U.S. patent application number 10/218542 was filed with the patent office on 2003-05-08 for expression of cyclin g1 in tumors.
This patent application is currently assigned to University of Southern California. Invention is credited to Anderson, W. French, Gordon, Erlinda M., Hall, Frederick L..
Application Number | 20030086927 10/218542 |
Document ID | / |
Family ID | 26746581 |
Filed Date | 2003-05-08 |
United States Patent
Application |
20030086927 |
Kind Code |
A1 |
Gordon, Erlinda M. ; et
al. |
May 8, 2003 |
Expression of cyclin G1 in tumors
Abstract
A method of treating a tumor (in particular osteosarcoma or
Ewing's sarcoma) in a host by administering to a host or to the
tumor cells an agent which inhibits cyclin G1 protein in an amount
effective to inhibit cyclin G1 protein in tumor cells of the host.
The agent may be an antisense polynucleotide which is complementary
to at least a portion of a polynucleotide encoding cyclin G1
protein, or an antibody or fragment or derivative thereof which
recognizes cyclin G1 protein. Also contemplated within the scope of
the present invention are (i) the immortalization of cell lines by
transducing cells with a polynucleotide encoding cyclin G1 protein;
(ii) increasing the receptiveness of cells to retroviral infection
by transducing cells with a polynucleotide encoding cyclin G1
protein; and (iii) the detection of cancer by detecting cyclin G1
protein or a polynucleotide encoding cyclin G1 protein in cells. In
addition, the present invention provides expression vehicles, such
as, for example, retroviral vectors and adenoviral vectors, which
include polynucleotides which encode agents which inhibit cyclin G1
protein, and expression vehicles which include a polynucleotide
encoding cyclin G1 protein.
Inventors: |
Gordon, Erlinda M.;
(Glendale, CA) ; Hall, Frederick L.; (Glendale,
CA) ; Anderson, W. French; (San Marino, CA) |
Correspondence
Address: |
Raymond J. Lillie
CARELLA, BYRNE, BAIN, GILFILLAN, CECCHI,
STEWART & OLSTEIN
Six Becker Farm Road
Roseland
NJ
07068
US
|
Assignee: |
University of Southern
California
|
Family ID: |
26746581 |
Appl. No.: |
10/218542 |
Filed: |
August 14, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10218542 |
Aug 14, 2002 |
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09066294 |
Oct 26, 1998 |
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09066294 |
Oct 26, 1998 |
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PCT/US96/17442 |
Oct 31, 1996 |
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PCT/US96/17442 |
Oct 31, 1996 |
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08551486 |
Nov 1, 1995 |
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Current U.S.
Class: |
424/146.1 ;
424/155.1; 514/44R |
Current CPC
Class: |
A61K 48/00 20130101;
G01N 2333/4739 20130101; A01K 2227/105 20130101; C07K 14/4738
20130101; A61K 38/00 20130101; A01K 2267/0331 20130101; A01K
2217/00 20130101; A01K 2207/15 20130101; C12N 2799/027 20130101;
C07K 14/4746 20130101; C12N 15/113 20130101; A01K 67/0271 20130101;
A01K 2217/058 20130101; C07K 16/18 20130101 |
Class at
Publication: |
424/146.1 ;
424/155.1; 514/44 |
International
Class: |
A61K 039/395; A61K
048/00 |
Claims
What is claimed is:
1. A method of treating a tumor in a host comprising: administering
to a host an agent which inhibits cyclin G1 protein, said agent
being administered in amount effective to inhibit cyclin G1 protein
in tumor cells of said host.
2. The method of claim 1 wherein said agent is an antisense
polynucleotide which is complementary to at least a portion of a
polynucleotide encoding cyclin G1 protein.
3. The method of claim 1 wherein said agent is an antibody or
fragment or derivative thereof which recognizes cyclin G1
protein.
4. The method of claim 3 wherein said antibody is a single-chain
antibody.
5. The method of claim 3 wherein said antibody is a monoclonal
antibody.
6. The method of claim 3 wherein said antibody is a polyclonal
antibody.
7. The method of claim 2 wherein said antisense polynucleotide is
administered to said host by transducing tumor cells of said host
with an expression vehicle including a polynucleotide encoding an
antisense polynucleotide which is complementary to at least a
portion of a polynucleotide encoding cyclin G1 protein.
8. The method of claim 7 wherein said expression vehicle is a
retroviral vector.
9. The method of claim 1 wherein said tumor is a cancerous
tumor.
10. The method of claim 9 wherein said cancerous tumor is
osteogenic sarcoma.
11. The method of claim 9 wherein said cancerous tumor is Ewing's
sarcoma.
12. The method of claim 3 wherein said antibody or fragment of
derivative thereof which recognizes cyclin G1 protein is
administered to said host by transducing tumor cells of said host
with an expression vehicle including a polynucleotide encoding said
antibody or fragment or derivative thereof which recognizes cyclin
G1 protein.
13. The method of claim 12 wherein said expression vehicle is a
retroviral vector.
14. A method of immortalizing non-tumor cells comprising:
transducing said non-tumor cells with a polynucleotide encoding
cyclin G1 protein or a derivative or analogue thereof.
15. The method of claim 14 wherein said polynucleotide encoding
cyclin G1 or a derivative or analogue thereof is contained within a
retroviral vector.
16. A method of enhancing transduction of cells with a retroviral
vector that includes a polynucleotide encoding a therapeutic agent,
comprising: transducing said cells with a first expression vehicle
that includes a polynucleotide encoding cyclin G1 protein, wherein
said first expression vehicle is not a retroviral vector; and
transducing said cells with a second expression vehicle that
includes a polynucleotide encoding a therapeutic agent, said second
expression vehicle being a retroviral vector.
17. The method of claim 16 wherein said first expression vehicle is
an adenoviral vector.
18. The method of claim 16 wherein said cells are transduced with
said first expression vehicle and said second expression vehicle in
vivo.
19. The method of claim 16 wherein said cells are transduced with
said first expression vehicle and said second expression vehicle in
vitro.
20. The method of claim 16 wherein said cells are transduced with
said first expression vehicle prior to transduction of said cells
with said second expression vehicle.
21. The method of claim 16 wherein said cells are transduced with
said first expression vehicle and said second expression vehicle
concurrently.
22. An expression vehicle including a polynucleotide encoding an
agent selected from the group consisting of (i) an agent which
inhibits cyclin G1 protein; and (ii) cyclin G1 protein.
23. The expression vehicle of claim 22 wherein said expression
vehicle is a viral vector.
24. The vector of claim 23 wherein said vector is a retroviral
vector.
25. The vector of claim 24 wherein said agent is an agent which
inhibits cyclin G1 protein.
26. The vector of claim 25 wherein said agent which inhibits cyclin
G1 protein is an antisense polynucleotide which is complementary to
at least a portion of a polynucleotide encoding cyclin G1
protein.
27. The vector of claim 25 wherein said agent which inhibits cyclin
G1 protein is an antibody or fragment or derivative thereof which
recognizes cyclin G1 protein.
28. The vector of claim 24 wherein said agent is cyclin G1
protein.
29. The vector of claim 23 wherein said vector is an adenoviral
vector.
30. The vector of claim 29 wherein said agent is an agent which
inhibits cyclin G1 protein.
31. The vector of claim 30 wherein said agent which inhibits cyclin
G1 protein is an antisense polynucleotide which is complementary to
at least a portion of a polynucleotide encoding cyclin G1
protein.
32. The vector of claim 30 wherein said agent which inhibits cyclin
G1 protein is an antibody or fragment or derivative thereof which
recognizes cyclin G1 protein.
33. The vector of claim 29 wherein said agent is cyclin G1
protein.
34. A producer cell which produces the retroviral vector of claim
24.
35. A method of detecting cancer, comprising: contacting cells with
an agent which binds to (i) cyclin G1 protein and/or (ii) a
polynucleotide encoding cyclin G1 protein; and determining binding
of said agent to said cyclin G1 protein and/or said polynucleotide
encoding cyclin G1 protein.
36. The method of claim 35 wherein said agent is a polynucleotide
which hybridizes to a polynucleotide encoding cyclin G1
protein.
37. The method of claim 35 wherein said agent is an antibody or a
fragment or derivative thereof which recognizes cyclin G1
protein.
38. The method of claim 35 wherein said cancer is osteogenic
sarcoma.
39. The method of claim 35 wherein said cancer is Ewing's
sarcoma.
40. A method of preventing restenosis, comprising: administering to
a host an agent which inhibits cyclin G1 protein, said agent being
administered in an amount effective to prevent restenosis in a
host.
Description
[0001] This application is a continuation-in-part of application
Ser. No. 08/551,486, filed Nov. 1, 1995, the contents of which are
hereby incorporated by reference.
[0002] This invention relates to the expression of cyclin G1 in
tumors. More particularly, this invention relates to: (i) the
treatment of tumors such as osteogenic sarcoma or Ewing's sarcoma,
by inhibiting cyclin G1 protein in the tumor cells; (ii) the
prevention of restenosis by inhibiting cyclin G1 protein in cells
at the site of an invasive vascular procedure or vascular lesion;
(iii) the immortalization of cells by transducing such cells with a
polynucleotide encoding cyclin G1 protein; (iv) making cells more
receptive to infection or transduction by a retroviral vector by
transfecting the cells with a polynucleotide encoding cyclin G1
protein, prior to or concurrently with said retroviral transduction
or infection; and (v) a method of detecting cancer by determining
the level of expression of cyclin G1 protein in cells. This
invention also relates to expression vehicles, preferably
retroviral vectors and adenoviral vectors, which include
polynucleotides encoding agents which inhibit cyclin G1 protein,
such as antisense polynucleotides, and antibodies or fragments or
derivatives thereof which recognize cyclin G1 protein, and to
expression vehicles which include a polynucleotide encoding cyclin
G1 protein.
BACKGROUND OF THE INVENTION
[0003] Genes encoding a new class of proteins known as cyclins have
been identified as a new class of protooncogenes, and
cyclin-dependent kinase (or Cdk) inhibitors have been identified as
tumor suppressors, thereby uniting the molecular mechanisms of
cellular transformation and tumorigenesis with the enzymology
governing cell cycle control. (Hall, et al., Curr. Opin. Cell
Biol., Vol. 3, pgs. 176-184 (1991); Hunter, et al., Cell, Vol. 55,
pgs. 1071-1074 (1991); Hunter, et al., Cell, Vol. 79; pgs. 573-582
(1994); Elledge, et al., Curr. Opin. Cell Biol., Vol 6, pgs.
874-878 (1994); Peter, et al., Cell, Vol. 79, pgs. 181-184 (1994)).
The sequential expression of specific cyclins and the essential
functions of specific Cdk complexes have been defined (Wu, et al.,
Int. J. Oncol., Vol. 3, pgs. 859-867 (1993); Pines, et al., New
Biolocist, Vol. 2, pgs 389-401 (1990); Pines, Cell Growth and
Differentiation, Vol. 2, pgs. 305-310 (1991); Reed, Ann. Rev. Cell
Biol., Vol. 8, pgs. 529-561 (1992); Sherr, Cell, Vol. 79, pgs.
551-555 (1994)), thereby providing direct links to the fundamental
mechanisms of DNA replication, transcription, repair, genetic
instability, and apoptosis. (D'Urso, et al., Science, Vol. 250,
pgs. 786-791 (1990); Wu, et al., Oncogene, Vol. 9, pgs 2089-2096
(1994); Roy, Cell, Vol. 79, pgs. 1093-1101 (1994); Meikrantz, et
al., Proc. Nat. Acad. Sci., Vol. 91, pgs. 3754-3758 (1994)). Both
the universal Cdk inhibitor p21/WAF1/CIP1 (Xiong, et al., Nature,
Vol. 366, pgs. 701-704 (1993); Harper, et al., Mol. Biol. Cell,
Vol. 6, pgs. 387-400 (1995)), and cyclin G1 (Wu, et al., Oncol.
Resorts, Vol. 1, pgs, 705-711 (1994)) are induced by the wild-type
p53 tumor suppressor protein in the initiation of DNA repair and/or
apoptosis. (El-Deiry, et al., Cell, Vol. 75, pgs 817-825 (1993);
El-Deiry, et al., Cancer Res., Vol. 54, pgs. 1169-1174 (1994)).
Thus, the molecular components regulating critical cell cycle
checkpoints represent strategic targets for potential therapeutic
intervention in the treatment of cell proliferation disorders,
including pediatric bone cancers, in which the Rb and the p53 tumor
suppressor genes often are inactivated. (Hansen, et al., Proc. Nat.
Acad. Sci., Vol. 82, pgs. 6216-6220 (1985); Toguchida, et al.,
Nature, Vol. 338, pgs. 156-158 (1989); Toguchida, et al., Cancer
Res., Vol. 48, pgs. 3939-3943 (1988); Diller, et al., Mol. Cell.
Biol., Vol. 10, pgs. 5772-5781 (1990)). Previous studies have
characterized the progressive profile of cyclin expression and Cdk
activation (Wu, 1993; Carbonaro-Hall, et al., Oncogene, Vol. 8, pgs
1649-1659 (1993); Hall, et al., Oncogene, Vol. 8, pgs. 1377-1384
(1993); Williams, et al., J. Biol. Chem., Vol. 268, pgs. 8871-8880
(1993); Albers, et al., J. Biol. Chem., Vol. 268, pgs. 22825-22829
(1993)), as well as the p53-independent induction of p21/WAF1/CIP1
(Wu, et al., Oncol. Reports, Vol. 2, pgs 227-231 (1995)), in MG-63
osteosarcoma cells. Also, antisense oligonucleotide strategies
directed against cyclin D1 effectively inhibit cell cycle
progression in these osteosarcoma cells. (Wu, 1993).
[0004] Metastatic carcinoma is an important target for gene therapy
as it is associated with poor outcome. Colorectal cancer, for
example, is the second leading cause of cancer death in the United
States after lung cancer, followed by breast and pancreatic cancer
(Silberberg et al., Cancer Clin., Vol. 40, pgs. 9-26 (1990)). Of
these carcinomas, pancreatic cancer has the worst prognosis. The
median survival of patients with metastatic pancreatic cancer is
three to six months and virtually all the patients are dead within
a year (Merrick et al., Gastroenterol. Clin. N. Amer., Vol. 19,
pgs. 935-962 (1990)). Approximately 40% of patients will have
metastatic disease either to the liver or the peritoneal cavity or
both at the time of diagnosis. Chemotherapy for metastatic disease
is ineffective despite multimodal therapy. Hence, alternative
approaches to metastatic carcinoma would be desirable.
[0005] Wu, et al. (Oncol. Reports, Vol. 1, pgs. 705-711 (1994)),
hereinabove mentioned, discloses the deduced amino acid sequence
and cDNA sequence for human cyclin G1 protein. Wu, et al., also
disclose that higher levels of cyclin G1 expression were found in
osteosarcoma cells and in Ewing's sarcoma cells than in normal
diploid human fibroblasts. Although Wu, et al., state that the
overexeression As cyclin G1 protein in human osteosarcoma cells
provides a potential link to cancer, Wu, et al., do not disclose
the treatment of cancer by interfering with or inhibiting the
function of cyclin G1 protein in cancer cells.
SUMMARY OF THE INVENTION
[0006] Applicants have discovered that by interfering with and/or
inhibiting the function or expression of cyclin G1 protein in
cancer cells, one may inhibit, prevent, or destroy the growth
and/or survival of such cancer cells. Thus, the present invention
is directed to the treatment of a tumor (preferably a cancerous
tumor) by inhibiting cyclin G1 protein, preferably through the
administration of antisense oligonucleotides to a polynucleotide
encoding cyclin G1 protein, or antibodies to cyclin G1 protein.
[0007] In addition, the present invention is directed to (i) the
prevention of restenosis by inhibiting cyclin G1 protein in cells
at the site of an invasive vascular procedure or vascular lesion;
(ii) the immortalization of cells by transducing cells with a
polynucleotide encoding cyclin G1 protein; (iii) the transducing of
cells with a polynucleotide encoding cyclin G1 protein in order to
make cells more receptive to transduction or infection with a
retroviral vector; and (iv) a cancer assay which involves detection
of cyclin G1 protein and/or a polynucleotide encoding such
protein.
