U.S. patent application number 11/141678 was filed with the patent office on 2006-01-05 for induction of apoptic or cytotoxic gene expression by adenoviral mediated gene codelivery.
This patent application is currently assigned to Board of Regents, The University of Texas System. Invention is credited to Elizabeth M. Bruckheimer, Bingliang Fang, Lin Ji, Timothy J. McDonnell, Jack A. Roth, Mona G. Sarkiss, Stephen G. Swisher.
Application Number | 20060002895 11/141678 |
Document ID | / |
Family ID | 34594159 |
Filed Date | 2006-01-05 |
United States Patent
Application |
20060002895 |
Kind Code |
A1 |
McDonnell; Timothy J. ; et
al. |
January 5, 2006 |
Induction of apoptic or cytotoxic gene expression by adenoviral
mediated gene codelivery
Abstract
The present invention generally relates to viral vectors and
their use as expression vectors for transforming human cells, both
in vitro and in vivo. More particularly, the present invention
relates to adenoviral vectors containing propapoptotic genes and
their use in cancer therapy.
Inventors: |
McDonnell; Timothy J.;
(Houston, TX) ; Swisher; Stephen G.; (Fresno,
TX) ; Fang; Bingliang; (Houston, TX) ;
Bruckheimer; Elizabeth M.; (Houston, TX) ; Sarkiss;
Mona G.; (Houston, TX) ; Ji; Lin; (Sugar Land,
TX) ; Roth; Jack A.; (Houston, TX) |
Correspondence
Address: |
FULBRIGHT & JAWORSKI L.L.P.
600 CONGRESS AVE.
SUITE 2400
AUSTIN
TX
78701
US
|
Assignee: |
Board of Regents, The University of
Texas System
|
Family ID: |
34594159 |
Appl. No.: |
11/141678 |
Filed: |
May 31, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09266465 |
Mar 11, 1999 |
6899870 |
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11141678 |
May 31, 2005 |
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60077541 |
Mar 11, 1998 |
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Current U.S.
Class: |
424/93.2 ;
514/109; 514/18.9; 514/19.3; 514/19.4; 514/19.5; 514/269; 514/27;
514/283; 514/34; 514/410; 514/44R; 514/8.9 |
Current CPC
Class: |
C12N 2800/40 20130101;
C12N 15/86 20130101; C12N 2830/002 20130101; C12N 2830/008
20130101; C07K 14/4747 20130101; C12N 2710/10343 20130101 |
Class at
Publication: |
424/093.2 ;
514/008; 514/027; 514/034; 514/044; 514/109; 514/410; 514/283;
514/269 |
International
Class: |
A61K 48/00 20060101
A61K048/00; A61K 38/16 20060101 A61K038/16; A61K 31/704 20060101
A61K031/704; A61K 31/7048 20060101 A61K031/7048 |
Claims
1-38. (canceled)
39. A method for treating a subject with cancer comprising the
steps of: (i) providing an adenoviral expression construct
comprising a first nucleic acid encoding a proapoptotic member of
the Bcl-2 gene family and a first promoter functional in eukaryotic
cells wherein said nucleic acid is under transcriptional control of
said first promoter; and (ii) contacting said expression construct
with cancer cells of said subject in a manner that allows the
uptake of said expression construct by said cells, wherein
expression of said proapoptotic gene results in the treatment of
said cancer.
40. The method of claim 39, further comprising contacting said
cancer cell with a further cancer therapeutic agent.
41. The method of claim 40, wherein said cancer therapeutic agent
is selected from the group consisting of tumor irradiation,
chemotherapeutic agent, a second nucleic acid encoding a cancer
therapeutic gene.
42. The method of claim 41, wherein said chemotherapeutic agent is
a DNA damaging agent selected from the group consisting of
verapamil, podophyllotoxin, carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,
melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin,
etoposide (VP16), tamoxifen, taxol, transplatinum, 5-fluorouracil,
vincristin, vinblastin and methotrexate.
43. The method of claim 41, wherein said radiation is selected from
the group consisting of X-ray radiation, UV-radiation,
.gamma.-radiation, or microwave radiation.
44. The method of claim 40, wherein said cancer therapeutic agent
comprises a second nucleic acid.
45. The method of claim 44, wherein said second nucleic acid is a
cDNA or genomic DNA.
46. The method of claim 44, wherein said second nucleic acid is a
second gene operatively linked to a promoter in said first
expression construct.
47. The method of claim 44, wherein said second nucleic acid is a
second gene operatively linked to a promoter in a second expression
construct.
48. The method of claim 47, wherein said second expression
construct is selected from the group consisting of an adenovirus,
an adeno-associated virus, a vaccinia virus and a herpes virus.
49. The method of claim 39, wherein said contacting is effected by
regional delivery of the expression construct.
50. The method of claim 39, wherein said contacting is effected by
local delivery of the expression construct.
51. The method of claim 39, wherein said contacting is effected by
direct injection of a tumor with said expression construct.
52. The method of claim 39, wherein said contacting comprise
delivering said expression construct endoscopically,
intratracheally, intralesionally, percutaneously, intravenously,
subcutaneously or intratumorally.
53. The method of claim 39, further comprising the step, prior to
said contacting, of tumor resection.
54. The method of claim 39, wherein said cancer is selected from
the group consisting of lung, breast, melanoma, colon, renal,
testicular, ovarian, lung, prostate, hepatic, germ cancer,
epithelial, prostate, head and neck, pancreatic cancer,
glioblastoma, astrocytoma, oligodendroglioma, ependymomas,
neurofibrosarcoma, meningia, liver, spleen, lymph node, small
intestine, blood cells, colon, stomach, thyroid, endometrium,
prostate, skin, esophagus, bone marrow and blood.
55. A method of inhibiting the growth of a cell comprising the
steps of: (i) providing an adenoviral expression construct
comprising a first nucleic acid encoding a proapoptotic member of
the Bcl-2 gene family and promoter functional in eukaryotic cells
wherein said nucleic acid is under transcriptional control of said
first promoter; and (ii) contacting said expression construct with
said cell in an amount effective to inhibit the growth of said
cell; wherein expression of said proapoptotic gene by said cell
results in slower growth of said cell relative to the growth of
said cell in the absence of said proapoptotic gene.
56. The method of claim 55, wherein said cell is a cancer cell.
57. The method of claim 56, wherein said inhibition of growth
comprises killing of said cancer cell.
58. The method of claim 56, wherein said cancer cell is selected
from the group consisting of lung, breast, melanoma, colon, renal,
testicular, ovarian, lung, prostate, hepatic, germ cancer,
epithelial, prostate, head and neck, pancreatic cancer,
glioblastoma, astrocytoma, oligodendroglioma, ependymomas,
neurofibrosarcoma, meningia, liver, spleen, lymph node, small
intestine, blood cells, colon, stomach, thyroid, endometrium,
prostate, skin, esophagus, bone marrow and blood.
59. The method of claim 56, wherein said cell is located within a
mammal.
60. The method of claim 59, wherein said inhibition of growth
comprises an inhibition of metastatic growth of said cancer
cell.
61. A method of inducing apoptosis in a cell comprising the steps
of: (i) providing an adenoviral expression construct comprising a
first nucleic acid encoding a proapoptotic member of the Bcl-2 gene
family and promoter functional in eukaryotic cells wherein said
nucleic acid is under transcriptional control of said first
promoter; and (ii) contacting said expression construct with said
cell in an amount effective to kill said cell; wherein expression
of said proapoptotic gene by said cell results in an increase in
the rate of death of said cell relative to the growth of said cell
in the absence of said proapoptotic gene.
62-67. (canceled)
68. A method for expressing a polypeptide in a target cell
comprising introducing into said target cell: (a) a first vector
comprising a coding region for said polypeptide under the control
of a first promoter inducible by an inducer polypeptide not
expressed in said target cell; and (b) a second vector comprising a
coding region for said inducer polypeptide under the control of a
second promoter active in said target cell.
69. The method of claim 68, wherein said first and said second
vectors are viral vectors.
70. The method of claim 68, wherein said first and said second
vectors are non-viral vectors.
71. The method of claim 68, wherein said first vector is a viral
vector and said second vector is a non-viral vector, or said first
vector is a non-viral vector and said second vector is a viral
vector.
72. The method of claim 68, wherein said second promoter is a
constitutive promoter, an inducible promoter or a tissue specific
promoter.
73. The method of claim 69, wherein said viral vectors are the same
or different and selected from the group consisting of an
adenoviral vector, a herpesviral vector, a retroviral vector, an
adeno-associated viral vector, a vaccinia viral vector or a polyoma
viral vector.
74. The method of claim 68, wherein said first vector and said
second vector are introduced into said target cell at a ratio of
1:1, respectively.
75. The method of claim 68, wherein said first vector and said
second vector are introduced into said target cell at a ratio of
2:1, respectively.
76. The method of claim 68, wherein said first vector is introduced
at 900 MOI and said second vector at 1500 MOI into said target
cell.
77. The method of claim 68, wherein the first promoter is GAL4 and
the inducer polypeptide is GAL4/VP16, respectively.
78. The method of claim 68, wherein the target cell is a
hyperproliferative cell.
79. The method of claim 78, wherein said cell is a pre-malignant
cell.
80. The method of claim 78, wherein said cell is a malignant
cell.
81. The method of claim 80, where said cell is a lung cancer cell,
a prostate cancer cell, a brain cancer cell, a liver cancer cell, a
breast cancer cell, a skin cancer cell, an ovarian cancer cell, a
testicular cancer cell, a stomach cancer cell, a pancreatic cancer
cell, a colon cancer cell, an esophageal cancer cell, head and neck
cancer cell.
82. The method of claim 68, wherein said first and second vectors
are introduced into said target cell at the same time.
83. The method of claim 68, wherein said first vector is introduced
into said target cell prior to said second vector.
84. The method of claim 83, wherein said second vector is
introduced into said target cell within 24 hours of said first
vector.
85. The method of claim 83, wherein said second vector is
introduced into said target cell within 12 hours of said first
vector.
86. The method of claim 83, wherein said second vector is
introduced into said target cell within 6 hours of said first
vector.
87. The method of claim 83, wherein said second vector is
introduced into said target cell within 3 hours of said first
vector.
88. The method of claim 83, wherein said second vector is
introduced into said target cell within 1 hour of said first
vector.
89. The method of claim 68, wherein said second vector is
introduced into said target cell prior to said first vector.
90. The method of claim 89, wherein said first vector is introduced
into said target cell within 24 hours of said second vector.
91. The method of claim 89, wherein said first vector is introduced
into said target cell within 12 hours of said second vector.
92. The method of claim 89, wherein said first vector is introduced
into said target cell within 6 hours of said second vector.
93. The method of claim 89, wherein said first vector is introduced
into said target cell within 3 hours of said second vector.
94. The method of claim 89, wherein said first vector is introduced
into said target cell within 1 hour of said second vector.
95. The method of claim 78, wherein said target cell is further
contacted with a DNA damaging agent.
96. The method of claim 95, wherein said DNA damaging agent is
radiotherapy.
97. The method of claim 95, wherein said DNA damaging agent is
chemotherapy.
98. The method of claim 72, wherein said promoter is an inducible
promoter and the inducing factor is present in said target
cell.
99. The method of claim 72, wherein said promoter is an inducible
promoter and the inducing factor is added to said target cell.
100. The method of claim 68, wherein one or both of said vectors
further comprise a polyadenylation signal.
101. The method of claim 68, wherein said polypeptide expressed in
said target cell is cytotoxic.
102. The method of claim 101, wherein said cytotoxic polypeptide is
selected from the group consisting of an inducer of apoptosis, a
cytokine, a toxin, a single chain antibody, a protease and a
antigen.
103. The method of claim 102, wherein said inducer of apoptosis is
selected from the group consisting of Bax, Bak, Bik, Bim, Bid, Bad
and Harakiri.
104. The method of claim 103, wherein said inducer of apoptosis is
Bax.
105. The method of claim 102, wherein said toxin is selected form
the group consisting of ricin A-chain, diptheria toxin A-chain,
pertussis toxin A subunit, E. coli enterotoxin A subunit, cholera
toxin A subunit and pseudomonas toxin c-terminal.
106. The method of claim 105, wherein said toxin is diptheria toxin
A-chain.
107. The method of claim 102, wherein said cytokine is selected
form the group consisting of oncostatin M, TGF-.beta., TNF-.alpha.
and TNF-.beta..
108-110. (canceled)
111. A method of treating a disease comprising introducing into
cells of a subject having said disease: (a) a first vector
comprising a coding region for said therapeutic polypeptide under
the control of a first promoter inducible by an inducer polypeptide
not expressed in said target cell; and (b) a second vector
comprising a coding region for said inducer polypeptide under the
control of a second promoter active in said target cell.
112. The method of claim 111, wherein said disease is selected from
the group consisting of lung cancer, prostate cancer, brain cancer,
liver cancer, breast cancer, skin cancer, ovarian cancer,
testicular cancer, stomach cancer, pancreatic cancer, colon cancer,
esophageal cancer and head and neck cancer.
113. The method of claim 111, wherein said therapeutic polypeptide
is selected from the group consisting of Bax, Bak, Bik, Bim, Bid,
Bad, Harakiri, ricin A-chain, diptheria toxin A-chain, pertussis
toxin A subunit, E. coli enterotoxin A subunit, cholera toxin A
subunit, pseudomonas toxin c-terminal, IL-1, IL-2, IL-3, IL-4,
IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF and G-CSF.
Description
BACKGROUND OF THE INVENTION
[0001] This application claims priority to and specifically
incorporates by reference, the content of U.S. Provisional
Application Ser. No. 60/077,541 filed Mar. 11, 1998. The entire
text of each of the above-referenced disclosures is specifically
incorporated by reference herein without disclaimer. The government
owns rights in the present invention pursuant to grant number
CA70907 from the National Institutes of Health.
[0002] 1. Field of the Invention
[0003] The present invention relates generally to viral vectors and
their use as expression vectors for transforming human cells, both
in vitro and in vivo. More specifically, the invention relates to
adenoviral expression constructs comprising a proapoptotic member
of the Bcl-2 gene family.
[0004] 2. Description of Related Art
[0005] Adenoviral vectors have become one of the leading vectors
for gene transfer, particularly in gene therapy contexts. These
vectors have been studied rigorously in both in vitro and in vivo
contexts because of the ability to generate high titer stocks,
their high transduction efficiency and their ability to infect a
variety of tissue types in different species. In addition, the
availability of cell lines to complement defects in adenoviral
replication functions provides for the use of replication defective
mutants carrying, in the place of selected structural genes,
recombinant inserts of interest.
[0006] Several studies have demonstrated the ability of
adenovirus-mediated wild-type p53 replacement gene therapy to
induce a G.sub.1 cell cycle arrest and/or apoptosis in malignant
cells carrying p53 gene mutations. Though the mechanism of G.sub.1
arrest via p21 and the cyclin-dependent kinase pathway has been
widely studied, little is known of the mechanisms by which
wild-type p53 induces apoptosis. It appears that p53 induces
apoptosis, at least in part, by up-regulating proapoptotic members
of the Bcl-2 family of proteins.
[0007] The Bcl-2 family of proteins and ICE-like proteases have
been demonstrated to be important regulators and effectors of
apoptosis in other systems. Apoptosis, or programmed cell death, is
an essential occurring process for normal embryonic development,
maintaining homeostasis in adult tissues, and suppressing
carcinogenesis (Kerr et al., 1972). The Bcl-2 protein, discovered
in association with follicular lymphoma, plays a prominent role in
controlling apoptosis and enhancing cell survival in response to
diverse apoptotic stimuli (Bakhshi et al., 1985; Cleary and Sklar,
1985; Cleary et al., 1986; Tsujimoto et al., 1985; Tsujimoto and
Croce, 1986). The evolutionarily conserved Bcl-2 protein now is
recognized to be a member of a family of related proteins which can
be categorized as death agonists or death antagonists.
[0008] Subsequent to its discovery, it was shown that Bcl-2 acts to
suppress cell death triggered by a variety of stimuli which will be
discussed in detail. Also, it now is apparent that there is a
family of Bcl-2 cell death regulatory proteins which share in
common structural and sequence homologies. These different family
members have been shown to either possess similar functions to
Bcl-2 or counteract Bcl-2 function and promote cell death.
[0009] One such family member having Bcl-2 counteracting function
is Bax. Bax, Bcl-2 associated X protein, is a death agonist member
of the Bcl-2 family of proteins (Oltvai et al., 1993). It has been
suggested that Bax may function as a primary response gene in a p53
regulated apoptotic pathway (Miyashita et al., 1994). Indeed, it
has been shown that there is a p53 consensus binding region in the
promoter region of the proapoptotic Bax gene (1995). Bax mRNA and
protein expression are increased following induction of p53.
However, the observed induction of p53-dependent apoptosis in Bax
knock out mice clearly indicates that other pathways or proteins
are involved. Bak, a Bcl-2 homologue, is expressed in a variety of
tissues and has been demonstrated to induce program cell death
independent of Bax expression (Krajewski et al., 1996; Chittenden
et al., 1995). The accumulation of Bak protein in cells infected
with Adp53, may be an additional mechanism by which p53 can induce
programmed cell death.
[0010] However, a recent report has demonstrated an increase in
Bcl-x.sub.L expression following wild-type p53 expression in the
human colorectal cancer cell line HT29 (Merchant et al., 1996). The
authors hypothesize that this increase expression may lead to an
inhibition of program cell death pathways and accounted for lack of
p53-induced apoptosis observed in these cells. Another potential
problem with p53 therapy is that the amount of viral material
administered provides risks of host cell toxicity and/or immune
response. Thus, any method which would increase the effect of p53
at low doses, or circumvent the need for high viral doses, would be
advantageous.
[0011] Given that p53 gene therapy is a powerful tool in the fight
against cancer, therapeutic compositions that may augment or
complement p53 will serve to improve the currently available cancer
therapy regimens. Indeed, compositions that provide the apoptotic
effect of p53 without the need for p53 itself would be additionally
useful.
SUMMARY OF THE INVENTION
[0012] The present invention generally is related the use of viral
vectors containing propapoptotic genes and their use in cancer
therapy, in order to induce an apoptotic effect in cancer cells to
either augment, complement or bypass the need for p53 based
therapy.
[0013] In order to achieve the objectives of the present invention,
a particular embodiment provides an adenoviral expression construct
comprising a nucleic acid encoding a proapoptotic member of the
Bcl-2 gene family and a first promoter functional in eukaryotic
cells wherein the nucleic acid is under transcriptional control of
the first promoter. In particularly preferred embodiments, the
proapoptotic Bcl-2 gene is a Bax, Bak, Bim, Bik, Bid or Bad gene.
In certain embodiments, it is contemplated that the adenoviral
expression construct may further comprise a second nucleic acid
encoding a second gene. In particular instances the second nucleic
acid is under the control of the first promoter.
[0014] In particularly preferred embodiments, the proapoptotic
Bcl-2 gene and the second nucleic acid are separated by an IRES. In
alternative embodiments, the second nucleic acid is under the
control of a second promoter operative in eukaryotic cells. It is
contemplated that the promoter employed herein may be any promoter
used in the production of expression constructs. In particularly
preferred embodiments the promoter may be selected from the group
consisting of CMV IE, SV40 IE, RSV, .beta.-actin, tetracycline
regulatable and ecdysone regulatable.
