriction digestion of the PCR product gives the appropriate termini. Either way
The generated promoter fragment is then subjected to standard cloning procedures (Sambrook
1989, supra)) and linked to the multiple cloning site of the reporter vector.
Is tied. Prior to transgenic animal production, PCR
It is recommended to confirm the DNA sequence of the promoter fragment obtained. Thus obtained
The reporter gene construct to be generated is a reporter gene such as GFP or lacZ cDNA.
Putative promoter fragment located upstream of the gene's open reading frame
Contains.       [0161]XII. Analysis of osteocalcin transcription regulatory sequence using transgenic mouse   Transgenic animals using mammalian osteocalcin transcriptional regulatory sequences
Expression, especially reporter coding sequence, homologous or heterologous gene expression
, Can be ordered. For the production of transgenic animals,
Animals can also be used, including, but not limited to, mice, rats, rabbits,
Guinea pigs, pigs, mini-pigs, goats, sheep, and non-human primates (eg,
Baboons, monkeys and chimpanzees). As used herein
The term "transgenic" refers to a heterologous osteocalcin transcription regulatory sequence.
Non-human animals that express (eg, rat or human osteocalcin genetics)
Mouse expressing osteocalcin transcription regulatory sequence from offspring), and endogenous (
Osteocalcin transcription regulatory sequence
Animals that have been engineered or that have knocked out certain sequences
An animal that has been genetically engineered.       [0162]   In one embodiment, the present invention relates to an osteocalcin transcription regulatory sequence or the same.
Transgenes (such as reporter genes) under the control of
Transgenic animals containing (in all cells) and (in all cells)
Animals that contain the transgene in some cells, ie, mosaic animals
I will provide a. Transgenes can be single transgenes or concatenations.
(Eg, head-to-head or head-to-tail). Transge
Also described, for example, in Lasko et al. (1992, Proc. Natl. Acad. Sci. USA
89: 6232-6236) and selectively introduced and activated in specific cell types.
obtain. Ensure that the transgene integrates into the chromosomal site of the corresponding endogenous gene.
If is desired, gene targeting is performed. Simply put,
When such techniques are used, the nucleotide sequence of the endogenous gene (chromosome sequence
(By homologous recombination with) to disrupt the function of the nucleotide sequence
, A vector containing several nucleotide sequences homologous to an endogenous gene is designed
.       [0163]   Control of osteocalcin transcriptional regulatory sequences using any technique known in the art
Introduce the transgene into the animal below and find the transgenic animal founder
Lineage can be created. Such techniques are not limiting
Is a pronuclear microinjection (Hoppe & Wagner, 1989, US Patent No. 4,873,
No. 191); Derivation from cultured embryonic cells, fetal cells or adult cells induced to the stationary phase
Nuclear transfer to nuclear oocytes (Campbell et al., 1996, Nature 380: 64-66; Wilmut et al., Natu
re 385: 810-813); Retroviral gene transfer into the germ line (Van der Putten et al.,
1985, Proc. Natl. Acad. Sci. USA82: 6148-6152); Gene targeting to embryonic stem cells
Getting (Thompson et al., 1989, Cell 65: 313-321); Electroporation of embryos
(Lo, 1983, Mol. Cell. Biol. 31: 1803-1814); and sperm-mediated inheritance
Transgenic Ani (Lavitrano et al., 1989, Cell 57: 717-723; Gordon, 1989, Transgenic Ani
mals, Intl. Rev. Cytol. 115: 171-229).       [0164]XIII. Screening Assay   Prostate cancer cells, prostate stromal cells, perivascular cells, proliferative cancer-related osteoblasts, and
Of bone and stromal cells that cannot express PSA and androgen receptor (AR)
Compounds that interfere with normal function and / or proliferation may be abnormal in prostate related disorders
Therapeutic methods targeted at are provided. Such compounds are used in prostate-related disorders, prostate cancer
, Brain cancer, ovarian cancer, thyroid cancer, various tumors, osteosarcoma, ocular melanoma, lung cancer or breast cancer
Can be used to prevent the onset or progression of Stimulates or suppresses promoter activity
The controlling compound can be used to ameliorate the symptoms of a prostate-related disorder.       [0165]   Osteocalcin transcription regulatory sequence operably linked to a reporter gene
Or a transgenic animal or cell containing a fragment thereof
For screening substances that modulate the activity of transcriptional regulatory sequences
Can be used. Therefore, it modulates the activity of the osteocalcin transcription regulatory sequence.
Such substances include prostate cancer cells, prostate stromal cells, perivascular cells,
Does not express cancer-associated osteoblasts and PSA or androgen receptor (AR)
It can be used to develop new treatments for prostate cancer and related bone stromal cells.
In addition, transgenic mice containing osteocalcin transcriptional regulatory sequences
Prostate cancer, brain cancer, ovarian cancer, thyroid cancer, various tumors, osteosarcoma, ocular
Target drugs to stop melanoma, lung or breast cancer progression
In vivo and i.v. to develop new treatments for such disorders
It becomes an experimental model in vitro.       [0166]   The present invention provides compounds that modulate the activity of osteocalcin transcription regulatory sequences.
Includes screening assays designed to identify. The present invention provides
Itro and cell-based assays and for transgenic animals
In vivo assays. Try to test as described below
Such compounds include, but are not limited to, oligonucleotides, peptides,
Examples include proteins, low-molecular organic or inorganic compounds, and antibodies.
.       [0167]   Examples of compounds include, but are not limited to, soluble peptides (limited
(Including Ig-terminal addition fusion peptides) and random peptides
Library members (eg, Lam et al., 1991, Nature 354: 82-84; Houghten
Et al., 1991, Nature 354: 84-86), and the D- and / or L-configuration.
Combinatorial chemistry-derived molecular library consisting of amino acids, phosphorus
Oxidized peptides (including but not limited to random or partially degenerated fingers
Directed phosphorylated peptide libraries and the like; eg, Songyang et al., 1993
, Cell 72: 767-778), antibodies (but not limited to, polyclonal)
, Monoclonal, humanized, anti-idiotype, chimeric or single-chain antibodies,
And their FAb, F (ab ')_TwoAnd FAb expression library fragments and epitopes
And small organic or inorganic molecules.       [0168]   Such compounds are further known to improve the symptoms of prostate related disorders
Compounds (especially drugs or members of drug classes or families)
I can see.       [0169]   Such compounds include, but are not limited to, lithium salts, carbama
Zepine, valproic acid, lysergic acid diethylamide (LSD), p-chlorophenyla
Lanine, dithiocarbamic acid p-propyl dopasetamide derivative (eg, FLA 63
)); Anxiolytics such as diazepam; iproniazide, clorgyline
Monoamine oxidases (MAO, phenelzine and isocarboxazide)
) Inhibitors; tricyclic antidepressants (eg, desipramine, imipramine and amitri
Bitin amine uptake blocking agent such as petitin); serotonin reversal such as fluoxetine
Uptake inhibitors; phenothiazine derivatives (eg, chlorpromazine (trazine) and
And trifluoropromazine), butyrophenones (eg, haloperidol (Hald
ol)), thioxanthene derivatives (eg, chlorprothixene) and dibenzodi
Antipsychotics such as azepines (eg clozapine); benzodiazepines; dopami
Agonists and antagonists (eg, L-DOPA, cocaine, amp
Tamine, α-methyl-tyrosine, reserpine, tetrabenazine, benzotropine,
Pargyline); noradrenergic agonists and antagonists (eg,
(Eg clonidine, phenoxybenzamine, phentolamine, tropolone); monoxide
Nitrovasodilators (eg nitroglycerin, nitroprusside)
And NO synthase enzymes); and growth factors (eg, VEGF, FGF,
Opoetin and endostatin).       [0170]   In one preferred embodiment, a female operably linked to a heterologous gene.
Using primary cultures of germ cells containing the mammalian osteocalcin transcriptional regulatory sequence,
Screen for compounds that can suppress or enhance column-specific DNA-protein interactions.
Develop an assay system for leaning. Such a method is osteocal
Transforms into a cell that expresses a gene under the control of a thin transcription regulatory sequence or its transcriptionally active fragment
Contact with the mixture and measure the level of gene expression or gene product activity.
The bell is used to determine the gene expression or inheritance provided by the cell in the absence of the compound.
Comparing the level of product activity with the level of product obtained in the presence of the compound.
If osteocalcin transcription is different from that obtained in its absence, mammalian osteocalcin transcription
Compounds that can modulate the expression of regulatory sequences have been identified. Genetic
The change in the current level may be by any method known to those skilled in the art.
For example, assaying the activity of a reporter gene, mR on cell lysates
Assaying NA transcripts, e.g., by Northern analysis, or
Using other methods known in the art to assay for the expressed genetic product
May be due to       [0171]   Compounds that suppress or enhance the activity of osteocalcin transcription regulatory sequences have been identified
Once it has been tested in an animal-based assay, the compound is
, Prostate cancer, brain cancer, ovarian cancer, thyroid cancer, various tumors, osteosarcoma, ocular melanoma, lung cancer
Or ameliorate and / or suppress breast cancer symptoms and / or prostate cancer cells,
Prostate stromal cells, perivascular cells, proliferative cancer-related osteoblasts, and PSA or
Prostate cancer not expressing androgen receptor (AR) and associated bone stromal cell proliferation
It can be determined whether or not it shows the ability to act to suppress multiplication.       [0172]   The present invention is illustrated by the following non-limiting examples.       [0173]XIV. Example 1: Systemic for the Treatment of Androgen-Independent Prostate Cancer Bone Metastasis
Osteocalcin (OC) promoter-driven replicable adenovirus Overview   Osteocalcin (OC) is a non-collagenous bone matrix protein,
It is expressed at high levels by alveoli, calcifying benign and malignant tumors.
In this study, a conditional OC replication-competent adenovirus (Ad
) Efficient systemic therapy for the treatment of hormone refractory prostate cancer bone metastases using vectors
Developed treatment. Recombinant Ad vector containing OC promoter-driven E1a gene Ad-OC
-E1a was built. Human prostate cancer cell lines (LNCaP, C4-2, PC-3, DU145, ARCaP)
Inhibition of growth of human bone cell line (MG-63) and prostate fibroblast cell line (9096F)
Was evaluated in vitro for the efficacy of Ad-OC-E1a. Ad-OC-E1a
Subcutaneous PC-3 and intraosseous C4-2 human prostate cancer xenograft in athymic and SCID / bg mice
Evaluation was also made by intratumoral and systemic administration to the model. Immunohistochemical research
Indicate that OC is widely expressed in both primary and metastatic prostate cancer
If the OC strain is positive, both the tumor epithelial cell compartment and the prostate or bone stromal cell compartment
It was shown to be found in. Prostate cancer cell line PSA-secreted (LNCaP, C
4-2, ARCaP) or non-secretory (PC-3, DU145) and bone (MG-63
) And prostate (9096F) stromal cell line growth was caused by Ad-OC-E1a
It was significantly suppressed by the lytic activity. Subcutaneous androgen receptor-negative PC-3 xenograft
In athymic nude mice bearing a piece, a single intratumoral injection of Ad-OC-E1a (2 × 10⁹ PFU) suppressed tumor growth. Intraosseous androgen receptor-positive C4-2 xenotransfer
In SCID / bg mice harboring the implant, a single intravenous administration of Ad-OC-E1a (2 × 10⁹PFU
) Resulted in no increase in PSA in 100% of the treated mice. Then Ad-OC-E1
Serum PSA rebound in 80% (4/5) of mice by systemic administration of a
Was suppressed. 40% (2/5) of the mice subsequently had tumors in the skeleton without PSA rebound
Cells were considered "healed" without being found. Therefore, systemic
OC promoter-driven conditionally replicable adenovirus has been tested in experimental models
Established earlier by cell lysis in both the tumor epithelium and the stroma that supports it
Tumor regression in hormonally refractory primary prostate cancer and its skeletal metastases
Very effective for guiding.       [0174]Materials and methods Cells and cell culture   LNCaP, an androgen responsive and positive for the androgen receptor
PSA-secreting human prostate cancer cell line is available from Horosewicz et al.²⁶Cervical lymph node metastasis
Derived from From this parental cell line, a series of androgen-independent (supporting stroma)
Inoculated into ischemic athymic male mice without cells or extracellular matrix
Defined as cells that can form PSA-secreting solid tumors) and lineage-related
LNCaP substrain^27,28Was induced. One of the substrains, C4-2, is an androgen receptor and
And PSA remain positive for either subcutaneous or orthotopic transplantation.