[0008] The present invention also is directed to expression
vehicles which include polynucleotides encoding agents which
inhibit cyclin G1 protein, and to expression vehicles which include
a polynucleotide encoding cyclin G1 protein. Such expression
vehicles include, but are not limited to, viral vectors such as
retroviral vectors and adenoviral vectors.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] The invention now will be described with respect to the
drawings, wherein:
[0010] FIG. 1 is a nucleotide sequence of human cyclin G1 cDNA;
[0011] FIG. 2 depicts the staining of MG-63 osteogenic sarcoma
cells following transduction of such cells with a retroviral vector
including a B-galactosidase, or lacZ gene;
[0012] FIG. 3 is a graph of the degrees of confluency (%) in
mixtures of MG-63 cells which were transduced with a retroviral
vector including a Herpes Simplex Virus thymidine kinase (TK) gene,
and cells which were not transduced with such vector;
[0013] FIG. 4 is a schematic of the retroviral vectors G1aD1SvNa,
G1aG1SvNa, G1p21SvNa, and G1XSvNa;
[0014] FIG. 5 is a graph of the cell counts in cultures of MG-63
cells transduced with G1XSvNa, G1aD1SvNa, G1aG1SvNa, or
G1p21SvNa;
[0015] FIG. 6 is a Western Blot of expression of p29 cyclin G1
protein in MG-63 cells transduced with G1XSvNa, G1aG1SvNa, or
G1aD1SvNa;
[0016] FIG. 7 depicts the morphological appearance of MG-63 cells
by light microscopy at 72 hours after transduction of such cells
with G1XSvNa, G1aG1SvNa, G1aD1SvNa, or G1p21SvNa;
[0017] FIG. 8 depicts the detection of apoptotic cells in cultures
of MG-63 cells transduced with G1XSvNa, G1aG1SvNa, G1aD1SvNa, or
G1p21SvNa;
[0018] FIG. 9A depicts FACS analysis of PI-stained nuclei 48 hours
after transduction of VX2 carcinoma cells with a retroviral vector
bearing antisense cyclin G1 (G1aG1SvNa), compared with that of the
control (G1XSvNa) vector.
[0019] FIG. 9B depicts FACS analysis of PI-stained nuclei 48 hours
after transduction of MG-63 osteosarcoma cells with retroviral
vectors bearing antisense cyclin G1 (G1aG1SvNa) compared with the
control (G1XSvNa) vector.
[0020] FIG. 10: Cytostatic effects of retroviral vectors bearing
antisense cyclin G1 and wild type p53 in transduced VX2
undifferentiated carcinoma cells. Cell densities were measured, by
cell counting, in cell cultures of VX2 cells at serial intervals
after retroviral vector transduction prior to G418 selection.
[0021] FIG. 11: Morphological appearance of VX2 cells 10 days after
transduction with retroviral vectors bearing antisense cyclin G1
(G1aG1SvNa), wild type p53 (G1p53SvNa) or the control (G1XSvNa)
vector after G418 selection.
[0022] FIG. 12: Inhibition of VX2 tumor growth in nude mice
following intratumoral injection retroviral vector bearing
antisense cyclin G1. The percentage increase in tumor size, plotted
on the vertical axis, is expressed as a function of time (days),
plotted on the horizontal axis.
[0023] FIG. 13A: Gross appearance of representative VX2
tumor-bearing mice one week after treatment with retroviral vectors
bearing antisense cyclin G1 (G1aG1SvNa) or the control vector
(G1XSvNa).
[0024] FIG. 13B: Hematoxylin-eosin stain of formalin-fixed tumor
sections one week following treatment with retroviral vectors
bearing antisense cyclin G1 (G1aG1SvNa) or the control vector
(G1XSvNa). 40.times. magnification.
[0025] FIG. 14 is a graph of tumor sizes in mice injected with
MNNG/HOS cells, followed by injection of the retroviral vectors
G1XSvNa or G1aG1SvNa. Tumor volumes are measured at 0, 4, 6, 8, 10,
and 12 days after injection of the retroviral vectors.
[0026] FIG. 15: (A) Aortic smooth muscle cells expressing
nuclear-targeted .beta.-galactosidase (cells with blue nuclei)
following transduction with the G1nBgSvNa vector; (B) Cytostatic
and cytocidal effects of antisense cyclin G1 and wild type p53 in
transduced aortic SMC. Cell densities were measured by direct cell
counting in cultures of aortic SMC harvested at serial intervals
after transduction with retroviral vectors bearing antisense G1
(G1aG1SvNa) and wild type p53 (G1pS3SvNa) as well as the control
vector (G1XSvNa); (C) .sup.3H-thymidine incorporation in cultured
aortic SMC after transduction with retroviral vectors (n=3 each
group). Radioactivity is expressed as dpm per well. Results are
expressed as arithmetic mean.+-.1 standard deviation;
[0027] FIG. 16: The morphological appearance of aortic SMC,
observed by light microscopy at 24 hrs after transduction with
control and antisense cyclin G1 retroviral vectors (A G1XSvNa
control vector; B-D=G1aG1SvNa). Detection of apoptosis in vascular
SMC after antisense cyclin G1 retroviral vector transduction; (E)
G1XSvNa control vector-transduced cells, (F) G1aG1SvNa antisense
cyclin G1 vector-transduced cells. The dark-staining apoptotic
bodies are noted both within and out of the syncytial cells;
[0028] FIG. 17: Cytocidal "bystander" effect in antisense cyclin G1
vector-transduced aortic SMC. Incorporation of non-transduced,
fluorescently labeled aortic SMC into multicellular syncytia when
overlaid onto an SMC culture previously transduced with an
antisense cyclin G1 vector. A and B, low magnification; C and D,
high magnification; A and C, phase contrast; B and D, UV light. A
representative multinuclear syncytium incorporating cells
containing the fluorescent label is identified by the arrow. (E)
Quantification of syncytia formation over time in vascular SMC
transduced with retroviral vectors: G1XSvNa, control vector;
G1aG1SvNa, vector bearing antisense cyclin G1 gene; G1pS3SvNa,
vector bearing wild type pS3;
[0029] FIG. 18: (A) High density cultures of aortic SMC scraped
with a 200 .mu.l pipet tip to create a 1 mm track devoid of cells,
(B) The appearance of the "wound" margin immediately upon scraping
and washing to remove detached cells, (C) Aortic SMC expressing
nuclear targeted .beta.-galactosidase along the margins of the
track, (D) Proliferation and migration of G1XSvNa control
vector-transduced aortic SMC into the track at 24 hrs after injury,
(E) Apoptotic and degenerative changes in G1aG1SvNa
vector-transduced aortic SMC with marked syncytia formation;
and
[0030] FIG. 19: Test of efficacy of an antisense cyclin G1 vector
in the rat carotid artery injury model of restenosis. The elastin
layer of the tunica media is identified (in A-D) by Verhoeff's
stain. The neointima, comprised of proliferating smooth muscle
cells (reddish yellow staining cells), is identified by Siris red
stain. A and C=non-treated arterial segments; B and D=antisense
cyclin G1 vector-treated arterial segments. E and F=higher
magnification of non-treated and aG1-treated arterial segments,
respectively; G=Analysis of neointima to media ratios of
non-treated (NT), control (GIX) and antisense cyclin G1
(aG1)-treated arterial segments are represented as vertical
bars.
DETAILED DESCRIPTION OF THE INVENTION
[0031] In accordance with an aspect of the present invention, there
is provided a method of treating a tumor in a host. The method
comprises administering to a host or to the tumor an agent which
inhibits cyclin G1 protein. The agent is administered in an amount
effective to inhibit cyclin G1 protein in the tumor cells.
[0032] The term "treating a tumor" as used herein means that one
provides for the inhibition, prevention, or destruction of the
growth of the tumor cells.
[0033] The term "inhibit cyclin G1 protein" as used herein, means
that the agent inhibits or prevents the expression of a
polynucleotide encoding cyclin G1 protein, or inhibits or prevents
the function of cyclin G1 protein.
[0034] Agents which inhibit cyclin G1 protein which may be employed
include, but are not limited to, polynucleotides (including
antisense oligonucleotides or polynucleotide fragments or sequences
which are complementary to at least a portion of a polynucleotide
encoding cyclin G1 protein) which bind to a polynucleotide encoding
cyclin G1 protein to prevent expression of a polynucleotide
encoding cyclin G1 protein, and antagonists to cyclin G1 protein,
such as, for example, antibodies or fragments or derivatives
thereof which recognize cyclin G1 protein, and cyclin-dependent
kinase inhibitors.
[0035] In one embodiment, the agent which inhibits cyclin G1
protein is a polynucleotide which binds to a polynucleotide
encoding cyclin G1 protein, and in particular is an antisense
polynucleotide which is complementary to at least a portion of a
polynucleotide encoding cyclin G1 protein. A nucleotide cDNA (FIG.
1) and deduced amino acid sequence of human cyclin G1 protein is
described in Wu, et al., Oncolocy Reports, Vol. 1, pgs. 705-711
(1994), which is incorporated herein by reference.
[0036] The term "polynucleotide" as used herein means a polymeric
form of nucleotide of any length, and includes ribonucleotides and
deoxyribonucleotides. Such term also includes single- and
double-stranded DNA, as well as single- and double-stranded RN. The
term also includes modified polynucleotides such as methylated or
capped polynucleotides.
[0037] In general, the antisense polynucleotide which is
complementary to at least a portion of a polynucleotide encoding
cyclin G1 protein includes at least 15 nucleotides, preferably at
least 18 nucleotides, and more preferably from 18 to 20
nucleotides. In one embodiment, the antisense polynucleotide is
complementary to the entire length of the polynucleotide encoding
cyclin G1 protein.
[0038] In one embodiment, the antisense polynucleotide is
complementary to, and thus capable of binding or hybridizing to, at
least a portion of mRNA encoding cyclin G1 protein, thereby
inhibiting translation of such mRNA. In another embodiment, the
antisense polynucleotide is complementary to, and thus capable of
binding or hybridizing to, single-stranded or double-stranded DNA
encoding cyclin G1 protein, thereby preventing the transcription of
such DNA to mRNA, or inhibiting the replication of such DNA. The
antisense polynucleotide may bind to any portion of the DNA or mRNA
encoding cyclin G1 protein, but preferably such antisense
polynucleotide binds at the 5' end of the DNA or mRNA.
[0039] In another embodiment, the antisense polynucleotide may be a
ribozyme that promotes the cleavage of mRNA encoding cyclin G1. As
used herein, the term "ribozyme" means any single strand of
polynucleotide that forms a secondary structure which promotes the
catalytic cleavage of a target mRNA molecule once specific
sequence-based recognition of the target mRNA is achieved.
[0040] The antisense oligonucleotide may be synthesized according
to techniques known to those skilled in the art, such as, for
example, by an automatic oligonucleotide synthesizer. The antisense
oligonucleotide then is administered to a host in an amount
effective to inhibit the expression of a polynucleotide encoding
cyclin G1 protein in tumor cells of a host. The antisense
oligonucleotide may be administered in an amount of from about 0.1
.mu.M to about 10 .mu.M, preferably from about 1 .mu.M to about 5
.mu.M. The host may be an animal host, and in particular a
mammalian host, including human and non-human primate hosts. The
antisense oligonucleotide in general is administered to the host
systemically in conjunction with an acceptable pharmaceutical
carrier, such as physiological saline. Alternatively, the antisense
oligonucleotides may be contained within liposomes, which are
administered to the host systemically in conjunction with an
acceptable pharmaceutical carrier. Such systemic administration may
be, for example, by intravenous, intraarterial, or intraperitoneal
administration. Alternatively, the antisense oligonucleotide may be
administered directly to the tumor.
[0041] The antisense oligonucleotides may be modified in order to
stabilize the oligonucleotide against degradation by nucleases
and/or to enhance the ability of the antisense oligonucleotide to
penetrate the tumor cells. Such modification may be accomplished by
substituting at least one of the phosphodiester bonds of the
antisense oligonucleotide with a structure which provides for
increased stabilization of the antisense oligonucleotide against
degradation by nucleases and/or enhances the ability of the
antisense oligonucleotide to penetrate the tumor cells. Such
substitutions may include phosphorothioate and phosphorodithioate
bonds, phosphotriesters, alkyl or aryl phosphonate bonds, such as
methylphosphonate bonds, short chain alkyl or cycloalkyl structures
or short chain heteroatomic or heterocyclic structures, such as,
for example, CH.sub.2--NH--O--CH.sub.2,
CH.sub.2--N(CH.sub.3)--O--CH.- sub.2,
CH.sub.2--O--N(CH.sub.3)--CH.sub.2,
CH.sub.2--N(CH.sub.3)--N(CH.sub- .3)--CH.sub.2, and
O--N(CH.sub.3)--CH.sub.2--CH.sub.2, as well as morpholino
structures. Examples of such modifications are described in PCT
Application No. WO93/05182, published Mar. 18, 1993, and in U.S.
Pat. No. 5,034,506, issued Jul. 23, 1991. Examples of alkyl or aryl
phosphonate bonds also are described in U.S. Pat. Nos. 4,469,863
and 4,511,713. Alternatively, at least one nucleotide of the
antisense oligonucleotide may be conjugated to a moiety which may
be an amino acid; a dipeptide mimic, a sugar; a sugar phosphate; a
neurotransmitter; a hydrophilic polymer such as
polyhydroxypropylmethacrylamide, dextrans, polymaleic anhydride, a
cyclodextrin, a starch, or polyethyleneimine. Examples of such
moieties are described in PCT Application No. WO94/01448, published
Jan. 20, 1994. Further examples of moieties which may be employed
in modifying the antisense oligonucleotide include, but are not
limited to, alkyl- or arylphosphorates, carbamates, sulfamates, and
(thio)formacetal.
[0042] The above modifications may be made to the antisense
oligonucleotide during synthesis of the antisense oligonucleotide
by means known to those skilled in the art. In a preferred
embodiment, when the antisense oligonucleotide is administered
directly or in a liposome, the antisense oligonucleotide includes
at least one phosphorothioate or phosphorodithioate linker moiety,
which may be attached to the backbone of the antisense
oligonucleotide during synthesis by techniques known to those
skilled in the art.
[0043] In another embodiment, the antisense oligonucleotide is
administered to the host by transducing tumor cells of the host
with a polynucleotide encoding an antisense polynucleotide which is
complementary to at least a portion of a polynucleotide encoding
cyclin G1 protein. The polynucleotide encoding an antisense
polynucleotide which is complementary to at least a portion of a
polynucleotide encoding cyclin G1 protein may be contained within
an appropriate expression vehicle which is transduced into the
tumor cell. Such expression vehicles include, but are not limited
to, plasmids, eukaryotic vectors, prokaryotic vectors (such as, for
example, bacterial vectors), and viral vectors.
[0044] In one embodiment, the vector is a viral vector. Viral
vectors which may be employed include RNA virus vectors (such as
retroviral vectors), and DNA virus vectors (such as adenoviral
vectors, adeno-associated virus vectors, Herpes Virus vectors, and
vaccinia virus vectors). When an RNA virus vector is employed, in
constructing the vector, the polynucleotide encoding the antisense
polynucleotide is in the form of MM. When a DNA virus vector is
employed, in constructing the vector, the polynucleotide encoding
the antisense polynucleotide is in the form of DNA.
[0045] In one embodiment, the viral vector is a retroviral vector.
Examples of retroviral vectors which may be employed include, but
are not limited to, Moloney Murine Leukemia Virus, spleen necrosis
virus, and vectors derived from retroviruses such as Rous Sarcoma
Virus, Harrey Sarcoma Virus, avian leukosis virus, human
immunodeficiency virus, myeloproliferative sarcoma virus, and
mammary tumor virus. The vector is generally a replication
incompetent retrovirus particle.
[0046] Retroviral vectors are useful as agents to mediate
retroviral-mediated gene transfer into eukaryotic cells. Retroviral
vectors are generally constructed such that the majority of
sequences coding for the structural genes of the virus are deleted
and replaced by the gene(s) of interest. Most often, the structural
genes (i.e., gag, pol, and env), are removed from the retroviral
backbone using genetic engineering techniques known in the art.
This may include digestion with the appropriate restriction
endonuclease or, in some instances, with Bal 31 exonuclease to
generate fragments containing appropriate portions of the packaging
signal.
[0047] These new genes have been incorporated into the proviral
backbone in several general ways. The most straightforward
constructions are ones in which the structural genes of the
retrovirus are replaced by a single gene which then is transcribed
under the control of the viral regulatory sequences within the long
terminal repeat (LTR). Retroviral vectors have also been
constructed which can introduce more than one gene into target
cells. Usually, in such vectors one gene is under the regulatory
control of the viral LTR, while the second gene is expressed either
off a spliced message or is under the regulation of its own,
internal promoter. Alternatively, two genes may be expressed from a
single promoter by the use of an Internal Ribosome Entry Site.
[0048] Efforts have been directed at minimizing the viral component
of the viral backbone, largely in an effort to reduce the chance
for recombination between the vector and the packaging-defective
helper virus within packaging cells. A packaging-defective helper
virus is necessary to provide the structural genes of a retrovirus,
which have been deleted from the vector itself.