[0015] In certain defined aspects, the second gene may encode a
protein selected from the group consisting of a tumor suppressor, a
cytokine, a receptor, inducer of apoptosis, and differentiating
agents. By "differentiating agents," the present application refers
to the function of bcl-2 family members in the induction of
differentiation in cells. Thus, the cells are not induced to die
via apoptosis, but terminally differentiate and stop growing, which
is equally effective as a cancer treatment. In particularly
preferred embodiments, the tumor suppressor may be selected from
the group consisting of p53, p16, p21, MMAC1, p73, zac1, C-CAM,
BRCAI and Rb. In certain embodiments, the inducer of apoptosis is
selected from the group consisting of Harakiri, Ad E1B and an
ICE-CED3 protease. In those embodiments employing a cytokine, the
cytokine may be selected from the group consisting of IL-2, IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12,
IL-13, IL-14, IL-15, TNF, GMCSF, .beta.-interferon and
.gamma.-interferon. In those embodiments where the second gene is a
receptor, the receptor may be selected from the group consisting of
CFTR, EGFR, VEGFR, IL-2 receptor and the estrogen receptor. It is
contemplated that the second nucleic acid may be an antiapoptotic
member of the Bcl-2 gene family or an oncogene, the second nucleic
acid being positioned in an antisense orientation with respect to
the promoter. In more preferred embodiments, the antiapoptotic
member of the Bcl-2 gene family is Bcl-2 or Bcl-x.sub.L. In
embodiments in which the second gene is an oncogene, the oncogene
may be selected from the group consisting of ras, myc, neu, raf
erb, src, fins, jun, trk, ret, gsp, hst, and abl.
[0016] In defined embodiments, the expression construct is a
replication-deficient adenovirus. In preferred aspects, the
adenovirus lacks at least a portion of the E1 region. In other
embodiments, the adenovirus further lacks the E3 coding region. In
preferred embodiments, the expression construct further comprises a
polyadenylation signal. In particular embodiments, the nucleic acid
may be a cDNA, or genomic DNA.
[0017] In particularly preferred embodiments, the proapoptotic
member of the Bcl-2 family is Bax. In other preferred embodiments,
the proapoptotic member of the Bcl-2 family is Bak. In more
preferred embodiments, the Bax gene expresses a truncated Bax
protein. In more preferred embodiments, the truncated Bax protein
comprises an intact death domain. In other preferred embodiments,
the truncated Bax protein comprises SEQ ID NO:2. In other preferred
embodiments, the truncated Bax protein comprises a BH3 region.
[0018] Also contemplated by the present invention is a
pharmaceutical composition comprising a first adenoviral expression
construct comprising a promoter functional in eukaryotic cells and
a first nucleic acid encoding a proapoptotic member of the Bcl-2
gene family, wherein the first nucleic acid is under
transcriptional control of the promoter and a pharmaceutically
acceptable buffer, solvent or diluent.
[0019] In particularly preferred embodiments, the proapoptotic
Bcl-2 family gene is a Bax, Bak, Bik, Bid, or Bad gene. In other
preferred embodiments, the promoter may be selected from the group
consisting of CMV IE, SV40 IE, RSV, .beta.-actin, tetracycline
regulatable and ecdysone regulatable. In other embodiments, the
pharmaceutical composition may further comprise a second expression
construct encoding a second nucleic acid encoding a second gene
operatively linked to a second promoter. In certain aspects, the
expression construct encoding the proapoptotic gene further
comprises a second nucleic acid encoding a second gene. The second
nucleic acid may be under the control of the first promoter. In
alternative embodiments, the second nucleic acid is under the
control of a second promoter operative in eukaryotic cells. The
second gene may encode a protein selected from the group consisting
of a tumor suppressor, a cytokine, a receptor, inducer of
apoptosis, and differentiating agents. In particularly preferred
embodiments, the second nucleic acid is an antiapoptotic member of
the Bcl-2 gene family or an oncogene, the second nucleic acid being
positioned in an antisense orientation with respect to the
promoter.
[0020] In preferred embodiments, the present invention further
contemplates a method for treating a subject with cancer comprising
the steps of providing an adenoviral expression construct
comprising a nucleic acid encoding a proapoptotic member of the
Bcl-2 gene family and a first promoter functional in eukaryotic
cells wherein the nucleic acid is under transcriptional control of
the first promoter; and contacting the expression construct with
cancer cells of the subject in a manner that allows the uptake of
the expression construct by the cells, wherein expression of the
proapoptotic gene results in the treatment of the cancer. By
"treatment," the present invention refers to any event that
decreases the growth, kills or otherwise abrogates the presence of
cancer cells in a subject. Such a treatment may also occur by
inhibition of the metastatic potential or inhibition of
tumorigenicity of the cell so as to achieve a therapeutic
outcome.
[0021] In other preferred aspects, the method further comprises
contacting the cancer cell with a further cancer therapeutic agent.
In particularly preferred embodiments, the cancer therapeutic agent
may be selected from the group consisting of tumor irradiation,
chemotherapeutic agent, a second nucleic acid encoding a cancer
therapeutic gene. In defined embodiments, the chemotherapeutic
agent is a DNA damaging agent selected from the group consisting of
verapamil, podophyllotoxin, carboplatin, procarbazine,
mechlorethamine, cyclophosphamide, camptothecin, ifosfamide,
melphalan, chlorambucil, bisulfan, nitrosurea, dactinomycin,
daunorubicin, doxorubicin, bleomycin, plicomycin, mitomycin,
etoposide (VP16), tamoxifen, taxol, transplatinum, 5fluorouracil,
vincristin, vinblastin and methotrexate. In alternative
embodiments, the radiation is selected from the group consisting of
X-ray radiation, UV-radiation, .gamma.-radiation, or microwave
radiation. In other defined embodiments, the cancer therapeutic
agent comprises a second nucleic acid. The second nucleic acid may
be a cDNA or genomic DNA.
[0022] In particular embodiments of the present invention, the
second expression construct is selected from the group consisting
of an adenovirus, an adeno-associated virus, a vaccinia virus and a
herpesvirus. In other embodiments, the contacting is effected by
regional delivery of the expression construct. In alternative
embodiments, the contacting is effected by local delivery of the
expression construct. In still further embodiments, the contacting
may be effected by direct injection of a tumor with the expression
construct. In particularly preferred embodiments, the contacting
comprises delivering the expression construct endoscopically,
intratracheally, intralesionally, percutaneously, intravenously,
subcutaneously or intratumorally to said subject. In certain
embodiments, the method may further comprise the step of tumor
resection. The tumor resection may occur prior to or after the
contacting. The tumor resection may be performed one, two, three or
more times. In particularly preferred embodiments, the cancer being
treated may be selected from the group consisting of lung, breast,
melanoma, colon, renal, testicular, ovarian, lung, prostate,
hepatic, germ cancer, epithelial, prostate, head and neck,
pancreatic cancer, glioblastoma, astrocytoma, oligodendroglioma,
ependymomas, neurofibrosarcoma, meningia, liver, spleen, lymph
node, small intestine, blood cells, colon, stomach, thyroid,
endometrium, prostate, skin, esophagus, bone marrow and blood.
[0023] The present invention also provides a method of inhibiting
the growth of a cell comprising the steps of providing an
adenoviral expression construct comprising a nucleic acid encoding
a proapoptotic member of the Bcl-2 gene family and promoter
functional in eukaryotic cells wherein the nucleic acid is under
transcriptional control of the first promoter; and contacting the
expression construct with the cell in an amount effective to
inhibit the growth of the cell wherein expression of the
proapoptotic gene by the cell results in a decrease in the growth
of the cell relative to the growth of the cell in the absence of
the proapoptotic gene.
[0024] In preferred embodiments, the cell is a cancer cell. In
other preferred embodiments, the inhibition of growth comprises
killing of the cancer cell. In other embodiments, the inhibition of
growth comprises an inhibition of metastatic growth of the cancer
cell. In defined embodiments, the cancer cell may be selected from
the group consisting of lung, breast, melanoma, colon, renal,
testicular, ovarian, lung, prostate, hepatic, germ cancer,
epithelial, prostate, head and neck, pancreatic cancer,
glioblastoma, astrocytoma, oligodendroglioma, ependymomas,
neurofibrosarcoma, meningia, liver, spleen, lymph node, small
intestine, blood cells, colon, stomach, thyroid, endometrium,
prostate, skin, esophagus, bone marrow and blood. In other
embodiments, the cell is located within a mammal.
[0025] The present invention also provides a method of inducing
apoptosis in a cell comprising the steps of providing an adenoviral
expression construct comprising a nucleic acid encoding a
proapoptotic member of the Bcl-2 gene family and promoter
functional in eukaryotic cells wherein the nucleic acid is under
transcriptional control of the first promoter; and contacting the
expression construct with the cell in an amount effective to kill
the cell; wherein expression of the proapoptotic gene by the
results in an increase in the rate of death of said cell relative
to the growth of said cell in the absence of said proapoptotic
gene. In particularly preferred embodiments, the proapoptotic
member of the Bcl-2 gene family is a Bax, Bak, Bim, Bik, Bid or Bad
gene. In more preferred embodiments, the proapoptotic member of the
Bcl-2 gene family is a truncated Bax gene. In other preferred
embodiments, the proapoptotic member of the Bcl-2 gene family is a
truncated Bak gene.
[0026] Also contemplated by the present invention is a nucleic acid
encoding a truncated Bax gene. In particular embodiments, the Bax
gene comprises a nucleic acid sequence of SEQ ID NO:1. In other
embodiments, the Bax gene encodes a protein having an amino acid
sequence of SEQ ID NO:2. In particularly preferred aspects the
truncated Bax gene encodes a protein comprising a BH3 region. In
alternative preferred embodiments, the truncated Bax gene encodes a
protein comprising an intact death domain.
[0027] In yet another embodiment, the present invention further
contemplates an adenoviral expression construct comprising a
nucleic acid encoding a truncated Bax gene and a first promoter
functional in eukaryotic cells wherein the nucleic acid is under
transcriptional control of the first promoter. The adenoviral
expression construct may further comprise a second nucleic acid
encoding a second gene. The second gene may be under the control of
the first promoter. In alternative embodiments, the second gene may
be under the transcriptional control of a second promoter. In a
further alternative, the truncated Bax gene and the second nucleic
acid may be separated by an IRES.
[0028] In yet another embodiment, the present invention further
contemplates an adenoviral expression construct comprising a
nucleic acid encoding a bak gene and a first promoter functional in
eukaryotic cells wherein the nucleic acid is under transcriptional
control of the first promoter. The adenoviral expression construct
may further comprise a second nucleic acid encoding a second gene.
The second gene may be under the control of the first promoter. In
alternative embodiments, the second gene may be under the
transcriptional control of a second promoter. In a further
alternative, the truncated Bak gene and the second nucleic acid may
be separated by an IRES.
[0029] In other embodiments there is provided, a method for
expressing a polypeptide in a target cell comprising introducing
into the target cell a first vector comprising a coding region for
a polypeptide under the control of a first promoter inducible by an
inducer polypeptide not expressed in the target cell and a second
vector comprising a coding region for the inducer polypeptide under
the control of a second promoter active in the target cell. In
certain embodiments, the first and second vectors are viral
vectors. In other embodiments, the first and said second vectors
are non-viral vectors. In yet other embodiments, the first vector
is a viral vector and the second vector is a non-viral vector, or
the first vector is a non-viral vector and the second vector is a
viral vector. It is contemplated that the second promoter is a
constitutive promoter, an inducible promoter or a tissue specific
promoter.
[0030] In certain embodiments, the viral vectors are the same or
different and may be selected from the group consisting of an
adenoviral vector, a herpesviral vector, a retroviral vector, an
adeno-associated viral vector, a vaccinia viral vector or a polyoma
viral vector.
[0031] It is contemplated in one embodiment that the first vector
and the second vector are introduced into the target cell at a
ratio of 1:1, respectively. In other embodiments, the first vector
and the second vector are introduced into the target cell at a
ratio of 2:1, respectively. In still other embodiments, the first
vector is introduced at 900 MOI and the second vector at 1500 MOI
into the target cell.
[0032] In another embodiment, the first promoter is GAL4 and the
inducer polypeptide is GAL4/VP16, respectively. It is contemplated
in other embodiments, that the first promoter can be selected from
the group consisting of the ecdysone-responsive promoter, and
Tet-On.TM. and the inducer ecdysone or muristeron A and
doxycycline, respectively.
[0033] In particular embodiments, the target cell is a
hyperproliferative cell, a pre-malignant cell or a malignant cell.
In embodiments where the target cell is malignant, it is
contemplated that the malignant cell may be selected form the group
consisting of a lung cancer cell, a prostate cancer cell, a brain
cancer cell, a liver cancer cell, a breast cancer cell, a skin
cancer cell, an ovarian cancer cell, a testicular cancer cell, a
stomach cancer cell, a pancreatic cancer cell, a colon cancer cell,
an esophageal cancer cell, head and neck cancer cell.
[0034] In certain embodiments, the first and second vectors are
introduced into the target cell at the same time. In one
embodiment, the first vector is introduced into the target cell
prior to the second vector. In other embodiments, the second vector
is introduced into the target cell within 24 hours, within 12
hours, within 6 hours, within 3 hours or within 1 hour of the first
vector. In another embodiment, the second vector is introduced into
the target cell prior to the first vector. It is contemplated, that
the first vector is introduced into the target cell within 24
hours, within 12 hours, within 6 hours, within 3 hours or within 1
hour of the second vector.
[0035] In other embodiments, the target cell is further contacted
with a DNA damaging agent. It is contemplated that the DNA damaging
agent may be radiotherapy or chemotherapy.
[0036] In one embodiment, the second promoter is an inducible
promoter and the inducing factor is present in the target cell. In
another embodiment, the second promoter is an inducible promoter
and the inducing factor is added to the target cell. In particular
embodiments, it is contemplated that one or both of the vectors
further comprise a polyadenylation signal.
[0037] In certain embodiments, the polypeptide expressed in the
target cell is cytotoxic. It is contemplated that the cytotoxic
polypeptide may selected from the group consisting of an inducer of
apoptosis, a cytokine, a toxin, a single chain antibody, a protease
and an antigen. It is further contemplated that the inducer of
apoptosis may be selected from the group consisting of Bax, Bak,
Bik, Bim, Bid, Bad and Harakiri. In preferred embodiments, the
inducer of apoptosis is Bax. In other embodiments, it is
contemplated that the cytokine may be selected form the group
consisting of oncostatin M, TGF-.beta., TNF-.alpha. and TNF-.beta..
In yet other embodiments, the toxin may be selected form the group
consisting of ricin A-chain, diphtheria toxin A-chain, pertussis
toxin A subunit, E. coli enterotoxin A subunit, cholera toxin A
subunit and pseudomonas toxin c-terminal. In particularly preferred
embodiments, the toxin is diphtheria toxin A-chain.
[0038] In one embodiment, a kit comprising a first vector
comprising a first promoter, inducible by an inducer polypeptide, a
multipurpose cloning site 3' to the first promoter in a suitable
container and a second vector comprising a coding region for the
inducer polypeptide under the control of a second promoter active
in the target cell in suitable container. In another embodiment,
the first vector further comprises a region coding for a
polypeptide under control of the first promoter. In yet another
embodiment, the second promoter is an inducible promoter and the
kit further comprises an agent that induces the second promoter in
a suitable container means.
[0039] Also contemplated is a method of treating a disease
comprising introducing into cells of a subject having a disease a
first vector comprising a coding region for the therapeutic
polypeptide under the control of a first promoter inducible by an
inducer polypeptide not expressed in the target cell and a second
vector comprising a coding region for the inducer polypeptide under
the control of a second promoter active in the target cell. In one
embodiment, the disease may be selected from the group consisting
of lung cancer, prostate cancer, brain cancer, liver cancer, breast
cancer, skin cancer, ovarian cancer, testicular cancer, stomach
cancer, pancreatic cancer, colon cancer, esophageal cancer and head
and neck cancer. In another embodiment, the therapeutic polypeptide
may be selected from the group consisting of Bax, Bak, Bik, Bim,
Bid, Bad, Harakiri, ricin A-chain, diphtheria toxin A-chain,
pertussis toxin A subunit, E. coli enterotoxin A subunit, cholera
toxin A subunit, pseudomonas toxin c-terminal, IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF
oncostatin M, TGF-.beta., TNF-.alpha., TNF-.beta. and G-CSF.
[0040] Other objects, features and advantages of the present
invention will become apparent from the following detailed
description. It should be understood, however, that the detailed
description and the specific examples, while indicating preferred
embodiments of the invention, are given by way of illustration
only, since various changes and modifications within the spirit and
scope of the invention will become apparent to those skilled in the
art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0041] The following drawings form part of the present
specification and are included to further demonstrate certain
aspects of the present invention. The invention may be better
understood by reference to one or more of these drawings in
combination with the detailed description of specific embodiments
presented herein.
[0042] FIG. 1 Schematic depiction of the protein structures of the
Bcl-2 family members. BH1, BH2, BH3, and BH4 are the conserved
homology domains. TM indicates the transmembrane domain, NH2
indicated the amino terminal domain, and the PEST domain represents
the region which is correlated to an early response gene product
and is associated with rapid protein turnover. GRS is grouped with
the anti-apoptotic family members, however, its role in apoptosis
is not currently known.
[0043] FIG. 2A and FIG. 2B. Western blot analysis of CPP32 and Parp
expression. Control non-infected cells and cells following
infection with Ad5/CMB/p53 were collected and subjected to western
blot analysis using monoclonal antibody against CPP32 (FIG. 2B) or
polyclonal antibody against parp (FIG. 2A). Fifty micrograms of
protein was analyzed by SDS-PAGE and visualized by western blotting
using the ECL chemiluminescence system. Image shown is an optical
scan of a representative film exposure from one of three studies.
The arrows indicate expected location of CPP32 and Parp cleavage
product.
[0044] FIG. 3A and FIG. 3B. Effect of Ad5/CMV/p53 gene transfer on
cell cycle regulation and induction of apoptosis. Cell cycle
analysis and TUNEL were performed on cells which were treated with
control vector DL312 or PBS or infected with Ad5/CMV/p53 and
collected at 6 h intervals following infection. Cells were
tripsinized at the reported time point fixed and analyzed for DNA
content by perpidium iodine staining and analyzed for TUNEL
labeling by fluorescence using flow cytometry. Infection with
Ad5/CMV/p53 resulted in a increase in G.sub.1 population of cells
and an increase in the 2N population of cells (FIG. 3A).
Additionally infection with Ad5/CMV/p53 resulted in an increased
population of TUNEL-labeled cells consistent with increases in
apoptotic death (FIG. 3B).
[0045] FIG. 4A, FIG. 4B, and FIG. 4C. FACS analysis to measure
apoptosis in MCF-7 cells (FIG. 4A), SKBr3 cells (FIG. 4B) and
MDA-MD-468 cells (FIG. 4C). Cells were either uninfected, infected
with an empty adenoviral vector control, an adenovirus vector
containing the truncated bax gene.
[0046] FIG. 5. Plasmid map of the Supercos vector.
[0047] FIG. 6. Plasmid map of pCOS/LJ07.
[0048] FIG. 7. Plasmid map of pCOS/Ad/LJ17.
[0049] FIG. 8. Plasmid map of pCMV/Bak.
[0050] FIG. 9. Plasmid map of pCOS/Ad-Bak.
[0051] FIG. 10. Schematic of cloning adenovirus genome into
cosmid.
[0052] FIG. 11. Schematic of construction of recombinant adenovirus
in E. coli.
[0053] FIG. 12. Schematic of production of recombinant
adenovirus.
[0054] FIG. 13. Schematic of adenovirus-mediated gene co-transfer.
The expression cassettes for the transgene (bax) and the
transactivator (GAL4/VP16) are cloned into separate vectors. The
expression of the transgene is then induced after co-infecting a
target cell with the two vectors.
[0055] FIG. 14. Apoptosis profiles after induction of bax gene
expression. Nuclear fragmentation detected by staining with Hoechst
33432. The treatment for each sample is indicated above each
panel.
[0056] FIG. 15. In vivo induction of bax gene expression. Nuclear
fragmentation detected by hematoxylin and eosin staining of liver
sections from mice treated with (a) PBS, (b) Ad/GT-Bax+Ad/CMV-GFP,
(c) Ad/GT-Bax+Ad/PGK-GV16, and (d) Ad/GT-LacZ+Ad/CMV-GV16.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0057] Cancer accounts the death of over half a million people each
year in the United States alone. The causes for cancer are
multifactorial, however, it is known that aberrations in controlled
cell death result in uncontrolled cell proliferation and hence
contribute to many cancer.