Acquired bone metastatic ability^27,28. ARCaP was established by our laboratory
Human progenitor with androgen-repressed androgen receptor and PSA low expression
Adenocarcinoma cell line. This cell line is highly tumorigenic and metastatic and has an advanced form
Human prostate cancer model²⁹. PC-3 is described in Kaighn et al. [Kaighn, M.E., Narayan,
 K.S., Ohnuki, Y., Lechner, J.F. and Jones, L.W.
acterization of a human prostatic carcinoma cell line (PC-3)
Establishment and Characterization of a Cancer Cell Line (PC-3). Invest. Urol., 17:16, 1979]
Androgen established from bone marrow aspirate from patients with confirmed metastatic disease
Human prostate cancer cells are independent and negative for androgen receptor and PSA
Is a stock. DU-145 has been developed by Stone et al. [Stone, K.R., Mickey, D.D., Wunderli, H., Mi
ckey, G.H. and Paulson, D.F.Isolation of a human prostate carcinoma ce
ll line (DU145) (Isolation of human prostate cancer cell line (DU145)). Int. J. Cancer, 2
1: 274, 1978] and was established from a patient with prostate cancer who has metastasized to the brain, Andro
Gen-independent human prostate cancer negative for androgen receptor and PSA
Cell line. Lovo is a colon carcinoma cell line that has been localized by Drewinko et al.
Established from tissue samples, the University of Texas, M.D. Anderson Cancer Center (Univers
and L. Y. Yang of the University of Texas M.D.Anderson Cancer Center (Huston, TX)
[Drewinko, B., Romsdahl, M.M., Yang, L.Y., Ah
earn, M.J. and Trujillo, J.M.Establishmen of human carcinoembryonic an
tigen-producing colon adenocarcinoma cell line
Establishment of intestinal adenocarcinoma cell line). Cancer Res., 36: 467, 1976]. WH is a human bladder transitional cell
A cell line derived from a cancer specimen, Zhau et al. [Zhau, H.E., Hong, S.J. and Ch.
ung, L.W.K.A fetal rat urogenital sinus mesenchymal cell line (rUGM): a
ccelerated growth and conferral of androgen-induced growth responsivenes
s upon a human bladder cancer epithelial cell line in vivo (fetal rat urine
Reproductive sinus mesenchymal cell line (rUGM): In vivo in human bladder cancer epithelial cell line
Int. J, Cancer., 56: 706, 199.
4]. 293 supports adenovirus replication by Graham et al.
A transformed human embryo established as having a complementing adenovirus E1 region
[Graham, F.L.Growth of 293 cells in suspension culture]
(Proliferation of 293 cells in suspension culture). J. Gen. Virol., 68: 937, 1987]. Hi
9096F, a prostate fibroblast cell line, has been approved by our lab for surgical prostate
Ozen, M., Multani, A.S., Kuniyasu, H., Chung, L.
W.K., von Eschenbach, A.C. and Pathak, S. Specific histologic and cytog
enetic evidence for in vivo malignant transformation of murine host cell
s by three human prostate cancer cell lines
Specific histological and in vivo transformation of mouse host cells in vivo
Oncol. Res., 9: 433, 1997]. Human bone stromal cell line
MG-63 was established from a sample of osteosarcoma and
Lucher Collection (American Type Culture Collection) (ATCC, Rockvi
lle, MD). PC-3, DU-145 and 293 cell lines were also obtained from ATCC. This
In the study of C4-2 and 9096F cells, T medium (Life Tec) containing 10% FBS was used.
hnologies, Inc.) [Gotoh, A., Ko
, S.C., Shirakawa, T., Cheon, J., Kao, C., Miyamoto, T., Gardner, T.A.,
Ho, L.J., Cleutjens, C.B., Trapman, J., Graham, F.L. and Chung, L.W.K.
Development of prostate-specific antigen promoter-based gene therapy for
 androgen-independent human prostate cancer
Development of gene therapy based on prostate specific antigen promoter for prostate cancer). Urol., 1
60: 220, 1998; Ozen, M., Multani, A.S., Kuniyasu, H., Chung, L.W.K., von
Eschenbach, A.C. and Pathak, S. Specific histologic and cytogenetic evi
dence for in vivo malignant transformation of murine host cells by three
 human prostate cancer cell lines
Specific histological and cellular regressions for in vivo malignant transformation of host cells
Oncol. Res., 9: 433, 1997]. LNCaP, PC-3, DU-145, ARCaP, WH
And MG-63 cells were all T medium containing 5% FBS (Life Technologies, Inc.
[Gotoh, A., Ko, S.C., Shirakawa, T., Cheon, J., Kao, C.,
Miyamoto, T., Gardner, T.A., Ho, L.J., Cleutjens, C.B., Trapman, J., Gra
ham, F.L. and Chung, L.W.K.Development of prostate-specific antigen pr
omoter-based gene therapy for androgen-independent human prostate cancer
 (Based on prostate-specific antigen promoter in androgen-independent human prostate cancer
J. Urol., 160: 220, 1998; Zhau, H.Y., Chang, S.M.,
Chen, B.Q., Wang, Y., Zhang, H., Kao, C., Sang, Q.A., Pathak, S.J. and
Chung, L.W.K.Androgen-repressed phenotype in human prostate cancer
Proc. Natl. Acad. Sci. USA.,
 93: 15152, 1996; Ko, S.C., Cheon, J., Kao, C., Gotoh, A., Shirakawa, T.,
 Sikes, R.A., Karsenty, G. and Chung, L.W.K.Osteocalcin promoter-based
 toxic gene therapy for the treatment of osteosarcoma in experimental mo
dels (as an osteocalcin promoter for the treatment of osteosarcoma in experimental models)
Cancer Res., 56: 4614, 1996]. Lovo cells, 10% FB
Maintained in a F-12 nutrient mixture containing S (Life Technologies, Inc.). 293
Cells were maintained in MEM (Life Technologies, Inc.) containing 10% FBS. This
These cells were supplied with fresh growth medium three times a week and 5% CO 2_TwoMaintained at 37 ° C in
Was.       [0175]   Construction and production of replicable Ad-OC-E1a   All plasmids were prepared using standard published protocols [Bett, A.J., Haddara,
 W., Prevec, L. and Graham, F.L.An efficient and flexible system for c
onstruction of adenovirus vectors with inserts or deletions in early rea
gions 1 and 3 (adenovirus vectors with insertions or deletions in early regions 1 and 3)
Natl. Acad. Sci. USA Proc. Natl. Acad. Sci. USA
., 91: 8802-1994]. Simply put, the 549th of Ad5 vector
The BamHI-XcaI fragment containing the backbone of the 5795792 base was ligated into plasmid pXC1.
The derivative pXC548C [McKinnon, R.D., Bacchetti, S. and Graham, F.L.
 mutagenesis of the transforming genes of human adenovirus type 5 (human
Tn5 mutagenesis of the transforming gene of adenovirus type 5).
 19:33, 1982] and digest pyE1sp1B [Dr. Frank Graham (Mac Master Universi
ty, Hamilton, Ontario, Canada). BamHI site and XcaI
A pyBPAEII shuttle vector was created by inserting between the sites. pOCE1a is Ad5
To drive the E1a gene, a 1370 bp fragment of the mouse OC promoter (pII1.5TK or
Cleaved with XhoI and SalI enzymes) into the XhoI site of pyBPAEII
Built by that. The shuttle pOCE1a vector was cloned as shown in FIG.
N- [1- (2,3-dioleoyloxyl) propyl sulfate together with the damaged recombinant Ad vector pJM17
Ru] -N, N, N-trimethylammonium methyl (Boehringer Mannheim Biochemicals
) Mediated transfection method [Zhang, W-W., Fang, X., Branch, C.D., Mazur
, W., French, B.A. and Roth, J.A.Generation and identification of reco
mbinant adenovirus by liposome-mediated transfection and PCR analysis (
Recombinant adenovirus by liposome-mediated transfection and PCR analysis
Biotechniques, 15: 868, 1993].
Transfectable Adenovirus Ad-OC-E1 transfected and partially deleted for E3
a was prepared. Ad-OC-E1a thus obtained is a cell expressing OC promoter activity.
It was shown to replicate in a restricted manner only in the vesicles. Complete cytotoxicity
The culture medium of 293 cells showing was recovered and centrifuged at 1,000 × g for 10 minutes. Pool
An aliquot of the supernatant was taken and stored at -80 ° C as the primary virus stock. C
The ills stock is grown in 293 cells and the selected Ad-OC-E1a virus clone is
, Graham and Pervec [Graham, F.L. and Prevec, L. Manipulation of
 adenovirus vectors. Volume 7, pages 109-128,
Clifton, NJ: The Humana Press, Inc., 1991].
Was. Pick one of the virus clones, grow in 293 cells, 36-40 hours after infection
Later collected, pelleted, resuspended in PBS and dissolved. Centrifuge cells
To remove the cell debris and remove the virus in the cell lysate by CsCl_TwoGradient
Purified by centrifugation. Dialyze the concentrated virus, take an aliquot, and
Saved in. Virus titers were determined by plaque assay as described previously [Go
toh, A., Ko, S.C., Shirakawa, T., Cheon, J., Kao, C., Miyamoto, T., Gard
ner, T.A., Ho., L.J., Cleutjens, C.B., Trapman, J., Graham, F.L. and Ch.
ung, L.W.K.Development of prostate-specific antigen promoter-based gene
 therapy for androgen-independent human prostate cancer
Of gene therapy based on prostate-specific antigen promoter for dependent human prostate cancer
J. Urol., 160: 220, 1998]. Controls used in this study
The ils Ad-CMV-pA and Ad-CMV-β-gal are similar to those described above.
Procedure [Ko, S.C., Gothoh, A., Thalmann, G.N., Zhau, H.E., Johnston, D.A.,
Zhang, W.W., Kao, C. and Chung, L.W.K.Molecular therapy with recombina
nt p53 adenovirus in an androgen-independent, metastatic human prostate
cancer model (recombination in androgen-independent metastatic human prostate cancer model)
Molecular therapy using p53 adenovirus). Hum. Gene Ther., 7: 1683, 1996]
Were constructed, plaque purified, and grown in 293 cells.       [0176]   Immunohistochemical staining of primary and metastatic human prostate cancer specimens   Deparaffinized primary human prostate cancer specimen and lymph node and bone metastasis
Samples were from the University of Virginia School of Medicin
e, Charlottesville, VA), Department of Urology and Pathology and McGill University (Mc
Gill University, Montreal, Quebec, Canada). 3% H tissue_TwoO_Two
And block with SuperBlock (Scytek Laboratories, Logan, Utah)
And a monoclonal OC antibody (OC4-30: Takara Shuzo, Otsu, Japan)
Antibody staining signal is converted to biotinylated peroxidase-conjugated
Amplification was performed using a vidin system (Bio-Genex Laboratories, San Ramen, CA). OC staining
Converts conjugated peroxidase to AEC chromogen (3-amino-9-ethylcarbamate).