[0049] Examples of retroviral vectors which may be employed include
retroviral vectors generated from retroviral plasmid vectors
derived from retroviruses including, but not limited to, Moloney
Murine Leukemia Virus vectors such as those described in Miller, et
al., Biotechniques, Vol. 7, pgs. 980-990 (1989), and in Miller, et
al., Human Gene Therapy, Vol. 1, pgs. 5-14 (1990).
[0050] In a preferred embodiment, the retroviral plasmid vector may
include at least four cloning, or restriction enzyme recognition
sites, wherein at least two of the sites have an average frequency
of appearance in eukaryotic genes of less than once in 10,000 base
pairs; i.e., the restriction product has an average DNA size of at
least 10,000 base pairs. Preferred cloning sites are selected from
the group consisting of NotI, SnaBI, SalI, and XhoI. In a preferred
embodiment, the retroviral plasmid vector includes each of these
cloning sites. Such vectors are further described in U.S. patent
application Ser. No. 08/340,805, filed Nov. 17, 1994, and in PCT
Application No. WO91/10728, published Jul. 25, 1991, and
incorporated herein by reference in their entireties.
[0051] When a retroviral plasmid vector including such cloning
sites is employed, there may also be provided a shuttle cloning
vector which includes at least two cloning sites which are
compatible with at least two cloning sites selected from the group
consisting of NotI, SnaBI, SalI, and XhoI located on the retroviral
vector. The shuttle cloning vector also includes at least one
desired gene which is capable of being transferred from the shuttle
cloning vector to the retroviral vector.
[0052] The shuttle cloning vector may be constructed from a basic
"backbone" vector or fragment to which are ligated one or more
linkers which include cloning or restriction enzyme recognition
sites. Included in the cloning sites are the compatible, or
complementary cloning sites hereinabove described. Genes and/or
promoters having Ends corresponding to the restriction sites of the
shuttle vector may be ligated into the shuttle vector through
techniques known in the art.
[0053] The shuttle cloning vector can be employed to amplify DNA
sequences in prokaryotic systems. The shuttle cloning vector may be
prepared from plasmids generally used in prokaryotic systems and in
particular in bacteria. Thus, for example, the shuttle cloning
vector may be derived from plasmids such as pBR322; pUC 18;
etc.
[0054] The retroviral plasmid vector includes one or more
promoters. Suitable promoters which may be employed include, but
are not limited to, the retroviral LTR; the SV40 promoter; and the
human cytomegalovirus (CMV) promoter described in Miller, et al.,
Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other promoter
(e.g., cellular promoters such as eukaryotic cellular promoters
including, but not limited to, the histone, pol III, and
.beta.-actin promoters). Other viral promoters which may be
employed include, but are not limited to, adenovirus promoters, TK
promoters, and B19 parvovirus promoters. The selection of a
suitable promoter will be apparent to those skilled in the art from
the teachings contained herein.
[0055] The retroviral plasmid vector then is employed to transduce
a packaging cell line to form a producer cell line. Examples of
packaging cells which may be transfected include, but are not
limited to, the PE501, PA317, .psi.-2, .psi.-AM, PA12, T19-14X,
VT-19-17-H2, .psi. CRE, .psi. CRIP, GP+E-86, GP+envAm12, and DAN
cell lines, as described in Miller, Human Gene Therapy, Vol. 1,
pgs. 5-14 (1990). The retroviral plasmid vector containing the
polynucleotide encoding the antisense polynucleotide, which is
complementary to at least a portion of a polynucleotide encoding
cyclin G1 protein, transduces the packaging cells through any means
known in the art. Such means include, but are not limited to,
electroporation, the use of liposomes, and CaPO.sub.4,
precipitation.
[0056] The packaging cells thus become producer cells which
generate retroviral vectors which include a polynucleotide encoding
an antisense polynucleotide which is complementary to at least a
portion of a polynucleotide encoding cyclin G1 protein. Such
retroviral vectors then are transduced into the tumor cells,
whereby the transduced tumor cells will produce the antisense
polynucleotide, which is complementary to at least a portion of the
polynucleotide encoding cyclin G1 protein.
[0057] The retroviral vectors are administered to a host in an
amount which is effective to inhibit, prevent, or destroy the
growth of the tumor cells through inhibition of the expression of
the polynucleotide encoding cyclin G1 protein in the tumor cells.
Such administration may be by systemic administration as
hereinabove described, or by direct injection of the retroviral
vectors in the tumor. In general, the retroviral vectors are
administered in an amount of at least 1.times.10.sup.5 cfu/ml, and
in general, such an amount does not exceed 1.times.10.sup.9 cfu/ml.
Preferably, the retroviral vectors are administered in an amount of
from about 1.times.10.sup.6 cfu/ml to about 1.times.10.sup.8
cfu/ml. The exact dosage to be administered is dependent upon a
variety of factors including the age, weight, and sex of the
patient, and the size and severity of the tumor being treated.
[0058] The retroviral vectors also may be administered in
conjunction with an acceptable pharmaceutical carrier, such as, for
example, saline solution, protamine sulfate (Elkins-Sinn, Inc.,
Cherry Hill, N.J.), water, aqueous buffers, such as phosphate
buffers and Tris buffers, or Polybrene (Sigma Chemical, St. Louis,
Mo.). The selection of a suitable pharmaceutical carrier is deemed
to be apparent to those skilled in the art from the teachings
contained herein.
[0059] In another alternative, retroviral producer cells, such as
those derived from the packaging cell lines hereinabove described,
which include a polynucleotide encoding an antisense
polynucleotide, which is complementary to at least a portion of a
polynucleotide encoding cyclin G1 protein, may be administered to a
host. Such producer cells may, in one embodiment, be administered
systemically (e.g., intravenously or intraarterially) at a point in
close proximity to the tumor, or the producer cells may be
administered directly to the tumor. The producer cell line then
produces retroviral vectors including a polynucleotide encoding an
antisense polynucleotide which is complementary to a polynucleotide
encoding cyclin G1 protein, in vivo, whereby such retroviral
vectors then transduce the tumor cells.
[0060] In another embodiment, the agent which inhibits cyclin G1
protein is an antagonist to cyclin G1 protein which binds to and
inhibits cyclin G1 protein. Examples of antagonists to cyclin G1
protein include, but are not limited to, antibodies or fragments or
derivatives thereof which recognize cyclin G1 protein, and small
molecules, such as, for example, cyclin-dependent kinase inhibitors
which bind to and inhibit the function of cyclin G1 protein.
[0061] In one embodiment the antagonist is an antibody or fragment
or derivative thereof which recognizes cyclin G1 protein. The term
"fragment or derivative thereof," means an antibody having
deletions and/or substitutions of amino acid residues with respect
to the unmodified antibody, yet such fragment or derivative
recognizes cyclin G1 protein. Such antibody may be a monoclonal or
polyclonal antibody. In one embodiment, the antibody is a single
chain antibody.
[0062] Preferably, the antibody is administered to the host such
that the antibody or fragment or derivative thereof enters the
tumor cells. In a preferred embodiment, the antibody or fragment or
derivative thereof which recognizes cyclin G1 protein is
administered to the host by transducing tumor cells of the host
with a polynucleotide encoding the antibody or fragment or
derivative thereof which recognizes cyclin G1 protein. The
polynucleotide may be contained in an appropriate expression
vehicle such as those hereinabove described. In one embodiment, the
polynucleotide is contained in a retroviral vector, which may be a
retroviral vector as hereinabove described.
[0063] The vector, which includes the polynucleotide encoding an
antibody or fragment or derivative thereof which recognizes cyclin
G1 protein is administered to the host in an amount effective to
inhibit the function of the cyclin G1 protein in the tumor cells in
the host. When a retroviral vector is employed, such retroviral
vector is administered in an amount of from about 1.times.10.sup.6
cfu/ml to about 1.times.10.sup.8 cfu/ml. Such vector may be
administered systemically (such as, for example, by intravenous,
intraarterial, or intraperitoneal administration) or,
alternatively, the vector may be administered directly to the
tumor. The vectors then transduce the tumor cells, whereby the
antibody or fragment or derivative thereof which recognizes cyclin
G1 protein is expressed in the tumor cells. Such antibody or
fragment or derivative thereof will bind to the cyclin G1 protein
in the tumor cells, thereby inhibiting the function of the cyclin
G1 protein in the tumor cells.
[0064] Tumors which may be treated in accordance with the present
invention, through the inhibition of cyclin G1 protein, include
non-malignant, as well as malignant, or cancerous tumors. Cancerous
tumors which may be treated include, but are not limited to,
osteogenic sarcoma and Ewing's sarcoma and other neoplastic
disorders in which cyclin G1 is expressed, such as, glioblastoma,
neuroblastoma, breast cancer, prostate cancer, leukemias, lymphomas
(includincg Hodgkin's and non-Hodgkin's fibrosarcoma,
rhabdomyosarcoma, colon cancer, pancreatic cancer, liver cancers
such as hepatocellular carcinoma, and adenocarcinomas.
[0065] The above treatments, in which cyclin G1 is inhibited, also
may be employed in combination with other treatments of tumors,
such as, for example, (i) radiation; (ii) chemotherapy; or (iii)
the transduction of the tumor cells with a polynucleotide encoding
a negative selective marker, such as, for example, a viral
thymidine kinase gene, followed by the administration of an
interaction agent, such as, for example, ganciclovir, which kills
the cells transduced with the polynucleotide encoding the negative
selective marker.
[0066] In one embodiment, an agent which inhibits cyclin G1 protein
is administered to a host in accordance with one of the methods
hereinabove described. The growth of any tumor cells which contain
the agent will be inhibited, prevented or destroyed. In addition,
the tumor cells are transduced with a polynucleotide encoding a
negative selective marker or "suicide" gene. The polynucleotide
encoding the negative selective marker may be contained in an
expression vehicle such as those hereinabove described. Once the
tumor cells are transduced with the polynucleotide encoding the
negative selective marker, an interaction agent is administered to
the host, whereby the interaction agent interacts with the negative
selective marker in order to prevent, inhibit, or destroy the
growth of the tumor cells.
[0067] Negative selective markers which may be employed include,
but are not limited to, thymidine kinase, such as Herpes Simplex
Virus thymidine kinase, cytomegalovirus thymidine kinase, and
varicella-zoster virus thymidine kinase; and cytosine
deaminase.
[0068] In one embodiment, the negative selective marker is a viral
thymidine kinase selected from the group consisting of Herpes
Simplex Virus thymidine kinase, cytomegalovirus thymidine kinase,
and varicella-zoster virus thymidine kinase. When such viral
thymidine kinases are employed, the interaction or chemotherapeutic
agent preferably is a nucleoside analogue, for example, one
selected from the group consisting of ganciclovir, acyclovir, and
1-2-deoxy-2-fluoro-.beta.- -D-arabinofuranosil-5-iodouracil (FIAU).
Such interaction agents are utilized efficiently by the viral
thymidine kinases as substrates, and such interaction agents thus
are incorporated lethally into the DNA of the tumor cells
expressing the viral thymidine kinases, thereby resulting in the
death of the tumor cells.
[0069] In another embodiment, the negative selective marker is
cytosine deaminase. When cytosine deaminase is the negative
selective marker, a preferred interaction agent is
5-fluorocytosine. Cytosine deaminase converts 5-fluorocytosine to
5-fluorouracil, which is highly cytotoxic. Thus, the tumor cells
which express the cytosine deaminase gene convert the
5-fluorocytosine to 5-fluorouracil and are killed.
[0070] The interaction agent is administered in an amount effective
to inhibit, prevent, or destroy the growth of the transduced tumor
cells. For example, the interaction agent may be administered in an
amount from 5 mg to 10 mg/kg of body weight, depending on overall
toxicity to a patient. The interaction agent preferably is
administered systemically, such as, for example, by intravenous
administration, by parenteral administration, by intraperitoneal
administration, or by intramuscular administration.
[0071] When an expression vehicle, such as those hereinabove
described, including a negative selective marker is administered to
tumor cells, a "bystander effect" may result, i.e., tumor cells
which were not originally transduced with the nucleic acid sequence
encoding the negative selective marker may be killed upon
administration of the interaction agent. Although Applicants do not
intend to be limited to any theoretical reasoning, the transformed
tumor cells may be producing a diffusible form of the negative
selective marker that either acts extracellularly upon the
interaction agent, or is taken up by adjacent, non-transformed
tumor cells, which then become susceptible to the action of the
interaction agent. It also is possible that one or both of the
negative selective marker and the interaction agent are
communicated between tumor cells.
[0072] Agents which inhibit cyclin G1 protein also may prevent
vascular restenosis after invasive vascular procedures such as
angioplasty, vascular grafts, such as arterial grafts, or coronary
bypass surgery. Thus, in accordance with another aspect of the
present invention, there is provided a method of preventing
restenosis which comprises administering to a host, or to the site
of an invasive vascular procedure or vascular lesion, an agent
which inhibits cyclin G1 protein. The agent is administered in an
amount effective to prevent restenosis in a host. The agent may be
administered during or after the invasive vascular procedure. The
term "invasive vascular procedure" as used herein means any
procedure which involves repair, removal, replacement and/or
redirection (e.g., bypass or shunt) of a portion of the vascular
system including but not limited to arteries and veins. Such
procedures include, but not limited to, angioplasty, vascular
grafts such as arterial grafts, removals of blood clots, removals
of portions of arteries or veins, and coronary bypass surgery.
[0073] Agents which inhibit cyclin G1 protein which may be employed
include, but are not limited to, those hereinabove described.
Preferably, the agent which inhibits cyclin G1 protein is an
antisense polynucleotide which is complementary to, and thus is
capable of binding or hybridizing to, at least a portion of a
polynucleotide encoding cyclin G1 protein as hereinabove described.
Such antisense oligonucleotide may have a length as hereinabove
described and be administered in an amount effective to prevent
restenosis. Such amount may be as hereinabove described. The
antisense oligonucleotide may be administered intravascularly and
may be administered directly to the site of the invasive vascular
procedure or the vascular lesion.
[0074] In a preferred embodiment, the antisense oligonucleotide is
administered to the host by transducing vascular cells at the site
of an invasive vascular procedure or a vascular lesion with a
polynucleotide encoding an antisense polynucleotide which is
complementary to at least a portion of a polynucleotide encoding
cyclin G1 protein. Such polynucleotide encoding the antisense
polynucleotide may be contained in an appropriate expression
vehicle as hereinabove described, which is transduced into the
cells of the site of an invasive vascular procedure or vascular
lesion. In one embodiment, the expression vehicle is a viral vector
such as those hereinabove described. In one embodiment, the viral
vector is a retroviral vector, which may be as hereinabove
described.
[0075] When a retroviral vector is employed, such retroviral vector
is administered in an amount hereinabove described, and is
administered intravascularly. In one embodiment, the retroviral
vector is administered to the site of the invasive vascular
procedure or vascular lesion. The vectors transduce the vascular
cells at the site of the invasive vascular procedure or vascular
lesion, whereby the antisense oligonucleotide is produced in such
cells, thereby inhibiting the expression of a polynucleotide
encoding cyclin G1 in such cells and thus preventing restenosis by
preventing the proliferation of such cells.
[0076] In another embodiment, the agent which inhibits cyclin G1
protein is an antagonist to cyclin G1 protein which binds to and
inhibits cyclin G1 protein as hereinabove described, and in one
embodiment may be an antibody or fragment or derivative thereof
which recognizes cyclin G1 protein.
[0077] The antibody is administered to the host such that the
antibody or fragment or derivative thereof enters the cells of the
site of the invasive vascular procedure or vascular lesion.
Preferably, the antibody or fragment or derivative thereof which
recognizes cyclin G1 protein is administered by transducing cells
at the site of the invasive vascular procedure or of a vascular
lesion with a polynucleotide encoding the antibody or fragment or
derivative thereof which recognizes cyclin G1 protein. The
polynucleotide may be contained in an appropriate expression
vehicle such as those hereinabove described. In one embodiment, the
expression vehicle is a retroviral vector as hereinabove described,
which may be administered in an amount as hereinabove described.
Such vector is administered intravascularly as hereinabove
described, and may be administered directly to the site of an
invasive vascular procedure or vascular lesion.
[0078] This method is applicable to the prevention and treatment of
restenosis and the prevention or treatment of vascular lesions
following a variety of invasive vascular procedures, including but
not limited to, cardiovascular angioplasty, arterial grafts, and
coronary bypass surgery. This method also applies to the prevention
and treatment of vascular lesions including, but not limited to,
lesions of the femoral, carotid, or renal arteries, particularly
renal arteries associated with renal dialysis fistulas.