[0058] The p53 gene is well-recognized as possessing tumor
suppressor capabilities and mutations in wild-type p53 are
correlated to a variety of cancers. However, the interaction of p53
with other cellular factors is not well characterized, in fact,
many of these factors remain undefined. It is not surprising that,
in light of the lack of significant information on p53 function,
there is an incomplete understanding of the pathways through which
p53 regulates tumor development. Nevertheless, p53-based gene
therapy has been remarkably effective in inducing cell cycle arrest
and/or apoptosis in malignant cells carrying p53 gene
mutations.
[0059] There now is a great deal of evidence that the apoptotic
effect of p53 is mediated through the members of the proapoptotic
Bcl-2 family. It has been shown that the p53 dependent expression
of Bax is induced in slow-growing apoptotic tumors. Further, tumor
growth appears accelerated, and apoptosis is decreased, in
Bax-deficient mice. This suggests that Bax is required for a full
p53-mediated response (Yin et al., 1997). The present invention,
for the first time, provides evidence that proapoptotic Bcl-2 genes
in adenoviral vectors can be used to decrease, diminish, inhibit or
otherwise abrogate the growth of cancer cells.
[0060] The present invention employs, in one embodiment, an
adenoviral expression construct comprising a gene that encodes a
truncated Bax protein. As discussed herein below, the Bcl-2 family
of proteins consists of death antagonists and death agonists that
regulate apoptosis and compete through dimerization. All members of
the Bcl-2 family of proteins contain one or more Bcl-2 homology
domains (BH). It appears that there are at least 4 BH domains,
referred to as BH1, BH2, BH3 and BH4. The competition between the
proapoptotic and antiapoptotic members is mediated at least in
part, by competitive dimerization between selective pairs of
antagonists and agonist molecules. Mutagenesis studies revealed
that intact BH1 and BH2 domains of antagonists are required for
repression of cell death. Conversely, the BH3 domain of Bax is the
domain responsible for conferring the death agonist activity to Bcl
proteins. Thus, in preferred embodiments, the present invention
uses a truncated Bax protein having an intact "death domain." Of
course other Bcl proteins such as Bak, Bid, Bik, that comprise the
death domain will also be useful in the adenoviral constructs of
the present invention.
[0061] In the present invention, the overexpression of the
proapoptotic mediator Bax has been demonstrated in cancer cell
lines transduced with an adenoviral Bax construct. Morphologically,
apoptosis was seen within 4 days post-transduction. Thus, the
present invention demonstrates that Bax induces apoptosis in cancer
cell lines and provides evidence that adenoviral constructs
containing Bax and/or other proapoptotic Bcl-2 gene family members
will be useful components of a cancer therapy regimen. Methods of
producing and using such compositions are discussed in further
detail below.
[0062] In another embodiment, an adenoviral-mediated gene
co-transfer system is described, that permits the regulated
expression of cytotoxic gene products for use in treating
hyperproliferative disease. In one embodiment, a first vector
carrying a gene encoding a toxic product is under the control of a
promoter, not active in the target source. A second vector,
comprises a transactivator gene, wherein the transactivator protein
product activates transcription from the promoter in the first
expression vector. The choice of promoter on the second expression
vector can be selected for use on an as needed basis (e.g., tissue
specificity). It is contemplated further, that the co-transfer
system can be used with any expression vector or combination
thereof (e.g., viral, plasmid, plasmid shuttle vector, cosmid),
introduced via any method of gene transfer desired (i.e., viral or
non-viral) and used for both in vivo and in vitro.
A. The Bcl-2 Gene Family and Apoptosis
[0063] Apoptosis is an essential process required for normal
embryonic development, maintenance of adult tissue homeostasis and
the suppression of carcinogenesis. Apoptosis has been defined as a
type of cell death which complements mitosis in the regulation of
cell populations (Kerr et al., 1972). Apoptosis can occur as a
result of both physiologic and pathologic conditions and is
believed to be, in many developmental contexts, a programmed event.
The sequence of events begins with nuclear and cytoplasmic
condensation and ends with the release and phagocytosis of
apoptotic bodies (Kerr et al., 1972).
[0064] A major advance in understanding the regulation of apoptosis
came with the discovery of the Bcl-2 proto-oncogene from the
t(14;18) chromosomal translocation breakpoint in follicular
lymphoma (Bakhshi et al., 1985; Cleary and Sklar, 1985; Cleary et
al., 1986; Tsujimoto et al., 1985; Tsujimoto and Croce, 1986).
Bcl-2 acts to suppress cell death triggered by a variety of stimuli
and, it is now apparent that there is a family of Bcl-2 cell death
regulatory proteins which share in common structural and sequence
homologies. These different family members have been shown to
either possess similar functions to Bcl-2 or counteract Bcl-2
function and promote cell death. These cell death regulators are
discussed in further detail herein below.
[0065] In mammalian development, Bcl-2 and Bcl-2 family members
have been shown to play a role in morphogenesis and normal
development. During murine fetal development Bcl-2 is expressed in
tissues derived from all three germ layers; however, as the fetus
matures, Bcl-2 expression becomes restricted (Novack and Korsmeyer,
1994). Similar observations were seen in human fetal tissues in
that Bcl-2 was expressed in a wide variety of tissue types and
expression became restricted as the fetus matured (LeBrun et al.,
1993; Chandler et al., 1994). Bcl-2 was detected in the human fetal
thymus, hematopoietic cells, endocrine glands, and hormonally
regulated tissues and differential expression of Bcl-2 family
members occurs during neuronal differentiation. Bcl-x.sub.L and
Bcl-2 are both expressed in neurons of the developing human fetus,
however, Bcl-x.sub.L expression persists throughout fetal
development and into adulthood whereas Bcl-2 expression diminishes
between wk 20-39 of gestation (Yachnis et al., 1997).
[0066] Although Bcl-2 protein is widely expressed in embryonic
tissues (Novack and Korsmeyer, 1994; Lu et al., 1993), absence of
Bcl-2 protein in Bcl-2 null mice does not interfere with normal
prenatal development (Veis et al., 1993). However, postnatally,
these mice display growth retardation, smaller ears, and polycystic
kidneys, and most die within several months due to kidney failure.
In the Bcl-2 deficient mice, which eventually become ill, the
thymus and spleen are atrophic due to massive lymphocyte apoptosis.
Also, Bcl-2 null thymocytes are more susceptible to undergo
apoptosis following .gamma.-irradiation or treatment with
dexamethasone (Kamada et al., 1995; Nakayama et al., 1994).
[0067] The tissue distribution of Bcl-2 expression also suggests
that Bcl-2 plays a role in survival in various cell types
(Hockenbery et al., 1991). Immunohistochemistry reveals that Bcl-2
is expressed in cells that regenerate such as the stem cells or in
cells that are long lived. In the lymphatic system, Bcl-2 is
strongly expressed in the thymic medulla where the T-cells which
have survived negative and positive selection reside, and in the
areas of lymph nodes associated with maintenance of plasma cells
and memory B-cells (Hockenbery et al., 1991; Nunez et al., 1991).
In non-hematopoietic tissues, Bcl-2 is restricted to cells that
undergo self renewal such as the basal layer of the skin, the crypt
cells of the small and large intestine, and in long lived cells
such as the neurons. Bcl-2 also is expressed in tissues such as
breast duct epithelium and prostate epithelium which undergo
hyperproliferation or involution at the influence of the hormone or
growth factors (Hockenbery et al., 1991; McDonnell et al.,
1992).
[0068] The Bcl-2 family continues to expand with the discovery of
new members. Table 1 summarizes the Bcl-2 family of cell death
regulators. TABLE-US-00001 TABLE 1 Human Bcl-2 Family Members
Genbank Family Gene mRNA Amino Acid Protein Size Chromosome
Sequence Member Size (Kb) Size (Kb) Residues (kD) Localization
Function Identifiers Bcl-2 230 6.5 239 25 18q21 Anti-Apoptotic
M14745 Bcl-x.sub.L ND 2.7 233 31 ND Anti-Apoptotic Bcl-w 22 3.7 193
22 ND Anti-Apoptotic Mcl-1 ND 3.8 350 37.3 1q21 Anti-Apoptotic Al
ND 1.4* 172 20** ND Anti-Apoptotic Bfl-1 ND 0.6* 175 20*** ND
Anti-Apoptotic Bax .alpha. 4.5 1.0 192 21 19q13.3-13.4
Pro-Apoptotic L22473 Bax .beta. 1.5 218 24 ND L22474 Bax .gamma.
41**** 4.5** ND L22475 Bax .delta. 1.5 143**** ? ND U19599 ND
Bcl-x.sub.s ND 1.0 170 19 ND Pro-Apoptotic Z23116; L20122 Bak 1 6
2.4 211 23 6 Pro-Apoptotic U16811; U23765 Bak 2 20 U16812; U23765
Bak3 11 Bad ND 1.1* 204 22 ND Pro-Apoptotic AF031523 Bid ND 1.1*
195 23 ND Pro-Apoptotic U75506 Bik ND 1.0 160 18 ND Pro-Apoptotic
U34584 GRS ND 0.8 175 ? 15q24-25 ND Harakiri ND 0.7 91 16***** ND
Pro-Apoptotic U76376 ND, not determined. *cDNA; **predicted size of
protein; ***size of Ha-Tagged protein; ****predicted amino acid
length; *****size of the Flagged protein. The known Genbank
sequences are listed and are specifically incorporated herein by
reference in order to provide additional disclosure of the
sequences of the genes of the Bcl family.
[0069] It is commonly accepted that tumorigenesis is a multistep
process which may involve chromosomal abnormalities and the
deregulated expression of proto-oncogenes (Bishop, 1991). This is
particularly evident in hematolymphoid neoplasms where chromosomal
translocations may result in the activation of a proto-oncogene.
Translocations involve the breakage and reunion of chromosomes
where part of one chromosome breaks off and becomes reattached to
another chromosome. Such translocations are described by a notation
that indicates which two chromosomes have been recombined. For
example, t(9:22) indicates that a translocation has occurred
between chromosome 9 and chromosome 22. Further delineation of the
exact regions or genes that are involved in the translocation lead
to the identification of the resulting gene fusions or
proto-oncogenes involved in each particular translocation event.
Certain chromosomal translocations are associated with activation
of oncogenes that lie near the breakpoint of the chromosome.
[0070] Characterization of the t(9;22) and t(8;14) translocations
in chronic myelogenous leukemia (Nowell and Hungerford, 1960;
Rowley, 1973) and Burkitt's lymphoma (Manalov and Manolova 1972;
Zech et al., 1976), respectively, provided a paradigm for the
deregulation of proto-oncogenes during multistep
carcinogenesis.
B. Bcl-2 Family Members
[0071] Many Bcl-2 family member proteins have now been identified
(FIG. 1). These Bcl-2 homologues can be broadly categorized as
death antagonists and death agonists. The growing list of Bcl-2
gene family members all share highly conserved domains referred to
as Bcl-2 homology domain 1 and 2 (BH1 and BH2) (Oltvai et al.,
1993; Yin et al., 1994; Yin et al., 1995) or domains B and C,
respectively (Hanada et al., 1995; Tanaka et al., 1993). These
homology domains seem to be important for Bcl-2 to form
heterodimeric complexes with the family members and to carry out
its anti-apoptotic function (Yin et al., 1994; Hanada et al., 1995;
Yang et al., 1995a; Korsmeyer et al., 1993; Sedlak et al., 1995).
For example, mutations in BH1 and BH2 prevent Bcl-2 from forming
heterodimeric complexes with the Bcl-2 homologue Bax and can
abrogate the survival function of Bcl-2 (Yin et al., 1994). The
Bcl-2 protein can also form homodimers with itself via its NH2
terminal region called the BH4 domain which spans residues 11
through 33 (Hanada et al., 1995).
[0072] Thus, as stated earlier, the Bcl-2 family members are
divided into proapoptotic and antiapoptotic genes. The proapoptotic
genes include Bax, Bak, Bcl-x.sub.S, Bad, Bik, Bid and Harakiri.
The antiapoptotic genes include Bcl-2, Bcl-x.sub.L, Mcl-1, A1,
Bcl-w and GRS (FIG. 1). Each of these genes are discussed in
further detail herein below.
[0073] i. Bcl-2
[0074] The t(9;22) results in the formation of a bcr-abl fusion
gene and chimeric protein (Shrivelman et al., 1985) while the
t(8;14) results in the inappropriate expression of c-myc
(Dalla-Favera et al., 1982; Taub et al., 1984; Nishikura et al.,
1983). Both of these molecular events result in augmented cellular
proliferation (Langdon et al., 1986).
[0075] Bcl-2 was discovered as a novel transcriptional element by
its association with the t(14;18) reciprocal chromosomal
translocation commonly found in follicular lymphoma (Bakhshi et
al., 1985; Cleary and Sklar, 1985, Tsujimoto et al., 1984). Bcl-2
was shown to be a unique oncogene in that its deregulation did not
result in an increase in cell proliferation, but rather enhancement
of cell survival (Vaux et al., 1988; Hockenbery et al., 1990;
McDonnell et al., 1989). Thus, Bcl-2 represents a class of oncogene
that enables neoplastic growth by suppressing cell death
(McDonnell, 1993a).
[0076] The Bcl-2 gene, is comprised of three exons and spans
approximately 230 Kb. The open reading frame is in exon 2 and 3,
and encodes a 25 kD integral membrane protein (Seto et al., 1988;
Zutter, et al., 1991). The message can be alternatively spliced to
give two transcripts, Bcl-2.alpha. and the truncated Bcl-2.alpha.
that lacks the C-terminus region (Tsujimoto and Croce, 1986). Bcl-2
possesses a very hydrophobic stretch of 23 amino acids at the
C-terminus which serve as a transmembrane domain (Hockenbery et
al., 1990). Bcl-2 protein localizes to the nucleus, rough ER, and
mitochondria. In mitochondria, the protein is localized to the
contact zone of the inner and outer membranes of the mitochondrial
membrane where the transport of materials from the cytosol into the
mitochondrial matrix occurs (Hockenbery et al., 1990; deJong et
al., 1992).
[0077] Bcl-2 is normally expressed in pro and mature B-cells, but
is downregulated in pre and immature B lymphocytes (Merino et al.,
1994). This differential expression points to the survival role of
Bcl-2 in B lymphocyte development. High levels of Bcl-2 are needed
to ensure the survival of pro-B-cells and mature B-cells in order
to maintain a population of functional lymphocytes. But low levels
of Bcl-2 are necessary for cells, which do not express functional
surface Ig or are self reactive, to undergo apoptosis. Also in
T-cells, Bcl-2 is expressed at low levels in double positive
thymocytes undergoing negative and positive selection, and at high
levels in mature single positive T-cells which have survived the
selection (Gratiot-Deans et al., 1993). Thus, Bcl-2 seems to have
an important role in lymphocyte development (McDonnell et al.,
1989; McDonnell et al., 1990; McDonnell and Korsmeyer, 1991;).
[0078] The Bcl-2-Ig transgenic mouse model demonstrates that
deregulation of Bcl-2 gene causes initially a polyclonal expansion
of mature B-cells which can progress to an aggressive monoclonal
malignancy with an acquisition of additional gene deregulation,
thus confirming the multistep nature of carcinogenesis (McDonnell,
1993b). In humans also, follicular lymphoma can progress to a high
grade lymphoma following the acquisition of t(8; 14) translocations
and c-myc gene deregulation, albeit this appears to be an uncommon
event (Gawerky et al., 1988).
[0079] It also has been demonstrated that Bcl-2 plays a role in the
suppression of p53-mediated cell death. Splenic mononuclear cells
obtained from Bcl-2-Ig mice, which possess wild-type p53, displayed
rates of apoptosis comparable to cells obtained from p53 knockout
mice following .gamma.-irradiation (Marin et al., 1994). Together,
these results and the results of others utilizing transformed cell
lines indicate that Bcl-2 is capable of blocking p53 mediated cell
death induction (Marin et al., 1994; Wang et al., 1993; Chiou et
al., 1994).
[0080] Mutations in the conserved domains of p53 were uncommon in
the lymphomas arising in the Bcl-2-Ig transgenic mice suggesting
that there is no selective advantage of acquiring p53 mutations
when Bcl-2 is overexpressed (Marin et al., 1994). Additionally, the
Bcl-2-Ig transgenic and p53 knockout murine models were further
utilized to determine the extent of genetic complementation between
p53 and Bcl-2. In p53 KO/Bcl-2 hybrid mice, tumor latency and
incidence were unchanged when compared to individual parental
strains of mice (Marin et al., 1994). Many human tumors, such as
breast and prostate, also demonstrate that there is an inverse
correlation between the presence of p53 mutations and Bcl-2
expression (Silvstrini et al., 1994; McDonnell et al., 1997).
[0081] ii. Bax
[0082] Bax (SEQ ID NO:3=cDNA; SEQ ID NO:4=wild-type protein),
"Bcl-2 associated X protein", is a death agonist member of the
Bcl-2 family of proteins. Discovered by co-immunoprecipitation with
Bcl-2, it was the first Bcl-2 homologue to be identified (Oltvai et
al., 1993). The 4.5 Kb Bax gene maps to 19q13.3-13.4 and is
comprised of six exons (Apte et al., 1995). It shares 21% identity
and 43% similarity with Bcl-2. The most conserved regions between
the two molecules are within the BH1 and BH2 domains encoded by
exons 4 and 5, respectively (Oltvai et al., 1993).
[0083] Multiple forms of Bax protein can result from various
splicing alternatives. The most prevalent from is Bax-.alpha.,
whose 1.0 Kb RNA encodes a 192 amino acid, 21 kD transmembrane
protein. The 24 kD cytosolic Bax-.beta. lacks the transmembrane
segment and is encoded by 1.5 Kb RNA transcript. A third form,
Bax-.gamma. lacks the exon 2 and can undergo alternative splicing
of intron S to yield 1.0 and 1.5 Kb RNA transcripts (Olsen et al.,
1996). Yet another alternatively spliced form of Bax, Bax.delta.,
has the C-terminal transmembrane anchor as well as the BH1 and BH2
domains (Apte et al., 1995). The functional role of these Bax
variants remains to be elucidated.
[0084] The Bax gene promoter contains four p53 binding sites and
the expression of Bax is upregulated at the transcriptional level
by p53 (Miyashita and Reed, 1995). A temperature sensitive p53
mutant transfected into a myeloid cell line was associated with
increased Bax mRNA after shifting to the permissive temperature
(Zhan et al., 1994). Also in cells obtained from p53-null mice, the
level of Bax proteins was found to be lower (Miyashita et al.,
1994). Moreover, following apoptosis induction by ionizing
radiation, the Bax mRNA was upregulated only in the cell line that
possesses wild-type p53 (Zhan et al., 1994). These data suggest
that Bax may function as a primary response gene in a p53 regulated
apoptotic pathway (Miyashita et al., 1994). However, thymocytes
from the Bax knockout mice were not diminished in their capacity to
undergo apoptosis after .gamma.-irradiation, a pathway driven by
p53 (Knudson et al., 1995). Bax expression can also be modulated by
other factors. The mRNA level has been shown to be downregulated in
myeloid leukemia cell lines treated with IL-6 and/or dexamethasone
(Lotem and Sachs, 1995). The half life of Bax mRNA can be increased
in cell lines expressing higher levels of Bcl-2 (Miyashita et al.,
1995). However, this increase in stability of Bax mRNA by Bcl-2
protein appears to be tissue specific.
[0085] Mutational analysis has shown that the BH1 and BH2 domains
of Bax are not required for heterodimerization with Bcl-2, nor is
the NH.sub.2 terminal amino acids needed for Bax homodimerization,
unlike the homodimerization requirement for Bcl-2. Rather a stretch
of amino acids spanning residues 59-101 in the BH3 domain was shown
to be essential in both the homodimerization and heterodimer
complex formation with Bcl-2 (Zha et al., 1996a). Additionally, in
contrast to Bcl-2, Bax can function in its monomeric form to
accelerate cell death (Simonian et al., 1996). Bax can
heterodimerize with other Bcl-2 related proteins, including
Bcl-x.sub.L, Mcl-1, and A1 (Sedlak et al., 1995). The "rheostat"
model has been proposed to explain the role of Bcl-2 family member
interactions in controlling cell death. This model suggests that
the relative amounts of Bcl-2 and Bax may determine the
susceptibility of a cell to undergo apoptosis (Korsmeyer et al.,
1993). According to this model, when Bcl-2 is in excess, Bcl-2/Bax
heterodimers predominate and cell death is inhibited. Conversely,
when Bax is in excess, Bax homodimers predominate and the cell
becomes susceptible to cell death induction following exposure to
an apoptotic stimulus.