Sol) and visualized.       [0177]   In vitro cell proliferation assay   5 × 10^ThreeLNCaP, C4-2, PC-3, DU-145, ARCaP, 293, WH, Lovo, MG-63 or
Plated 9096F cells into 24-well plates. 24 hours later, the cells were ad-OC
-E1a at 0.01-5 MOI (or pfu / cell: this is 0.2-100 virus particles / cell)
(Estimated) for 2 hours. Ad-CMV-pA or Ad-CMV-
Cells infected with β-gal served as negative controls (FIGS. 17A-D). Three days later, the cell count was
Automated E-Max spectrophotometric platelet with crystal violet assay
(Molecular Devices Corp., Sunnyvale, CA).
[Ko, S.C., Cheon, J., Kao, C., Gotoh, A., Shirakawa, T., Si
kes, R.A., Karsenty, G. and Chung, L.W.K.Osteocalcin promoter-based to
xic gene therapy for the treatment of osteosarcoma in experimental model
s (based on osteocalcin promoter for treatment of osteosarcoma in experimental models
Cancer Res., 56: 4614, 1996].       [0178]   Virus yield in prostate and bladder cancer cell lines infected with Ad-OC-Ea1
Evaluation of   2 × 10^FiveIndividual C4-2 or 293 cells were plated in duplicate in 6-well plates.
Was. Twenty-four hours later, the medium is aspirated and Ad-OC-E1a, Ad-CMV-pA or wild-type Ad vector
Either was replaced with 0.5 ml of T or MEM medium containing a MOI of 2 pfu / cell. negative
As a control, WH cells cultured in T medium were similarly infected with the test virus. 37
Two hours after infection with the Ad vector at 0 ° C, the cells are washed twice with PBS and
2 ml of medium was added. The cell culture medium is collected, diluted, added to 293 cells, and plated.
Workout assays were performed in triplicate at intervals of 0-72 hours. 100 μl for this assay
The diluted cell culture medium with 0.75% semi-solid agarose medium.
Addition to fluent 293 cell cultures. 5 days later, 0.5% crystal
The number of plaques was visualized and counted by staining with violet
rum, F.D. and Ornelles, D.A. p53 status does not determine outcome of E
1B 55-kilodalton mutant adenovirus lytic infection (p53 status is E1B 55
The results of a Rodalton mutant adenovirus lytic infection are not determined). J. Viol.,
72: 9479, 1998].       [0179]   Evaluation of adenovirus infectivity in mouse and human bone   To determine if normal mouse or healthy human bone is susceptible to Ad infection
In addition, two studies were conducted. First, Ad-CMV-β-gal (1 × 10⁹ pfu) adult ma
Injected into the femur of a mouse, and three days later the bone was harvested and the method established previously [Ko,
S.C., Choen, J., Kao, C., Gotoh, A., Shirakawa, T., Sikes, R.A., Karsent
y, G and Chung, L.W.K.Osteocalcin promoter-based toxic gene therapy fo
r the treatment of osteosarcoma in experimental models
Osteocalcin promoter-based toxin gene therapy for the treatment of osteosarcoma
Cancer Res., 56: 4614, 1996] was used for histochemical analysis of β-gal activity.
. Second, a normal bone specimen taken from a broken 69-year-old man was 0.6% cold
Cultured in T medium containing heavens. Infection of human prostate cancer PC-3 xenograft in parallel
And reserved as a control. Tissue samples were collected from Ad-CMV-β-gal (1 × 10⁹ pfu)
3 days after infection. After removing the bone and prostate cancer specimens, the tissue is first
Washed in PBS and fixed in 0.05% glutaraldehyde at 4 ° C. for 24 hours. Bone
Specimens were placed in PBS for 24 hours after fixation, and 0.25 M EDTA in PBS (pH 7.4) was used.
Decalcification was performed at 4 ° C. for 5 days. After decalcification, the sample was reconstituted at 1 mg / ml X-gal (5-bromo-
4-chloro-3-indolyl-β-D-galactopyranoside), 5 mM K_ThreeFe (CN)₆, 5mM K_Four Fe (CN)₆And 2mM MgCl_TwoWas stained overnight in a solution of PBS. Prostate cancer specimen
Was treated as described above and tested for β-galactosidase activity.
Stained as described above [Ko, S.C., Cheon, J., Kao, C.
, Gotoh, A., Shirakawa, T., Sikes, R.A., Karsenty, G. and Chung, L.W.K.
 Osteocalcin promoter-based toxic gene therapy for the treatment of oste
osarcoma in experimental models
Toxin gene therapy based on the steocalcin promoter). Cancer Res., 56: 4614
, 1996].       [0180]   In vivo animal experiments   Athymic mice (20-25g) to determine tumor specificity of Ad-OC-E1a lytic activity
1 × 10 6 suspended in 100 μl of T medium containing 5% FBS⁶PC-3 cells or Lo
vo cells were inoculated subcutaneously. Once the tumor is palpable (4-5mm in diameter)
These animals were randomly divided into two experimental groups: Group 1, Ad-OC-E1a; Group 2, Ad-OC-E1a.
CMV-β-gal. Single dose of virus (2 x 10⁹ pfu) was injected intratumorally into mice.
Four weeks after administration of the test virus, tumor size was measured and recorded.       [0181]   For the evaluation of Ad-OC-E1a in an intraosseous prostate cancer model, 1 × 10⁶C4-2 cells,
Previously published procedures [Wu, T.T., Sikes, R.A., Cui, Q., Thalmann, G.N.,
Kao, C., Murphy, C.F., Yang, H., Zhau, H.E., Balian, G. and Chung, L.W.
K. Establishing human prostate cancer cell xenografts in bone: induction
 of ostaoblastic reaction by prostate-specific antigen-producing tumors
in athymic and SCID / bg mice using LNCaP and lineage-derived metastatic s
ublines (Establishment of human prostate cancer cell xenografts in bone: LNCaP and lineage derived
Prostate-Specific Antigen Production in Athymic and SCID / bg Mice Using Metastatic Sublines
Induction of osteoblast response by live tumor). Int. J. Cancer, 77: 887, 1998].
Castrated male SCID / bg mice were injected into the medullary cavity of the right tibia. For the PSA assay,
A blood sample (about 100 μl) was collected from the tail vein once a week. Serum PSA is a fine-particle solid-phase yeast
Immunoassay (MEIA) using Abbott Imx instrument (Abbott Park, IL)
did. After an increase in serum PSA was detected, a single dose of 25 μl of Ad-OC-E1a [1 animal
2 × 10 per⁹ pfu (ie 4 × 10^TenVirus particles)] into mice intravenously
Was administered. When serum PSA rebound occurs, animals are
Treated by giving a second or third intravenous injection of the same dose of test virus
did. Serum PSA was monitored weekly and tumors were sacrificed at the time of animal sacrifice.
And X-rays were routinely evaluated.       [0182]result Immunohistochemical staining of OC in primary and metastatic prostate cancer specimens   OC has been shown to be a specific marker indicator of osteoblast lineage cell differentiation
[Hoffmann, H.M., Catron, K.M., Wijnen, A.F.V., McCabe, L.R., Lian,
J.B., Stein, G.S. and Stein, J.L.Transcriptional control of the tissue
-specific, developmentally regulated osteocalcin gene requires a binding
 motif for the Msx family of homeodomain proteins
Regulation of the osteocalcin gene is regulated by homeodomain proteins
Requires the binding motif of the Msx family). Proc. Natl. Acad. Sci. USA.,
91: 12887, 1994]. In addition, OC is composed of calcified normal tissue [Bini, A., Mann, K.G.,
Kudryk, B.J. and Schoen, F.J.Noncollagenous bone matrix proteins, calc
ification, and thrombosis in carotid artery atherosclerosis
Non-collagenous bone matrix protein in sclerosis, calcification and thrombosis)
Arterioscler Thromb. Vasc. Biol., 19: 1852, 1999], perivascular cells [Reill
y, T.M., Seldes, R., Luchetti, W. and Brighton, C.T.
e phenotypic expression of pericytes and bone cells
Similarity of osteocyte phenotypic expression). Clin. Orthop., 346: 95, 1998] and benign tumors
Ulcers [Lantuejous, S., Issac, S., Pinel, N., Negoescu, A., Guibert, B. and
And Brambilla, E. Clear cell tumor of the lung: an immunohistochemical and
 ultrastructural study supporting a pericytic differentiation
Alveolar tumors: immunohistochemical and ultrastructural studies supporting differentiation of perivascular cells
Mod. Pathol., 10: 1001, 1997]. Immunohistochemical
Using the method, primary prostate cancer matrix (FIG. 2a) and tumor epithelium and stroma (FIG. 2b)
), The expression of OC was observed. Positive OC strains account for 85% of primary prostate cancer specimens (23/27
) And 100% of metastatic lymph node and bone specimens (12/12 lymph nodes, 10
/ 10). Representative immunohistochemical staining of lymph nodes and bone
The feel is shown in FIGS. 2d and 2e, respectively. In primary prostate cancer and bone metastases
Background immunohistochemical staining of OC is shown in FIGS. 2c and 2f, respectively.       [0183]   Ad-OC-E1a cytotoxicity against prostate cancer cell lines in vitro: endogenous PSA and And AR state independence   To evaluate the cytotoxicity of Ad-OC-E1a, several human prostate cancer cell lines, LNC
aP, C4-2, PC-3, DU-145 and ARCaP were converted to a wide range of Ad-OC-
Exposure to E1a vector. Human 293 cells or WH and Lovo cells
[Graham, F.L.Growth of 293 cells in su
spension culture (growth of 293 cells in suspension culture). J. Gen. Virol., 68:
937, 1987; Ko, S.C., Cheon, J., Kao, C., Gotoh, A., Shirakawa, T., Sikes
, R.A., Karsenty, G. and Chung, L.W.K.Osteocalcin promoter-based toxic
 gene therapy for the treatment of osteosarcoma in experimental models (
Based on osteocalcin promoter for treatment of osteosarcoma in experimental model
Cancer Res., 56: 4614, 1996]. LNCaP cells and C4-2 cells
Exposure to 5 MOI of Ad-OC-E1a inhibited the growth of these cells by 70%.
However, the same dose of Ad-OC-E1a was used in WH cells and Lovo cells (these were
(Showing no or no motor activity).
(Fig. 3a). Control virus (insert (Ad-CMV-P
A) or cells lacking either Ad-CMV-β-gal)
Exposure to the virus at 5 MOI was found to be unaffected (FIG. 3a).
Second, the potency of Ad-OC-E1a is reduced to very low levels (eg, ARCaP) or to undetectable
Some others express PSA and AR only at levels (eg, PC-3 and DU-145)
Of human prostate cancer cell lines. From Figure 3b, all human prostates tested
Cancer cell lines are tested in vitro, regardless of their native PSA and AR expression levels.
Were susceptible to Ad-OC-E1a-induced cell lysis. In addition, in vitro
The effect of Ad-OC-E1a on the growth of prostate and bone fibroblast cell lines was also evaluated.
As shown in FIG. 3c, Ad-OC-E1a infection resulted in cultured human prostate fibroblasts (eg,
For example, 9096F) and bone fibroblasts (MG-63).
Upset.       [0184]   Ad-OC-E1a replication in androgen-independent human prostate cancer cell lines   The OC promoter drives the expression of the E1a transgene (as a result,
May cause adenovirus replication in tent human prostate cancer cell lines)
C4-2 after infection with either Ad-OC-E1a or Ad-CMV-PA to determine
Viral activity in the supernatant of a cell line (androgen-independent human prostate cancer cell line)
The value was evaluated. 293 cells and WH cells were used as positive and negative controls, respectively.