[0079] In accordance with another aspect of the present invention,
there is provided a method of immortalizing non-tumor cells which
comprises transducing the non-tumor cells with a polynucleotide
encoding cyclin G1 protein or a derivative or analogue thereof. The
term "derivative or analogue thereof" as used herein means that the
protein may be a protein which has deletions and/or substitutions
of amino acid residues with respect to the native cyclin G1 protein
sequence, yet retains the same biological properties as native, or
unmodified cyclin G1 protein. Although the scope of this aspect of
the present invention is not intended to be limited to any
theoretical reasoning, Applicants have discovered that
overexpression of cyclin G1 protein in non-tumor cells, would
contribute to cell immortalization and permanent cell lines that
would retain the ability to respond to subsequent cell cycle
events, and avoiding the use of viral oncogenes which cause cell
transformation.
[0080] The polynucleotide encoding cyclin G1 protein or a fragment
or derivative thereof may be contained in an appropriate expression
vehicle, which may be as hereinabove described. In one embodiment,
the expression vehicle is a retroviral vector, which may be as
hereinabove described.
[0081] Non-tumor cells which may be transduced in accordance with
this aspect of the present invention include, but are not limited
to, fibroblasts, hepatocytes, muscle cells, endothelial cells, and
epithelial cells.
[0082] In accordance with yet another aspect of the present
invention, there is provided a method of enhancing transduction of
cells with a retroviral vector. The method comprises transducing
the cells with a first expression vehicle including a
polynucleotide encoding cyclin G1 protein. The first expression
vehicle is not a retroviral vector. The cells also are transduced
with a second expression vehicle which preferably includes a
polynucleotide encoding a therapeutic agent. The second expression
vehicle is a retroviral vector. This method can be used to
transduce cells in vivo or ex vivo or in vitro.
[0083] The first expression vehicle may be any expression vehicle
which is not a retroviral vector. Such expression vehicles include,
but are not limited to, plasmid vectors, eukaryotic vectors,
prokaryotic vectors (such as, for example, bacterial vectors), and
viral vectors other than retroviral vectors, including, but not
limited to, adenoviral vectors, adeno-associated virus vectors, He
e virus vectors, and vaccinia virus vectors.
[0084] In a preferred embodiment, the first expression vehicle is
an adenoviral vector. Although this embodiment is not to be limited
to any theoretical reasoning, cyclin G1 protein is induced in very
early G1 phase, when cell activation occurs. The transduction of
the cells with an adenoviral vector including a polynucleotide
encoding cyclin G1 protein provides transient overexpression of
cyclin G1 protein in the cells, thereby activating the cells, and
enabling increased integration of the retroviral vector including
the polynucleotide encoding the therapeutic agent into the cells.
Such method is applicable particularly to the introduction of
retroviral vectors into cells with low replication indices and low
transduction efficiency.
[0085] The adenoviral vector which is employed may, in one
embodiment, be an adenoviral vector which includes essentially the
complete adenoviral genome (Shenk et al., Curr. Top. Microbiol.
Immunol., 111(3): 1-39 (1984). Alternatively, the adenoviral vector
may be a modified adenoviral vector in which at least a portion of
the adenoviral genome has been deleted.
[0086] In the preferred embodiment, the adenoviral vector comprises
an adenoviral 5' ITR; an adenoviral 3' ITR; an adenoviral
encapsidation signal; a DNA sequence encoding cyclin G1 protein;
and a promoter controlling the DNA sequence encoding cyclin G1
protein. The vector is free of at least the majority of adenoviral
E1 and E3 DNA sequences, but is not free of all of the E2 and E4
DNA sequences, and DNA sequences encoding adenoviral proteins
promoted by the adenoviral major late promoter.
[0087] In one embodiment, the vector also is free of at least a
portion of at least one DNA sequence selected from the group
consisting of the E2 and E4 DNA sequences.
[0088] In another embodiment, the vector is free of at least the
majority of the adenoviral E1 and E3 DNA sequences, and is free of
a portion of the other of the E2 and E4 DNA sequences.
[0089] In still another embodiment, the gene in the E2a region that
encodes the 72 kilodalton binding protein is mutated to produce a
temperature sensitive protein that is active at 32.degree. C., the
temperature at which the viral particles are produced. This
temperature sensitive mutant is described in Ensinger et al., J.
Virology, 10:328-339 (1972), Van der Vijet et al., J. Virology,
15:348-354 (1975), and Friefeld et al., Virology, 124:380-389
(1983).
[0090] Such a vector, in a preferred embodiment, is constructed
first by constructing, according to standard techniques, a shuttle
plasmid which contains, beginning at the 5' end, the "critical left
end elements," which include an adenoviral 5' ITR, an adenoviral
encapsidation signal, and an E1a enhancer sequence; a promoter
(which may be an adenoviral promoter or a foreign promoter); a
multiple cloning site (which may be as herein described); a poly A
signal; and a DNA segment which corresponds to a segment of the
adenoviral genome. The vector also may contain a tripartite leader
sequence. The DNA segment corresponding to the adenoviral genome
serves as a substrate for homologous recombination with a modified
or mutated adenovirus, and such sequence may encompass, for
example, a segment of the adenovirus 5 genome no longer than from
base 3329 to base 6246 of the genome. The plasmid may also include
a selectable marker and an origin of replication. The origin of
replication may be a bacterial origin of replication.
Representative examples of such shuttle plasmids include pAvS6,
which is described in published PCT Application Nos. WO94/23582,
published Oct. 27, 1994, and WO95/09654, published Apr. 13, 1995.
The DNA sequence encoding cyclin G1 protein may then be inserted
into the multiple cloning site to produce a plasmid vector.
[0091] This construct is then used to produce an adenoviral vector.
Homologous recombination is effected with a modified or mutated
adenovirus in which at least the majority of the E1 and E3
adenoviral DNA sequences have been deleted. Such homologous
recombination may be effected through co-transfection of the
plasmid vector and the modified adenovirus into a helper cell line,
such as 293 cells, by CaPO.sub.4 precipitation. Upon such
homologous recombination, a recombinant adenoviral vector is formed
that includes DNA sequences derived from the shuttle plasmid
between the Not I site and the homologous recombination fragment,
and DNA derived from the E1 and E3 deleted adenovirus between the
homologous recombination fragment and the 3' ITR.
[0092] In one embodiment, the homologous recombination fragment
overlaps with nucleotides 3329 to 6246 of the adenovirus 5 (ATCC
VR-5) genome.
[0093] Through such homologous recombination, a vector is formed
which includes an adenoviral 5' ITR, an adenoviral encapsidation
signal; an E1a enhancer sequence; a promoter; a DNA sequence
encoding cyclin G1 protein protein; a poly A signal; adenoviral DNA
free of at least the majority of the E1 and E3 adenoviral DNA
sequences; and an adenoviral 3' ITR. The vector also may include a
tripartite leader sequence. The vector may then be transfected into
a helper cell line, such as the 293 helper cell line (ATCC No.
CRL1573), which will include the E1a and E1b DNA sequences, which
are necessary for viral replication, and to generate adenoviral
particles. Transfection may take place by electroporation, calcium
phosphate precipitation, microinjection, or through
proteoliposomes.
[0094] The vector hereinabove described may include a multiple
cloning site to facilitate the insertion of the DNA sequence
encoding the cyclin G1 protein into the cloning vector. In general,
the multiple cloning site includes "rare" restriction enzyme sites;
i.e., sites which are found in eukaryotic genes at a frequency of
from about one in every 10,000 to about one in every 100,000 base
pairs. An appropriate vector is thus formed by cutting the cloning
vector by standard techniques at appropriate restriction sites in
the multiple cloning site, and then ligating the DNA sequence
encoding cyclin G1 protein into the cloning vector.
[0095] The DNA sequence encoding cyclin G1 protein is under the
control of a suitable promoter, which may be selected from those
herein described, or such DNA may be under the control of its own
native promoter.
[0096] In one embodiment, the adenovirus may be constructed by
using a yeast artificial chromosome (or YAC) containing an
adenoviral genome according to the method described in Ketner, et
al., PNAS, Vol. 91, pgs. 6186-6190 (1994), in conjunction with the
teachings contained herein. In this embodiment, the adenovirus
yeast artificial chromosome is produced by homologous recombination
in viva between adenoviral DNA and yeast artificial chromosome
plasmid vectors carrying segments of the adenoviral left and right
genomic termini. A DNA sequence encoding cyclin G1 protein then may
be cloned into the adenoviral DNA. The modified adenoviral genome
then is excised from the adenovirus yeast artificial chromosome in
order to be used to generate adenoviral vector particles as
hereinabove described.
[0097] The retroviral vector, which is the second expression
vehicle, may be as hereinabove described. Such retroviral vector
includes a polynucleotide encoding a therapeutic agent. The term
"therapeutic" is used in a generic sense and includes treating
agents, prophylactic agents, and replacement agents.
[0098] Polynucleotides encoding therapeutic agents which may be
contained in the retroviral plasmid vector include, but are not
limited to, polynucleotides encoding tumor necrosis factor (TNF)
genes, such as TNF-.alpha.; genes encoding interferons such as
Interferon-.alpha., Interferon-.beta., and Interferon-.gamma.;
genes encoding interleukins such as IL-1, IL-16, and Interleukins 2
through 14; genes encoding GM-CSF; genes encoding adenosine
deaminase, or ADA; genes which encode cellular growth factors, such
as lymphokines, which are growth factors for lymphocytes; genes
encoding epidermal growth factor (EGF), and keratinocyte growth
factor (KGF); genes encoding soluble CD4; Factor VIII; Factor IX;
cytochrome b; glucocerebrosidase; T-cell receptors; the LDL
receptor, ApoE, ApoC, ApoAI and other genes involved in cholesterol
transport and metabolism; the alpha-1 antitrypsin (.alpha.1AT)
gene; the insulin gene; the hypoxanthine phosphoribosyl transferase
gene; the CFTR gene; negative selective markers or "suicide" genes,
such as viral thymidine kinase genes, such as the Herpes Simplex
Virus thymidine kinase gene, the cytomegalovirus virus thymidine
kinase gene, and the varicella-zoster virus thymidine kinase gene;
Fc receptors for antigen-binding domains of antibodies, antisense
sequences which inhibit viral replication, such as antisense
sequences which inhibit replication of hepatitis B or hepatitis
non-A non-B virus; antisense c-myb oligonucleotides; and
antioxidants such as, but not limited to, manganese superoxide
dismutase (Mn-SOD), catalase, copper-zinc-superoxide dismutase
(CuZn-SOD), extracellular superoxide dismutase (EC-SOD), and
glutathione reductase; tissue plasminogen activator (tPA); urinary
plasminogen activator (urokinase); hirudin; the phenylalanine
hydroxylase gene; nitric oxide synthetase; vasoactive peptides;
angiogenic peptides; the dopamine gene; the dystrophin gene; the
.beta.-globin gene; the .alpha.-globin gene; the HbA gene;
protooncogenes such as the ras, src, and bcl genes;
tumor-suppressor genes such as p53 and Rb; the LDL receptor; the
heregulin-.alpha. protein gene, for treating breast, ovarian,
gastric and endometrial cancers; monoclonal antibodies specific to
epitopes contained within the e-chain of a T-cell antigen receptor;
the multi-drug resistance (MDR) gene; polynucleotides encoding
ribozymes; antisense polynucleotides; genes encoding secretory
peptider which act as competitive inhibitors of angiotensin
converting enzyme, of vascular smooth muscle calcium channels, or
of adrenergic receptors, and polynucleotides encoding enzymes which
break down amyloid plaques within the central nervous system. It is
to be understood, however, that the scope of the present invention
is not to be limited to any particular therapeutic agent.
[0099] The polynucleotide encoding the therapeutic agent is under
the control of a suitable promoter. Suitable promoters which may be
employed include, but are not limited to, the retroviral LTR; the
SV40 promoter; the cytomegalovirus (CMV) promoter; the Rous Sarcoma
Virus (RSV) promoter; the histone promoter; the polIII promoter,
the .beta.-actin promoter; inducible promoters, such as the MMTV
promoter, the metallothionein promoter; heat shock promoters;
adenovirus promoters; the albumin promoter; the ApoAI promoter; B19
parvovirus promoters; human globin promoters; viral thymidine
kinase promoters, such as the Herpes Simplex thymidine kinase
promoter; retroviral LTRs; human growth hormone promoters, and the
MxIFN inducible promoter. The promoter also may be the native
promoter which controls the polynucleotide encoding the therapeutic
agent. It is to be understood, however, that the scope of the
present invention is not to be limited to specific foreign genes or
promoters.
[0100] The first expression vehicle, which preferably is an
adenoviral vector, which includes a DNA sequence encoding cyclin G1
protein or an analogue derivative thereof, and the retroviral
vector, which includes a polynucleotide encoding therapeutic agent,
may transduce cells in vivo or in vitro.
[0101] In one embodiment the cells are transduced with the first
expression vehicle, which preferably is an adenoviral vector, prior
to transduction of the cells with the second expression vehicle
(i.e., the retroviral vector). In another embodiment, the cells are
transduced with the first expression vehicle and the second
expression vehicle concurrently.
[0102] When administered in vivo, the adenoviral vector is
administered in an amount effective to transduce the desired cells
with the polynucleotide encoding cyclin G1 protein. The adenoviral
vector may be administered systemically, such as, for example, by
intravenous, intraarterial, or intraperitoneal administration.
Alternatively, the adenoviral vector may be administered by direct,
nonsystemic injection to a desired tissue, organ, or mass of cells,
such as, for example, a tumor. In general, the adenoviral vector is
administered at a multiplicity of infection of from about 1 to
about 10.
[0103] The retroviral vector is administered to the animal host in
vivo in an amount effective to produce a therapeutic effect in the
animal.
[0104] The animal may be a mammal, including human and non-human
primates. The retroviral vectors may be administered systemically,
for example, intravenously or intraarterially or intraperitoneally,
or by direct nonsystemic injection into a desired tissue, organ or
mass of cells, such as, for example, a tumor.
[0105] The retroviral vectors are administered to an animal in an
amount effective to produce a therapeutic effect in the animal. In
general, the retroviral vectors are administered in an amount of at
least 1.times.10.sup.5 cfu/ml, and in general such amount does not
exceed 1.times.10.sup.9 cfu/ml. Preferably, the retroviral vectors
are administered in an amount of from about 1.times.10.sup.6 cfu/ml
to about 1.times.10.sup.4 cfu/ml. The exact dosage to be
administered is dependent upon various factors, including the age,
height, weight, and sex of the patient, the disorder being treated,
and the severity thereof.
[0106] The retroviral vectors and the adenoviral vectors each are
administered to the patient in a pharmaceutically acceptable
carrier, such as, for example, a physiological saline solution.
Other pharmaceutical carriers include, but are not limited to,
mineral oil, alum, and lipid vesicles such as liposomes. The
selection of a suitable pharmaceutical carrier is deemed to be
within the scope of those skilled in the art from the teachings
contained herein.
[0107] In one embodiment, the eukaryotic cells which are transduced
in vivo with the retroviral and adenoviral vectors are primary
human cells. The gene encoding a therapeutic agent can be any gene
having clinical usefulness, for example, therapeutic or marker
genes. Preferably, the primary human cells are blood cells. The
term "blood cells" as used herein is meant to include all forms of
nucleated blood cells as well as progenitors and precursors
thereof.
[0108] The gene carried by the blood cells can be any gene which
directly enhances the therapeutic effects of the blood cells. The
gene carried by the blood cells can be any gene which allows the
blood cells to exert a therapeutic effect that it would not
ordinarily have, such as a gene encoding a clotting factor (e.g.,
Factor VIII or Factor IX) useful in the treatment of hemophilia.
The gene can encode one or more products having therapeutic
effects. Examples of suitable genes include those that encode
cytokines such as TNF, interleukins (interleukins 1-12),
interferons (.alpha., .beta., .gamma.-interferons), T-cell receptor
proteins and Fc receptors for binding to antibodies.
[0109] The retroviral vectors are useful in the treatment of a
variety of diseases including but not limited to adenosine
deaminase deficiency, sickle cell anemia, thalassemia, hemophilia,
diabetes, .alpha.-antitrypsin deficiency, brain disorders such as
Alzheimer's disease, and other illnesses such as growth disorders
and heart diseases, for example, those caused by alterations in the
way cholesterol is metabolized and defects of the immune
system.
[0110] In one embodiment, the retroviral vectors may include a
negative selectable marker, such as, for example, a viral thymidine
kinase gene, and more particularly, the Herpes Simplex Virus
thymidine kinase (TK) gene. Such retroviral vectors may be
administered in conjunction with the adenoviral vectors hereinabove
described to tumor cells (in particular to cancer cells) in a human
patient in vivo. The adenoviral vectors and the retroviral vectors
then transduce the tumor cells. After the retroviral vectors have
transduced the tumor cells, the patient is given an interaction
agent, such as gancyclovir or acyclovir, which interacts with the
protein expressed by the negative selectable marker in order to
kill all replicating cells (i.e., the tumor cells) which were
transduced with the retroviral vector including the negative
selectable marker.