[0086] The tissue distribution of Bax protein is more widespread
than Bcl-2. (Krajewski et al., 1994a). The immunohistochemical
staining of murine tissues has revealed that the expression of
Bcl-2 and Bax overlap in some tissues, and that Bax is not always
expressed at high levels in compartments marked by a high turnover
rate. For example, Bax, as well as Bcl-2, are expressed in the
thymic medulla but not in the thymic cortex, despite high numbers
of cortical thymocytes which undergo apoptosis. Also, a high level
of Bax protein is observed in neurons, cells that have a long life.
However, in certain tissues such as colonic epithelium, gastric
glands, and secretory epithelial cells of prostate, Bax expression
corresponds to the cells that are susceptible to undergoing
apoptotic cell death (Krajewski et al., 1994a).
[0087] Evidence that apoptosis is not absolutely dependent on the
expression of Bax is also apparent from an analysis of the Bax
knockout mice. In these mice the absence of Bax is associated with
either tissue specific hyperplasia or hypoplasia (Knudson et al.,
1995). For example, there was an increase in number of resting
mature B-cells and thymocytes causing hyperplasia in the spleen and
thymus. However, the male Bax knockout mice were infertile due to
atrophic testes, resulting from the abrogation of spermatogenesis
(Knudson et al., 1995).
[0088] Recent evidence suggests that Bax may play a role as a tumor
suppressor. Normally Bax-.alpha. is expressed at high levels in
breast tissue but is not detectable, or expressed at low levels, in
breast cancers (Bargou et al., 1995). Furthermore, in metastatic
breast cancer, patients with reduced Bax expression showed poor
response to chemotherapy (Krajewski et al., 1995a). Transgenic mice
have been generated, which express a truncated form of the SV40 T
antigen (Tgl21) resulting in inactivation of the retinoblastoma
protein but not p53. Tgl21 mice bearing targeted disruptions of
either the p53 gene or the Bax gene exhibited an increased rate of
brain tumor formation compared to Tgl21 mice with intact p53 or Bax
genes (Yin et al., 1997). Also frequent frame shift mutations of
Bax were found in microsatellite mutator phenotype (MMP) colon
adenocarcinomas, suggesting that the wild-type Bax gene may play a
tumor suppressor role in colorectal carcinogenesis (Rampino et al.,
1997).
[0089] iii. Bcl-x
[0090] Bcl-x was initially isolated from chicken lymphoid cells
using a murine Bcl-2 cDNA probe under low stringency conditions
(Boise et al., 1993). The Bcl-x gene shares 44% identity with
Bcl-2. Bcl-x was shown to interact with other members of the Bcl-2
family in a manner similar to that shown for Bcl-2 when analyzed by
the yeast two-hybrid system (Sato et al., 1994). Two human Bcl-x
cDNAs have been cloned (Boise et al., 1993). Bcl-x.sub.L (long
form) is a 31 kD protein, with an open reading frame of 233 amino
acid. This form of Bcl-x contains the BH1 and BH2 domains. The
Bcl-x.sub.L cDNA was found to be co-linear with the genomic
sequence denoting the absence of mRNA splicing. Bcl-x.sub.s (short
form) encodes a 170 amino acid, 19 kD protein. The carboxy-terminal
63 amino acids encoding the BH1 and BH2 domains are deleted from a
5' splice site within exon 1 of the Bcl-x gene (Boise et al.,
1993). A third alternative splice variant of Bcl-x has been
isolated from a murine cDNA library, Bcl-x.sub..beta.,
(Gonzalez-Garcia et al., 1994). Bcl-x.sub..beta., encodes a 209
amino acid protein that results from an unspliced first coding exon
and lacks the carboxy-terminal 19 hydrophobic amino acids necessary
for transmembrane insertion.
[0091] Both the level and pattern of expression of Bcl-x differ
from that of Bcl-2. The levels of Bcl-x expression are generally
higher than Bcl-2 in all tissues examined except for the lymph
nodes where Bcl-2 is predominant (Krajewski et al., 1994a).
Bcl-x.sub.L is mainly expressed in the cells of the central nervous
system, kidney, and bone marrow (Gonzalez-Garcia et al., 1994,
Rouayrenc et al., 1995). Both Bcl-x.sub.L and Bcl-x.sub.s, but not
Bcl-2 are expressed in CD34.sup.+, CD38.sup.-, lin.sup.-
hematopoietic precursors (Park et al., 1995). However, the
subcellular distribution of Bcl-x protein is similar to Bcl-2 in
that it localizes to mitochondria and the nuclear envelope. This
suggests that the function of the two proteins may be similar
(alez-Garcia et al., 1994,).
[0092] Further insight into the role of Bcl-x during development
was obtained from Bcl-x deficient mice (Motoyama et al., 1995).
Heterozygous mice developed normally while homozygous, knockout
mutants die at approximately day 13 of gestation. The Bcl-x
knockout embryos display extensive apoptosis involving post-mitotic
neurons of the developing brain, spinal cord, dorsal root ganglia,
and hematopoietic cells in the liver. Additionally, lymphocytes
from Bcl-x deficient mice showed diminished maturation. The life
span of immature lymphocytes but not mature lymphocytes was
shortened. This data indicates that Bcl-x is required for the
embryonic development of the nervous and hematopoietic systems.
[0093] Similar to Bcl-2, Bcl-x.sub.L was shown to confer resistance
to apoptosis induction following growth factor deprivation.
However, Bcl-x.sub.s counteracted the ability of Bcl-2 to block
apoptosis (Boise et al., 1993). Although Bcl-x.sub.L and Bcl-2
initially seemed to have the same functions, several observations
suggest that biologically these two proteins are not completely
overlapping. The tissue distribution of Bcl-2 and Bcl-x are not
identical and the phenotypes' of the corresponding knockout strains
of mice are substantially different. Furthermore, it has been shown
that WEHI-231 cells can be protected from apoptosis induced by
surface IgM cross-linking by enforced Bcl-x.sub.L expression while
enforced Bcl-2 expression exerts no such protective effect (Choi
and Boise, 1995; Gottschalk et al., 1994).
[0094] The crystalline structure of Bcl-x has expanded the
inventors' insight into the potential mechanisms of function of
Bcl-2 family members (Muchmore et al., 1996). Bcl-x structure was
shown to consist of two central hydrophobic .alpha. helices
surrounded by two amphipathic helices (Muchmore et al., 1996).
Interestingly, the conserved BH1, BH2 and BH3 domains were in
spatial proximity and formed a hydrophobic cleft. This cleft is
believed to form a binding site for other Bcl-2 family members
(Muchmore et al., 1996). Evidence in favor of this hypothesis was
provided when Bcl-x and a 16 residue bak peptide derived from the
BH3 domain were co-crystallized. The heterodimeric crystal
structure revealed that the bak BH3 domain interacts with the
hydrophobic cleft made by the BH1, BH2, and BH3 domains of Bcl-x
(Sattler et al., 1997). The crystal structure of Bcl-x was also
found to resemble the translocation domain of the diphtheria toxin
and colicins (Muchmore et al., 1996). This similarity in structure
implies similarity in function and indicates that Bcl-2 family
members can be considered channel forming proteins capable of
regulating the transmembrane trafficking of molecules involved in
signaling cell death.
[0095] iv. Bak
[0096] Bak (Bcl-2-homologous antagonist/killer) was first cloned
from human heart and Epstein-Barr transformed human B-cell cDNA
libraries (Chittenden et al., 1995; Kiefer et al., 1995; Farrow et
al., 1995). There are three closely related bak genes (bak-1, 2,
and 3) which are located on chromosome 6 (bak-1), chromosome 20
(bak-2) and chromosome 11 (bak-3). The bak genes contain at least
three exons and span 6 Kb. Bak is a 211 amino-acid, 23 kD protein
which shares 53% amino-acid identity with Bcl-2. It possesses the
same hydrophobic carboxy-terminal domain as Bcl-2 and Bcl-x.sub.L,
which suggests that bak is an integral membrane protein. In
contrast to Bcl-2, bak is expressed at high levels in the kidney,
pancreas, liver, and fetal heart, as well as adult brain (Kiefer et
al., 1995). Similar to Bax in the intestine, bak expression is
strongest in the cells in the luminal surface where most apoptosis
is occurring. However, in a colorectal carcinoma cell line, only
bak expression was shown to be modulated following apoptosis
induction, indicating that bak may play a primary role in
enterocyte apoptosis (Moss et al., 1996). This contention is
further supported by the observation that bak expression is reduced
in colorectal adenocarcinoma samples. Therefore, a downregulation
of bak may facilitate the accumulation of neoplastic cells in the
early stages of colorectal tumorigenesis (Krajewski et al., 1996).
Bak was shown to accelerate cell death following IL-3 withdrawal
(Chittenden et al., 1995; Kiefer et al., 1995), but inhibits
apoptosis induced by serum withdrawal and menadione treatment
(Chittenden et al., 1995).
[0097] v. Bad
[0098] Bad (Bcl-x.sub.L/Bcl-2 associated death promoter homologue)
a novel member of the Bcl-2 family that was identified as a Bcl-2
interacting protein using the yeast two hybrid system (Yang et al.,
1995b). The full-length Bad cDNA sequence encodes a novel 204 amino
acid protein with a predicted molecular weight of 22 kD. Bad shares
only limited homology with known Bcl-2 family members in the BH1
and BH2 domains. However, the functionally significant W/YGR
triplet in BH1, the W at position 183, the WD/E at the exon
junction in BH2 and the spacing between BH1 and BH2 domains is
conserved. Unlike many other Bcl-2 family members, Bad does not
contain a transmembrane anchor domain.
[0099] Bad was shown to heterodimerize with Bcl-2 and Bcl-x in vivo
using co-immunoprecepitation. Bad's interaction with either Bcl-2
or Bcl-x can displace Bax from the heterodimers. Significantly,
this was shown to reverse the death repressor activity of Bcl-x,
but not of Bcl-2. However, Bad does not appear to interact with
Bax, Mcl-1, or A1 nor, apparently, does Bad form homodimers (Yang
et al., 1995b). Recent studies have shown that Bad may function in
intracellular signal transduction pathways. Upon IL-3 stimulation
of an IL-3 dependent hematopoietic cell line, Bad becomes rapidly
phosphorylated at two serine residues and is prevented from forming
heterodimeric complexes with Bcl-x.sub.L. The phosphorylated Bad is
found to be complexed with 14-3-3, a phosphoserine binding protein
which regulates protein kinases, and is sequestered in cytosol (Zha
et al., 1996b). Therefore, only the non-phosphorylated Bad is
heterodimerized with the membrane bound Bcl-x.sub.L and counters
the anti-apoptotic activity of Bcl-x.sub.L. One of the models to
explain the apoptotic activity of Bad is that in its
non-phosphorylated form, Bad binds to membrane associated
Bcl-x.sub.L which releases Bax to enhance cell death (Zha et al.,
1996b). Another link between the phosphorylation event and the
apoptotic pathway was shown when it was found that in vitro, Bad is
phosphorylated by mitochondrial membrane targeted Raf-1, but not by
the plasma membrane targeted Raf-1. Moreover, Bcl-2 was shown to
target Raf-I to mitochondrial membrane which resulted in
phosphorylation of Bad and the subsequent enhancement of cell
survival (Wang et al., 1996a).
[0100] vi. Mcl-1
[0101] Mcl-1 (human myeloid cell differentiation protein) was
identified by differentially screening cDNA library of the human
myeloid leukemia cell line, ML-1, following induction by phorbol
12-myristate 13-acetate (TPA) (Kozopas et al., 1993). Mcl-1 has
also been detected in normal peripheral blood B cells after
treatment with IL4 and anti-IgM. Mcl-1 is an early response gene,
that reduces its expression immediately following differentiation
induction (Kozopas et al., 1993; Yang et al., 1995). A study done
using a yeast two-hybrid assay indicates that Mcl-1 interacts
strongly and selectively with Bax, but not with any other Bcl-2
family members (Sedlak et al., 1995; Sato et al., 1994).
[0102] Mcl-1 shares sequence homology to Bcl-2 in the BH1 and BH2
domains and has a carboxy-terminal transmembrane anchor domain
(Yang et al., 1995). In addition, the Mcl-1 protein possesses PEST
sequences (Kozopas et al., 1993), which correlate with the its role
as an early response gene product (Yang et al., 1995). The human
Mcl-1 gene maps to chromosome 1 band q21 (Craig et al., 1994), an
area often involved in chromosomal abnormalities in neoplastic and
preneoplastic diseases (Atkin, 1986; Gendler et al., 1990; Testa,
1990).
[0103] Mcl-1 protects against apoptosis induced by constitutive
expression of c-myc or Bax (Reynolds et al., 107). However, in the
5AHSmyc cell line, Mcl-1 overexpression is not as effective as
Bcl-2 overexpression in preventing myc-mediated cell death
(Reynolds et al., 1994). It has been proposed that Mcl-1 may
function as an alternative to Bcl-2 in situations where Bcl-2
cannot block apoptosis or in tissues lacking Bcl-2 expression. For
example, in normal peripheral blood B cells treated with agents
which promote survival (IL-4, anti-.mu., and TPA) or enhance rates
of cell death (TGF.beta.1 and forskolin), upregulation of Mcl-1
correlates with cell survival and downregulation of Mcl-1 precedes
cell death. In contrast, levels of Bcl-2 expression are not
modulated under the same experimental conditions (Lomo et al.,
1996).
[0104] Additionally, the tissue distribution of Mcl-1 and Bcl-2
expression show significant differences such as brain and spinal
cord neurons in which Bcl-2 predominates compared to skeletal
muscle, cardiac muscle, cartilage and liver where Mcl-1
predominates over Bcl-2 (Krajewski et al., 1995b). Similarly, Mcl-1
levels in normal lymph nodes are highest in germinal centers, where
the rate of apoptosis is high. In contrast, Bcl-2 is most intense
in the mantle zone. It has been postulated that Mcl-1 temporarily
blocks cell death until suppression such as Bcl-2 are upregulated
(Krajewski et al., 1994b).
[0105] vii. A1
[0106] A1 was identified by differentially screening a cDNA library
of normal peripheral blood B cells and after treatment with IL-4
and anti-IgM. The A1 cDNA was isolated from murine macrophages
after GM-CSF induction of differentiation (Lin et al., 1993). A1 is
an early response gene that decreases its level of expression
immediately following differentiation induction (Lin et al., 1993).
Yeast two-hybrid assay indicates that A1 interacts strongly and
selectively with Bax, with but not with any other Bcl-2 family
member (Sedlak et al., 1995; Sato et al., 1994). A1 shares homology
with Bcl-2 in the BH1 and BH2 domains, but does not possess the
carboxy-terminal transmembrane domain (Lin et al., 1993).
[0107] The correlation of GM-CSF and LPS-induced differentiation
with A1 upregulation suggest A1 could potentially function as a
cell death suppressor (Lin et al., 1993). Later reports has shown
that A1 protects against TNF induced apoptosis in the presence of
actinomycin D in a human microvascular endothelial cells (Karsan et
al., 1996). A1 could also inhibit ceramide induced cell death in
these endothelia cells (Karsan et al., 1996). A1 expression
displays a rather limited tissue distribution and appears to be
confined to hematopoietic tissues, including helper T-cells,
macrophages, and neutrophils (Lin et al., 1993).
[0108] viii. Bft-1I
[0109] Bfl-1 (Bcl-2 related gene expressed in human fetal liver)
was identified during a random cDNA sequencing project (Choi et
al., 1995). It was found to be homologous to Bcl-2 family members
with the highest homology to the A.sub.1 gene. The main region of
homology was in the conserved BH1, BH2, and BH3 domains. Bfl-1 is
mainly expressed in bone marrow while low levels of expression are
detected in lung, spleen, esophagus, and liver. Bfl-1 mRNA was
detected at relatively high levels in six out of eight stomach
cancer tumors and metastasis when compared to normal stomach tissue
from the same patients (Choi et al., 1995). Bfl-1 protein
suppresses apoptosis induced by p53 in the BRK cell line to the
same extent Bcl-2, Bcl-X.sub.L. Bfl-1 was also shown to cooperate
with E1a in the transformation of primary rodent epithelial cells
(D'Sa-Eipper et al., 1996).
[0110] ix. GRS
[0111] GRS was incidentally cloned during the cloning of fibroblast
growth factor 4 (FGF-4) from a patient with chronic myelogenous
leukemia (Lucas et al., 1994). The FGF-4 gene was truncated by a
DNA rearrangement with a novel gene named GRS (Glasgow Rearranged
Sequence) with a breakpoint 30 nucleotides downstream from the
translation termination codon of FGF-4. The full length cDNA of GRS
was then cloned from human activated T cell cDNA library. The GRS
cDNA is 824 nucleotides (Kenny et al., 1997). Sequence analysis of
GRS revealed 71% identity to the murine A1 protein at the amino
acid level.
[0112] Northern blot analysis showed a high level of expression of
GRS in hematopoietic cells and to a lesser extent in lung and
kidney (Kenny et al., 1997). GRS also is expressed in cell lines of
hematopoietic origin including HL-60 (promyelocytic leukemia), Raji
(Burkitt lymphoma) and K-562 (chronic myeloid leukemia). However
GRS is not expressed in MOLT-4 T lymphoblastic leukemia and T-cells
prior to activation. The melanoma cell line G-361 also expressed
high levels of GRS. GRS is localized to chromosome 15q24-25. This
location positions GRS adjacent to t(15;17) region translocation
frequently observed in acute promyelocytic leukemia. The GRS
location also places it in the breakpoint described in Fanconi
anemia that is associated with high incidence of acute
leukemia.
[0113] x. Bid
[0114] Bid (BH3 interacting domain death agonist) was initially
identified as a protein that interacts with both Bcl-2 and Bax
proteins. The labeled Bax and Bcl-2 proteins were used to screen a
.lamda.EXlox expression library constructed from the murine T-cell
hybridoma line 2B4 (Wang et al., 1996c). Bid is a 23 kD, 195 amino
acid protein. Sequence analysis of Bid revealed that Bid shares
homology only with the BH3 domain of the Bcl-2 family and that it
lacks the carboxy-terminus transmembrane hydrophobic domain. A
human homologue of Bid has also been identified. Human Bid shares
72.3% sequence homology to the murine Bid and has a 195 amino acid
open reading frame (Wang et al., 1996c).
[0115] In adult mouse tissue, Bid is mainly expressed in the
kidneys but is also present in brain, spleen, liver, testis and
lung (Wang et al., 1996c). Low levels of expression are detected in
the heart and skeletal muscle. The mouse hematopoietic cell line,
FL5.12, was also found to express high levels of Bid. Subcellular
fractionation has revealed that Bid is predominantly localized to
the cytosol (90%) with a small fraction in the membrane fraction
(Wang et al., 1996c).
[0116] Expression of Bid in the IL-3 dependent FL5.12 cell line
could induce a subtle but consistent enhancement of apoptosis
following IL-3 withdrawal (Wang et al., 1996c). Bid inducible
expression as well as transient transfections of Bid in Rat-1
fibroblasts and Jurkat T-cells, results in reducing cell viability
to <40% at 48 h (Wang et al., 1996c). Bid could also restore
apoptosis in FL5.12 clones overexpressing Bcl-2. The level of
apoptosis was intermediate between the parental and Bcl-2
overexpressing clones. The degree of cell death in all cases
corresponded to the level of Bid protein expression as detected by
Western blot analysis. Bid induced apoptosis could be inhibited by
zVAD-fmk, an irreversible inhibitor particularly effective against
the CPP32-like subset of proteases. This suggests that Bid induced
cell death involves activation of CPP32-like proteases (Wang et
al., 1996c).