[Graham, F.L.Growth of 293 cells in suspension culture
J. Gen. Virol., 68: 937, 1987; Ko, S.C., Cheon, J.
, Kao, C., Gotoh, A., Shirakawa, T., Sikes, R.A., Karsenty, G. and Chun
g, L.W.K.Osteocalcin promoter-based toxic gene therapy for the treatmen
t of osteosarcoma in experimental models
Toxin Gene Therapy Based on the Osteocalcin Promoter for Cancer Res.,
 56: 4614, 1996]. From the results of these studies, the supernatants of all tested cell lines were
The virus titer in infected cells was C4-2 cells, WH cells and 293 cells when infected with Ad-CMV-PA.
0 pfu / cell, 0 pfu / cell and 170 ± 60 pfu / cell for cells, respectively
It was shown that. When infected with Ad-OC-E1a, the replication of adenovirus is C4-2
Cells and 293 cells (15 ± 8 and 7.7 ± 3.2 pfu / cell, respectively).
No significant level of replication was detected (0.047 ± 0.021 pfu / cell). Ad-OC-E
1a is very potent and in replication in C4-2 cells, the number of infectious
It turned out to be similar to that seen in the replicable 293 cell line.       [0185]   Specificity of intratumoral Ad-OC-E1a in disrupting subcutaneous PC-3 tumor growth in vivo   To evaluate the specificity of Ad-OC-E1a in inhibiting the growth of prostate cancer in vivo
In addition, the activity of this virus against the growth of the previously established subcutaneous PC-3 human prostate cancer
Sex was compared to that of a control, Lovo human colon cancer. From FIG. 4, Ad-OC-E1a is
Intratumoral injection effectively suppresses PC-3 tumor growth but suppresses Lovo tumor growth
Not shown. These data show that OC promoter activity is present in PC-3
But not in Lovo cells (data not shown).
You.       [0186]   Ad-OC-E1a to mice with previously established C4-2 human prostate cancer in the skeleton
Intravenous administration of   Systemic Ad-OC-E1a in previously established intraskeletal prostate cancer xenografts
As a model to evaluate efficacy, androgen-independent and PSA-secreting C
4-2 Human prostate cancer cell line was selected [Wu, T.T., Sikes, R.A., Cui, Q., Thalmann,
 G.N., Kao, C., Murphy, C.F., Yang, H., Zhau, H.E., Balian, G. and Chun
g, L.W.K.Establishing human prostate cancer cell xenografts in bone: in
duction of osteoblastic reaction by prostate-specific antigen-producing
tumors in athymic and SCID / bg mice using LNCaP and lineage-derived metas
Establishment of human prostate cancer cell xenografts in bone: LNCaP and
Prostate in athymic and SCID / bg mice using a lineage-derived metastatic substrain
Induction of osteoblast response by gland-specific antigen-producing tumors). Int. J. Cancer, 77: 887.
, 1998]. The standard protocol is for tumor growth in the tibia to increase over serum PSA.
Start intravenous Ad-OC-E1a treatment if positively indicated
To do it. Check serum PSA once a week and if PSA rebound occurs
Animals were repeatedly subjected to Ad-OC-E1a treatment. In this study, a total of six animals
evaluated. From FIG. 5a, in one control untreated mouse (# 1), serum PSA was 6 weeks
Within 10 ng / ml significantly above basal level within 630 ng / ml at 15 weeks
The exponential logarithm is shown to increase. Profile of this rapid PSA rise
Are consistent with our earlier report [Wu, H.C., Hsieh, J.T., Gleave, M.E.,
 Brown, N.M., Pathak, S. and Chung, L.W.K.Delivetion of andgrogen-inde
pendent human LNCaP prostatic cancer cell subline: role of bone stromal
Induction of androgen-independent human LNCaP prostate cancer cell line: bone stromal cells
Role). Int. J. Cancer, 57: 406, 1994; Thalmann, G.N., Anezinis, P.E., Ch.
ang, S.M., Zhau, H.E., Kin, E.E., Hopwood, V.L., Pathak, S., von Eschenb
ach, A.C. and Chung, L.W.K.Androgen-independent cancer progression and
 bone metastasis in the LNCaP model of human prostate cancer
Androgen-independent cancer progression and bone metastasis in the LNCaP model of cancer). Cancer Re
s., 54: 2577, 1994; Wu, T.T., Sikes, R.A., Cui, Q., Thalmann, G.N., Kao,
C., Murphy, C.F., Yang, H., Zhau, H.E., Balian, G. and Chung, L.W.K.Es
tablishing human prostate cancer cell xenografts in bone: induction of o
steoblastic reaction by prostate-specific antigen-producing tumors in at
hymic and SCID / bg mize using LNCaP and lineage-derived metastatic sublin
es (Establishment of human prostate cancer cell xenografts in bone: LNCaP and lineage-derived translocations
Prostate-specific antigen in athymic and SCID / bg mice using transgenic sublines
Induction of Osteoblast Response by Producing Tumor). Int. J. Cancer, 77: 887, 1998]. Ad
Serum PSA profiles of -OC-E1a treated mice are shown in FIGS. Some PSA responses
1) Mouse # 2 and # 3 were twice
Responded to intravenous Ad-OC-E1a treatment, the skeletal tumors completely regressed (FIGS. 6a, 6b), and 1
After 5 weeks, the PSA reached the lowest level (Figs. 5b and 5c). These two mice are all
Considered "healed" by physical Ad-OC-E1a. 2) Mouse # 4 and # 5
Responded to systemic Ad-OC-E1a and showed a marked and rapid decrease in PSA. These mau
The PSA minimum is maintained for various periods of time, ranging from 1 to 6 weeks.
(FIGS. 5d and 5e). 3) Mouse # 6 initially responds favorably to systemic Ad-OC-E1a
(PSA bottoms lasted 5 weeks), but the second and third systemic Ad-OC-E
At the time of 1a administration treatment, systemic Ad-OC-E1a growth inhibitory effect was gradually impaired (
Figure 5f).       [0187]   Macroscopic morphology, histopathology and growth of prostate cancer xenografts in mouse tibia And immunohistochemistry: Effect of Ad-OC-E1a   Figure 6a shows macroscopic anatomical differences between control and Ad-OC-E1a-treated mice
did. As shown by X-ray and gross anatomy, when compared to untreated controls
Systemic Ad-OC-E1a showed a marked regression of prostate cancer growth in the tibia. this
Improvements were achieved using histopathological sections of tumors from control and Ad-OC-E1a treated animals.
This was confirmed by inspection (FIG. 6b, compare panels A and C). Control test
Positive PSA staining was observed in the body (FIG. 6b, panel B), whereas the Ad-OC-E1a treated specimen
No PSA staining was detected (data not shown). Adenovirus infectivity is also
, In situ in mouse bone, and human bone and human prostate PC-3 xenograft
The pieces were compared in vitro. From the results of this study, it was found that Ad-CMV-β-gal
Injection efficiently infects bone cells in mice and does not affect cortical bone
(FIG. 6c, panel A). Ad-CMV-β-gal is a PC-3 tumor in vitro
But it does not infect human bone that is maintained as an explant in soft agar.
(FIG. 6c, compare panels B and C, respectively).       [0188]Consideration   Cancer treatment using adenovirus vectors is divided into two broad categories:
Heise, C. and Kirn, D.H. Replic
ation-selective adenoviruses as oncolytic agents (replication as an oncolytic agent)
Selective adenovirus). J. Clin. Invest., 105: 847, 2000]. All cancer cells
Since it is difficult to infect adenovirus vectors into
Viral gene expression in competent tumor cells without damaging normal tissues
/ Various forms of viral architecture with the primary goal of increasing the efficiency of replication
I'm designing a structure. One such approach is to use replication defective adenovirus vectors.
"Bystander" genes (eg, HSV-TK or cytosine
Aminase) and its bystander gene is prodola.
Transforming the egg into a biologically active anti-proliferative product and characterizing it with a virus-borne gene
Induces efficient cell killing even in untransfected cells [Gotoh, A.,
 Ko, S.C., Shirakawa, T., Cheon, J., Kao, C., Miyamoto, T., Gardner, T.A
., Ho, L.J., Cleutjens, C.B., Trapman, J., Graham, F.L. and Chung, L.W.
K. Development of prostate-specific antigen promoter-based gene therapy
for androgen-independent human prostate cancer
Development of gene therapy based on prostate-specific antigen promoter for prostate cancer). J. U
rol., 160: 220, 1998; Eastham, J.A., Chen, S.H., Sehgal, I., Yang, G., Ti
mme, T.L., Hall, S.J., Woo, S.L. and Thompson, T.C.Prostate cancer gen
e therapy: herpes simplex virus thymidine kinase gene transduction follo
wed by ganciclovir in mouse and human prostate cancer models
Genetic Therapy: Herpes Simplex Virus Chi in Mouse and Human Prostate Cancer Models
Hum. Gene Ther., 7: 515,
1996; Koeneman, K.S., Kao, C., Ko, S.C., Yang, L., Wada, Y., Kallmes, D.
A., Gillenwater, J.Y., Zhau, H.E., Chung, L.W.K and Gardner, T.A. Osteo
calcin directed gene therapy for prostate cancer bone metastasis
Osteocalcin-directed gene therapy for bone metastasis of cancer). World J. Urol., 18: 102, 20
00; Gardner, T.A., Ko, S.C., Kao, C., Shirakawa, S., Cheon, J., Gotoh, A
., Wu, T.T., Sikes, R.A., Zhau, H.E., Cui, Q., Balian, G. and Chung, L.
.W.K.Exploiting stromal-epithelial interaction for model development an
d new strategies of gene therapy for prostate cancer and osteosarcoma me
tastasis (Development of a model for metastasis of prostate cancer and osteosarcoma and novel gene therapy
Utilization of stromal-epithelial interaction for the strategy of Gene). Gene Ther. Mol. Biol., 2:41, 1
998; Blackburn, R.V., Galoforo, S.S., Corry, P.M and Lee, Y.J. Adenovir
al-mediated transfer of a heat-inducible double suicide gene into prosta
te carcinoma cells (Adenovirus, a heat-induced double suicide gene for prostate cancer cells)
Cancer Res., 58: 1358, 1998]. E1b (55 kDa protein) missing
The construction of highly replicable ONYX-015 is conceptually useful for tumor cells that lack functional p53 protein.
[Heise, C., Williams, A., Xue, S., Propst, M. and Kirn
, D. Intravenous administration of ONYX-015, a selectively-replicating a
denovirus, induces antitumoral efficiency (selective replicating adenovirus ONY
Intravenous administration of X-015 induces antitumor efficiency). Cancer Res., 59: 2623, 1999].
. Conditional activation of viral gene expression and replication is controlled by a tissue-specific promoter
Has been achieved using e.g. PSA in the case of prostate cancer^9-11And liver cancer
Α-fetal protein [Kanai, F., Lan, K.H., Shiratori, Y.,
Tanaka, T., Ohashi, M., Okudaira, T., Yoshida, Y., Wakimoto, H., Hamada
, H., Nakabayashi, H., Tamaoki, T. and Omata, M. In vivo gene therapy f
or alpha-fetoprotein-producing hepatocellular carcinoma by adenovirus-me
diated transfer of cytosine deaminase gene
In vivo inheritance of α-fetoprotein producing hepatocellular carcinoma by adenovirus-mediated transfer
Child Res.). Cancer Res., 57: 461, 1997]. Adenovirus vector virus
The adenoviral killing protein (adenoviral death p
rotein) has been shown to significantly increase virus replication efficiency
[Doronin, K., Toth, K., Kuppuswamy, M., Ward, P., Tollefson, A.E. and
Wold, W.S.Tumor-specific, replication-competent adenovirus vectors over
expressing the adenovirus death protein
Overexpressing tumor-specific replicable adenovirus vector). J. Virol., 74:61
47, 2000]. In this study, we investigated adenovirus expression in cells containing OC promoter activity.