[0111] The adenoviral vectors and the retroviral vectors mentioned
hereinabove also may be administered in an animal model for
determining the effectiveness of a gene therapy treatment. For
example, an adenoviral vector including a polynucleotide encoding
cyclin G1 protein and a retroviral vector including a
polynucleotide encoding a therapeutic agent, may be administered to
animals of the same species. The retroviral vector is administered
to the animals in varying amounts. From determining the
effectiveness of the gene therapy treatment in the animal, one may
determine an effective amount of the retroviral vector to be
administered to a human patient.
[0112] In another embodiment, the adenoviral vectors, which include
a DNA sequence encoding cyclin G1 protein, are administered in vivo
to a patient in conjunction with retroviral producer cells which
generate retroviral vectors including a polynucleotide encoding a
therapeutic agent.
[0113] Such an embodiment is applicable particularly to the
treatment of tumors (including malignant and non-malignant tumors)
such as, for example, liver tumors, bone tumors, and lung tumors.
For example, the producer cells may include a retroviral plasmid
vector including a negative selectable marker. The adenoviral
vectors and the retroviral producer cells then are administered to
the tumor, whereby the producer cells generate retroviral vector
particles including the polynucleotide encoding the negative
selectable marker. The adenoviral vectors and the retroviral vector
particles generated by the retroviral producer cells transduce the
tumor cells, whereby the tumor cells produce the negative
selectable marker. Upon administration of an interaction agent to
the patient, the transduced tumor cells are killed.
[0114] Alternatively, the adenoviral vectors and the retroviral
vector may transduce eukaryotic cells, in vitro, whereby the
eukaryotic cells are cultured in vitro for the in vitro production
of the therapeutic agent, or, alternatively, the transduced
eukaryotic cells may be administered to a host as part of a gene
therapy procedure, whereby the transduced eukaryotic cells express
the therapeutic agent in vivo in a host.
[0115] As stated hereinabove, the above methods of the present
invention may be accomplished through the use of appropriate
expression vehicles containing either a polynucleotide encoding an
agent which inhibits cyclin G1 protein (when one desires to treat a
tumor by inhibiting cyclin G1 protein), or a polynucleotide
encoding cyclin G1 protein (when one desires to immortalize a cell
line or enhance retroviral transduction of cells). Thus, in
accordance with another aspect of the present invention, there is
provided an expression vehicle which includes a polynucleotide
encoding an agent which, in one embodiment, is an agent which
inhibits cyclin G1 protein. Such agents include those hereinabove
described, such as, for example, antisense polynucleotides or
antibodies or fragments or derivatives thereof which recognize
cyclin G1 protein, or a cyclin-dependent kinase inhibitor. In
another embodiment, the polynucleotide encodes cyclin G1
protein.
[0116] The expression vehicle may be selected from those
hereinabove described, and preferably may be a viral vector,
including RNA virus vectors and DNA virus vectors as hereinabove
described.
[0117] In one embodiment, the viral vector is an RNA virus vector,
and preferably is a retroviral vector, such as those hereinabove
described. In another embodiment, the viral vector is a DNA virus
vector, and preferably is an adenoviral vector, such as those
hereinabove described.
[0118] In accordance with a further aspect of the present
invention, there is provided a method of detecting cancer by
detection of increased expression of cyclin G1 protein, with such
increased expression being detected by detecting increased amounts
of polynucleotides encoding cyclin G1 protein, or by detecting
increased amounts of cyclin G3 protein, as compared with normal,
non-cancerous cells. The method comprises contacting cells with an
agent which binds to (i) cyclin G1 protein and/or (ii) a
polynucleotide encoding cyclin G1 protein. Binding of the agent to
cyclin G1 protein and/or a polynucleotide encoding cyclin G1
protein then is determined.
[0119] The cyclin G1 protein is expressed intracellularly, and to
assay for the increased expression of cyclin G1 protein,
appropriate procedures are employed prior to contacting the cells
with agent which binds to cyclin G1 protein and/or a polynucleotide
encoding cyclin G1 protein, to enable binding of the agent in the
assay. Such procedures include, but are not limited to, the
fixation of a histological sample of cells prior to the assay.
[0120] Agents which may be employed in this aspect include, but are
not limited to, polynucleotides (e.g., DNA or RNA probes) which
hybridize to a polynucleotide encoding cyclin G1 protein, and
antibodies or fragments or derivatives thereof which recognize
cyclin G1 protein.
[0121] In one embodiment, the agent is a polynucleotide which
hybridizes to a polynucleotide encoding cyclin G1 protein.
[0122] In another embodiment, the agent is an antibody or fragment
or derivative thereof which recognizes cyclin G3 protein. Such
antibodies include, but are not limited to, monoclonal antibodies,
polyclonal antibodies, and single chain antibodies.
[0123] Certain properties of cancer may be determined through the
analysis of the amount of binding to cyclin G1 protein expressed in
the cells, or the amount of binding to a polynucleotide encoding
cyclin G1 protein present in the cells. A determination an elevated
amount of binding of the agent to cyclin G1 protein as compared to
that observed in normal cells, or to a polynucleotide encoding
cyclin G1 protein may be indicative of the presence of cancer
cells, cancers which may be determined in accordance with this
method include osteogenic sarcoma and Ewing's sarcoma, and other
neoplastic disorders in which cyclin G1 is expressed, such as those
hereinabove described.
[0124] The determination of binding of the agent to cyclin G1
protein or to a polynucleotide encoding cyclin G1 protein may be
determined by a variety of assay methods known to those skilled in
the art. Such assays include, but are not limited to, direct and
indirect sandwich assays, calorimetric assays and ELISA assays.
[0125] In the above assays, the agent which binds to the cyclin G1
protein or to the polynucleotide which encodes cyclin G1 protein,
or a binder which binds to the agent when an indirect sandwich
assay is employed, is coupled to a detectable label or marker. Such
labels or markers include, but are not limited to, radioactive
isotopes of, for example, iodine, cobalt or tritium; an enzyme; a
fluorescent dye; an absorbing dye; a chemiluminiscent substance; a
spin label; biotin; hematoxylin; a colored particle or any other
labeling substance known to one skilled in the art.
[0126] In one embodiment, fixed cells, suspected of being cancer
cells, are contacted with an antibody which recognizes cyclin G1
protein. Detection of bound antibody may be determined by an
indirect sandwich assay employing a biotin-avidin complex, such as
a biotin-streptavidin complex which is bound to the antibody. The
avidin is bound to an enzyme, such as, for example, alkaline
phosphatase. The sample is contacted with a substrate for the
enzyme, which produces a colored reaction product. By measuring the
development of the colored reaction product, the amount of cyclin
G1 protein in the sample of cells may be determined, thereby
determining the presence of cancer and/or the extent and severity
thereof.
EXAMPLES
[0127] The invention now will be described with respect to the
following examples, however, the scope of the present invention is
not intended to be limited thereby.
Example 1
Materials and Methods
[0128] Cloning of Antisense Cyclin G1, Antisense Cyclin D1, and
p21/WAF1/CIP1 Expression Constructs.
[0129] The full coding regions of human cyclin G1 (FIG. 1) (Wu, et
al., Oncol. Reports, Vol. 1, pgs. 705-711 (1994)), cyclin D1
(Xiong, et al., Cell, Vol. 65, pgs. 691-699 (1991)), and
p21/WAF1/CIP1 (Harper, et al., Mol. Biol. Cell., Vol. 6, pgs.
387-400 (1995); El-Deiry, et al., Cell, Vol. 75, pgs. 817-825
(1993)), including the stop codons, were prepared by
primer-directed RT-PCR amplification. To create the antisense
cyclin G1 (aG1) expression construct, the 586 bp N-terminal
fragment, including -65 bp of the untranslated region, was released
by double digestion with XbaI/HpaI from the CYCG1 gene originally
isolated from a human WI-38 fibroblast (ATCC, Rockville, Md.) cDNA
library, and then cloned by blunt ligation into the pcDNA3 vector
(Invitrogen, San Diego, Calif.) at the EcoRV site. The 605
N-terminal region of cyclin D1 (antisense orientation, aD1) and the
495 bp full coding region of WAF1/CIP1 (p21) were released by
digestion with NdeI/NcoI and NdeI/EcoRI, respectively, followed by
blunt end cloning into the pcDNA3 vector at the EcoRV site. The
structure of each construct was confirmed by manual DNA sequence
analysis, using a modified dideoxy chain termination method (United
States Biochemicals).
[0130] Construction of Retroviral Vectors Bearing Cell Cycle
Control Genes (G1aG1SvNa, G1aD1SvNa, and G1p21SvNa: Retroviral
Vector Source, pG1XSvNa; Insert Source, pcDNA3aG1, pcDNA3aD1,
pcDNA3p21).
[0131] To create each retroviral vector, pG1XSvNa (Genetic Therapy,
Inc., Gaithersburg, Md.) was digested with NotI, the 5' phosphates
were removed by treatment with calf intestinal alkaline
phosphatase, and the resulting fragment was then gel-purified (1%
agarose), excised, and electroeluted. pG1XSvNa is a retroviral
plasmid vector derived from pG1 (described in PCT Application No.
WO91/10728 published Jul. 25, 1991), and which includes a
retroviral 5' LTR, a retroviral 3' LTR, a multiple cloning region
and a neomycin resistance gene under control of the SV40 promoter.
pG1XSvNa is described further in PCT Application No. WO95/09654,
published Apr. 13, 1995. This procedure generated a 5856 bp long
fragment of DNA which cannot relegate or re-circularize. To isolate
the aG1, aD1 and p21 insert fragments, the respective plasmid DNAs
were double digested with HindIII/NotI for aG1 and EcoRI/NotI for
aD1 and p21, respectively. These digests were resolved on it
agarose gels yielding the 597 bp HindIII/NotI fragment of aG1, the
632 bp EcoRI/NotI fragment of aD1, and the 522 bp EcoRI/NotI
fragment of p21. These bands were then excised from the agarose
gels and electroeluted. The NotI end of each insert was ligated to
the NotI end of the digested pG1XSvNa vector, and isolated on 1%
agarose gels yielding 6453, 6488 and 6378 bp long fragments for
aG1, aD1 and p21 respectively. Each fragment was then electroeluted
and treated with the Klenow fragment to generate blunt ends, and
then ligated to generate closed plasmid DNA including the
respective genes of interest. Successful cloning and insert
orientation were determined by restriction analysis. The expected
DNA fragments generated by digestion with BstEII and NotI were 920,
955 and 845 bp for aG1, aD1 and p21 inserts respectively, and
.+-.5500 bp for the vector DNA.
[0132] Retroviral Vector Supernatants and Producer Cell Lines.
[0133] The .beta. galactosidase and HStk expression vectors were
kindly provided as high titer PA317 packaging cell clones (titers:
1.3.times.10.sup.6 and 4.9.times.10.sup.6 G418.sup.r colony-forming
units, cfu/ml for .beta. galactosidase and HStk vectors
respectively) by Genetic Therapy, Inc. (Gaithersburg, Md.). The 3
experimental retroviral plasmid vectors bearing cell cycle control
enzyme cDNAs were packaged in PA317 cells (Miller, et al., Mol.
Cell Biol., Vol. 6, pgs. 2895-2902 (1986)) and tested as pooled
vector supernatants (vector titer: 1.times.10.sup.6 cfu/ml each).
The vectors are referred to as G1BgSvNa, G1TK1SvNa.7, G1p21SvNa,
G1aD1SvNa and G1aG1SvNa to indicate the order of promoters and
coding regions contained in each vector (G1 vector, Moloney Murine
Leukemia Virus long terminal repeat (LTR) sequences; Bg, .beta.
galactosidase or lacZ gene; HStk, Herpes Simplex thymidine kinase
gene; aG1, antisense human cyclin G1; aD1, antisense cyclin D1;
p21, Cdk inhibitor p21/Waf1/Cip1 gene; Sv, SV40 early region
enhancer/promoter; and Na.7, neo.sup.r gene, clone 7). Retroviral
vector G1TK1SvNa 7 is described further in PCT Application No.
WO95/09654, published Apr. 13, 1995. Retroviral vector G1BgSvNa was
generated from the plasmid pG1BgSvNa. pG1BgSvNa was constructed by
digesting pSvNa (PCT Application No. WO95/09654) and pG1Bg (PCT
Application No. WO91/10728) with SalI and HindIII. The SalI-HindIII
fragment of pSvNa containing the SV40 promoter and a neomycin
resistance gene was ligated to the SalI/HindIII digested pG1Bg to
form The vector source, G1XSvNa, containing only the SV40
promoter-driven neo gene was used as a control for the effects of
gene transduction and G418 selection.
[0134] Cells, Cell Culture Conditions and Transduction of Cells
with lacZ, Cell Cycle Control Genes, and HStk Vectors.
[0135] Human osteogenic sarcoma (MG-63, ATCC No. CRL 1427) cells
and primary normal diploid human fibroblasts (of hepatic origin)
were cultured at a plating density of 2.5.times.10.sup.4 cells in
each of six-well plates, in DMEM supplemented with 10% FBS (D10).
After overnight attachment, the cells were exposed to 1 ml of the
respective retroviral vector in the presence of Polybrene (8
.mu.g/ml) for 2 hours, after which 1 ml of fresh D10 was added to
each well. Forty-eight hours after transduction with the lacZ
vector, gene transfer efficiency was measured by determining the
percentage of lacZ positive cells, upon X-gal staining and light
microscopy.
[0136] Ganciclovir (GCV) Cytotoxicity/Bystander Effects in HStk
Vector Transduced MG-63 Cells.
[0137] Initial dose-response studies determined the sensitivity of
MG-63 cells and the optimal concentrations of G418 used to select
transduced cells. Upon G418 selection, varying proportions of
HStk-transduced and non-transduced MG-63 cells (plating density
2.5.times.10.sup.4 cells) were exposed to 20 .mu.g GCV/ml D10 in
each of six-well plates, for 10 days. Hence, the bystander effects
of GCV in HStk-transduced MG-63 were measured by determining the
degree of confluency of cells in each well in 10 day cultures.
Bystander effects of GCV treatment were compared to those in
HStk-transduced NIH 3T3 cells (ATCC No. CRL 1658).
[0138] Evaluation of Cell Growth, Protein Expression, and Apoptosis
in MG-63 Cells Bearing Chimeric Retroviral Vectors.
[0139] To assess the cytostatic effects of retroviral vectors
bearing cell cycle modulators, the cells that were transduced with
control vectors or vectors expressing cell cycle modulators were
evaluated for their proliferative potential by counting the number
of viable cells in each culture at serial intervals (0, 24, 48, 72,
144 and 192 hrs) after transduction. Western analysis of protein
expression was performed as described previously (Williams, et al.,
J. Biol. Chem., Vol. 268, pgs. 8871-8880 (1993); Wu, et al., Oncol.
Reports, Vol. 2, pgs. 227-231 (1995)), using a polyclonal
anti-peptide antibody recognizing the C-terminal 18 amino acids of
human cyclin G1 (Wu, et al., 1994). To analyze the comparative
efficacy of antisense G1, antisense D1, and p21 expression in the
induction of apoptosis in MG-63 cells, the cells initially were
examined by light microscopy for morphologic changes associated
with apoptosis (cell shrinkage, cytolysis, nuclear fragmentation,
and condensation of chromatin). The relative number of apoptotic
cells were further confirmed and quantified using the Apoptag Plus
in situ apoptosis detection kit (Oncor, Gaithersburg, Md.), which
specifically detects the nascent 3'-OH DNA ends generated by
endonuclease-mediated DNA fragmentation. The significance of
differences among retroviral vectors bearing aG1, aD1, and p21
inserts, and control vectors was determined by analysis of
variance.
Results
[0140] Hagen Osteogenic Sarcoma as a Target for Gene Therapy Using
Retroviral Vectors.
[0141] Initial studies were aimed at characterizing the
transduction efficiency of human osteosarcoma cells, using the
G1BgSvNa retroviral vector construct. The apparent transduction
efficiency of the retroviral vector was relatively high,
approaching 80-90% for the transformed MG-63 cells, as compared to
normal diploid fibroblasts in which transduction efficiencies of
20-30% were observed. FIG. 2 shows the .beta.-galactoisidase
staining MG-63 cells following transduction with the lacZ vector.