[0117] Bid interacts with both death agonists and antagonists
members of the Bcl-2 family. Bid can interact with. Bcl-2, Bcl-x,
and Bax but does not form homodimers. Bid was unable to form
trimolecular complexes with Bcl-2/Bax heterodimers suggesting that
Bid interacts with monomeric or homodimeric Bcl-2 or Bax. Several
mutants of Bcl-2, Bax, and Bid were examined to detect the regions
of each molecule required for their interactions. The BH3 domain of
Bid was essential for interaction with Bax and Bcl-2. Differential
specificity of these mutants was also detected as mutant (M97A,
D98A) could bind Bax but not Bcl-2, mutant (G97A) could bind Bcl-2
but not Bax while other mutants did not bind either protein.
Noteworthy is that all BH3 mutants of Bid were impaired in their
ability to counter Bcl-2 protection including mutants that could
still bind Bcl-2. However, Bid mutant (M97A, D98A) that can still
bind Bax but not Bcl-2, retained its activity. Conversely, the BH1
domain of Bcl-2 and Bax were shown to be required for their
interaction with Bid. It is suggested that the a helix BH3 domain
of Bid interacts with the hydrophobic cleft contributed by the BH1
domain of Bcl-x. This interaction might result in a conformational
change in Bid, Bcl-2, or Bax that signals cell death.
[0118] xi. Bik
[0119] Bik (Bcl-2 interacting killer) is a novel Bcl-2 family
member that was detected when a human B-cell line cDNA library was
used in a yeast two hybrid screen for proteins that interact with
Bcl-2 (Boyd et al., 1995). Bik is a 160 amino acid protein and has
a predicted molecular weight of 18 kD encoded by 928 bp cDNA and 1
Kb mRNA. Bik shares homology only within the BH3 domain of the
Bcl-2 family and has a carboxy-terminal transmembrane hydrophobic
domain. Bik was found to localize to the nuclear envelope and
cytoplasmic membrane structures.
[0120] Transient co-transfection of Bik and .beta.-galactosidase
expression plasmids in Rat-1 fibroblasts resulted in a dramatic
reduction in the number of blue cells, consistent with reduced
viability of Bik transfected cells (Boyd et al., 1995).
Co-transfection of Bik and Bcl-2, Bcl-x, adenovirus E1B-19 kDa, or
EBV-BHRF1 resulted in an increase in blue cell number indicating
the ability of these proteins to reverse cell death by Bik.
Deletion of the BH3 domain of Bik resulted in loss of its
pro-apoptotic activity. Bik induced apoptosis was shown to be
inhibited by zVAD-fmk. However, CrmA could not inhibit Bik induced
cell death. This suggests that Bik induced cell death involves
selective activation of CPP32-like proteases (Orth and Dixit,
1997).
[0121] Interactions between Bik and other Bcl-2 family members was
examined using the yeast two hybrid system, GST-fusion protein
capture on glutathione agarose beads, and transient co-transfection
of tagged Bik with other anti-apoptotic Bcl-2 family members (Boyd
et al., 1995). These in vitro and in vivo studies revealed
interactions between Bik and Bcl-2, Bcl-x, adenovirus E1B-19 kDa,
and EBV-BHRF1. Bik also interacts with Bcl-x, a death promoting
protein that lacks BH1 and BH2 domains. This suggests that Bik does
not require BH1 and BH2 domain for its interaction with Bcl-2
family members. Bcl-2 residues 43-48 and E1B-19 kDa residues 90-96
were shown to be essential for interaction with Bik. Noteworthy is
that these residues are not within the conserved regions of Bcl-2
family members.
[0122] xii. Bcl-w
[0123] Bcl-w was cloned using degenerate primers to the conserved
BH1 and BH2 domains in a low stringency PCR.TM. reaction (Gibson et
al., 1996). These primers were used to amplify cDNA from mouse
macrophage and mouse brain cell lines. The PCR.TM. product was then
used to screen cDNA libraries from mouse brain, spleen, and myeloid
cell lines. Bcl-w is a 22 Kb gene with a 3.7 Kb mRNA which encodes
a 22 kD protein. Human Bcl-w was then isolated from an adult human
brain cDNA library. Bcl-w possesses the BH1, BH2, and BH3 domains.
The human and mouse genes are 99% identical at the amino acid level
and 94% at the nucleotide level.
[0124] Bcl-w mRNA is expressed at high levels in brain, colon, and
salivary gland. Surprisingly, Bcl-w expression is not detected in
T- and B-lymphoid cell lines. However, mRNA was detected in myeloid
cell lines of macrophage, megakaryocyte, erythroid, and mast cell
origin. Bcl-w also has a hydrophobic C-terminal transmembrane
domain. The cytoplasmic localization of Bcl-w is similar to that of
Bcl-2. Bcl-w resides in the central region of mouse chromosome 14
and human chromosome 14 at q11.2. Hematopoetic cell lines
expressing Bcl-w were resistant to apoptosis induction to the same
extent as Bcl-2 and Bcl-x stable transfectants. However, Bcl-w did
not protect CHI B-lymphoma cells from CD95-induced apoptosis while
Bcl-2 and Bcl-x.sub.L were able to do so (Gibson et al., 1996).
[0125] xiii. Harakiri
[0126] The Harakiri gene and its protein product Hrk was identified
by a yeast two hybrid screen of a HeLA cDNA library to detect
proteins that bind to Bcl-2 (Inohara et al., 1997). A 9-wk human
embryo cDNA library was used to obtain the full length Hrk cDNA.
Hrk was detected as a 716 bp cDNA that was confirmed by the
northern blot analysis using both human and mouse tissue as 0.7 Kb
mRNA. The cDNA encodes an open reading flame of 91 amino acids. Hrk
shares homology with Bcl-2 family member BH3 domain, however, the
rest of the protein has no significant homology to any other
protein or Bcl-2 family. A region of 28 hydrophobic amino acids
that may serve as a membrane-spanning domain was also identified at
the COOH-terminus of Hrk.
[0127] Northern blot analysis demonstrates high levels of Hrk
expression in all lymphoid tissues examined including the bone
marrow and spleen. Hrk is also expressed in the pancreas and at low
levels in the kidney, liver, lung, and brain (Inohara et al.,
1997). Hrk was seen as a cytosolic granular staining by confocal
microscopy of transiently transfected cells with flagged Hrk. This
staining is similar to the previously reported localization of
Bcl-2 and Bcl-x.
[0128] Transient transfections of Harakiri in 293T cells, HeLa, and
FL5.12 progenitor B-cells resulted in a dramatic decrease in cell
viability by 36 h post-transfection. However co-expression of Bcl-2
and Bcl-x could inhibit the death promoting activity of Hrk.
Interestingly, Hrk appears to interact only with Bcl-2 and
Bcl-x.sub.L but not with the other pro-apoptotic family members
Bax, Bak, and Bcl-x.sub.s. Deletion mutants of Hrk lacking 16 amino
acids including the BH3 domain were unable to interact with Bcl-2
and Bcl-x. This mutant also failed to induce cell death in 293T
cells. Deletion analysis has also revealed the requirement of BH1
and BH2 domains of Bcl-2 and Bcl-x to interact with Hrk.
C. Interactions of Bcl-2 Family Members and Mechanisms of
Function
[0129] One of the reasons for the modest understanding of the
mechanisms by which Bcl-2 homologues execute their cellular roles
stems from a lack of identifiable sequence motifs in the Bcl-2
family which would implicate a mechanism of action. What have been
defined, however, are shared domains designated as Bcl-2 homology
domain 1, 2, 3 and 4. The BH1 domain spans amino acid residues
136-155 of the Bcl-2 protein, BH2 spans resides 187-202, BH3 spans
resides 93-107 and BH4 spans residues 10-30. The BH3 domain, for
its pan appears to be involved in selective interactions between
Bcl-2 family members.
[0130] The BH3 domain appears to be required for the death
promoting activity of Bax and bak are required for their
interaction with two death-repressing members, Bcl-2 and
Bcl-x.sub.L (Zha et al., 1996a; Chittenden et al., 1995).
[0131] The BH1 and BH2 domains serve equally important functions.
The creation of point mutations in either domain, can effectively
abolish the death repression function of Bcl-2 (Yin et al., 1994).
Recent evidence suggests, however, that the formation of
heterodimers is not required for function of family members (Cheng
et al., 1996). These same BH1 and BH2 domain mutants of Bcl-2 fail
to heterodimerize with Bax, although they do homodimerize well (Yin
et al., 1994). Some of the most compelling evidence that the BH3
motif represents a "death domain" comes from studies of Bid (Wang
et al., 1996c). Bid possesses only the BH3 domain, lacks the
carboxy-terminal signal-anchor segment, and localizes to both
cytosolic and membrane compartments. Importantly, ectopic
expression of Bid abrogates the pro-survival effect of Bcl-2.
Additionally, expression of Bid, without another death stimulus,
induces ICE-like proteases and apoptosis. An intact BH3 domain of
Bid was required to bind the BH1 domain of either Bcl-2 or Bax.
[0132] The BH4 domain, which is located at the amino-terminus has
been far less characterized. To date, it has been reported that
deletion of the BH4 domain of Bcl-2 nullifies anti-apoptotic
function and homodimerization, but does not impair Bcl-2/Bax
heterodimerization (Reed et al., 1996). There is some evidence
which indicates that the BH4 domain may mediate interactions of
Bcl-2 family member protein with non-Bcl-2-related proteins such as
calcineurin (Shibasaki et al., 1997). Thus the BH4 domain may serve
as an tethering domain that bridges Bcl-2 and Bcl-2-related
proteins to important signal transduction proteins.
[0133] Perhaps, at its simplest level, the expression of various
Bcl-2 related proteins may determine whether a cell responds to an
applied stress by initiating a cell death program or surviving.
However, another hypothesis, that has substantial experimental
evidence based on a mutational analysis of the BH domains, suggests
that the cellular response to injury may be a function of the
multiple heterodimerization and homodimerization states between
members of this protein family. This model, commonly known as the
"rheostat model" has been advocated by Dr. Stanley Korsmeyer's
group (Oltvai et al., 1993; Korsmeyer et al., 1993). In this
scenario, the relative levels of dimerization partners available
shifts the balance of cell fate in favor of viability (e.g.,
Bcl-2/Bcl-2 homodimers favoring cell survival) or cell death (e.g.,
Bax/Bax homodimers favoring cell death) following exposure to an
appropriate stress. This ability of Bcl-2-related proteins to
hereto- and homodimerize in vivo, is perhaps one of the most
important features of the family.
[0134] Complicating the picture further are reports of the ability
of several Bcl-2 family members to physically interact with several
signaling protein complexes containing p21 ras (Chen and Faller,
1996), Raf-1 kinase (Wang et al., 1996b) and p23 R-ras proteins
(Wang et al., 1995). Another feature, is the conservation of a
hydrophobic membrane targeting sequence in the carboxy-terminal
tail of most members of the Bcl-2 family. The targeting domain most
likely ensures that the various members are correctly routed to the
appropriate intracellular organelle. Perhaps, this routing domain
ensures that the various Bcl-2-related proteins are localized in
close proximity to secure proper physical interactions should the
appropriate stress be detected.
[0135] The mechanisms of programmed cell death are far from being
completely elucidated. At present, many different factors such as
protease activation (Yuan, et al., 1993; Fraser and Evan, et al.,
1996; Chinnaiyan et al., 1997), DNA cleavage, and calcium signaling
(Lam et al., 1994; Marin et al., 1996; Minn et al., 1997) are known
to participate in apoptosis. The placement of Bcl-2 and Bcl-2
family members in cell death regulatory pathways is now being
elucidated. It is now known that Bcl-x.sub.L can form ion channels
and it may be that other Bcl-2 family members function in a similar
manner. The specific interactions that Bcl-2 family proteins have
with various signaling molecules and within the Bcl-2 family itself
are active areas of investigation.
D. Engineering Expression Constructs
[0136] In certain embodiments, the present invention involves the
manipulation of genetic material to produce expression constructs
that encode therapeutic genes. Such methods involve the generation
of expression constructs containing, for example, a heterologous
DNA encoding a gene of interest and a means for its expression,
replicating the vector in an appropriate helper cell, obtaining
viral particles produced therefrom, and infecting cells with the
recombinant virus particles.
[0137] The gene will be a therapeutic gene such as one or more of
the proapoptotic genes discussed herein above, or the gene may be a
second therapeutic gene or nucleic acid useful in the treatment of,
for example cancer cells. In the context of gene therapy, the gene
will be a heterologous DNA, meant to include DNA derived from a
source other than the viral genome which provides the backbone of
the vector. Finally, the virus may act as a live viral vaccine and
express an antigen of interest for the production of antibodies
thereagainst. The gene may be derived from a prokaryotic or
eukaryotic source such as a bacterium, a virus, a yeast, a
parasite, a plant, or even an animal. The heterologous DNA also may
be derived from more than one source, i.e., a multigene construct
or a fusion protein. The heterologous DNA also may include a
regulatory sequence which may be derived from one source and the
gene from a different source.
[0138] i. Additional Therapeutic Genes
[0139] The present invention contemplates the use of a variety of
different genes in combination with adenoviral Bax and the
proapoptotic Bcl-2 gene constructs. For example, genes encoding
enzymes, hormones, cytokines, oncogenes, receptors, tumor
suppressors, transcription factors, drug selectable markers, toxins
and various antigens are contemplated as suitable genes for use
according to the present invention. In addition, antisense
constructs derived from oncogenes are other "genes" of interest
according to the present invention.
[0140] a. p53
[0141] As stated earlier, p53 currently is recognized as a tumor
suppressor gene. High levels of mutant p53 have been found in many
cells transformed by chemical carcinogenesis, ultraviolet
radiation, and several viruses. The p53 gene is a frequent target
of mutational inactivation in a wide variety of human tumors and is
already documented to be the most frequently-mutated gene in common
human cancers. It is mutated in over 50% of human NSCLC (Hollstein
et al., 1991) and in a wide spectrum of other tumors.
[0142] The p53 gene encodes a 393-amino acid phosphoprotein that
can form complexes with host proteins such as large-T antigen and
E1B. The protein is found in normal tissues and cells, but at
concentrations which are minute by comparison with transformed
cells or tumor tissue. Interestingly, wild-type p53 appears to be
important in regulating cell growth and division. Overexpression of
wild-type p53 has been shown in some cases to be anti-proliferative
in human tumor cell lines. Thus, p53 can act as a negative
regulator of cell growth (Weinberg, 1991) and may directly suppress
uncontrolled cell growth or indirectly activate genes that suppress
this growth. Thus, absence or inactivation of wild-type p53 may
contribute to transformation. However, some studies indicate that
the presence of mutant p53 may be necessary for full expression of
the transforming potential.
[0143] Wild-type p53 is recognized as an important growth regulator
in many cell types. Missense mutations are common for the p53 gene
and are essential for the transforming ability of the oncogene. A
single genetic change prompted by point mutations can create
carcinogenic p53. Unlike other oncogenes, however, p53 point
mutations are known to occur in at least 30 distinct codons, often
creating dominant alleles that produce shifts in cell phenotype
without a reduction to homozygosity. Additionally, many of these
dominant negative alleles appear to be tolerated in the organism
and passed on in the germ line. Various mutant alleles appear to
range from minimally dysfunctional to strongly penetrant, dominant
negative alleles (Weinberg, 1991).
[0144] Casey and colleagues have reported that transfection of DNA
encoding wild-type p53 into two human breast cancer cell lines
restores growth suppression control in such cells (Casey et al.,
1991). A similar effect has also been demonstrated on transfection
of wild-type, but not mutant, p53 into human lung cancer cell lines
(Takahasi et al., 1992). p53 appears dominant over the mutant gene
and will select against proliferation when transfected into cells
with the mutant gene. Normal expression of the transfected p53 does
not affect the growth of cells with endogenous p53. Thus, such
constructs might be taken up by normal cells without adverse
effects. It is thus proposed that the treatment of p53-associated
cancers with wild-type p53 or other therapies described herein will
reduce the number of malignant cells or their growth rate,
alternatively the treatment will result in the decrease of the
metastatic potential of the cancer cell, a decrease in tumor size
or a halt in the growth the tumor.
[0145] b. p16
[0146] The major transitions of the eukaryotic cell cycle are
triggered by cyclin-dependent kinases, or CDK's. One CDK,
cyclin-dependent kinase 4 (CDK4), regulates progression through the
G.sub.1. The activity of this enzyme may be to phosphorylate Rb at
late G.sub.1. The activity of CDK4 is controlled by an activating
subunit, D-type cyclin, and by an inhibitory subunit, the
p16.sup.INK4 has been biochemically characterized as a protein that
specifically binds to and inhibits CDK4, and thus may regulate Rb
phosphorylation (Serrano et al., 1993; Serrano et al., 1995). Since
the p16.sup.INK4 protein is a CDK4 inhibitor (Serrano, 1993),
deletion of this gene may increase the activity of CDK4, resulting
in hyperphosphorylation of the Rb protein. p16 also is known to
regulate the function of CDK6.
[0147] p16.sup.INK4 belongs to a newly described class of
CDK-inhibitory proteins that also includes p16.sup.B, p21.sup.WAF1,
and p27.sup.KIP1. The p16.sup.INK4 gene maps to 9p21, a chromosome
region frequently deleted in many tumor types. Homozygous deletions
and mutations of the p16.sup.INK4 gene are frequent in human tumor
cell lines. This evidence suggests that the p16.sup.INK4 gene is a
tumor suppressor gene. This interpretation has been challenged,
however, by the observation that the frequency of the p16.sup.INK4
gene alterations is much lower in primary uncultured tumors than in
cultured cell lines (Caldas et al., 1994; Cheng et al., 1994;
Hussussian et al., 1994; Kamb et al., 1994; Kamb et al., 1994; Mori
et al., 1994; Okamoto et al., 1994; Nobori et al., 1995; Orlow et
al., 1994; Arap et al., 1995). Restoration of wild-type
p16.sup.INK4 function by transfection with a plasmid expression
vector reduced colony formation by some human cancer cell lines
(Okamoto, 1994; Arap, 1995).
[0148] c. C-CAM
[0149] C-CAM is expressed in virtually all epithelial cells (Odin
and Obrink, 1987). C-CAM, with an apparent molecular weight of 105
kD, was originally isolated from the plasma membrane of the rat
hepatocyte by its reaction with specific antibodies that neutralize
cell aggregation (Obrink, 1991). Recent studies indicate that,
structurally, C-CAM belongs to the immunoglobulin (Ig) superfamily
and its sequence is highly homologous to carcinoembryonic antigen
(CEA) (Lin and Guidotti, 1989). Using a baculovirus expression
system, Cheung et al. (1993) demonstrated that the first Ig domain
of C-CAM is critical for cell adhesive activity.
[0150] Cell adhesion molecules, or CAM's are known to be involved
in a complex network of molecular interactions that regulate organ
development and cell differentiation (Edelman, 1985). Recent data
indicate that aberrant expression of CAM's maybe involved in the
tumorigenesis of several neoplasms; for example, decreased
expression of E-cadherin, which is predominantly expressed in
epithelial cells, is associated with the progression of several
kinds of neoplasms (Edelman and Crossin, 1991; Frixen et al., 1991;
Bussemakers et al., 1992; Matsura et al., 1992; Umbas et al.,
1992). Also, Giancotti and Ruoslahti (1990) demonstrated that
increasing expression of .alpha..sub.5.beta..sub.1 integrin by gene
transfer can reduce tumorigenicity of Chinese hamster ovary cells
in vivo. C-CAM now has been shown to suppress tumors growth in
vitro and in vivo.
[0151] d. Other Tumor Suppressors
[0152] Other tumor suppressors that may be employed according to
the present invention include RB, APC, DCC, NF-1, NF-2, WT-1,
MEN-I, MEN-II, zac1, p73, VHL, MMAC1, FCC and MCC. Additional
inducers of apoptosis in addition to those of the Bcl-2 family,
such as, Ad E1B and ICE-CED3 proteases, similarly could find use
according to the present invention.