Tissue-specific (ie, osteoblast-specific) and tumor-specific to drive lupus replication
OC (ie, limited expression in calcified benign and malignant tumors)
The possibility of using a promoter was investigated. This form of the adenovirus vector comprises
Tumor epithelium and stromal cells supporting it, such as fibromuscular stromal cells expressing OC
[Gardner, T.A., Ko, S.C., Kao, C., Shirakawa, S., Cheon, J., Gotoh, A.,
 Wu, T.T., Sikes, R.A., Zhau, H.E., Cui, Q., Balian, G. and Chung, L.W
.K. Exploiting stromal-epithelial interaction for model development and
new strategies of gene therapy for prostate cancer and osteosarcoma meta
stasis (Development of a model for metastasis of prostate cancer and osteosarcoma and the development of novel gene therapy
Utilization of stromal-epithelial interaction for strategy). Gene Ther. Mol. Biol., 2:41, 199.
8; Koeneman, K.S., Yeung, F. and Chung, L.W.K. Osteomimetic properties
of prostate cancer cell: a hypothesis supporting the predilection of pro
state cancer metastasis and growth in the bone environment
Osteomimetic properties of bone: prediction of metastasis and growth of prostate cancer in bone environment
Prostate, 39: 246, 1999] and perivascular cells [Doherty, M.
.J., Ashton, B.A., Walsh, S., Beresford, J.N., Grant, M.E. and Canfield
, A.E.Vascular pericytes express osteogenic potential in vitro and in v
ivo (perivascular cells express osteoblastic potential in vitro and in vivo). J. Bone
Miner. Res., 13: 828, 1998] to enable virus replication.
The purpose is. Thus, Ad-OC-E1a is primarily a virus in tumor epithelium
Secondly, replication leads to cytolysis in prostate fibroblasts and perivascular cells.
By disrupting the intracellular communication between the tumor and stroma by inducing
It is believed that it can result in significant cell killing. In vitro experimental co-culture studies
And HSV-TK / acyclovir (ACV) in chimeric tumor models in vivo
Induced osteoblast death significantly suppressed prostate cancer cell growth (unpublished)
Findings). OC expression is very restricted to osteoblasts during maturation [Hoffma
nn, H.M., Catron, K.M., Wijnen, A.F.V., McCabe, L.R., Lian, J.B., Stein,
 G.S. and Stein, J.L.Transcriptional control of the tissue-specific, d
evelopmentally regulated osteocalcin gene requires a binding motif for t
he Msx family of homeodomain proteins (regulated during tissue-specific development
Regulation of osteocalcin gene transcription is the Msx family of homeodomain proteins
Natl. Acad. Sci. USA., 91: 12887, 1
994], 43 Ad-OC-E1a damages bone and alters the rate of bone resorption and deposition
It seems to be possible. We have addressed this issue, but we have summarized our findings below.
I will. First, both mouse and human cortical bone are restricted to adenovirus infection.
It is constant. Mouse bone marrow is very susceptible to adenovirus infection,
Human bone (including mature osteoblasts) is more resistant to adenovirus infection
Seemed to be high. Therefore, replication of Ad-OC-E1a is
Is restricted to proliferating and mature osteoblasts expressing OC promoter activity
Could be Second, intraosseous administration of Ad-OC-HSVTK to non-castrated adult mice
And ACV administered intraperitoneally did not cause any skeletal abnormalities [Gardner
, T.A., Ko, S.C., Kao, C., Shirakawa, S., Cheon, J., Gotoh, A., Wu, T.T.
, Sikes, R.A., Zhau, H.E., Cui, Q., Balian, G. and Chung, L.W.K.Explo
iting stromal-epithelial interaction for model development and new strat
egies of gene therapy for prostate cancer and osteosarcoma metastasis (
Development of models for metastasis of prostate cancer and osteosarcoma and development of novel gene therapy strategies
Utilization of stromal-epithelial interaction). Gene Ther. Mol. Biol., 2:41, 1998]. Thing
In fact, OC inhibits the growth of mineral crystal growth in in vitro assays.
[Romberg, R.W., Werness, P.G.,
Riggs, B.L. and Mann, K.G.Inhibition of hydroxyapatite crystal growth
by bone-specific and other calcium-binding proteins
Inhibition of hydroxyapatite crystal growth by calcium-binding protein).
Biochemistry, 25: 1176, 1986]. This role of OC plays a role in the expression of OC-expressing cells by HSV-TK.
Transgenics whose destruction results in increased bone mass and bone formation
It is consistent with the OC "knockout" mouse model [Ducy, P., Desbois, C., Boy
ce, B., Pinero, G., Story, B., Dunstan, C., Smith, E., Bonadio, J., Gold
Stein, S., Gundberg, C., Bradley, A. and Karsenty, G. Increased bone fo
rmation in osteocalcin-deficient mice
Nature, 382: 448, 1996].       [0189]   OC is PSA enhancer [Rodrigez, R., Schuur, E.R., Lim, H.Y., Henderso
n, G.A., Simons, J.W. and Henderson, D.R.Prostate attenuated replicati
on competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate
-specific antigen-positive prostate cancer cells
Denovirus (ARCA) CN706: selective for prostate-specific antigen-positive prostate cancer
Cell Residue). Cancer Res., 57: 2559, 1997; Yu, D-.C., Chen, Y., Seng, M., D
illey, J. and Henderson, D.R.The addition of adenovirus type 5 region
E3 enables calydon virus 787 to eliminate distant prostate tumor xenogra
fts (calidone virus 787 is distantly established by the addition of the E3 region of adenovirus type 5
Adenocarcinoma xenografts disappear). Cancer Res., 59: 4200, 1999], human calik
Rain 2 (hK2) [Yu, D-.C., Sakamoto, G.T. and Henderson, D.R. Identifi
cation of the transcriptional regulatory sequences of human kallikrein 2
 and their use in the construction of calydon virus 764, an attenuated r
eplication competent adenovirus for prostate cancer therapy (human and potassium)
Identification of transcriptional regulatory sequences for crane 2 and calidone virus for the treatment of prostate cancer
764 (attenuated replication-competent adenovirus) and their use in the construction). Cance
r Res., 59: 1498, 1999] or other prostate features such as prostate specific membrane antigen (PSMA).
Tissue-specific and tumor-specific promoters with potential advantages over heterologous promoters
The concept of being a motor is described below. OC is primary and metastatic in humans
Is widely expressed in prostate cancer and its expression is associated with the tumor epithelium and its surroundings
It is found in both interstitial compartments (see Figure 2) [Gardner, T.A., Ko, S.C., K
ao, C., Shirakawa, S., Cheon, J., Gotoh, A., Wu, T.T., Sikes, R.A., Zhau
, H.E., Cui, Q., Balian, G. and Chung, L.W.K.Exploiting stromal-epith
elial interaction for model development and new strategies of gene thera
py for prostate cancer and osteosarcoma metastasis
-Epithelial interaction for the development of metastasis models and novel gene therapy strategies
Gene Ther. Mol. Biol., 2:41, 1998; Koeneman, K.S., Yeung, F.
And Chung, L.W.K.Osteomemetic properties of prostate cancer cell: a h
ypothesis supporting the predilection of prostate cancer metastasis and
growth in the bone environment
A Hypothesis Supporting Prediction of Metastasis and Growth of Prostate Cancer. Prostate, 39: 246, 1999].
OC expression is not limited to prostate cancer and is benign and malignant with other calcifications.
Plaques associated with various tissues, such as heart valves and blood vessels [Doherty, M.J., A
shton, B.A., Walsh, S., Beresford, J.N., Grant, M.E. and Canfield, A.E.
 Vascular pericytes express osteogenic potential in vitro and in vivo (
Perivascular cells express osteoblastic potential in vitro and in vivo.). J. Bone Miner
Res., 13: 828, 1998], osteosarcoma, brain tumor, thyroid, breast, lung and ovarian cancer.
(Unpublished results) also expressed independently of their PSA and AR status
Turned out to be. This is important. Because about 20% of prostate cancer patients
Now, despite this disease being detected and progressing, the rise in PSA
[Carter, H.B., Pearson, J.D., Metter, E.J.,
 Brant, L.J., Chan, D.W., Andres, R., Fozard, J.L. and Walsh, P.C.Long
itudinal evaluation of prostate-specific antigen levels in men with and
without prostate disease (prostate in humans with or without prostate disease)
Longitudinal assessment of gland-specific antigen levels). JAMA, 267: 2215, 1992]. In addition, AR genetics
Offspring amplification and overexpression were detected in nearly 30% of clinical prostate cancer specimens
[Visakorpi, T., Hyytinen, E., Koivisto, P., Tanner, M., Keinanen, R., P
almberg, C., Palotie, A., Tammela, T., Isola, J. and Kallioniemi, O.P.
In vivo amplification of the androgen receptor gene and progression of h
uman prostate cancer (in vivo amplification of androgen receptor gene and pre-human
Nat. Genet., 9: 401, 1995], nevertheless AR-sudden changes
Atypical or AR-null prostate cancer cells and tissues were usually seen [Gaddipati, J. et al.
P., McLeod, D.G., Heidenberg, H.B., Sesterhenn, I.A., Finger, M.J., Moul
, J.W. and Srivastava, S. Frequent detection of codon 877 mutation in t
he androgen receptor gene in advanced prostate cancers
Frequency of mutation at codon 877 in the androgen receptor gene in the rat)
r Res., 54: 2861, 1994]. Based on these findings, PSA and / or AR
Negative tumors are involved in Ad-OC-E1a-induced cell lysis, but not in Ad-PSA-E1a-induced cells.
May not be involved in vesicle lysis. In some earlier publications, Ad vectors were poisonous
Mediates the HSV-TK gene (the expression of which is driven by the OC promoter)
Tumor growth [Gardner, T.A., Ko, S.C., Kao, C., Shirakawa, S., Cheon, J., Go
toh, A., Wu, T.T., Sikes, R.A., Zhau, H.E., Cui, Q., Balian, G. and Ch.
ung, L.W.K.Exploiting stromal-epithelial interaction for model developm
ent and new strategies of gene therapy for prostate cancer and osteosarc
oma metastasis (Development of a model for metastasis of prostate cancer and osteosarcoma and novel inheritance
Utilization of stromal-epithelial interaction for strategies of child therapy). Gene Ther. Mol. Biol., 2
: 41, 1998; Ko, S.C., Cheon, J., Kao, C., Gotoh, A., Shirakawa, T., Sikes
, R.A., Karsenty, G. and Chung, L.W.K.Osteocalcin promoter-based toxic
 gene therapy for the treatment of osteosarcoma in experimental models (
Based on osteocalcin promoter for treatment of osteosarcoma in experimental model
Cancer Res., 56: 4614, 1996; Cheon, J., Ko, S.C., Gardn.
er, T.A., Shirakawa, T., Gotoh, T., Kao, C. and Chung, L.W.K.Chemogene
 therapy: Osteocalcin promote-based suicide gene therapy in combination
with methotrexate in a murine osteosarcoma model (chemical gene therapy: mouse
Osteocalcin promoter combined with methotrexate in an osteosarcoma model
Cancer Gene Ther. 4: 359, 1997] and its transformation.
[Shirakawa, T., Ko, S.C., Gardner, T.A., Cheon, J., Miyamoto, T., Got
oh, A., Chung, L.W.K. and Kao, C. In vivo suppression of osteosarcoma p
ulmonary metastasis with intravenous osteocalcin promoter-based toxic ge
ne therapy (intravenous osteocalcin promoter-based toxin gene therapy
In vivo suppression of osteosarcoma pulmonary metastasis by cancer). Cancer Gene Ther., 5: 274, 1998]
Control the growth of prostate cancer both in vivo and in vitro [G
ardner, T.A., Ko, S.C., Kao, C., Shirakawa, S., Cheon, J., Gotoh, A., Wu
, T.T., Sikes, R.A., Zhau, H.E., Cui, Q., Balian, G. and Chung, L.W.K.