Next, potential "bystander" cytocidal effects by mixing cells
transduced with the Herpes Simplex thymidine kinase (HStk) gene
with non-transduced cells followed by exposure to 20 .mu.g/ml
ganciclovir (GCV) was examined. FIG. 3 is a graph which shows the
degree of confluency (%) in mixtures of HStk+ and HStk- MG-63 cells
cultured for 10 days in the presence of GCV (20 .mu.g/ml). The
non-transduced cultures containing 100% HStk- cells showed 75%
confluency. In contrast, the cultures containing 10% HStk+/90%
HStk- and 30% HStk+/70% HStk- cells showed only 15% confluency,
while cultures containing 50% HStk+/50% HStk- cells achieved 10%
confluency, and cultures with greater than 50% HStk+ cell cultures
achieved <10% confluency. The non-linearity of the survival
curve demonstrates a significant bystander effect of GCV in mixed
cultures of MG-63 cells. Both the high transduction efficiency of
retroviral vectors and the occurrence of pronounced bystander
effects to HStk+/GCV treatment attest to the feasibility of gene
therapy for human osteogenic sarcoma using retroviral vectors.
[0142] Cytostatic and Cytocidal Effects of the Antisense Cyclin G1
Retroviral Vector in Cultured Human osteogenic Sarcoma Cells.
[0143] The structure of the experimental retroviral vector
constructs are presented diagrammatically in FIG. 4, including the
location of the neomycin phosphotransferase (neo.sup.r) gene
positioned downstream of the respective genes for 3 cell cycle
control proteins, two of which are truncated fragments engineered
in antisense orientation. The expected sizes of the transcripts for
antisense cyclin G1, antisense cyclin D1, and p21 expression
vectors are 3421, 3456, and 3346 base pairs, respectively.
Transduction of MG-63 cells with each of the test vectors (FIG. 5)
revealed a marked reduction in the number of viable cells observed
at 24 to 168 hours post-transduction, when compared to transduced
cultures containing the control vector expressing only the
neo.sup.r gene. Cell densities were measured, by cell counting, in
cultures of MG-63 cells at serial intervals after transduction with
the retroviral vectors bearing antisense cyclin G1 (G1aG1),
antisense cyclin D1 (G1aD1), and p21(G1p21), as well as the control
vector G1XSvNa (G1X).
[0144] As shown in FIG. 6, the comparative expression of the p29
cyclin G1 protein was analyzed by Western blotting, and found to be
significantly reduced in MG-63 cells bearing the antisense cyclin
G1 vector.
[0145] Antisense Knock-Out Cyclin G1 Induces Apoptosis in Human
Osteogenic Sarcoma Cells.
[0146] The morphological appearance of MG-63 cells was observed by
light microscopy at 72 hours after transduction of retroviral
vectors bearing antisense cyclin G1, antisense cyclin D1, p21
inhibitor, and control vector constructs (FIG. 7). In addition to
significant decreases in cell densities observed in cultures
transduced with vectors containing antisense cyclin G1, as well as
the antisense cyclin D1 and p21 constructs (see FIG. 5),
morphological evidence of apoptotic changes were noted, including
cell shrinkage, nuclear segmentation, chromatin condensation, and
nuclear fragmentation (Arends, et al., Int. Rev. Exp. Pathol., Vol.
32, pgs. 223-254 (1991); Wyllie, et al., International Review of
Cytology, Vol. 68, pgs. 251-306 (1980)), in cells transduced with
each of these cell cycle control elements. To investigate further
the mechanism of cell death, a molecular/immunocytochemical
approach (Arends, et al., Amer. J. Path., Vol. 136, pgs. 593-608
(1990); Gavrieli, et al., J. Cell Biol., Vol. 119, pgs. 493-501
(1992)) was employed to detect the endonuclease-mediated DNA
cleavage fragments that are characteristic of apoptosis (Bursch, et
al., Biochem. Cell Biol., Vol. 68, pgs. 1071-1074 (1990); Compton,
Canc. Metast., Vol. 11, pgs. 105-119 (1992)). FIG. 8 shows the
detection of apoptotic cells by immunocytochemical analysis of DNA
fragmentation in cultures bearing the chimeric vectors containing
antisense cyclin G1, antisense cyclin D1, and p21 constructs. The
induction of apoptosis in each of the cultures transduced with the
cell cycle control vectors was determined to be highly significant
(antisense cyclin G1, mean incidence=38.8.+-.5.0%, n=6, p<0.001;
antisense cyclin Dl, mean incidence=37.4.+-.24.4%, n=6, p<0.01;
and p21, mean incidence=37.5.+-.8.2%, n=6; p<0.001) when
compared to cultures transduced with the control vector (mean
incidence=3.6.+-.4.1%, n=6). These results confirm that the
observed cytocidal effects of these retroviral-mediated cell cycle
blockades result from apoptosis.
[0147] Metastatic osteogenic sarcoma is a target for experimental
gene therapies as it is invariably associated with a fatal outcome.
This type of sarcoma tends to recur locally, spread to other bones
or to lungs, which are surgically accessible sites. In fact, recent
studies have reported increased survival time in patients who have
undergone aggressive metastasectomy (Damron, et al., Oncology, Vol.
9, pgs. 327-340 (1995)). The safety and efficacy of therapeutic
vectors bearing specific cell cycle control enzymes or HStk could
be evaluated by intratumoral injection of producer cells or vector
supernatant into metastatic foci, followed by metastasectomy and
histologic examination for evidence of apoptosis, cytolysis or
overt cytodifferentiation. The present study reveals a relatively
high transduction efficiency of MG-63 osteosarcoma cells for the
above-mentioned retroviral vectors in comparison with normal
diploid fibroblasts. Interestingly, the apparent transduction
efficiency of these cells (80-90%) is far greater than the
percentage of cells in S phase in asynchronous cultures
(Carbonaro-Hall, et al., Oncogene, Vol. 8, pgs. 1649-1659 (1993)).
Non-transduced MG-63 cells exhibited significant "bystander"
cytocidal effects of ganciclovir, when mixed with HStk+ transduced
cells, which, together with retroviral transduction susceptibility,
affirm the feasibility of developing gene therapy approaches in the
clinical management of metastatic disease.
[0148] Previous studies characterized the precise sequence of
cyclin expression in MG-63 osteosarcoma cells (Wu, et al., Int. J.
Oncol., Vol. 3, pgs. 859-867 (1993); Carbanaro-Hall, 1993; Hall, et
al., Oncogene, Vol. 8, pgs. 1377-1384 (1993); Williams, et al., J.
Biol. Chem., Vol. 268, pgs. 8871-8880 (1993)), enabling the
temporal localization of a novel Cdk-associated cell cycle block
point revealed by the antiproliferative agent rapamycin (Albers, et
al., J. Biol. Chem., Vol. 268, pgs. 22825-22829 (1993)). The
results of the present study with retroviral vectors confirms the
results of previous studies using penetrant antisense
oligonucleotides (Wu, 1993): that antisense strategies directed
against the cyclin D1 locus effectively inhibit osteosarcoma cell
proliferation. The mechanism of cell death observed in cells
transduced with each of the experimental constructs (i.e., aG1,
aD1, and p21) was determined to be apoptosis, which is of
considerable importance in terms of therapeutic efficacy in
vivo.
[0149] The physiological function of cyclin G1 and its therapeutic
potential is of particular interest, in that this candidate
protooncogene (CYCG1) was first linked to cancer pathogenesis in
human osteosarcomas (Wu, 1994). Moreover, a recent study suggests
that cyclin G1, like p21, is a transcriptional target of the p53
tumor suppressor protein (Okamoto, et al., EMBO J., Vol. 13, pgs.
4816-4822 (1994)). However, the initial hypothesis that cyclin G1
might counterintuitively function as an inhibitory subunit of
cyclin-dependent kinases in a p53-mediated pathway to prevent
tumorigenesis was discounted by experiments in which enforced
overexpression of cyclin G1 failed to cause cell cyclin arrest in
either normal or neoplastic cell lines (Okamoto, 1994). In
contrast, the present study represents the first demonstration that
cyclin G1 is essential for the survival and/or growth of
osteosarcoma cells. These new data support the concept that cyclin
G1 is involved in cell activation and or "competence" (Wu, 1994),
and that blockade of cyclin G1 expression by antisense constructs
exert profound cytocidal as well as cytostatic effects.
Example 2
Materials and Methods
[0150] Retroviral Vector Supernatants and Producer Cell Lines.
[0151] The .beta.-galactosidase and p53 expression vectors were
kindly provided as high titer PA317 packaging cell clones (titers:
1.3.times.10.sup.4 and 2.times.10.sup.4 colony-forming units,
cfu/ml for .beta.-galactosidase and p53 vectors, respectively) by
Genetic Therapy, Inc. (Gaithersburg, Md.). The experimental vector
bearing antisense cyclin G1 cDNA was packaged in PA317 cells and
grown to high titer clones (vector titer: 1.times.10.sup.4 cfu/ml
each). The vectors are referred to as G1BgSvNa, G1p53SvNa.7, and
G1aG1SvNa to indicate the order of promoters and coding regions
contained in each vector (G1 vector, Moloney murine leukemia virus
long terminal repeat (LTR) sequences; Bg, .beta.-galactosidase or
lacZ gene; p53, p53 tumor suppressor gene; aG1, antisense human
cyclin G1; Sv, SV40 early region enhancer/promoter; and Na,
neo.sup.r gene). The vector source, G1XSvNa, containing only the
SV40 promoter-driven neo.sup.r gene was used as a control for the
effects of gene transduction and G418 selection.
[0152] The vector G1p53SvNa.7 was constructed from pG1XSvNa and the
plasmid pp53. Plasmid pp53 was constructed from pBSK-SN3, obtained
from PharmaGenics (Allendale, N.J.), which contains a 1.8 kb XbaI
fragment that includes the wild type p53 open reading frame as well
as 5' and 3' untranslated rections cloned into the XbaI site of
pBluescriptSK (Stratagene, LaJolla, Calif.). pBSK-SN3 was digested
with SmaI and partially digested with NcoI to generate a 1,322 bp
fragment containing the p53 open reading frame. The fragment was
gel purified and ligated into plasmid pBg (described in published
PCT Application No. WO91/10728, published Jul. 25, 1991), in place
of the 6-galactosidase gene between the NcoI and the XhoI sites to
yield plasmid pp53.
[0153] Plasmid pG1XSvNa was digested with SnaBI and NotI. The SnaBI
and NotI sites are located in the polylinker region of the plasmid.
The digest generated a fragment having a length of 5,848 base
pairs. The ends of the fragment were treated with calf intestinal
alkaline phosphatase.
[0154] Plasmid pp53 was digested with NotI and SmaI, the digest
generated a 2,081 base pair fragment and a 1,400 base pair
fragment. The 1,400 base pair fragment contained the p53 gene. This
fragment was isolated and gel purified.
[0155] The 5,848 base pair fragment obtained from pG1XSvNa, and the
1,400 base pair fragment obtained from pp53, with each fragment
having sticky/blunt ends, were ligated to form pG1p53SvNa. The
resulting plasmid was identified and confirmed by several
diagnostic restriction analyses. The plasmid pG1p53SvNa then was
packaged in PA317 cells to generate the retroviral vector
G1p53SvNa.7.
[0156] Cells, Cell Culture Conditions and Transduction of Cells
with lacZ, Antisense Cyclin G1 and p53 Vectors.
[0157] Rabbit undifferentiated carcinoma (VX2) cells and other
primary and established cell lines were cultured at a plating
density of 2.5.times.10.sup.4 cells in each of six-well plates, in
DMEM supplemented with 10% FBS (D10). After overnight attachment,
the cells were exposed to 1 ml of the respective retroviral vector
in the presence of Polybrene (8 .mu.g/ml) for 2 hours, after which
1 ml of fresh D10 was added to each well. Forty-eight hours after
transduction with the lacZ vector, gene transfer efficiency was
measured by determining the percentage of lacZ positive cells, upon
X-gal staining and light microscopy.
[0158] Evaluation of Cell Proliferation and Cell, Cycle Kinetics in
VX2 Transduced with Retroviral Vectors Bearing Cell Cycle Control
Genes.
[0159] To assess the cytostatic effects of retroviral vectors
bearing cell cycle modulators, the cells that were transduced with
control vectors, or vectors expressing antisense cyclin G1 or p53
genes, were evaluated for their proliferative potential by counting
the number of viable cells in each culture at serial intervals
after transduction. The effect of cell cycle modulators on the cell
cycle kinetics of VX2 (carcinoma) as well as MG-63 (sarcoma) cells
was tested by FACS analysis. The survival of transduced VX2 cells
in the presence of G418 also was evaluated to determine to what
extent the antisense cyclin G1 was cytocidal to the transduced
cells.
[0160] Development of a Tumor Model in Athymic Nude Mice for in
vivo Gene Therapy Using Retroviral Vectors Bearing Cell Cycle
Modulators.
[0161] Undifferentiated carcinoma (VX2) tumors have been grown
successfully in nude mice by subcutaneous implantation of VX2
cells. These tumors grow rapidly within three weeks, and are
surgically accessible for evaluation of changes in tumor volume and
morphology. Briefly, VX2 tumors were grown over 5 weeks in athymic
nude mice by subcutaneous injection of 1.times.10.sup.7 VX2 cells.
When the tumors reached 100 mm.sup.3 in size, 100 .mu.l of
concentrated retroviral vector supernatant (G1aG1SvNa, bearing the
antisense cyclin G1 gene or the G1XSvNa control vector, bearing
only the neo.sup.r gene: vector titer, 1.times.10.sup.8 cfu/ml) was
injected intratumorally, under Metofane anesthesia, every day for 2
weeks. Tumor volume was measured every week using a Vernier
caliper, and the percentage change in tumor volume was estimated.
The significance of differences between the antisense cyclin G1
vector- and control vector-treated tumors was tested using the
Student's t test. Additionally, the formalin-fixed tumors were
stained with hematoxylin-eosin (H & E) for histologic
examination.
Results
[0162] A wide variety of cell lines were tested for sensitivity to
retroviral vectors bearing cell cycle modulators. The results of
such testing are given in Table I below.
1TABLE I In Vitro Transduction Efficiencies and Cytostatic Effects
of an Antisense Cyclin G1 Retroviral Vector in Cancer and Non-
cancer Cells Transduction Cytostatic Efficiency Effect Cell Line
Cell Type (GlBgSvNa) (GlaGlSvNa) Human Cancer MG-63 osteosarcoma
80% + HT29 colon carcinoma 13% + Bxpc-3 pancreatic carcinoma 9% +
Miapaca pancreatic carcinoma 19% + Mnng/Hos osteosarcoma 15% + EW-1
Ewing's Sarcoma 5% + MDA-MB 231 breast cancer <1% - Non-human
Cancer XC-6 (rat) osteosarcoma 22% + Km12C (rat) colon carcinoma
20% + Km12C4A (rat) colon carcinoma 15% + Km12SM (rat) colon
carcinoma 15% + C6 (rat) glioma 5% + VX-2 (rabbit) undifferentiated
CA 6% + Human Non-cancer primary bone marrow stroma 22% - primary
activated keratocyte 20% + primary hepatic fibroblast 23% - primary
keloid fibroblast 31% + primary dermal fibroblast 24% + ECU
endothelial 5% - Non-human Non-cancer A10 (rat) aortic smooth
muscle 45% + NIH3T3 (mouse) fibroblast 30% +
[0163] Of the cells tested, proliferation of 4 colon cancer cells
(HT-29, KK12C4A, KM12C and KM12SM), Ewings sarcoma (EW-1), C6
glioma, 2 pancreatic cancer (BxPc3, Miapaca) and 2 osteosarcoma
(MG-63, MnngHOS) was inhibited by the antisense cyclin G1
retroviral vector. The HT29, BxPc3, and KM12SM cells were also
sensitive to wild type p53. Among the non-cancer cell lines,
cytostasis was induced by antisense cyclin G1 and p53 in embryonic
rat aortic smooth muscle cells and human skin and keloid
fibroblasts, but not in normal human stromal, human liver-derived
fibroblasts or human endothelial cells.
[0164] FACS analysis was used to investigate the effect of the
antisense cyclin G1 retroviral vector on the cell cycle kinetics of
sarcomatous and carcinomatous tumor cells. VX2 undifferentiated
carcinoma cells transduced with retroviral vectors bearing
antisense cyclin G1 showed profound alterations of cell cycle
kinetics upon FACS analysis, exhibiting a broadening of peaks that
is indicative of nuclear fragmentation and a reduction of cells in
S phase (FIG. 9A). In comparison, FACS analysis of MG-63 cells
transduced with the antisense cyclin G1 vector showed accumulation
of cells in G1 phase, and a significant decrease in the number of
cells in S phase, suggesting that the mechanism of cytostasis in
these transduced cells accompanies a G1 phase cell cycle block
(FIG. 9B).