[0153] e. Enzymes
[0154] Various enzyme genes are of interest according to the
present invention. Such enzymes include cytosine deaminase,
hypoxanthine-guanine phosphoribosyltransferase,
galactose-1-phosphate uridyltransferase, phenylalanine hydroxylase,
glucocerbrosidase, sphingomyelinase, .alpha.-L-iduronidase,
glucose-6-phosphate dehydrogenase, HSV thymidine kinase and human
thymidine kinase.
[0155] f. Cytokines
[0156] Other classes of genes that are contemplated to be inserted
into the therapeutic expression constructs of the present invention
include interleukins and cytokines. Interleukin 1 (IL-1), IL-2,
IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12,
GM-CSF and G-CSF.
[0157] g. Antibodies
[0158] In yet another embodiment, the heterologous gene may include
a single-chain antibody. Methods for the production of single-chain
antibodies are well known to those of skill in the art. The skilled
artisan is referred to U.S. Pat. No. 5,359,046, (incorporated
herein by reference) for such methods. A single chain antibody is
created by fusing together the variable domains of the heavy and
light chains using a short peptide linker, thereby reconstituting
an antigen binding site on a single molecule.
[0159] Single-chain antibody variable fragments (Fvs) in which the
C-terminus of one variable domain is tethered to the N-terminus of
the other via a 15 to 25 amino acid peptide or linker, have been
developed without significantly disrupting antigen binding or
specificity of the binding (Bedzyk et al., 1990; Chaudhary et al.,
1990). These Fvs lack the constant regions (Fc) present in the
heavy and light chains of the native antibody.
[0160] Antibodies to a wide variety of molecules can be used in
combination with the present invention, including antibodies
against oncogenes, toxins, hormones, enzymes, viral or bacterial
antigens, transcription factors, receptors and the like.
[0161] ii. Antisense Constructs
[0162] Oncogenes such as ras, myc, neu, raf erb, src, fms, jun,
trk, ret, gsp, hst, and abl as well as the antiapoptotic member of
the Bcl-2 family also are suitable targets. However, for
therapeutic benefit, these oncogenes would be expressed as an
antisense nucleic acid, so as to inhibit the expression of the
oncogene. The term "antisense nucleic acid" is intended to refer to
the oligonucleotides complementary to the base sequences of
oncogene-encoding DNA and RNA. Antisense oligonucleotides, when
introduced into a target cell, specifically bind to their target
nucleic acid and interfere with transcription, RNA processing,
transport and/or translation. Targeting double-stranded (ds) DNA
with oligonucleotide leads to triple-helix formation; targeting RNA
will lead to double-helix formation.
[0163] Antisense constructs may be designed to bind to the promoter
and other control regions, exons, introns or even exon-intron
boundaries of a gene. Antisense RNA constructs, or DNA encoding
such antisense RNAs, may be employed to inhibit gene transcription
or translation or both within a host cell, either in vitro or in
vivo, such as within a host animal, including a human subject.
Nucleic acid sequences comprising "complementary nucleotides" are
those which are capable of base-pairing according to the standard
Watson-Crick complementary rules. That is, that the larger purines
will base pair with the smaller pyrimidines to form only
combinations of guanine paired with cytosine (G:C) and adenine
paired with either thymine (A:T), in the case of DNA, or adenine
paired with uracil (A:U) in the case of RNA.
[0164] As used herein, the terms "complementary" or "antisense
sequences" mean nucleic acid sequences that are substantially
complementary over their entire length and have very few base
mismatches. For example, nucleic acid sequences of fifteen bases in
length may be termed complementary when they have a complementary
nucleotide at thirteen or fourteen positions with only single or
double mismatches. Naturally, nucleic acid sequences which are
"completely complementary" will be nucleic acid sequences which are
entirely complementary throughout their entire length and have no
base mismatches.
[0165] While all or part of the gene sequence may be employed in
the context of antisense construction, statistically, any sequence
17 bases long should occur only once in the human genome and,
therefore, suffice to specify a unique target sequence. Although
shorter oligomers are easier to make and increase in vivo
accessibility, numerous other factors are involved in determining
the specificity of hybridization. Both binding affinity and
sequence specificity of an oligonucleotide to its complementary
target increases with increasing length. It is contemplated that
oligonucleotides of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20 or more base pairs will be used. One can readily determine
whether a given antisense nucleic acid is effective at targeting of
the corresponding host cell gene simply by testing the constructs
in vitro to determine whether the endogenous gene's function is
affected or whether the expression of related genes having
complementary sequences is affected.
[0166] In certain embodiments, one may wish to employ antisense
constructs which include other elements, for example, those which
include C-5 propyne pyrimidines. Oligonucleotides which contain C-5
propyne analogues of uridine and cytidine have been shown to bind
RNA with high affinity and to be potent antisense inhibitors of
gene expression (Wagner et al., 1993).
[0167] iii. Ribozyme Constructs
[0168] As an alternative to targeted antisense delivery, targeted
ribozymes may be used. The term "ribozyme" refers to an RNA-based
enzyme capable of targeting and cleaving particular base sequences
in oncogene DNA and RNA. Ribozymes either can be targeted directly
to cells, in the form of RNA oligo-nucleotides incorporating
ribozyme sequences, or introduced into the cell as an expression
construct encoding the desired ribozymal RNA. Ribozymes may be used
and applied in much the same way as described for antisense nucleic
acids.
[0169] iv. Selectable Markers
[0170] In certain embodiments of the invention, the therapeutic
expression constructs of the present invention contain nucleic acid
constructs whose expression may be identified in vitro or in vivo
by including a marker in the expression construct. Such markers
would confer an identifiable change to the cell permitting easy
identification of cells containing the expression construct.
Usually the inclusion of a drug selection marker aids in cloning
and in the selection of transformants. For example, genes that
confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT,
zeocin and histidinol are useful selectable markers. Alternatively,
enzymes such as herpes simplex virus thymidine kinase (tk) may be
employed. Immunologic markers also can be employed. The selectable
marker employed is not believed to be important, so long as it is
capable of being expressed simultaneously with the nucleic acid
encoding a gene product. Further examples of selectable markers are
well known to one of skill in the art and include reporters such as
EGFP, .beta.-gal or chloramphenicol acetyltransferase (CAT).
[0171] v. Multigene Constructs and IRES
[0172] In certain embodiments of the invention, the use of internal
ribosome binding sites (IRES) elements are used to create multigene
polycistronic messages. IRES elements are able to bypass the
ribosome scanning model of 5'-methylated, Cap-dependent translation
and begin translation at internal sites (Pelletier and Sonenberg,
1988). IRES elements from two members of the picanovirus family
(polio and encephalomyocarditis) have been described (Pelletier and
Sonenberg, 1988), as well an IRES from a mammalian message (Macejak
and Sarnow, 1991). IRES elements can be linked to heterologous open
reading frames. Multiple open reading frames can be transcribed
together, each separated by an IRES, creating polycistronic
messages. By virtue of the IRES element, each open reading frame is
accessible to ribosomes for efficient translation. Multiple genes
can be efficiently expressed using a single promoter/enhancer to
transcribe a single message.
[0173] Any heterologous open reading frame can be linked to IRES
elements. This includes genes for secreted proteins, multi-subunit
proteins, encoded by independent genes, intracellular or
membrane-bound proteins and selectable markers. In this way,
expression of several proteins can be simultaneously engineered
into a cell with a single construct and a single selectable
marker.
[0174] vi. Control Regions
[0175] A. Promoters
[0176] Throughout this application, the term "expression construct"
is meant to include any type of genetic construct containing a
nucleic acid coding for gene products in which part or all of the
nucleic acid encoding sequence is capable of being transcribed. The
transcript may be translated into a protein, but it need not be. In
certain embodiments, expression includes both transcription of a
gene and translation of mRNA into a gene product. In other
embodiments, expression only includes transcription of the nucleic
acid encoding genes of interest.
[0177] The nucleic acid encoding a gene product is under
transcriptional control of a promoter. A "promoter" refers to a DNA
sequence recognized by the machinery of the cell, or introduced
machinery, required to initiate the specific transcription of a
gene. The phrase "under transcriptional control" means that the
promoter is in the correct location and orientation in relation to
the nucleic acid to control RNA polymerase initiation and
expression of the gene.
[0178] The term promoter will be used here to refer to a group of
transcriptional control modules that are clustered around the
initiation site for RNA polymerase II. Much of the thinking about
how promoters are organized derives from analyses of several viral
promoters, including those for the HSV thymidine kinase (tk) and
SV40 early transcription units. These studies, augmented by more
recent work, have shown that promoters are composed of discrete
functional modules, each consisting of approximately 7-20 bp of
DNA, and containing one or more recognition sites for
transcriptional activator or repressor proteins.
[0179] At least one module in each promoter functions to position
the start site for RNA synthesis. The best known example of this is
the TATA box, but in some promoters lacking a TATA box, such as the
promoter for the mammalian terminal deoxynucleotidyl transferase
gene and the promoter for the SV40 late genes, a discrete element
overlying the start site itself helps to fix the place of
initiation.
[0180] Additional promoter elements regulate the frequency of
transcriptional initiation. Typically, these are located in the
region 30-110 bp upstream of the start site, although a number of
promoters have recently been shown to contain functional elements
downstream of the start site as well. The spacing between promoter
elements frequently is flexible, so that promoter function is
preserved when elements are inverted or moved relative to one
another. In the tk promoter, the spacing between promoter elements
can be increased to 50 bp apart before activity begins to decline.
Depending on the promoter, it appears that individual elements can
function either co-operatively or independently to activate
transcription.
[0181] The particular promoter employed to control the expression
of a nucleic acid sequence of interest is not believed to be
important, so long as it is capable of directing the expression of
the nucleic acid in the targeted cell. Thus, where a human cell is
targeted, it is preferable to position the nucleic acid coding
region adjacent to and under the control of a promoter that is
capable of being expressed in a human cell. Generally speaking,
such a promoter might include either a human or viral promoter.
[0182] In various embodiments, the human cytomegalovirus (CMV)
immediate early gene promoter, the SV40 early promoter, the Rous
sarcoma virus long terminal repeat, .beta.-actin, rat insulin
promoter and glyceraldehyde-3-phosphate dehydrogenase can be used
to obtain high-level expression of the coding sequence of interest.
The use of other viral or mammalian cellular or bacterial phage
promoters which are well-known in the art to achieve expression of
a coding sequence of interest is contemplated as well, provided
that the levels of expression are sufficient for a given purpose.
By employing a promoter with well-known properties, the level and
pattern of expression of the protein of interest following
transfection or transformation can be optimized.
[0183] Selection of a promoter that is regulated in response to
specific physiologic or synthetic signals can permit inducible
expression of the gene product. For example in the case where
expression of a transgene, or transgenes when a multicistronic
vector is utilized, is toxic to the cells in which the vector is
produced in, it may be desirable to prohibit or reduce expression
of one or more of the transgenes. Examples of transgenes that may
be toxic to the producer cell line are pro-apoptotic and cytokine
genes. Several inducible promoter systems are available for
production of viral vectors where the transgene product may be
toxic.
[0184] The ecdysone system (Invitrogen, Carlsbad, Calif.) is one
such system. This system is designed to allow regulated expression
of a gene of interest in mammalian cells. It consists of a tightly
regulated expression mechanism that allows virtually no basal level
expression of the transgene, but over 200-fold inducibility. The
system is based on the heterodimeric ecdysone receptor of
Drosophila, and when ecdysone or an analog such as muristerone A
binds to the receptor, the receptor activates a promoter to turn on
expression of the downstream transgene high levels of mRNA
transcripts are attained. In this system, both monomers of the
heterodimeric receptor are constituitively expressed from one
vector, whereas the ecdysone-responsive promoter which drives
expression of the gene of interest is on another plasmid.
Engineering of this type of system into the gene transfer vector of
interest would therefore be useful. Cotransfection of plasmids
containing the gene of interest and the receptor monomers in the
producer cell line would then allow for the production of the gene
transfer vector without expression of a potentially toxic
transgene. At the appropriate time, expression of the transgene
could be activated with ecdysone or muristeron A.
[0185] Another inducible system that would be useful is the
Tet-Off.TM. or Tet-On.TM. system (Clontech, Palo Alto, Calif.)
originally developed by Gossen and Bujard (Gossen and Bujard, 1992;
Gossen et al., 1995). This system also allows high levels of gene
expression to be regulated in response to tetracycline or
tetracycline derivatives such as doxycycline. In the Tet-On.TM.
system, gene expression is turned on in the presence of
doxycycline, whereas in the Tet-Off.TM. system, gene expression is
turned on in the absence of doxycycline. These systems are based on
two regulatory elements derived from the tetracycline resistance
operon of E. coli. The tetracycline operator sequence to which the
tetracycline repressor binds, and the tetracycline repressor
protein. The gene of interest is cloned into a plasmid behind a
promoter that has tetracycline-responsive elements present in it. A
second plasmid contains a regulatory element called the
tetracycline-controlled transactivator, which is composed, in the
Tet-Off.TM. system, of the VP16 domain from the herpes simplex
virus and the wild-type tertracycline repressor. Thus in the
absence of doxycycline, transcription is constituitively on. In the
Tet-On.TM. system, the tetracycline repressor is not wild type and
in the presence of doxycycline activates transcription. For gene
therapy vector production, the Tet-Off.TM. system would be
preferable so that the producer cells could be grown in the
presence of tetracycline or doxycycline and prevent expression of a
potentially toxic transgene, but when the vector is introduced to
the patient, the gene expression would be constituitively on.
[0186] In some circumstances, it may be desirable to regulate
expression of a transgene in a gene therapy vector. For example,
different viral promoters with varying strengths of activity may be
utilized depending on the level of expression desired. In mammalian
cells, the CMV immediate early promoter if often used to provide
strong transcriptional activation. Modified versions of the CMV
promoter that are less potent have also been used when reduced
levels of expression of the transgene are desired. When expression
of a transgene in hematopoetic cells is desired, retroviral
promoters such as the LTRs from MLV or MMTV are often used. Other
viral promoters that may be used depending on the desired effect
include SV40, RSV LTR, HIV-1 and HIV-2 LTR, adenovirus promoters
such as from the E1A, E2A, or MLP region, AAV LTR, cauliflower
mosaic virus, HSV-TK, and avian sarcoma virus.
[0187] Similarly tissue specific promoters may be used to effect
transcription in specific tissues or cells so as to reduce
potential toxicity or undesirable effects to non-targeted tissues.
For example, promoters such as the PSA, probasin, prostatic acid
phosphatase or prostate-specific glandular kallikrein (hK2) may be
used to target gene expression in the prostate. Similarly, the
following promoters may be used to target gene expression in other
tissues (Table 2). TABLE-US-00002 TABLE 2 Tissue specific promoters
Tissue Promoter Pancreas insulin elastin amylase pdr-1 pdx-1
glucokinase Liver albumin PEPCK HBV enhancer alpha fetoprotein
apolipoprotein C alpha-1 antitrypsin vitellogenin, NF-AB
Transthyretin Skeletal muscle myosin H chain muscle creatine kinase
dystrophin calpain p94 skeletal alpha-actin fast troponin 1 Skin
keratin K6 keratin K1 Lung CFTR human cytokeratin 18 (K18)
pulmonary surfactant proteins A, B and C CC-10 P1 Smooth muscle
sm22 alpha SM-alpha-actin Endothelium endothelin-1 E-selectin von
Willebrand factor TIE (Korhonen et al., 1995) KDR/flk-1 Melanocytes
tyrosinase Adipose tissue lipoprotein lipase (Zechner et al., 1988)
adipsin (Spiegelman et al., 1989) acetyl-CoA carboxylase (Pape and
Kim, 1989) glycerophosphate dehydrogenase (Dani et al., 1989)
adipocyte P2 (Hunt et al., 1986) Blood .beta.-globin
[0188] In certain indications, it may be desirable to activate
transcription at specific times after administration of the gene
therapy vector. This may be done with such promoters as those that
are hormone or cytokine regulatable. For example in gene therapy
applications where the indication is a gonadal tissue where
specific steroids are produced or routed to, use of androgen or
estrogen regulated promoters may be advantageous. Such promoters
that are hormone regulatable include MMTV, MT-1, ecdysone and
RuBisco. Other hormone regulated promoters such as those responsive
to thyroid, pituitary and adrenal hormones are expected to be
useful in the present invention. Cytokine and inflammatory protein
responsive promoters that could be used include K and T Kininogen
(Kageyama et al., 1987), c-fos, TNF-alpha, C-reactive protein
(Arcone et al., 1988), haptoglobin (Oliviero et al., 1987), serum
amyloid A2, C/EBP alpha, IL-1, IL-6 (Poli and Cortese, 1989),
Complement C3 (Wilson et al., 1990), IL-8, alpha-1 acid
glycoprotein (Prowse and Baumann, 1988), alpha-1 antitypsin,
lipoprotein lipase (Zechner et al., 1988), angiotensinogen (Ron et
al., 1991), fibrinogen, c-jun (inducible by phorbol esters,
TNF-alpha, UV radiation, retinoic acid, and hydrogen peroxide),
collagenase (induced by phorbol esters and retinoic acid),
metallothionein (heavy metal and glucocorticoid inducible),
Stromelysin (inducible by phorbol ester, interleukin-1 and EGF),
alpha-2 macroglobulin and alpha-1 antichymotrypsin.
[0189] It is envisioned that cell cycle regulatable promoters may
be useful in the present invention. For example, in a bi-cistronic
gene therapy vector, use of a strong CMV promoter to drive
expression of a first gene such as p16 that arrests cells in the G1
phase could be followed by expression of a second gene such as p53
under the control of a promoter that is active in the G1 phase of
the cell cycle, thus providing a "second hit" that would push the
cell into apoptosis. Other promoters such as those of various
cyclins, PCNA, galectin-3, E2F1, p53 and BRCA1 could be used.
[0190] Tumor specific promoters such as osteocalcin,
hypoxia-responsive element (HRE), MAGE-4, CEA, alpha-fetoprotein,
GRP78/BiP and tyrosinase may also be used to regulate gene
expression in tumor cells. Other promoters that could be used
according to the present invention include Lac-regulatable,
chemotherapy inducible (e.g. MDR), and heat (hyperthermia)
inducible promoters, Radiation-inducible (e.g., EGR (Joki et al.,
1995)), Alpha-inhibin, RNA pol III tRNA met and other amino acid
promoters, U1 snRNA (Bartlett et al., 1996), MC-1, PGK, -actin and
alpha-globin. Many other promoters that may be useful are listed in
Walther and Stein (1996).
[0191] It is envisioned that any of the above promoters alone or in
combination with another may be useful according to the present
invention depending on the action desired. In addition, this list
of promoters is should not be construed to be exhaustive or
limiting, those of skill in the art will know of other promoters
that may be used in conjunction with the promoters and methods
disclosed herein.
[0192] B. Enhancers
[0193] Enhancers are genetic elements that increase transcription
from a promoter located at a distant position on the same molecule
of DNA. Enhancers are organized much like promoters. That is, they
are composed of many individual elements, each of which binds to
one or more transcriptional proteins. The basic distinction between
enhancers and promoters is operational. An enhancer region as a
whole must be able to stimulate transcription at a distance; this
need not be true of a promoter region or its component elements. On
the other hand, a promoter must have one or more elements that
direct initiation of RNA synthesis at a particular site and in a
particular orientation, whereas enhancers lack these specificities.
Promoters and enhancers are often overlapping and contiguous, often
seeming to have a very similar modular organization.
[0194] Below is a list of promoters additional to the tissue
specific promoters listed above, cellular promoters/enhancers and
inducible promoters/enhancers that could be used in combination
with the nucleic acid encoding a gene of interest in an expression
construct (Table 3 and Table 4). Additionally, any
promoter/enhancer combination (as per the Eukaryotic Promoter Data
Base EPDB) could also be used to drive expression of the gene.