 Exploiting stromal-epithelial interaction for model development and new
 strategies of gene therapy for prostate cancer and osteosarcoma metasta
sis (prostate cancer and osteosarcoma metastasis model development and novel gene therapy warfare
Utilization of stromal-epithelial interaction for abbreviation). Gene Ther. Mol. Biol., 2:41, 1998.
]It has been shown. Most of these early studies used intratumoral Ad-OC-TK.
However, we found that intravenous administration of Ad-OC-TK resulted in significant remission of pulmonary metastasis of osteosarcoma.
And improved survival rates were observed [Shirakawa, T., Ko, S.C., Gardn
er, T.A., Cheon, J., Miyamoto, T., Gotoh, A., Chung, L.W.K. and Kao, C.
 In vivo suppression of osteosarcoma pulmonary metastasis with intraveno
us osteocalcin promoter-based toxic gene therapy (intravenous osteocalcin
In vivo suppression of lung metastasis of osteosarcoma by promoter-based toxin gene therapy.
cer Gene Ther., 5: 274, 1998]. Intravenous Ad-OC-TK does not cause liver toxicity
Because of its ability to show antitumor activity against osteosarcoma lung metastasis,
Tumor-specific to drive the expression of therapeutic genes or viral replication in
Or tissue-specific promoter selection.
Was. In this regard, replication-selective adenoviruses have introduced oncolytic virus
May have the advantage of expanding and helping to spread the drug to neighboring cells
It is clear [Rodrigez, R., Schuur, E.R., Lim, H.Y., Henderson
, G.A., Simons, J.W. and Henderson, D.R.Prostate attenuated replicatio
n competent adenovirus (ARCA) CN706: a selective cytotoxic for prostate-
specific antigen-positive prostate cancer cells
Denovirus (ARCA) CN706: Selective screening of prostate-specific antigen-positive prostate cancer cells
Vesicle injury). Cancer Res., 57: 2559, 1997; Yu, D-.C., Sakamoto, G.T. and H.
enderson, D.R.Identification of the transcriptional regulatory sequence
s of human kallikrein 2 and their use in the construction of calydon vir
us 764, an attenuated replication competent adenovirus for prostate canc
er therapy (Identification of transcriptional regulatory sequences of human kallikrein 2 and treatment of prostate cancer
Virus 764 (attenuated replication-competent adenovirus) for construction
Cancer Res., 59: 1498, 1999; Yu, D-.C., Chen, Y., Seng,
 M., Dilley, J. and Henderson, D.R.The addition of adenovirus type 5 r
egion E3 enables calydon virus 787 to eliminate distant prostate tumor x
enografts (addition of adenovirus type 5 E3 region causes calidone virus 787
Cancer Res., 59: 4200, 1999; Heise, C
And Kirn, D.H.Replication-selective adenoviruses as oncolytic agents
(Replication-selective adenovirus as an oncolytic). J. Clin. Invest., 105: 84.
7, 2000; Heise, C., Williams, A., Xue, S., Propst, M. and Kirn, D. Intr.
avenous administration of ONYX-015, a selectively-replicating adenovirus
, induces antitumoral efficiency (selectivity of the selective replicating adenovirus ONYX-015)
Intravenous administration induces antitumor efficiency). Cancer Res., 59: 2623, 1999]. This study
Now, systemic Ad for the treatment of androgen-independent prostate cancer skeletal xenografts
-Substantial efficacy of -OC-E1a was demonstrated. Pre-existing human prostate in bone
Repeated administration of Ad-OC-E1a is required to eliminate adenocarcinoma xenografts.
Rukoto has been shown. All mice initially responded to Ad-OC-E1a treatment (serum P
Only one mouse (20%) showed the effect of Ad-OC-E1a when judged by SA response)
Evidence was obtained that it gradually became non-responsive to Ad-OC-E1a. 40% (2/5)
In Ad-OC-E1a-treated mice, complete tumor regression occurred, indicating that
Deemed "healed" by Le. Mouse responds to Ad-OC-E1a
The reason for the loss of sex is currently unknown, but it is reasonable that these
In resistant tumors, decreased adenovirus receptor CAR on the tumor cell surface or systemic circulation
Ad-OC-E12a by rapid clearance of the Ad vector from the ring or tumor site
This suggests that the infectivity of the virus may have decreased. Current protocols are clinical
Considerations that may be applicable to the treatment of skeletal metastases of prostate cancer, but require observation
There are: 1) more extensive investigation of Ad-OC-E1a replication in normal human tissues
is necessary. Human bone and human prostate cancer chimeric xenografts grown subcutaneously
Seems to be ideal for this assessment. 2) Serum PSA response is a measure of tumor regression
Can be, but do not prove, [Thalmann, G.N., Siles, R.A., C
hang, S-M., Johnston, D.A., von Eschenbach, A.S., and Chung, L.W.K.Sur
amin-induced decrease in prostate-specific antigen expression with no ef
fect on tumor growth in the LNCaP model of human prostate cancer
Prostate-specific antigen release does not affect tumor growth in the LNCaP model of prostate cancer
J. Natl. Cancer Inst., 88: 794, 1996]. if
This may be a pitfall in using changes in serum PSA as a measure of antitumor activity.
If effective, serum PSA responses can improve survival, improve pain,
Level of normalization, normalization of bone-derived alkaline phosphatase
It is firmly recognized that there is a direct correlation with weight gain and improved behavioral status
[Millikan, R.E., Chemotherapy of advanced prostate carcinoma.
Chemontherapy for Prostate Cancer). Semin. Oncol., 26: 185, 1999]. Smith et al. For 8 weeks
A decrease in serum PSA levels by at least 50% in the meantime is associated with a significant increase in survival
[Smith, D.C., Dunn, R.L., Strawderman, M.S. and Pi.
enta, K.J.Change in serum prostate-specific antigen as a marker of resp
onse to cytotoxic therapy for hormone-refractory prostate cancer
Serum Prostate as a Marker of Response to Cytotoxic Therapy for Non-Refractory Prostate Cancer
Changes in specific antigens). J. Clin. Oncol., 16: 1835, 1998]. Such data
Change serum PSA levels with response parameters in trials of treatment of prostate cancer.
It is recognized that it is effective to use it. In this study, this PSA response
Parameters demonstrate the efficacy of systemic OC promoter-driven replicable gene therapy
Testify.       [0190]   In summary, the present inventors have determined that tissue-driven replication of adenovirus
Experimental human prostate cancer scaffold heterologous using specific, tumor-restricted OC promoter
Novel replicable adenowis for treating prostate cancer metastasis in a graft model
Lus treatment was established. Ad-OC-E1a can be used in human bone without side effects.
Effective in eliminating pre-existing androgen-independent prostate cancer in mice
It was shown to be. This study shows that prostate cancer and bone
Is that it is an effective means of destroying human prostate cancer in bone.
Was shown.       [0191]XV. Example 3: Effects on renal cancer RCC52 cells, PC3 cells, C4-2 cells and DU145 cells
Action of replicable Ad-hOC-E1 + vitamin D Materials and methods   To investigate the effect of vitamin D on the expression of vitamin D receptor (VDR)
Total RNA from normal prostate cancer cell lines (C4-2 and PC3) and kidney cancer cell line (RC52)
Released. RT-PCR targeting VDR (1.3 KB) was performed for 25 cycles. VDR expression
The effect of vitamin D was also evaluated. Expression of human OC (hOC) in various cell lines
The effect of vitamin D on cholesterol was also investigated.       [0192]   Human prostate cancer cell line (C4-2, PC3), human renal cancer cell line (RCC52), human osteosarcoma
Western blot analysis of vitamin D receptor (VDR) in blastocyst strain (MG-63)
Was. The reference track protein was loaded on the left track of the gel (min.
Quantities range from 20.6 to 122 kDa).       [0193]   Replication of Ad-hOC-E1 + Vitamin D against human renal carcinoma RCC52 cell line in vitro
The following procedure was used to assess cytotoxicity. Exposing cells to Ad-hOC-E1 infection
(Dose ranged from 0.01 to 5 MOI or pfu / cell;
2-100 cells / cell were estimated). Expose cells to Ad-hOC-E1 for 2 hours to remove virus
Remove the medium containing 5% fetal bovine serum in the presence or absence of vitamin D.
The medium was replaced with T medium. Standard protocols were used in this study, and
Are described below [ie, 5 nM (C4-2 cells) to 10 nM (DU145, PC3 and RC
C52 cells), containing new vitamin D on day 3 using vitamin D concentrations in the range
Medium was replaced). On days 1, 3, 5, and 7, cell numbers were determined by Chris.
Automated E-Max spectrophotometric plate reader with tall violet assay
It measured using. Data are as of Day 1, 3 after exposure to Ad-hOC-E1 and Vitamin D.
The relative cell numbers on days 5, 5 and 7 (uninfected control samples = 1.0) and
To represent. Vitamin D increased RCC52 cell killing with 5 MOI of Ad-OC-E1
Please pay attention to. Replicable Ad-sPSA-E1 + Vitamin D cytotoxicity on PC3 cells
Toxicity measurement, cytotoxicity of replicable Ad-sPSA-E1 + vitamin D on DU145 cells
Measurement, cytotoxicity of replicable Ad-sPSA-E1 + vitamin D on C4-2 cells
Measurement, determination of cytotoxicity of replicable Ad-hOC-E1 + vitamin D on PC-3 cells
Of cytotoxicity of replicable Ad-hOC-E1 on DU145 cells, and C4-2 cells
The same procedure was used to determine the cytotoxicity of Ad-hOC-E1 + vitamin D for
.       [0194]Results and Discussion   In this experiment, the PSA promoter and osteo-
The effects of the calcine promoter and vitamin D were relatively investigated. PC3 cell line
And DU145 cell lines do not express PSA or androgen receptor, whereas C4-2 cells express PSA.
Expresses both androgen receptors. As shown in Figure 1, vitamin D
It appears to suppress VDR mRNA expression in cancer cell lines but not renal cancer cell lines.
Human prostate cancer cell lines (C4-2, PC3), human renal cancer cell lines (RCC52), human osteosarcoma cells
From Western blot analysis of vitamin D receptor (VDR) in strain (MG-63),
Consistent with the reduction of mRNA due to vitamin D treatment in prostate cancer cell lines, VDR
It was also found that the expression of parkin was slightly reduced by the treatment with vitamin D (
(Fig. 2).       [0195]   The effect of vitamin D on hOC expression was determined using cultured human prostate cancer cell lines (C4-2 and PC3).
), Human renal cancer cell line (RC52), human osteosarcoma cell line (MG-63) and human transitional epithelium
When examined in cancer cell lines (WH), treatment with vitamin D showed PC3, RC52, MG-63 and
And WH cell lines increased hOC mRNA expression, but not C4-2 cell line
Was.       [0196]   Replicable Ad-hOC-E1 + Vitamin D or Replicatable Ad-sPSA for various cell lines
When the cytotoxic effect of -E1 + vitamin D was examined, the following results were obtained. i
Replication of Ad-hOC-E1 + Vitamin D on human renal cancer RCC52 cell line in vitro
For vesicle injury, vitamin D kills RCC52 cells at 5 MOI of Ad-OC-E1
Was increased (FIG. 4). Ad-sPSA-E1 + Vitamin D
With respect to vesicle injury, vitamin D has the lowest effect on PC3 cell proliferation.