[0165] Simultaneous with the altered cell cycle kinetics, the
antisense cyclin G1 as well as the p53 vectors inhibited
proliferation of VX2 carcinoma cells over 144 hours compared with
control vector-treated cells (FIG. 10). Upon selection of
transduced cells with G418, only 5% of the VX2 cells were
eliminated (FIG. 11), indicating that the vast majority of cells
bearing antisense cyclin G1 and wild type p53 had undergone cell
death, presumably via apoptosis. These data represent the first in
vitro demonstration that antisense cyclin G1 may exhibit antitumor
activity in cancers of epithelial origin.
[0166] FIG. 12 shows inhibition of VX2 tumor growth in nude mice by
intratumoral, injection of a retroviral vector bearing antisense
cyclin G1 (G1aG1) when compared to growth of VX2 tumors in mice
receiving the control vector (G1X; p<0.05 at 7 days; p<0.001
at 11 days; and p<0.05 at 21 days; n=3 mice each group). In 1 of
5 mice treated with antisense cyclin G1 vector, a 12% decrease in
tumor size was noted one week following treatment. In contrast,
tumor growth was not arrested in the mice treated with the control
vector.
[0167] The mice treated with the antisense cyclin G1 vector showed
grossly smaller tumors than control mice. FIG. 13A shows
representative antisense cyclin G1 versus control vector-treated
mice while FIG. 13B shows the histopathologic characteristics of
formalin-fixed and H&E stained VX2 tumor sections, harvested at
21 days (one week after completion of treatment). The sections of
tumors that were treated with the control vector showed areas of
increased cell density with anaplastic spindle-shaped cells and
numerous mitotic figures. In contrast, the sections of tumors that
were treated with the antisense cyclin G1 vector showed areas of
decreased cell density with less mitotic figures and notable
mononuclear cell infiltration. However, residual tumor cells were
noted in sections of tumor that received the antisense cyclin G1
vector, indicating that a population of tumor cells were not
effectively transduced. Taken together, the retroviral vector
expressing antisense cyclin G1 appears to exhibit antitumor effects
in vivo in this tumor model of undifferentiated carcinoma.
Discussion
[0168] Cancer is a leading target for gene therapy because patients
with cancer, particularly those with metastatic disease, often have
few or no treatment options, and would be eligible for experimental
therapies. The retroviral vector delivery system has been used in
76 of the 106 human trials approved. This vector system utilizes a
replication incompetent mouse retrovirus, and thus far, its use,
both ex vivo and in vivo, has not caused any major side
effects.
[0169] Other gene therapy strategies include 1) enhancement of the
immune response by injection of tumor vaccines containing
transduced irradiated tumor cells expressing cytokines, MHC Class 1
or B7 genes, 2) enforced expression of tumor suppressor genes, 3)
knock-out of protooncogene overexpression by antisense vectors, and
4) enforced expression of growth factor receptor genes. In recent
years, overexpression or amplification of various cell cycle
control genes have been reported in various malignant disorders,
indicating that antisense knock-out of these overexpressed genes
could be used to re-establish control of cell proliferation, induce
cytostasis, inhibit tumor growth and decrease tumor burden.
[0170] These concepts arise from initial studies of the budding
yeast S. cerevisiae wherein extracellular signals that modulate the
growth and differentiation act via regulation of a G1 control point
termed START (Hartwell, Science, Vol. 183, pgs. 46-51 (1974); Cross
et al., Ann. Rev. Cell Biol., Vol. 5, pgs. 341-395 (1989)), which
is loosely analogous to the G1 restriction point (R point) observed
in animal cells in culture (Pardee, Science, Vol. 246, pgs. 603-608
(1989)). Therefore, the finding that G1 cyclins (Clns) in S.
cerevisiae, in association with a Cdk subunit (Cdc28), were
required for cells to pass START led to the hypothesis that G1
specific cyclins may indeed function as upstream components of the
mammalian S phase Promoting Factor (Draetta, Trends Biochem. Sci.,
Vol. 15, pgs. 378-383 (1990); Reed, Trends in Genetics, Vol. 7,
pgs. 95-99 (1991)). Screening of human cDNA libraries for genes
that could serve to rescue Cln-deficient yeast cells led to the
identification and molecular cloning of three novel families of
human G1 cyclins (cyclins C, D, and E: Lew et al., Cell, Vol. 66,
pgs. 1197-1206 (1991); Koff et al., Cell, Vol. 66, pgs. 1217-1228
(1991); Xiong et al., Cell, Vol. 65, pgs. 691-699 (1991); Sherr,
Cell, Vol. 73, pgs.1059-1065 (1993)). Subsequent studies have
mapped the PRAD1/Cyclin D1 gene to chromosome 11q13, implicating
cyclin D1 as the BCL-1 oncogene that is translocated and
overexpressed in B cell neoplasms (Rosenberg et al., Proc. Nat.
Acad. Sci., Vol. 88, pg. 9638 (1991); Withers et al., Mol. Cell.
Biol., Vol. 11, pg. 4846 (1991)) and as the 11q13 oncogene that is
amplified and overexpresssed in squamous cell, breast, esophageal,
and bladder cancers (Lammie et al., Oncogene, Vol. 6, pg. 439
(1991); Jiang et al., Cancer Res., Vol. 52, pg. 2980 (1992);
Motokura et al., Curr. Opin. Genet. Dev., Vol. 3, pg. 5 (1993)).
Genetic amplification, increased expression, and altered metabolism
of cyclin E has also been observed in human cancer cells (Buckley
et al., Oncogene, Vol. 8, pg. 2127 (1993); Keyomarsi et al.,
Cancer. Res., Vol. 54, pg. 380 (1994)). More recently, a human
G-type cyclin, a G1 cyclin that was markedly overexpressed in a
subset of osteosarcoma cells was isolated (Wu et al., 1994). Taken
together, these findings affirm that constitutive, ectopic, or
deregulated expression of G1 cyclins, which normally link signal
transduction pathways to the enzymatic machinery of the cell cycle
(Hunter and Pines, Cell, Vol. 66, pgs. 1071-1074 (1991); Sherr,
(1993)), may play an important role in neoplastic transformation
and tumorigenesis (Hunter and Pines, Cell, Vol. 79; pgs. 573-382
(1994)), and could be used as strategic checkpoints for development
of novel gene therapy approaches to cancer and hyperproliferative
disorders.
[0171] In this study, the safety and efficacy of an antisense
cyclin G1 retroviral vector supernatant as a potential gene therapy
approach to cancer was tested. A wide variety of cancer cells
showed sensitivity to antisense knockout cyclin G1 in comparison to
wild-type p53. The proliferation of some non-cancerous cells also
was inhibited by the antisense cyclin G1 vector, suggesting its
potential utiliy in the management of non-malignant
fibroproliferative disorders as well. Hence, various cell types
showed differential sensitivity to cell cycle modulators. The
antisense cyclin G1 vector had profound effects on the cell cycle
kinetics of both carcinomatous and sarcomatous tumor cells, with a
net effect of decreased DNA synthesis, as evidenced by a reduction
of cells in S phase. These data suggest that the mechanism of
cytostasis in these transduced cells accompanies a G1 phase cell
cycle block. Upon selection of transduced cells with G418, only 5%
of the VX2 cells were eliminated, indicating that the vast majority
of cells bearing antisense cyclin G1 and wild type p53 had
undergone cell death, presumably via apoptosis.
[0172] Finally, in vivo tumor growth was inhibited dramatically by
successive intratumoral injection of a concentrated antisense
cyclin G1 retroviral vector supernatant. In contrast, tumor growth
was not arrested in the mice treated with the control vector.
Histologic examination of the tumors one week after cessation of
treatment showed areas of increased cell density with anaplastic
spindle-shaped cells and numerous mitotic figures in control
vector-treated tumors. In contrast, the sections of tumors that
were treated with the antisense cyclin G1 vector showed areas of
decreased cell density with less mitotic figures and notable
mononuclear cell infiltration. Taken together, these findings
represent the first demonstration of in viva antitumor activity of
a retroviral vector expressing antisense cyclin G1 in a model of
undifferentiated carcinoma.
Example 3
Inhibition of in vivo Tumor Growth by a Retroviral Vector Bearing
Antisense Cyclin G1 in Athymic Nude Mice
[0173] Osteosarcoma tumors were grown over two weeks in athymic
nude mice by subcutaneous injection of 1.times.10.sup.7 MNNG/HOS
cells. When the tumors reached 100 mm.sup.3 in size, 100 .mu.l of
concentrated retroviral vector supernatant (G1XSvNa control vector,
bearing only the neo.sup.R gene, or G1aG1SvNa, bearing the
antisense cyclin G1 gene: vector titers: each 1.times.10.sup.4
cfu/ml) were injected intratumorally every day for 10 days.
[0174] The tumor volume was measured at intervals of 0, 4, 6, 8,
10, and 12 days after vector injection. FIG. 14 shows the tumor
volume at each of the above-mentioned intervals. As shown in FIG.
14, the antisense cyclin G1 vector-treated mouse has a smaller
tumor than the control vector-treated mouse.
[0175] Hematoxylin and eosin staining of formalin-fixed MNNG/HOS
tumor sections for two days following the treatment with the
retroviral vectors bearing the antisense cyclin G1 gene (G1aG1SvNa)
or the control vector (G1XSvNa) shows decreased mitotic index (1%
for antisense cyclin G1-treated tumors versus 3.5% for control
vector-treated tumors), and increased stroma formation.
[0176] FACS analysis of PI-stained nuclei obtained from MNNG/HOS
tumors showed a dramatic decrease in the number of aneuploid cells
in the antisense cyclin G1 vector-treated tumors (2%) compared with
that in control vector treated tumors (45%). Further, the diploid
population of cells from the antisense cyclin G1 vector-treated
tumors showed a 77% accumulation of cells in G1 phase versus 49% in
G1XSvNa control vector-treated tumors, and a significant decrease
in the number of cells in S phase (15% versus 25%), which suggests
that the mechanism of cytostasis in the transduced tumors was
accompanied by a G1 phase cell cycle block.
Example 4
Materials and Methods
[0177] Retroviral Vectors, Vector Supernatants and Producer Cell
Lines.
[0178] The cDNA sequence encoding human cyclin G1 (Accession
#X77794) is as originally described by Wu et al., 1994. The
experimental vector bearing the antisense cyclin G1 cDNA (Wu, et
al., 1994) was packaged in PA317 cells and grown to high titer
clones (vector titer: 1.times.10.sup.4 cfu/ml each). The
.beta.-galactosidase and p53 expression vectors were provided
kindly as high titer PA317 packaging cell clones (titers:
5.times.10.sup.5 and 2.times.10.sup.6 colony-forming units, cfu/ml
for .beta.-galactosidase and p53 vectors respectively) by Genetic
Therapy, Inc. (Gaithersburg, Md.). The vectors are referred to as
G1nBgSvNa (described in PCT Application Nos. WO95/19427, published
Jul. 20, 1995 and WO96/22212, published Jul. 25, 1996),
G1pS3SvNa.7, and G1aG1SvNa to indicate the order of promoters and
coding regions contained in each vector (G1, Moloney murine
leukemia virus long terminal repeat (LTR) sequences; Bg,
.beta.-galactosidase gene; p53, p53 tumor suppressor gene; aG1,
antisense human cyclin G1; Sv, SV40 early region enhancer/promoter;
and Na, neo.sup.r gene). The retroviral vector supernatants were
concentrated further to a titer of 1.times.10.sup.8 cfu/ml by low
speed centrifugation. The vector backbone, G1XSvNa, containing only
the SV40 promoter-driven neo.sup.r gene was used as a control for
the effects of transduction and G418 selection.
[0179] Cells, Cell Culture Conditions, and Transduction with
Retroviral Vectors.
[0180] Rat aortic smooth muscle (A10) cells were obtained from ATCC
(Cat. #CRL1476) and maintained as monolayers at a plating density
of 2.5.times.10.sup.4 cells per well, in DMEM supplemented with 10%
fetal bovine serum (FBS;D10). After overnight attachment, the cells
were exposed to 1 ml of the respective retroviral vector in the
presence of Polybrene (8 .mu.g/ml) for 2 hours, with periodic
rocking, after which 1 ml of fresh D10 was added to each well.
Forty-eight hours after transduction with the .beta.-galactosidase
vector, gene transfer efficiency was measured by determining the
percentage of .beta.-galactosidase positive cells, upon exposure to
X-gal (.beta.-galactosidase) staining as described in Lal, et al.,
J. Histochem. Cytochem., Vol. 42, pgs. 953-956 (1994), and
visualization by light microscopy.
[0181] Analysis of Cell Proliferation, DNA Synthesis, Cyclin G1
Protein Expression and Apoptosis
[0182] To assess the cytostatic effects of retroviral vectors
bearing cell cycle modulators, the SMC that were transduced with
control vectors or vectors expressing antisense cyclin G1 (or p53)
gene(s) were evaluated for their proliferative potential by
counting the number of viable cells in each culture at serial
intervals after transduction. Values shown represent the mean of
triplicate.+-.standard deviation (S.D.) The effect of cell cycle
modulators on DNA synthesis was monitored by the incorporation of
.sup.3H-thymidine into DNA as described in Gordon, et al., Proc.
Nat. Acad. Sci., Vol. 93, pgs. 2174-2179 (1996). Briefly, 24 hrs,
after transduction with the antisense cyclin G1 or control
retroviral vector, the cell cultures were exposed to
.sup.3H-thymidine (1 .mu.Ci per well; specific activity, 6.7
Ci/mmol; 1 Ci=37 GBq; New England Nuclear) for 2 hrs. The cells
were then placed on ice, rinsed twice with cold phosphate-buffered
saline (PBS), and then rinsed three times with ice-cold 5t
trichloroacetic acid (TCA). The final TCA rinse was removed and the
TCA-precipitated material was solubilized with 0.2 ml of 1M sodium
hydroxide followed by neutralization with an equal volume of 1M
acetic acid. .sup.3H-thymidine incorporation into cellular
macromolecules was measured by liquid scintillation counting and
expressed as radioactivity units in dpm/well. The significance of
differences between untreated and vector-treated groups was
determined by analysis of variance (ANOVA).
[0183] Western Blot analysis of cyclin expression was performed as
described in Wu, et al., Int. J. Oncol., Vol. 3, pgs. 859-867
(1993) and Colton, Statistics in Medicine, pg. 99, Little, Brown
& Co., (1974), using a polyclonal antipeptide antibody
recognizing the C-terminal 18 amino acids of human cyclin G1 (Wu,
et al., 1994). The occurrence of apoptosis in transduced cell
cultures was evaluated with the Apoptag Plus in situ detection kit
(Oncor), which detects nascent 3'-OH DNA ends generated by
endonuclease-mediated DNA fragmentation utilizing enzymatic
(terminal deoxynucleotidyl transferase;TdT) addition of
digoxigenin-labeled nucleotides followed by immunocytochemical
detection of the modified DNA fragments (Skotzko, et al., Cancer
Res., Vol. 55, pgs. 5493-5498 (1995)).
[0184] Retrovirus-Mediated Transfer of the Antisense Cyclin G1 Gene
in a Rat Carotid Injury Model of Vascular Restenosis.
[0185] Under general anesthesia (ketamine, 10 mg/kg; rompun, 5
mg/kg), in accordance with a protocol approved by the USC
Institution Animal Care and Use Committee (IACUC), a 2F Intimax
arterial embolectomy catheter (Applied Medical Resources Corp.,
Laguna Hills Calif.) was used to denude the carotid artery
endothelium of Wistar rats (each weighing 400-500 gm). The catheter
was inserted into the external carotid artery which was ligated
distally, and passed into the common carotid artery. The balloon
was inflated to a volume of 10 .mu.l and passed 3 times along the
length of the common carotid artery. After balloon injury, the
embolectomy catheter was removed and the internal carotid artery
was ligated transiently just distal to the bifurcation. The distal
half of the injured segment was likewise transiently ligated, and
then exposed to the retroviral vectors for 15 minutes. Each group
of animals received an infusion of 100 .mu.l of concentrated high
titer antisense cyclin G1 vector (n=7) or a control vector bearing
only the neo.sup.r gene (n=4), after which the rats were allowed to
recover, and fed a regular mouse/rat diet and water ad libitum. For
purposes of analgesia, the animals were given buprenex, 0.2 mg/kg
s.c. every 12 hours for 72 hours post-operatively. The rats were
sacrificed 2 weeks after induction of vascular injury by an
overdose of sodium pentobarbital (120 mg/kg IM), and formalin-fixed
sections of both injured and non-injured contralateral carotid
arteries were stained with hematoxylin-eosin, Siris red-Verhoeff's
elastin stain. Histologic sections were examined by light
microscopy, and morphometric evaluation of the neointima versus
media surface areas was conducted using a digitizing system; the
extent of intimal hyperplasia following vascular injury is
expressed as neointima to media ratios. The significance of
differences between the neointima to media ratios of non-treated
and vector-treated vessels was determined by paired t-test (Colton,
1974).