Eukaryotic cells can support cytoplasmic transcription from certain
bacterial promoters if the appropriate bacterial polymerase is
provided, either as part of the delivery complex or as an
additional genetic expression construct. TABLE-US-00003 TABLE 3
ENHANCER Immunoglobulin Heavy Chain Immunoglobulin Light Chain
T-Cell Receptor HLA DQ .alpha. and DQ .beta. .beta.-Interferon
Interleukin-2 Interleukin-2 Receptor MHC Class II 5 MHC Class II
HLA-DR.alpha. .beta.-Actin Muscle Creatine Kinase Prealbumin
(Transthyretin) Elastase I Metallothionein Collagenase Albumin Gene
.alpha.-Fetoprotein .tau.-Globin .beta.-Globin e-fos c-HA-ras
Insulin Neural Cell Adhesion Molecule (NCAM) .alpha.1-Antitrypsin
H2B (TH2B) Histone Mouse or Type I Collagen
Glucose-RegulatedProteins (GRP94 and GRP78) Rat Growth Hormone
Human Serum Amyloid A (SAA) Troponin I (TN I) Platelet-Derived
Growth Factor Duchenne Muscular Dystrophy SV40 Polyoma Retroviruses
Papilloma Virus Hepatitis B Virus Human Immunodeficiency Virus
Cytomegalovirus Gibbon Ape Leukemia Virus
[0195] TABLE-US-00004 TABLE 4 Element Inducer MT II Phorbol Ester
(TPA) Heavy metals MMTV (mouse mammary tumor Glucocorticoids virus)
.beta.-Interferon poly(rI)X poly(rc) Adenovirus 5 E2 Ela c-jun
Phorbol Ester (TPA), H.sub.2O.sub.2 Collagenase Phorbol Ester (TPA)
Stromelysin Phorbol Ester (TPA), IL-1 SV40 Phorbol Ester (TPA)
Murine MX Gene Interferon, Newcastle Disease Virus GRP78 Gene
A23187 .alpha.-2-Macroglobulin IL-6 Vimentin Serum MHC Class I Gene
H-2kB Interferon HSP70 Ela, SV40 Large T Antigen Proliferin Phorbol
Ester-TPA Tumor Necrosis Factor FMA Thyroid Stimulating Hormone
.alpha. Thyroid Hormone Gene Insulin E Box Glucose
[0196] In preferred embodiments of the invention, the expression
construct comprises a virus or engineered construct derived from a
viral genome. The ability of certain viruses to enter cells via
receptor-mediated endocytosis and to integrate into host cell
genome and express viral genes stably and efficiently have made
them attractive candidates for the transfer of foreign genes into
mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988;
Baichwal and Sugden, 1986; Temin, 1986). The first viruses used as
gene vectors were DNA viruses including the papovaviruses (simian
virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988;
Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988;
Baichwal and Sugden, 1986). These have a relatively low capacity
for foreign DNA sequences and have a restricted host spectrum.
Furthermore, their oncogenic potential and cytopathic effects in
permissive cells raise safety concerns. They can accommodate only
up to 8 kB of foreign genetic material but can be readily
introduced in a variety of cell lines and laboratory animals
(Nicolas and Rubenstein, 1988; Temin, 1986).
[0197] C. Polyadenylation Signals
[0198] Where a cDNA insert is employed, one will typically desire
to include a polyadenylation signal to effect proper
polyadenylation of the gene transcript. The nature of the
polyadenylation signal is not believed to be crucial to the
successful practice of the invention, and any such sequence may be
employed such as human or bovine growth hormone and SV40
polyadenylation signals. Also contemplated as an element of the
expression cassette is a terminator. These elements can serve to
enhance message levels and to minimize read through from the
cassette into other sequences.
E. Methods of Gene Transfer
[0199] In order to mediate the effect transgene expression in a
cell, it will be necessary to transfer the therapeutic expression
constructs of the present invention into a cell. Such transfer may
employ viral or non-viral methods of gene transfer. This section
provides a discussion of methods and compositions of gene
transfer.
[0200] i. Viral Vector-Mediated Transfer
[0201] The proapoptotic Bcl-2 genes are incorporated into an
adenoviral infectious particle to mediate gene transfer to a cell.
Additional expression constructs encoding other therapeutic agents
as described herein may also be transferred via viral transduction
using infectious viral particles, for example, by transformation
with an adenovirus vector of the present invention as described
herein below. Alternatively, retroviral or bovine papilloma virus
may be employed, both of which permit permanent transformation of a
host cell with a gene(s) of interest. Thus, in one example, viral
infection of cells is used in order to deliver therapeutically
significant genes to a cell. Typically, the virus simply will be
exposed to the appropriate host cell under physiologic conditions,
permitting uptake of the virus. Though adenovirus is exemplified,
the present methods may be advantageously employed with other viral
vectors, as discussed below.
[0202] Adenovirus. Adenovirus is particularly suitable for use as a
gene transfer vector because of its mid-sized DNA genome, ease of
manipulation, high titer, wide target-cell range, and high
infectivity. The roughly 36 kB viral genome is bounded by 100-200
base pair (bp) inverted terminal repeats (ITR), in which are
contained cis-acting elements necessary for viral DNA replication
and packaging. The early (E) and late (L) regions of the genome
that contain different transcription units are divided by the onset
of viral DNA replication.
[0203] The E1 region (E1A and E1B) encodes proteins responsible for
the regulation of transcription of the viral genome and a few
cellular genes. The expression of the E2 region (E2A and E2B)
results in the synthesis of the proteins for viral DNA replication.
These proteins are involved in DNA replication, late gene
expression, and host cell shut off (Renan, 1990). The products of
the late genes (L1, L2, L3, L4 and L5), including the majority of
the viral capsid proteins, are expressed only after significant
processing of a single primary transcript issued by the major late
promoter (MLP). The MLP (located at 16.8 map units) is particularly
efficient during the late phase of infection, and all the mRNAs
issued from this promoter possess a 5' tripartite leader (TL)
sequence which makes them preferred mRNAs for translation.
[0204] In order for adenovirus to be optimized for gene therapy, it
is necessary to maximize the carrying capacity so that large
segments of DNA can be included. It also is very desirable to
reduce the toxicity and immunologic reaction associated with
certain adenoviral products. The two goals are, to an extent,
coterminous in that elimination of adenoviral genes serves both
ends. By practice of the present invention, it is possible achieve
both these goals while retaining the ability to manipulate the
therapeutic constructs with relative ease.
[0205] The large displacement of DNA is possible because the cis
elements required for viral DNA replication all are localized in
the inverted terminal repeats (ITR) (100-200 bp) at either end of
the linear viral genome. Plasmids containing ITR's can replicate in
the presence of a non-defective adenovirus (Hay et al., 1984).
Therefore, inclusion of these elements in an adenoviral vector
should permit replication.
[0206] In addition, the packaging signal for viral encapsidation is
localized between 194-385 bp (0.5-1.1 map units) at the left end of
the viral genome (Hearing et al., 1987). This signal mimics the
protein recognition site in bacteriophage .lamda. DNA where a
specific sequence close to the left end, but outside the cohesive
end sequence, mediates the binding to proteins that are required
for insertion of the DNA into the head structure. E1 substitution
vectors of Ad have demonstrated that a 450 bp (0-1.25 map units)
fragment at the left end of the viral genome could direct packaging
in 293 cells (Levrero et al., 1991).
[0207] Previously, it has been shown that certain regions of the
adenoviral genome can be incorporated into the genome of mammalian
cells and the genes encoded thereby expressed. These cell lines are
capable of supporting the replication of an adenoviral vector that
is deficient in the adenoviral function encoded by the cell line.
There also have been reports of complementation of replication
deficient adenoviral vectors by "helping" vectors, e.g., wild-type
virus or conditionally defective mutants.
[0208] Replication-deficient adenoviral vectors can be
complemented, in trans, by helper virus. This observation alone
does not permit isolation of the replication-deficient vectors,
however, since the presence of helper virus, needed to provide
replicative functions, would contaminate any preparation. Thus, an
additional element was needed that would add specificity to the
replication and/or packaging of the replication-deficient vector.
That element, as provided for in the present invention, derives
from the packaging function of adenovirus.
[0209] It has been shown that a packaging signal for adenovirus
exists in the left end of the conventional adenovirus map
(Tibbetts, 1977). Later studies showed that a mutant with a
deletion in the E1A (194-358 bp) region of the genome grew poorly
even in a cell line that complemented the early (E1A) function
(Hearing and Shenk, 1983). When a compensating adenoviral DNA
(0-353 bp) was recombined into the right end of the mutant, the
virus was packaged normally. Further mutational analysis identified
a short, repeated, position-dependent element in the left end of
the Ad5 genome. One copy of the repeat was found to be sufficient
for efficient packaging if present at either end of the genome, but
not when moved towards the interior of the Ad5 DNA molecule
(Hearing et al, 1987).
[0210] By using mutated versions of the packaging signal, it is
possible to create helper viruses that are packaged with varying
efficiencies. Typically, the mutations are point mutations or
deletions. When helper viruses with low efficiency packaging are
grown in helper cells, the virus is packaged, albeit at reduced
rates compared to wild-type virus, thereby permitting propagation
of the helper. When these helper viruses are grown in cells along
with virus that contains wild-type packaging signals, however, the
wild-type packaging signals are recognized preferentially over the
mutated versions. Given a limiting amount of packaging factor, the
virus containing the wild-type signals are packaged selectively
when compared to the helpers. If the preference is great enough,
stocks approaching homogeneity should be achieved.
[0211] Retrovirus. The retroviruses are a group of single-stranded
RNA viruses characterized by an ability to convert their RNA to
double-stranded DNA in infected cells by a process of
reverse-transcription (Coffin, 1990). The resulting DNA then stably
integrates into cellular chromosomes as a provirus and directs
synthesis of viral proteins. The integration results in the
retention of the viral gene sequences in the recipient cell and its
descendants. The retroviral genome contains three genes--gag, pol
and env--that code for capsid proteins, polymerase enzyme, and
envelope components, respectively. A sequence found upstream from
the gag gene, termed .PSI., functions as a signal for packaging of
the genome into virions. Two long terminal repeat (LTR) sequences
are present at the 5' and 3' ends of the viral genome. These
contain strong promoter and enhancer sequences and also are
required for integration in the host cell genome (Coffin,
1990).
[0212] In order to construct a retroviral vector, a nucleic acid
encoding a promoter is inserted into the viral genome in the place
of certain viral sequences to produce a virus that is
replication-defective. In order to produce virions, a packaging
cell line containing the gag, pol and env genes but without the LTR
and .PSI. components is constructed (Mann et al., 1983). When a
recombinant plasmid containing a human cDNA, together with the
retroviral LTR and .PSI. sequences is introduced into this cell
line (by calcium phosphate precipitation for example), the .PSI.
sequence allows the RNA transcript of the recombinant plasmid to be
packaged into viral particles, which are then secreted into the
culture media (Nicolas and Rubenstein, 1988; Temin, 1986; Mann et
al., 1983). The media containing the recombinant retroviruses is
collected, optionally concentrated, and used for gene transfer.
Retroviral vectors are able to infect a broad variety of cell
types. However, integration and stable expression of many types of
retroviruses require the division of host cells (Paskind et al.,
1975).
[0213] An approach designed to allow specific targeting of
retrovirus vectors recently was developed based on the chemical
modification of a retrovirus by the chemical addition of galactose
residues to the viral envelope. This modification could permit the
specific infection of cells such as hepatocytes via
asialoglycoprotein receptors, should this be desired.
[0214] A different approach to targeting of recombinant
retroviruses was designed in which biotinylated antibodies against
a retroviral envelope protein and against a specific cell receptor
were used. The antibodies were coupled via the biotin components by
using streptavidin (Roux et al., 1989). Using antibodies against
major histocompatibility complex class I and class II antigens, the
infection of a variety of human cells that bore those surface
antigens was demonstrated with an ecotropic virus in vitro (Roux et
al., 1989).
[0215] Adeno-associated Virus. AAV utilizes a linear,
single-stranded DNA of about 4700 base pairs. Inverted terminal
repeats flank the genome. Two genes are present within the genome,
giving rise to a number of distinct gene products. The first, the
cap gene, produces three different virion proteins (VP), designated
VP-1, VP-2 and VP-3. The second, the rep gene, encodes four
non-structural proteins (NS). One or more of these rep gene
products is responsible for transactivating AAV transcription.
[0216] The three promoters in AAV are designated by their location,
in map units, in the genome. These are, from left to right, p5, p19
and p40. Transcription gives rise to six transcripts, two initiated
at each of three promoters, with one of each pair being spliced.
The splice site, derived from map units 42-46, is the same for each
transcript. The four non-structural proteins apparently are derived
from the longer of the transcripts, and three virion proteins all
arise from the smallest transcript.
[0217] AAV is not associated with any pathologic state in humans.
Interestingly, for efficient replication, AAV requires "helping"
functions from viruses such as herpes simplex virus I and II,
cytomegalovirus, pseudorabies virus and, of course, adenovirus. The
best characterized of the helpers is adenovirus, and many "early"
functions for this virus have been shown to assist with AAV
replication. Low level expression of AAV rep proteins is believed
to hold AAV structural expression in check, and helper virus
infection is thought to remove this block.
[0218] The terminal repeats of the AAV vector can be obtained by
restriction endonuclease digestion of AAV or a plasmid such as
p201, which contains a modified AAV genome (Samulski et al. 1987),
or by other methods known to the skilled artisan, including but not
limited to chemical or enzymatic synthesis of the terminal repeats
based upon the published sequence of AAV. The ordinarily skilled
artisan can determine, by well-known methods such as deletion
analysis, the minimum sequence or part of the AAV ITRs which is
required to allow function, i.e., stable and site-specific
integration. The ordinarily skilled artisan also can determine
which minor modifications of the sequence can be tolerated while
maintaining the ability of the terminal repeats to direct stable,
site-specific integration.
[0219] AAV-based vectors have proven to be safe and effective
vehicles for gene delivery in vitro, and these vectors are being
developed and tested in pre-clinical and clinical stages for a wide
range of applications in potential gene therapy, both ex vivo and
in vivo (Carter and Flotte, 1996; Chatterjee et al., 1995; Ferrari
et al., 1996; Fisher et al., 1996; Flotte et al., 1993; Goodman et
al., 1994; Kaplitt et al., 1994; 1996, Kessler et al., 1996;
Koeberl et al., 1997; Mizukami et al., 1996; Xiao et al.,
1996).
[0220] AAV-mediated efficient gene transfer and expression in the
lung has led to clinical trials for the treatment of cystic
fibrosis (Carter and Flotte, 1996; Flotte et al., 1993). Similarly,
the prospects for treatment of muscular dystrophy by AAV-mediated
gene delivery of the dystrophin gene to skeletal muscle, of
Parkinson's disease by tyrosine hydroxylase gene delivery to the
brain, of hemophilia B by Factor 1.times.gene delivery to the
liver, and potentially of myocardial infarction by vascular
endothelial growth factor gene to the heart, appear promising since
AAV-mediated transgene expression in these organs has recently been
shown to be highly efficient (Fisher et al., 1996; Flotte et al.,
1993; Kaplitt et al., 1994; 1996; Koeberl et al., 1997; McCown et
al., 1996; Ping et al., 1996; Xiao et al., 1996).
[0221] Other Viral Vectors. Other viral vectors may be employed as
expression constructs in the present invention. Vectors derived
from viruses such as vaccinia virus (Ridgeway, 1988; Baichwal and
Sugden, 1986; Coupar et al., 1988) canary pox virus, and herpes
viruses may be employed. These viruses offer several features for
use in gene transfer into various mammalian cells.
[0222] ii. Non-Viral Transfer
[0223] DNA constructs of the present invention are generally
delivered to a cell, in certain situations, the nucleic acid to be
transferred is non-infectious, and can be transferred using
non-viral methods.
[0224] Several non-viral methods for the transfer of expression
constructs into cultured mammalian cells are contemplated by the
present invention. These include calcium phosphate precipitation
(Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al.,
1990) DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et
al., 1986; Potter et al., 1984), direct microinjection (Harland and
Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982;
Fraley et al., 1979), cell sonication (Fechheimer et al., 1987),
gene bombardment using high velocity microprojectiles (Yang et al.,
1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and
Wu, 1988).
[0225] Once the construct has been delivered into the cell the
nucleic acid encoding the therapeutic gene may be positioned and
expressed at different sites. In certain embodiments, the nucleic
acid encoding the therapeutic gene may be stably integrated into
the genome of the cell. This integration may be in the cognate
location and orientation via homologous recombination (gene
replacement) or it may be integrated in a random, non-specific
location (gene augmentation). In yet further embodiments, the
nucleic acid may be stably maintained in the cell as a separate,
episomal segment of DNA. Such nucleic acid segments or "episomes"
encode sequences sufficient to permit maintenance and replication
independent of or in synchronization with the host cell cycle. How
the expression construct is delivered to a cell and where in the
cell the nucleic acid remains is dependent on the type of
expression construct employed.
[0226] In a particular embodiment of the invention, the expression
construct may be entrapped in a liposome. Liposomes are vesicular
structures characterized by a phospholipid bilayer membrane and an
inner aqueous medium. Multilamellar liposomes have multiple lipid
layers separated by aqueous medium. They form spontaneously when
phospholipids are suspended in an excess of aqueous solution. The
lipid components undergo self-rearrangement before the formation of
closed structures and entrap water and dissolved solutes between
the lipid bilayers (Ghosh and Bachhawat, 1991). The addition of DNA
to cationic liposomes causes a topological transition from
liposomes to optically birefringent liquid-crystalline condensed
globules (Radler et al., 1997). These DNA-lipid complexes are
potential non-viral vectors for use in gene therapy.
[0227] Liposome-mediated nucleic acid delivery and expression of
foreign DNA in vitro has been very successful. Using the
.beta.-lactamase gene, Wong et al. (1980) demonstrated the
feasibility of liposome-mediated delivery and expression of foreign
DNA in cultured chick embryo, HeLa, and hepatoma cells. Nicolau et
al. (1987) accomplished successful liposome-mediated gene transfer
in rats after intravenous injection. Also included are various
commercial approaches involving "lipofection" technology.
[0228] In certain embodiments of the invention, the liposome may be
complexed with a hemagglutinating virus (HVJ). This has been shown
to facilitate fusion with the cell membrane and promote cell entry
of liposome-encapsulated DNA (Kaneda et al., 1989). In other
embodiments, the liposome may be complexed or employed in
conjunction with nuclear nonhistone chromosomal proteins (HMG-1)
(Kato et al., 1991). In yet further embodiments, the liposome may
be complexed or employed in conjunction with both HVJ and HMG-1. In
that such expression constructs have been successfully employed in
transfer and expression of nucleic acid in vitro and in vivo, then
they are applicable for the present invention.
[0229] Other vector delivery systems which can be employed to
deliver a nucleic acid encoding a therapeutic gene into cells are
receptor-mediated delivery vehicles. These take advantage of the
selective uptake of macromolecules by receptor-mediated endocytosis
in almost all eukaryotic cells. Because of the cell type-specific
distribution of various receptors, the delivery can be highly
specific (Wu and Wu, 1993).
[0230] Receptor-mediated gene targeting vehicles generally consist
of two components: a cell receptor-specific ligand and a
DNA-binding agent. Several ligands have been used for
receptor-mediated gene transfer. The most extensively characterized
ligands are asialoorosomucoid (ASOR) (Wu and Wu, 1987) and
transferring (Wagner et al., 1990). Recently, a synthetic
neoglycoprotein, which recognizes the same receptor as ASOR, has
been used as a gene delivery vehicle (Ferkol et al., 1993; Perales
et al., 1994) and epidermal growth factor (EGF) has also been used
to deliver genes to squamous carcinoma cells (Myers, EPO
0273085).