(FIG. 5). Replicative Ad-sPSA-E1 + Vitamins for DU145 cells
Regarding the cytotoxicity of vitamin D, vitamin D at the highest concentration (1 MOI)
Was observed to suppress the growth of DU145 cells (FIG. 6). C4-2 cells
As for the cytotoxicity of Ad-sPSA-E1 + vitamin D, vitamin D is 0.01MO
At doses higher than I, it has a significant growth inhibitory effect on C4-2 cells in vitro
(Fig. 7). Replicatable Ad-hOC-E1 + vitamin for PC-3 cells
Regarding the cytotoxicity of D, vitamin D was compared with Ad-sPSA-E1 in vitro.
Shows higher stimulatory effect on increasing Ad-OC-E1 cytotoxicity against PC3 cells
However, apparent growth inhibition was detected at Ad-hOC-E1 at a concentration higher than 1 MOI.
Observed (FIG. 8). About the cytotoxicity of replicable Ad-hOC-E1 on DU-145 cells
Although Ad-sPSA-E1 does not suppress the growth of DU145 cells in the absence of vitamin D,
(Data not shown), Ad-hOC-E1 was added to DU145 cells in the presence of vitamin D
If DU145 cells show in vitro a higher growth inhibition than Ad-sPSA-E1,
Was observed (FIG. 9). Finally, the details of Ad-hOC-E1 + vitamin D on C4-2 cells
For alveolar injury, Ad-hOC-E1 was expressed at 0.01 MOI in the presence of vitamin D.
Even observed to have very high efficacy in inhibiting the growth of C4-2 cells
(Fig. 10).       [0197]   From these results, in conclusion, PSA⁺Cell population positive for androgen receptor
And PSA⁻Of human prostate cancer with androgen receptor negative cell population
In particular, Ad-OC-E1a + vitamin D can be
Excellent cytotoxicity compared to Ad-sPSA-E1 + vitamin D when tested
You can see clearly that it was.       [0198]   The control resulted in the following: Ad-CMV-PA control virus without insert was in
It did not inhibit the growth of RCC52 cells in vitro (FIG. 11A). As expected, the inserter
Ad-CMV-PA control virus, which did not contain PCT cells, did not inhibit PC-3 cell growth (FIG. 1).
1B). As expected, the Ad-CMV-PA control virus without insert + vitamin D
Did not inhibit the growth of DU145 cells (FIG. 11C). Includes inserts as expected
No Ad-CMV-PA control virus did not suppress the growth of C4-2 cells (FIG. 11D). Forecast
As expected, wild-type Ad vectors higher than 0.1 MOI can be used to grow PC-3 cells in vitro.
Was suppressed (FIG. 11E). As expected, even the lowest dose of wild-type Ad
It inhibited the growth of RCC52 cells in vitro (FIG. 11F). As expected, wild type Ad vector
Has a very high potency in inhibiting the growth of C4-2 cells in vitro (
(Figure 11G).       [0199]XVI. Example 4: C4 of replicable Ad-MOC-E1 upon co-inoculation of osteogenic D1 stromal cells -2 Ability to induce cell cytotoxicity Materials and methods   Herpes simplex thimi in the presence of the prodrug ganciclovir (GCV)
In combination with a mouse pluripotent osteogenic D1 stromal cell line transduced with the gin kinase (TK) gene
When cultured, C4-2 cells (progenitors positive for androgen receptor and PSA)
Luciferase tag to determine its effect on the cytotoxicity of adenocarcinoma cells).
C4-2 cell killing was measured in the added C4-2 cell line (C4-2 luc). Luciferer
Ze specific activity was primarily correlated with C4-2 cell number. C4-2luc cells were placed in tissue culture dishes
Co-cultured with either 1 or D1-TK (total cell number = 4 × 10^Four/ Well).       [0200]   Fineness of C4-2 cells when co-inoculated with mouse pluripotent osteogenic D1 stromal cell line in vivo
C4-2 cells alone or together with D1-TK to determine their effect on cytotoxicity
Inject (2 × 10⁶) / Solid tumors were formed subcutaneously.       [0201]   In SCID / bg mice bearing C4-2 tumors in bone, Ad-ma
The effect of intravenous administration of mouse OC-E1a (Ad-mOC-E1a) was also examined.       [0202]Results and Discussion   Herpes simplex thymidine in the presence of the prodrug ganciclovir (GCV)
With mouse pluripotent osteogenic D1 stromal cell line transduced with TK kinase
When C4-2 cells are co-cultured, D1 or D1-TK cells
Stimulation of proliferation was observed. When GCV was added, the number of C4-2 cells (upper panel
) And a marked suppression of total cellular proteins (lower panel) (FIG. X).
These results indicate that C4-2 prostate cancer cells require stromal cells for their own survival.
Is shown.       [0203]   Inoculated with D1-TK alone or with C4-2 cells as an osteogenic cell line
Chimeric osteogenic prostate cancers similar to skeletal metastases of prostate cancer were formed (Panels A and
And B: X-ray images of subcutaneous tumors in athymic mice, respectively.
, And C4-2 / D1-TK chimeric tumors. ACV intraperitoneally
Upon administration, a significant reduction in tumor size was observed (see FIG. X, panel A),
A sharp drop in serum PSA occurred (see FIG. X, panel C).       [0204]    Furthermore, when Ad-mouse OC-E1a is administered intravenously, it carries C4-2 tumor in bone
In SCID / bg mice, tumor regression was observed by both radiography and macroscopic morphology.
Evidence has been obtained. FIG. X (Panel A) shows SCID / bg mouse bearing intraosseous C4-2 tumor.
The effect of the Ad-mouse OC-E1a adenovirus replicable vector on
ing. FIG. X (panel B) shows the Ad-mouse OC-E1a adenovirus replicable vector.
Control SCID / bg mice bearing intraosseous C4-2 tumors in the absence of
You.       [0205]   Therefore, the replication-competent Ad-mouse OC-E1a adenovirus vector is compatible with C4-2 tumor
In mice bearing bone in bone, such as, but not limited to, bone stromal cells
Has the ability to kill both MG-63 cells) and human prostate C4-2 cancer cells
It was very effective.       [0206]   Thus, from the experiments described above, the prostate cancer cell line, PSA secretory (
Without limitation, LNCaP, C4-2, ARCaP, etc. or non-secreted (limited)
(But not PC-3, DU145, etc.), and bone (MG-63) and anterior
Proliferation of stromal cell line (9096F) is induced by Ad-OC-E1a by viruolytic activity.
This is demonstrated to be significantly suppressed.       [0207]   All publications, patents and patent applications mentioned herein are hereby incorporated by reference.
Each publication, patent or patent application is hereby incorporated by reference.
To the same extent as is expressly stated to be incorporated herein.       [0208]Equivalent   Those skilled in the art will use only routine experimentation to
Many equivalents of particular embodiments of the invention will be understood or ascertainable.
You. Such equivalents are intended to be encompassed by the following claims. [Brief description of the drawings]     FIG.   Homologous pair of shuttle vector pOC-E1a and recombinant vector pJM17 in 293 cells
Construction of replicable adenovirus type 5 Ad-OC-E1a by recombination.     FIG. 2   Immunohistochemical demonstration of the presence of OC in primary and metastatic human prostate cancer specimens
. Positive OC staining indicates primary cancer-associated stroma (panel A) and both stroma and tumor epithelium
Note that this was detected (panel B). Positive immunostaining was performed in lymph nodes (panel
D) and bone (panel E) metastases were also found. Background immunostaining
Were found in control primary (panel C) and bone metastatic (panel F) prostate cancer.     FIG. 3   Human prostate cancer and stromal cells of bone and prostate by replicable Ad-OC-E1a
In vitro inhibition of proliferation. Cell growth is in vitro with Ad-OC-E1a, Ad-CMV-β-gal or Ad
-Evaluated in the presence of CMV-PA. The cell viability (%) is between the test virus (0.01 to 5 MOI).
Or pfu / cell) on day 3 after infection. The results of these tests are:
Showed: (A) Does Ad-CMV-PA or Ad-CMV-β-gal affect C4-2 proliferation?
However, Ad-OC-E1a suppressed cell growth of C4-2 and 293 in a virus concentration-dependent manner
. Ad-OC-E1a was not effective in suppressing cell proliferation of WH and Lovo cells.
This is because WH and Lovo cells lack OC promoter activity and OC expression.
You. (b) Ad in C4-2, LNCaP, PC-3, ARCaP and DU-145 human prostate cancer cell lines
Comparison of the effects of cell lysis induced by -OC-E1a. Strongly PSA and AR positive (LNCa
P, C4-2) and negative (PC-3, DU-145) and barely PSA and AR positive (ARCaP)
Human prostate cancer cell lines are sensitive to cell lysis induced by Ad-OC-E1a
Note that Both human prostate and osteofibroblasts are also Ad-OC-E1a
It is therefore sensitive to induced cell lysis. Data are standard deviation within 15% of average
Represents the average of three experiments, determined using differences.     FIG. 4   Differential in vivo differentiation of PC-3 and Lovo tumor growth after intratumoral injection of Ad-OC-E1a
Suppression. 1x10⁶PC-3 or Lovo cells were injected subcutaneously into athymic nude mice for 4 weeks
2x10 later in mice bearing tumors⁹ Ad-OC-E1a or Ad-CMV-β-gal of pfu once
Administered intratumorally. Ad-OC-E1a (2x10⁹ pfu) is significant for PC-3 cell proliferation
Did not show any significant suppression. Ad-OC-E1a suppressed tumor growth of PC-3 in vivo,
Vo, it did not curb.     FIG. 5   Intravenous Ad-O versus serum PSA concentration in SCID / bg mice injected with C4-2 cells intraosseously
Proof of the effect of C-E1a. (a) 1x10⁶Treated after intraosseous injection of C4-2 cells
Serum PSA concentration in no control mice. Blood in untreated mice (mouse # 1)
Note the exponential rise of Qing PSA. (b)-(f) (i.e., mouse # 2 to # 6)
 1x10⁶Serum PSA elevation was detected in mice injected intraosseously with C4-2 cells
Later, Ad-OC-E1a (2x10⁹ pfu) Serum PSA concentration in mice injected.
Arrows indicate when Ad-OC-E1a was administered intravenously (week). Systemic Ad-OC-E1a is blood
Ad-OC-E1a leads to a rapid decrease in Qing PSA and 4/5 (80%) of the animals are repeated
Significant response to treatment.     FIG. 6   All-morphological and X-ray of Ad-OC-E1a eradicates the growth of intraosseous human prostate tumor xenografts
And histopathological evidence. (a) This panel shows Ad-OC-E1a (2 × 10⁹ pfu
Shows that a single administration of) induced significant tumor regression in treated mice
(See left panel). (b) Prostate tumor regression due to Ad-OC-E1a
Tissues that look like cells (panel A) and those cells expressing PSA (panel B)
Supported by pathological evidence. In the treated sample, any tumor cells
Neither (panel C) nor PSA is observed in the skeleton (data not shown). (c) positive
Normal mouse and Ad-CMV-β-gal (1x10⁹ X-gal staining of human bone infected with pfu). Ma
Mouse myeloma cells are susceptible to Ad-CMV-β-gal infection, whereas mouse cortical bone is
Note that it is resistant to staining (Panel A). Panel B is on soft agar
Human PC-3 prostate tumors are susceptible to Ad-CMV-β-gal infection when maintained as explants
Sexuality. Normal human bone exposed to this virus has detectable β-g
normal human bone cells that do not result in al activity may be resistant to Ad vector infection
Suggests none (Panel C).     FIG. 7   RT-PCR of vitamin D receptor (VDR) in normal and neoplastic human cancer cell lines. Figure
Lane to the left represents marker RNA.     FIG. 8   Human prostate cancer (C4-2, PC3), human renal cancer (RCC52), and human osteosarcoma (MG-63
) Western blot of vitamin D receptor (VDR) in cell lines.     FIG. 9   RT-PCR of human osteocalcin (hOC) mRNA. Effect of vitamin D on hOC expression
Fruits were cultured from human prostate cancer (C4-2, PC3), human renal carcinoma (RC52), human osteosarcoma (M
G-63) and a human transitional cell carcinoma (WH) cell line.     FIG. 10   In vitro replication of Ad-hOC-E1 + vitamin D in human renal cancer RCC52 cell line
Vesicle injury.     FIG. 11   Cytotoxicity of replicable Ad-sPSA-E1 + vitamin D on PC3 cells.     FIG.   Cytotoxicity of replicable Ad-sPSA-E1 + vitamin D on DU145 cells.     FIG. 13   Cytotoxicity of replicable Ad-sPSA-E1 + vitamin D on C4-2 cells.     FIG. 14   Cytotoxicity of replicable Ad-hOC-E1 + vitamin D on PC-3 cells.     FIG.   Cytotoxicity of replicable Ad-hOC-E1 + vitamin D on DU-145 cells.     FIG. 16   Cytotoxicity of Ad-hOC-E1 + vitamin D on C4-2 cells.     FIG.   (A) Cytotoxicity of Ad-CMV-PA on RCC52 cells. (B) Ad-CMV-PA on PC-3 cells
Cytotoxicity. (C) Cytotoxicity of Ad-CMV-PA to DU145 cells. (D) Ad-CMV
-Cytotoxicity of PA to C4-2 cells. (E) Wild-type Ad vector inhibits PC-3 cell growth
Cytotoxicity. (F) Cytotoxicity of wild-type Ad vector on proliferation of RCC52 cells.