Results
[0186] Transduction of Aortic SMC with Retroviral Vectors Bearing
Cell Cycle Control Genes.
[0187] Using a nuclear-targeted .beta.-galactosidase vector
(G1nBgSvNa), the apparent transduction efficiency of rat (A10)
aortic SMC was about 45% (FIG. 15A), which was similar to murine
NIH3T3 cells, and somewhat greater than normal human fibroblasts or
scar-derived (keloid) fibroblasts in which transduction
efficiencies of 20 and 30, respectively, were observed.
Transduction of aortic SMC with vectors bearing antisense cyclin G1
(aG1) showed a marked decrease in the number of viable cells
observed at 24 to 144 hours post-transduction, when compared to
transduced cultures containing the empty (control) vector (FIG.
15B). Western Blot analysis confirmed down-regulation of cyclin G1
protein expression in aortic SMC transduced with antisense cyclin
G1 when compared to the control vector (not shown). Proliferation
of A10 cells was also inhibited by retroviral mediated
overexpression of the p53 tumor suppressor gene in sense
orientation. Both antisense cyclin G1 and p53 vectors inhibited
cell cycle progression, as determined by the incorporation of
.sup.3H-thymidine (p<0.001 for both aG1 and p53; FIG. 15C).
[0188] Antisense Cyclin G1 Induces Degeneration, Multicellular
Syncytia Formation, and Apoptosis in Aortic SMC.
[0189] The photomicrographs shown in FIG. 16 display the
morphological appearance of aortic SMC observed by light microscopy
at 24 hours after transduction with control and antisense cyclin G1
retroviral vectors. As shown in FIG. 16A, the cells transduced with
the control vector showed no significant morphologic changes. In
contrast, a significant decrease in cell density was observed in
cultures transduced with vectors bearing antisense cyclin G1,
associated with overt degenerative changes, increased multinuclear
syncytium formation, and cytolysis (FIGS. 16B, 16C, 16D).
Remarkably, the proportion of cells involved in the syncytia far
exceeded the transduction efficiency as determined by the
transduction and expression of .beta.-galactosidase. Syncytium
formation occurred in A10 cultures transduced with the antisense
cyclin G1 vector supernatants derived from three different high
titer clones, as well as the p53 vector to some extent, but not in
the control (G1XSvNa) or .beta.-galactosidase vectors. To further
investigate the mechanisms of cell death, a molecular and
immunocytochemical approach was employed to detect the
endonuclease-mediated DNA cleavage fragments that are
characteristic of apoptosis. As shown in FIGS. 16E and 16F, no
evidence of apoptosis was observed in cells transduced with the
control vector (FIG. 16E); however, a number of apoptotic cells
were observed in the antisense cyclin G1 vector-transduced cultures
(FIG. 16F). These results indicate that the cytocidal effects of
the antisense cyclin G1 vector in A10 aortic SMC result in part
from apoptosis, cell degeneration, and aberrant syncytium
formation.
[0190] Evidence for a Cytocidal "Bystander" Effect in Aortic SMC
Cultures Transduced with Antisense Cyclin G1 Retroviral
Vectors.
[0191] To confirm that non-transduced cells were incorporated into
the multicellular syncytia found in antisense cyclin G1-transduced
cultures, we loaded non-transduced A10 cells with a fluorescent
marker and overlaid the marked cells on previously transduced
cultures two hours after washout of the vector supernatant. The
incorporation of non-transduced, flourescently-labeled A10 smooth
muscle cells into multinuclear syncytia clearly was evident when
these marked cells were overlaid onto previously transduced A10
cultures (FIGS. 17A and 17B, low magnification; 17C and 17D, high
magnification; 17A and 17C, phase contrast; 17B and 17D, UV light).
A representative multinuclear syncytium incorporating cells
containing the flourescent label is identified by the arrow.
Twenty-four hours after co-culture with non-transduced,
fluorescently-labelled aortic SMC, a considerable number of the
multinucleated syncytia were also labelled with the fluorescent
dye, indicating that cell fusion between the transduced and
non-transduced cells had occurred. This finding provides additional
evidence of a novel cytocidal "bystander effect" distinguishable
from the classic "bystander effect" induced by the Herpes Simplex
Virus thymidine kinase/ganciclovir system and mediated by gap
junctions present in susceptible cells.
[0192] The phenomenology of cell fusion was followed over time
(FIG. 17E, revealing a significant increase in the number of
syncytia that increased over 4-8 hours in aortic SMC that were
transduced with the antisense cyclin G1 vector (G1aG1SvNa), when
compared to the cells transduced with the control vector (G1XSvNa;
p<0.001). An appreciable degree of syncytium formation also was
noted in cells that were transduced with the wild-type p53 vector
(G1p53SvNa) which also produced marked cytostasis in A10 cells.
However, the number of syncitia observed in p53 transduced cells
was significantly less than than that observed in aG1 transduced
cells at B, 12 and 24 hours (p<0.001).
[0193] The Antisense Cyclin G1 Vector Inhibits Proliferation and
Migration of Aortic Smooth Muscle Cells in an in vitro "Tissue"
Injury Model.
[0194] High density (confluent) monolayer cultures of A10 SMC
exhibiting contact inhibition of cell growth can be stimulated to
proliferate along a track of cell/tissue disturbance exhibiting a
characteristic "wound healing" response over a period of 7 days.
FIG. 11A shows high density cultures of aortic SMC scraped with a
200 .mu.l pipet tip to create a 1 mm track devoid of cells. FIG.
18B shows the appearance of the "wound" margin immediately upon
scraping and washing to remove the detached cells. As shown in FIG.
18C, subsequent transduction of the cell cultures (at t=24 hours)
with a nuclear-targeted .beta.-galactosidase vector was greatest at
the margins of the "wound", an area of activated SMC proliferation.
FIG. 18D shows proliferation and migration of aortic SMC into the
wound track at t=24 hours after injury. In contrast, apoptotic and
other degenerative changes were observed in the SMC that were
transduced with the antisense cyclin G1 vector (FIG. 18E). Notably,
these degenerative changes were marked by multicellular syncytia
formation that was not observed in either the control or
.beta.-galactosidase vector. Further, cell proliferation and overt
cell migration into the wound track was reduced markedly in the
antisense cyclin G1-transduced cell cultures, evidenced by delayed
closure of the wound track (about 7days) compared to the control
vector-treated cultures (about 3 days).
[0195] Inhibition of Neointima Formation in vivo by Infusion of
High Titer Antisense Cyclin G1 Vector Supernatant.
[0196] Previous studies demonstrated direct transfer of recombinant
marker genes into the arterial wall by retroviral vectors with
viral titers of 104-106 particles/ml (Nabel, et al., Science, Vol.
249, pgs. 1285-1288 (1990)), and a number of studies have
demonstrated the efficacy of cytostatic gene therapies delivered by
other methods in animal models of vascular restenosis. In this
study, high titer retroviral vector supernatants (viral titer:
1.times.10.sup.4 cfu/ml) were generated to test the efficacy of
antisense cyclin G1 delivered by highly concentrated retroviral
vectors in the rat carotid injury model of restenosis. Histologic
examination of stained sections obtained from balloon-injured
untreated arteries showed substantial neointima formation at 2
weeks, as evidenced by invasion of the tunica intima by
proliferating vascular SMC (FIGS. 19A and 19C). In contrast,
injured arterial segments that were treated with high titer
antisense cyclin G1 vector supernatants showed a significant
reduction in neointima formation (FIGS. 19B and 19D). Morphometric
analysis confirmed significant inhibition in neointima formation in
injured carotid arteries that were treated with the antisense
cyclin G1 retroviral vector (I:M ratio 0.4.+-.S.D. 0.4) compared to
the untreated arterial segments (I:M ratio 1.1.+-.0.4; p<0.001;
FIG. 19G). In control studies, there was no difference between the
extent of neointima formation in non-treated arterial segments (I:M
ratio 1.3.+-.SD 0.5) when compared with high titer vectors
containing only the neo.sup.r gene (I:M ratio 1.5.+-.0.2).
Discussion
[0197] Clinical trials based on the molecular blockade of
identified growth factors and/or growth factor receptors implicated
in the pathogenesis of intimal hyperplasia have not proven to be
effective vehicles for cytostatic vascular therapy (Faxon, et al.,
J. Amer. Coll. Cardiol., Vol. 25, pgs. 362-369 (1995)). Thus, it
has been suggested that approaches which target intracellular
signalling cascades that are shared by many growth regulatory
molecules may be more strategic (Gibbons, et al., Science, Vol.
272, pgs. 689-693 (1996)). Accordingly, novel gene therapy
approaches to inhibit SMC proliferation and neointima formation
have focused recently on cell cycle control mechanisms. Indeed,
antisense approaches against cell cycle regulatory genes has been
shown to be remarkably effective in limiting neointimal hyperplasia
in animal models of lesion formation following both bypass surgery
(Mann, et al., Proc. Nat. Acad. Sci., Vol. 92, pgs. 4502-4506
(1995)) and balloon angioplasty. A single intraluminal delivery of
antisense Cdc2 kinase or Cdk2 kinase produced significant
inhibition of neointimal hyperplasia Morishita, et al., Proc. Nat.
Acad. Sci., Vol. 90, pgs. 8474-8478 (1993); Morishita, et al., J.
Clin. Invest., Vol. 93, pgs. 1458-1464 (1994); Abe, Biochem.
Biophys. Res. Comm., Vol. 198, pgs. 16-24 (1994)). An adenoviral
vector bearing a nonphosphorylatable, constitutively active form of
Rb also was reported to inhibit SMC proliferation and neointima
formation following balloon angioplasty (Chang, et al., Science,
Vol. 267, pgs. 518-522 (1995)). Molecular strategies directed
against E2F also have been developed, as the concerted induction of
numerous cell cycle regulatory genes is regulated by this
transcription factor. Oligonucleotides containing the E2F cis
element sequence function as "decoys" that bind E2F within the cell
and inhibit neointimal lesion formation in vivo (Morishita, et al.,
Proc. Nat. Acad. Sci., Vol. 92, pgs. 5855-5859 (1995)). Further
support for the concept of cytostatic gene therapy based on the
inhibition of cell cycle control enzymes is provided by recent
findings that rapamycin, which inhibits the activation of cell
division/cycle enzymes (Albers, et al., Ann. New York Acad. Sci.,
Vol. 696, pgs. 54-62 (1993); Albers, et al., J. Biol. Chem., Vol.
268, pgs. 22825-22829 (1993); Jayaraman, et al., J. Biol. Chem.,
Vol. 34, pgs. 25385-25388 (1993), also inhibits vascular lesion
formation in both rat and porcine models (Gregory, et al.,
Transplantation, Vol. 59, pgs. 655-661 (1995); Marx, et al., Circ.
Res., Vol. 76, pas. 412-417 (1995)).
[0198] Cyclin G1 is a member of the so-called G1 family of cyclins
which act in concert with cyclin-dependent protein kinases during
the G1 phase of the cell cycle (Wu, et al., Int. J. Oncol., Vol. 3,
pgs. 859-867 (1993); Sherr, Cell, Vol. 79, pgs. 551-555 (1994)).
Induced in early G1 and suspected to participate in the molecular
mechanisms of cell activation (Wu et al., Oncol. Reports, Vol. 1,
pgs. 705-711 (1994)), cyclin G1 appears to be a transcriptional
target of the p53 tumor suppressor gene (Okamoto, et al., EMBO J.,
Vol. 13, pgs. 4816-4822 (1994)). Cyclin G1 overexpression was first
linked to cancer (Wu, et al., 1994) and, more recently,
down-regulation of cyclin G1 expression by retroviral vectors
bearing antisense CYCG1 was reported to inhibit the growth and
survival of human osteosarcoma (MG-63) cells (Skotzko, et al.,
1995).
[0199] In this example, the effects of retroviral vectors bearing
an antisense cyclin G1 construct on the proliferation of A10 rat
aortic smooth muscle cells were examined. Retroviral vectors
bearing the antisense cyclin G1 gene, as well as the p53 gene, in
sense orientation, inhibited the survival and proliferation of
transduced A10 cells in 2-6 day cultures. Cytostasis was associated
with decreased DNA synthesis and down-regulation of cyclin G1 in
vascular SMC transduced with the antisense cyclin G1 vector as
compared to those transduced with the control vector. Morphological
examination of the transduced SMC revealed cytolysis, giant
syncytia formation, and overt apoptotic changes evidenced by cell
shrinkage, nuclear fragmentation, and chromatin condensation
observed in both antisense cyclin G1 vector- and p53
vector-transduced A10 cells. However, the number of multinuclear
syncytia were found to be significantly higher in the cell cultures
treated with the antisense cyclin G1 vector. Pronounced "bystander"
effects were noted in A10 cells transduced with the antisense
cyclin G1 vector as determined by quantitative cell fusion assays
and the fluorescent labeling of non-transduced cells. These
findings indicate that the antisense cyclin G1 vector induces a
"fusion-promoting factor", possibly a protease or glycosylase, that
facilitates cell fusion and syncytia formation, perhaps by
augmenting mechanisms related to the fusogenic properties of the
MoMuLV envelope protein (Jones, et al., J. Virol., Vol. 67, pgs.
67-74 (1993)).
[0200] Cytostatic gene therapies for restenosis show promise of
additional therapeutic consequences in that the inhibition of cell
cycle regulatory genes is reported to trigger vascular cell
apoptosis (Gibbons, et al., 1996; Laird, et al., Circulation, Vol.
93, pgs. 529-536 (1996)). In mitotically activated SMC, as in
osteosarcoma cells (Skotzko, et al., 1995), the cytotoxicity of the
cyclin G1 blockade is attributable, at least in part, to the
activation of an apoptotic pathway (FIG. 16F). Furthermore, the
induction of cell cycle arrest in some circumstances also appears
to inhibit SMC migration and extracellular matrix production (Biro,
et al., Proc. Nat. Acad. Sci., Vol. 90, pgs. 654-658 (1993)). In
the in vitro "tissue injury" model, both the proliferation and
migration of A10 cells that were transduced with the antisense
cyclin G1 vector were inhibited in the area of cell injury (FIG.
18E). Taken together with the observations of marked cytotoxicity,
cell cycle blockade, and multicellular syncytia formation, these
findings lend additional support for the concept that cyclin G1 may
represent a strategic locus for therapeutic intervention in the
management of proliferative disorders.
[0201] Once a potential therapeutic gene has been identified, the
challenge remains to deliver the gene transfer vector efficiently
to the appropriate physiologic site. In the case of balloon
angioplasty, both the denudation of the endothelial lining and the
mitogenic activation of neighboring SMC provide favorable
conditions for the delivery of retroviral vectors, as the
therapeutic genes delivered by retroviral vectors are expressed
preferentially in mitotically active cells. In the present study,
very high titer supernatants (10.sup.8 cfu/ml) were generated to
enhance the transduction efficiency of vascular SMC, and hence, the
efficacy of retroviral vectors in this experimental model of
restenosis. Indeed, the in vitro studies of retroviral
vector-mediated gene delivery in embryonic A10 SMC, may be
particularly relevant to the physiology of restenosis, for numerous
reports have indicated that embryonic and neointimal SMC exhibit
similar responses to mitogenic signals (Schwartz, et al., The
Vascular Smooth Muscle Cell, Schwartz, et al., eds. pg. 81-139,
Academic Press, Inc., New York (1995)). This study in the rat
carotid artery injury model of restenosis demonstrates the efficacy
of this approach: Sections of balloon-injured carotid arteries that
were treated with an infusion of highly concentrated (10.sup.8
cfu/ml) antisense cyclin G1 retroviral vector supernatant showed a
signifcant reduction in neointima formation. Taken together, these
data support the utility of retroviral vectors bearing cyclin G1,
alone or in combination with p53 or the now-classic Herpes Simplex
Virus thymidine kinase/GCV approach, in the development of novel
gene therapy strategies to combat vascular restenosis.
[0202] The disclosures of all patents, publications, (including
published patent applications), database accession numbers, and
depository accession numbers referenced in this specification are
specifically incorporated herein by reference in their entirety to
the same extent as if each such individual patent, publication,
database accession number, and depository accession number were
specifically and individually indicated to be incorporated by
reference.
[0203] It is to be understood, however, that the scope of the
present invention is not to be limited to the specific embodiments
described above. The invention may be practiced other than as
particularly described and still be within the scope of the
accompanying claims.
* * * * *