(G) Cytotoxicity of wild-type Ad vector on proliferation of C4-2 cells.     FIG.   Herpes simplex thymidine in the presence of the prodrug ganciclovir (GCV)
Murine pluripotent osteogenic D1 stromal cell line transduced with the kinase (TK) gene
Cytotoxicity of C4-2 cells when co-cultured.     FIG.   Cytotoxicity of C4-2 cells co-inoculated in vivo with a murine pluripotent osteogenic D1 stromal cell line
sex.     FIG.   Radiography of tumor regression in SCID / bg mice bearing C4-2 tumor in bone
-And full morphological evidence.     FIG. 21   1370 bp mouse osteocalcin promoter nucleotide sequence (SEQ ID NO:
1).

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 35/00 A61P 35/04 35/04 43/00 105 43/00 105 C12N 1/15 C12N 1/15 1/19 1/19 1/21 1/21 7/00 5/10 C12Q 1/02 7/00 1/68 A C12Q 1/02 C12N 15/00 ZNAA 1/68 5/00 A (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG, AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW), EA (AM, AZ, BY, KG, K , MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU, CZ , DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI , SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventors Gardner, Thomas, A. United States 46077 Woods Edge Drive, Zion'sville, Indiana 4626 (72) Inventor Ko, Song-chu United States 22901 Charlottesville, Virginia Green Turtle Lane 141 6th F Term (Reference) 4B024 AA01 AA12 BA32 BA36 CA04 CA05 CA02 DA02 EA02 FA02 GA11 HA14 HA17 4B063 QA18 QQ43 QR32 QR55 QS34 4B065 AA90X AA99Y AB01 AC14 AC20 BA02 CA44 4C084 AA13 MA01 MA66 NA06 NA13 NA14 ZA811 ZB261 4C087 AA01 AA02 BC83 CA12 MA01 NA06 NA13 NA14 ZA81 ZABZ

Claims (1)

Claims 1. A replicable adenovirus vector comprising an adenovirus gene under the transcriptional control of an osteocalcin transcription regulatory sequence. 2. The adenovirus vector according to claim 1, wherein the adenovirus gene is a gene essential for virus replication. 3. The adenovirus vector according to claim 1, wherein the osteocalcin transcription regulatory sequence includes an osteocalcin gene-derived enhancer. 4. The adenovirus vector according to claim 1, wherein the osteocalcin transcription control sequence includes a promoter derived from an osteocalcin gene. 5. The adenovirus vector according to claim 1, wherein the osteocalcin transcription regulatory sequence includes a promoter derived from the osteocalcin gene and an enhancer derived from the osteocalcin gene. 6. The adenovirus vector according to claim 1, wherein the osteocalcin transcription control sequence comprises the sequence of SEQ ID NO: 1. 7. A composition comprising the adenovirus vector according to claim 1 and a pharmaceutically acceptable excipient. 8. The adenovirus vector of claim 1, further comprising at least one additional adenovirus gene under the transcriptional control of at least one additional prostate-specific transcription regulatory sequence. 9. The adenovirus vector according to claim 1, further comprising a heterologous gene under the transcriptional control of an osteocalcin transcription control sequence. 10. A host cell transformed with the adenovirus vector according to claim 1. 11. A method for detecting a cell in which a osteocalcin transcription regulatory sequence functions in a biological sample, which comprises conditions suitable for osteocalcin transcription regulatory sequence-mediated gene expression in a cell in which the osteocalcin transcription regulatory sequence functions. Contacting a biological sample with the adenovirus vector of claim 1 and determining whether an osteocalcin transcription regulatory sequence mediates gene expression in said biological sample, The above method, wherein the osteocalcin transcription regulatory sequence-mediated gene expression indicates the presence of a cell in which the osteocalcin transcription regulatory sequence functions. 12. A method for propagating an adenovirus specific to a cell that functions with an osteocalcin transcription regulatory sequence, comprising combining the adenovirus vector according to claim 1 with a cell that functions with an osteocalcin transcription regulatory sequence. The above method, wherein the method comprises growing the adenovirus. 13. A method of altering the genotype of a target cell, comprising contacting a cell that functions with an osteocalcin transcriptional regulatory sequence with the adenovirus of claim 1, whereby the adenovirus is transformed into the cell. The method as described above. 14. A method for inhibiting the growth of tumor cells, comprising contacting tumor cells and non-tumor cells with the adenovirus vector according to claim 1, so that the adenovirus vector enters the tumor cells. The above-described method, comprising displaying selective cytotoxicity against said tumor cells. A kit comprising the adenovirus vector according to claim 1. 16. A composition comprising the adenovirus vector according to claim 1 and a buffer. 17. The method of claim 2, wherein the adenovirus gene is an early gene.
2. The adenovirus vector according to item 1. 18. The adenovirus gene of claim 2, wherein the adenovirus gene is a late gene.
2. The adenovirus vector according to item 1. 19. The adenovirus early gene is E1A.
2. The adenovirus vector according to item 1. 20. The adenovirus early gene is E1B.
2. The adenovirus vector according to item 1. 21. The adenovirus vector of claim 8, wherein said at least one additional prostate-specific transcription control sequence comprises an osteocalcin transcription control sequence. 22. The adenoviral vector of claim 21, wherein said at least one additional prostate-specific transcription regulatory sequence comprises an osteocalcin promoter sequence as set forth in SEQ ID NO: 1. 23. The adenovirus vector of claim 8, wherein said at least one additional prostate-specific transcription regulatory sequence comprises a prostate-specific antigen (PSA) transcription regulatory sequence. 26. A composition comprising the adenovirus vector of claim 8 and a pharmaceutically acceptable excipient. 27. A method for propagating an adenovirus specific to a mammalian cell in which the osteocalcin transcription regulatory sequence functions, wherein the adenovirus vector according to claim 8 and a mammalian cell in which the osteocalcin transcription regulatory sequence functions. Combining the adenovirus, thereby growing the adenovirus. 28. A method of altering the genotype of a target cell, comprising contacting a cell that functions with an osteocalcin transcriptional regulatory sequence with the adenovirus of claim 8, whereby the adenovirus is transformed into the cell. The method as described above. 29. A method for inhibiting the growth of a tumor cell, comprising contacting the tumor cell with the adenovirus vector according to claim 8, and consequently, the adenovirus vector enters the tumor cell and binds to the tumor cell. The above method, which comprises causing the cell to show selective cytotoxicity. A kit comprising the adenovirus vector according to claim 8. 31. A composition comprising the adenovirus vector of claim 8 and a buffer. 32. The adenovirus vector of claim 8, wherein said additional adenovirus gene is a gene essential for virus replication. 33. A host cell transformed by the adenovirus vector according to claim 8. 34. The adenovirus vector according to claim 9, wherein said heterologous gene is a reporter gene. 35. The adenovirus vector of claim 9, wherein said heterologous gene is a conditionally required gene for cell survival. 36. The method of claim 11, wherein said cells function osteocalcin transcriptional regulatory sequences and do not express PSA or androgen receptor. 37. The additional adenovirus gene is an early gene.
An adenovirus vector according to claim 35. 38. The additional adenovirus gene is a late gene.
An adenovirus vector according to claim 35. 39. The adenovirus vector according to claim 37, wherein the early gene is E1A. 40. The adenovirus vector according to claim 37, wherein the early gene is E1B. 41. A method for imparting selective toxicity to a target cell, comprising contacting a cell that functions with an osteocalcin transcription regulatory sequence with an adenovirus vector containing an adenovirus gene that is under transcriptional control of the osteocalcin transcription regulatory sequence. Wherein the expression of the gene under the transcriptional control of an osteocalcin transcription regulatory sequence contributes to cytotoxicity. 42. The adenovirus gene is an early gene.
2. The method according to 1. 43. The adenovirus gene is a gene essential for replication.
42. The method of claim 41. 44. The method of claim 41, wherein said early gene is E1A. 45. The method of claim 42, wherein said early gene is E1B. 46. The adenovirus gene is an early gene.
2. The adenovirus vector according to item 1. 47. The additional adenovirus gene is an early gene.
An adenovirus vector according to claim 14. 48. The method of claim 41, wherein said adenovirus vector further comprises at least one additional adenovirus gene under the transcriptional control of at least one additional prostate-specific transcription regulatory sequence. 49. The method according to claim 14, wherein the adenovirus gene is a gene essential for virus replication. 50. The adenovirus gene is an early gene.
4. The method according to 4. 51. The adenovirus gene is a late gene.
4. The method according to 4. 52. A method of treating cancer in an individual, comprising administering to the individual a conditionally replicable adenovirus, Ad-OC-E1a. 53. The method of claim 52, wherein said cancer is in the form of a solid tumor. 54. The method of claim 52, wherein Ad-OC-E1a is administered by injection into a tumor. 55. The method of claim 52, wherein said administration is by an intravenous route or by direct injection of a conditionally replicable adenovirus Ad-OC-E1a. 56. A method of treating cancer in an individual, the method comprising conditionally administering to the individual an adenovirus Ad-OC-E1a capable of tissue-specific and tumor-replicating replication. An adenovirus Ad-OC-E1a vector that is conditionally tissue-specific and tumor-replicatable is specific for cells that function osteocalcin transcriptional regulatory sequences, and that the cells express PSA or androgen receptor (AR). Such a method, wherein the method is prostate cancer cells, prostate stromal cells, perivascular cells, proliferative cancer-related osteoblasts, and prostate cancer and related bone stromal cells that do not express. 57. The method of claim 56, wherein Ad-OC-E1a is administered by injection into a tumor. 58. The method of claim 56, wherein said administering is by an intravenous route or by direct injection of a conditionally replicable adenovirus Ad-OC-E1a. 59. A method for treating a disease associated with calcification in an individual, comprising conditionally administering to the individual an adenovirus Ad-OC-E1a capable of tissue-specific and tumor-replicating replication. Here, the adenovirus Ad-OC-E1a vector, which is conditionally tissue-specific and tumor-replicatable, is specific for cells that function osteocalcin transcription regulatory sequences, and the disease is benign prostate hyperplasia (BPH ) Or arteriosclerosis.