[0231] In other embodiments, the delivery vehicle may comprise a
ligand and a liposome. For example, Nicolau et al. (1987) employed
lactosyl-ceramide, a galactose-terminal asialganglioside,
incorporated into liposomes and observed an increase in the uptake
of the insulin gene by hepatocytes. Thus, it is feasible that a
nucleic acid encoding a therapeutic gene also may be specifically
delivered into a cell type such as prostate, epithelial or tumor
cells, by any number of receptor-ligand systems with or without
liposomes. For example, the human prostate-specific antigen (Watt
et al., 1986) may be used as the receptor for mediated delivery of
a nucleic acid in prostate tissue.
[0232] In another embodiment of the invention, the expression
construct may simply consist of naked recombinant DNA or plasmids.
Transfer of the construct may be performed by any of the methods
mentioned above which physically or chemically permeabilize the
cell membrane. This is applicable particularly for transfer in
vitro, however, it may be applied for in vivo use as well. Dubensky
et al. (1984) successfully injected polyomavirus DNA in the form of
CaPO.sub.4 precipitates into liver and spleen of adult and newborn
mice demonstrating active viral replication and acute infection.
Benvenisty and Neshif (1986) also demonstrated that direct
intraperitoneal injection of CaPO.sub.4 precipitated plasmids
results in expression of the transfected genes. It is envisioned
that DNA encoding a CAM may also be transferred in a similar manner
in vivo and express CAM.
[0233] Another embodiment of the invention for transferring a naked
DNA expression construct into cells may involve particle
bombardment. This method depends on the ability to accelerate DNA
coated microprojectiles to a high velocity allowing them to pierce
cell membranes and enter cells without killing them (Klein et al.,
1987). Several devices for accelerating small particles have been
developed. One such device relies on a high voltage discharge to
generate an electrical current, which in turn provides the motive
force (Yang et al., 1990). The microprojectiles used have consisted
of biologically inert substances such as tungsten or gold beads
F. Gene Delivery System for Toxic Gene Products
[0234] It is now known that programmed cell death, or apoptosis,
plays an important role in development, homeostasis, and disease
processes. It is contemplated in the present invention, that genes
involved in apoptotic pathways may be useful in the treatment of
diseases related to disorders in these pathways. In another
embodiment, the use of non-pro-apoptic, cytotoxic genes are
contemplated for use in treating hyperproliferative and other
disease states in which cell death would be therapeutic.
[0235] The use of proapoptotic genes to treat cancers was proposed
several years ago (Fisher, 1994; Thompson, 1995). However, the
expression of pro-apoptic genes often results in death of the host
cell if their expression is not regulated. In another embodiment of
the present invention, it is contemplated that a novel co-transfer
vector system is used to permit delivery of vectors that express
potentially toxic genes. For example, the expression of Bcl-2
family member via gene transfer may be valuable in the treatment of
a variety of hyperproliferative diseases, such as cancer. However,
constructing an adenoviral vector expressing a pro-apoptic gene
driven by a constitutive promoter becomes problematic in the
packaging cell, presumably because of its high apoptotic activity
(i.e., cell toxicity).
[0236] In one embodiment, the inventors demonstrate a system for
safely inducing the expression of the bax gene in a host cell by
adenovirus-mediated gene co-transfer. The system provides a first
adenoviral vector containing a human gene wherein the expression
product is cytotoxic. The cytotoxic gene is driven by a promoter,
not active in the host or target cell. A second adenoviral vector
is provided, wherein the gene, under the control of a promoter,
encodes a transactivating protein. Induction of the promoter
driving the expression of the transactivating protein, initiates
the expression of the cytotoxic gene product.
[0237] Experimental data demonstrate that the vector expresses a
minimal background level of bax protein in cultured mammalian cells
thus preventing apoptosis of packaging cells. The expression of the
bax gene was substantially induced both in vitro and in vivo by
transferring it into target cells along with of an adenoviral
vector expressing the transactivator, fusion protein GAL4/VP16.
Thus, adenovirus-mediated gene co-transfer permits the regulated
expression of bax via the inducible expression of the GAL4/VP16
gene. In other embodiments of the invention, the pro-apoptic genes
Bak, Bim, Bik, Bid, Bad and Harakiri are contemplated for use in
adenovirus-mediated gene co-transfer
[0238] Adenovirus-mediated gene co-transfer is not limited to
proapoptotic genes or a specific promoter. It also is contemplated
that co-transfer system could be used to treat various
hyperproliferative diseases, wherein regulating the expression of a
toxic gene product is desired. Depending on the tissue being
treated, a tissue specific promoter could be chosen to permit in
vivo transactivation only in the target tissue. For example,
co-transfer of a tumor suppressor gene linked to a promoter and a
vector expressing a transactivator that specifically binds to the
promoter, would be useful in treating hyperproliferative diseases.
Thus, in one embodiment of the invention, a promoter linked to a
particular gene can be selected to provide tissue specific
expression. Regulated co-transfer expression of other toxic gene
products also are contemplated and discussed below.
[0239] i. Vector Co-Transfer and Promoters
[0240] The use of proapoptotic genes to treat cancers via gene
therapy has not been reported, possibly due to the difficulty in
constructing vectors that can efficiently transduce target cells
with such genes. For example, Larregina et al. showed that
constructing an adenovirus expressing the Fas-Ligand (Fas-L) was
difficult because Fas-L induces apoptosis in 293 packaging cells,
(Larregina et al., 1998). Arai et al. achieved efficient antitumor
activity by adenovirus-mediated Fas-L gene transfer, but this
required the use of 293 cells resistant to Fas-L or caspase
inhibitor for vector production, (Arai et al., 1997).
[0241] The gene co-transfer system of the present invention
overcomes these obstacles, by providing a pro-apoptic gene linked
to a regulatable, promoter. The regulatable promoter prevents
expression of the pro-apoptic gene in the host or packaging cell,
which would result in cell death. The expression of the pro-apoptic
gene is induced by a transactivator protein, carried by a gene on a
second expression vector. In one embodiment of the present
invention, adenovirus-mediated gene co-transfer uses a first
adenovirus comprising human bax cDNA driven by a promoter
consisting of a heptamer of GAL4-binding sites and a TATA box. A
second adenovirus (i.e., co-transfer) comprising the GAL4/VP16
transactivator fusion protein linked to a regulatable promoter
operable in eukaryotic cells, is provided to selectively induce bax
expression. It is contemplated in other embodiments, that the first
promoter can be the ecdysone-responsive promoter or Tet-On.TM. and
the inducer of the first promoter ecdysone or muristeron A and
doxycycline, respectively. For a complete description of the
ecdysone system and Tet-On.TM. see section D, herein. It also is
contemplated, that that the first promoter can be the HIV-1 LTR or
HIV-2 LTR and the inducer of the first promoter tat. It is
contemplated in other embodiments, that yeast, E. coli and insect
promoters may also be useful in the present invention for regulated
expression of cytotoxic genes.
[0242] For human or mammalian cytotoxic gene therapy via
vector-mediated gene co-transfer, the Bcl-2 genes Bak, Bim, Bik,
Bid, Bad and Harakiri are contemplated as useful in the present
invention. Additional cytotoxic gene products contemplated as
useful in the present invention, are described below.
[0243] It is contemplated in the present invention that a gene
encoding a transactivating protein is supplied by a second vector.
In certain embodiments, the gene encoding the transactivating
protein can be linked to a constitutive promoter. In other
embodiments, the gene encoding the transactivating protein can be
under the control of an inducible promoter, permitting regulated
expression of the transactivating protein. Pancreatic, liver,
skeletal muscle, smooth muscle, skin, lung, endothelium and blood
are some examples of tissues in which tissue specific promoters
might be selected for use. Table 2, Table 3 and Table 4 provide a
list of some useful tissue specific promoters, promoter/enhancers
and inducible promoter/enhancers, respectively, that may be used in
combination and are considered useful in the present invention.
[0244] An important consideration when selecting a promoter to
drive the expression of the cytotoxic gene product on the first
vector, is that the transactivating protein (i.e., inducer) is not
active in the host cell. For example, if the host cell expresses a
transactivating protein, capable of activating the promoter on the
first expression vector, upregulation of the cytotoxic gene may
result.
[0245] The vector-mediated co-transfer system is particularly
useful in vivo. In such embodiments, it may be desirable that
transactivating protein also is not active in the target cell. The
presence of the transactivating protein in the target cell would
limit the temporal use of the co-delivery system, as the
transactivating protein would be present at the time of delivery of
the cytotoxic expression construct.
[0246] A variety of transactivating genes theoretically can be
chosen to express transactivating protein factors, to drive the
expression of a toxic gene on the first vector. Another
consideration in choosing a transactivating protein factor is its
efficacy of transcriptional activation in a given tissue type. It
may be that a particular tissue specific transactivating factor has
low levels of cross tissue activity, which could potentially be
cytotoxic to healthy, normal cell or tissue types.
[0247] ii. In Vitro and In Vivo Delivery of Vectors to Target
Cells
[0248] An adenoviral vector expressing a Blc-2 member gene, would
facilitate the therapeutic evaluation of the Bcl-2 member gene,
since such a vector would have potentially high transduction
efficiencies in a variety of tissues. However, constructing an
adenoviral vector that can express bax for example, has been
problematic, presumably because of the bax gene's high apoptotic
activity and its toxic effect on packaging 293 cells (Rosse et al.,
1998). It is contemplated that vector-mediated gene co-transfer of
the present invention will be useful for regulating both in vitro
and in vivo expression of potentially cytotoxic gene products
[0249] In one embodiment of the present invention, the in vitro
expression of therapeutic genes are considered. In one example,
shuttle plasmids in which bax cDNA was driven by a GAL4-responsive
promoter consisting of five GAL4-binding sites and a TATA box (GT)
were constructed. Recombinant viral vectors (Ad) were obtained
after a single in vitro transfection of 293 cells with pAd/GT-Bax
plus a 35-kb ClaI fragment from Ad/p53, (Zhang et al., 1993). Virus
from a single plaque was expanded in 293 cells, twice purified and
vector titer determined to be 3.3.times.10.sup.12 viral
particles/ml. Thus, the vector-mediated gene co-transfer system
allows the in vitro replication of Ad/GT-Bax particles
(3.3.times.10.sup.12 viral particles/ml) in the host cell (e.g.,
293 cells), without killing the host cell (i.e. no Bax expression).
The functionality of Ad/GT-Bax in vitro was documented by the
co-transfer of Ad/GT-Bax and the transactivator Ad/PGK-GV16 to the
cultured human lung carcinoma cell line H1299, demonstrating the
induction of Bax expression via co-transfer. The in vitro
expression of Bax in the vector-mediated gene co-transfer was also
demonstrated to promote apoptosis in human lung cancer cell
lines.
[0250] In other embodiments, the induction of therapeutic gene
expression in vivo is contemplated for use in the present
invention. Thus, in another example, to test whether bax gene
expression could be similarly induced by adenovirus-mediated gene
codelivery in vivo, adult Balb/c mice were infused via their tail
veins with Ad/GT-Bax plus Ad/PGK-GV16, at a total vector dose of
6.times.10.sup.10 particles/mouse and a vector ratio of 2:1. Mice
were then sacrificed at 24 h after treatment, after which liver
samples were harvested for western blot analysis and
histopathological examination. A 14-fold increase in bax protein
levels in animals treated with Ad/GT-Bax plus Ad/PGK-GV16 relative
to control animals was observed as well as apoptosis of normal
liver cells. These results demonstrate that bax expression can
regulated in vivo by expressing GAL4/VP16 protein via the
adenovirus-mediated gene co-transfer system.
[0251] In certain embodiments of the invention, the temporal
sequence of vector-mediated co-transfer delivery is contemplated.
In one embodiment, the vectors are delivered simultaneously. In
other embodiments, the expression vector encoding the cytotoxic
gene is delivered first, followed by the expression vector encoding
the transactivating protein. In still other embodiments, the
expression vector encoding the transactivating protein is delivered
first, followed by the expression vector encoding the cytotoxic
gene. The time between delivery of the first vector and the second
vector is dependent on various parameters. Parameters to be
considered when formulating a protocol include, but are not limited
to, vector transducing efficiency, transducing cell type,
efficiency of cytotoxic gene expression, efficiency of
transactivating gene expression, cytotoxic protein stability and
transactivating protein stability.
[0252] iii. Viral and Non-Viral Vectors
[0253] It is contemplated in the present invention, that gene
co-transfer can be employed using any vector (i.e., viral, plasmid,
shuttle vector). The therapeutic gene as described above, can be
incorporated into an adenoviral infectious particle to mediate gene
transfer to a cell. Alternatively, retrovirus, adeno-associated
virus, vaccinia virus, canary pox virus, herpes virus, canary pox
virus and reovirus also are contemplated as gene transfer vectors
for use in the present invention.
[0254] In certain embodiments, non-viral vectors, such as plasmids,
shuttle plasmids and cosmids are contemplated for use. Non-viral
methods for the transfer of expression constructs into cultured
mammalian cells include calcium phosphate precipitation (Graham and
Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990)
DEAE-dextran (Gopal, 1985), electroporation (Tur-Kaspa et al.,
1986; Potter et al., 1984), direct microinjection (Harland and
Weintraub, 1985), DNA-loaded liposomes (Nicolau and Sene, 1982;
Fraley et al., 1979), cell sonication (Fechheimer et al., 1987),
gene bombardment using high velocity microprojectiles (Yang et al.,
1990), and receptor-mediated transfection (Wu and Wu, 1987; Wu and
Wu, 1988). For a more detailed description of both viral and
non-viral methods and applications of gene transfer, refer to
section F.
[0255] iv. Other Genes Toxic to Host Cells
[0256] In other embodiments of the present invention, the use of
gene co-transfer system is contemplated for use in delivering
non-pro-apoptic therapeutic genes that express potentially
cytotoxic gene products. It is contemplated, that cancer,
hyperproliferative (e.g., psoriasis, cytys) and inflammatory
conditions (e.g. rheumatoid arthritis, allergies) could be treated
by using the gene co-transfer system, by targeting these cells with
genes that encode potentially cytotoxic products. It is
contemplated that genes encoding cytokines (e.g., interferons),
toxins antisense constructs, ribozymes, single chain antibodies,
proteases and antigens would be useful in particular therapies, and
that the co-transfer method will allow regulated expression of
these genes.
[0257] In certain embodiments, various toxins are contemplated to
be useful as part of the expression vectors of the present
invention, these toxins include bacterial toxins such as ricin
A-chain (Burbage, 1997), diphtheria toxin A (Massuda et al., 1997;
Lidor, 1997), pertussis toxin A subunit, E. coli enterotoxin toxin
A subunit, cholera toxin A subunit and pseudomonas toxin
c-terminal. Recently, it was demonstrated that transfection of a
plasmid containing the fusion protein regulatable diphtheria toxin
A chain gene was cytotoxic for cancer cells. Thus, gene transfer of
regulated toxin genes might also be applied to the treatment of
cancers or other hyperproliferative diseases (Massuda et al.,
1997).
[0258] In certain embodiments, cytokines such as IL-1, IL-2, IL-3,
IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 IL-12, GM-CSF,
oncostatin M, TGF-.beta., TNF-.alpha., TNF-.beta. and G-CSF are
contemplated for use in the vector-mediated co-transfer system.
[0259] In other embodiments, antisense constructs are contemplated
for use in the present invention. Antisense methodology takes
advantage of the fact that nucleic acids tend to pair with
"complementary" sequences. Antisense polynucleotides, when
introduced into a target cell, specifically bind to their target
polynucleotide and interfere with transcription, RNA processing,
transport, translation and/or stability. Antisense RNA constructs,
or DNA encoding such antisense RNA's, may be employed to inhibit
gene transcription or translation or both within a host cell,
either in vitro or in vivo, such as within a host animal, including
a human subject. Engineering antisense constructs is covered in
detail in Section D. Particular oncogenes that are targets for
antisense constructs are ras, myc, neu, raf erb, src, fms, jun,
trk, ret, hst, gsp and abl. Also contemplated to be useful will be
anti-apoptotic genes such as Bcl-2, Mcl-1, A1 and Bfl-1
[0260] In still other embodiments, ribozymes are considered for use
in the present invention. Although proteins traditionally have been
used for catalysis of nucleic acids, another class of
macromolecules has emerged as useful in this endeavor. Ribozymes
are RNA-protein complexes that cleave nucleic acids in a
site-specific fashion. Ribozymes have specific catalytic domains
that possess endonuclease activity (Kim and Cook, 1987; Gerlach et
al., 1987; Forster and Symons, 1987). For example, a large number
of ribozymes accelerate phosphoester transfer reactions with a high
degree of specificity, often cleaving only one of several
phosphoesters in an oligonucleotide substrate (Cook et al., 1981;
Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This
specificity has been attributed to the requirement that the
substrate bind via specific base-pairing interactions to the
internal guide sequence ("IGS") of the ribozyme prior to chemical
reaction.
[0261] Ribozyme catalysis has primarily been observed as part of
sequence-specific cleavage/ligation reactions involving nucleic
acids (Joyce, 1989; Cook et al., 1981). For example, U.S. Pat. No.
5,354,855 reports that certain ribozymes can act as endonucleases
with a sequence specificity greater than that of known
ribonucleases and approaching that of the DNA restriction enzymes.
Thus, sequence-specific ribozyme-mediated inhibition of gene
expression may be particularly suited to therapeutic applications
(Scanlon et al., 1991; Sarver et al., 1990). Recently, it was
reported that ribozymes elicited genetic changes in some cells
lines to which they were applied; the altered genes included the
oncogenes H-ras, c-fos and genes of HIV. Most of this work involved
the modification of a target mRNA, based on a specific mutant codon
that is cleaved by a specific ribozyme. Targets for this embodiment
will include oncogenes such as ras, myc, neu, raf, erb, src, fins,
jun, trk, ret, hst, gsp, bcl-2, EGFR, grb2 and abl. Other
constructs will include overexpression of antiapoptotic genes such
as bcl-2.
[0262] In yet another embodiment, one gene may comprise a
single-chain antibody. Methods for the production of single-chain
antibodies are well known to those of skill in the art. The skilled
artisan is referred to U.S. Pat. No. 5,359,046, (incorporated
herein by reference) for such methods and section D above.
[0263] Antibodies to a wide variety of molecules are contemplated,
such as oncogenes, growth factors, hormones, enzymes, transcription
factors or receptors.
[0264] In certain embodiments, it may be useful to express enzymes,
that are potentially cytotoxic. For example, the protease
caspase-7, has been implicated in apoptosis and thus potentially
useful in gene therapy (Marcelli et al., 1999). One could express a
variety of proteases, which have either been genetically engineered
to function at physiological pH and/or active without enzymatic
processing (Briand et al., 1999). Alternatively, proteases can be
cloned from thermostable or pH stable organisms (Choi et al., 1999;
Sundd et al., 1998). Thus, one could express a protease in a given
cell and potentially inactivate via proteolysis, key metabolic and
signaling proteins, needed for cell viability.
[0265] In another embodiment, treatment of protein folding
disorders via the gene co-transfer system are contemplated. For
example, Cruetzfeldt-Jakob disease, Kuru, the human transmissible
bovine spongiform encephalopathy (e.g., mad cow disease) and
scrappie in sheep, are diseases related to cellular prion protein
misfolding (Grandien and Wahren, 1998; Buschmann et al., 1998; Hill
et al., 1999) The disease state ensues when an individual is
exposesed to an infectious (mutated) form of the prion protein.
This infectious prion protein (PrP(Sc)) acts as a misfolding
catalyst or scaffold, and induces conformational changes in an
individuals native prion proteins (PrP(C)), leading to the
intraneuronal accumulation of a pathological prion isoform. Prions
replicate in lymphoreticular tissues before neuroinvasion and have
been demonstrated to be detectable via tonsil biopsy (Hill et al.,
1999). It might be possible using vector-mediated co-transfer, to
provide antisense mRNA to patients who test positive for PrP(Sc),
to prevent transcription of prion mRNA and thus block protein
synthesis. Alternatively, expression of cytokines could be targeted
to lymphoreticular tissues, expression of proteases or specific
antigens could be used to tag these cells for destruction, reducing
prion protein expression. It is contemplated further in the present
invention, that Alzheimer's disease could be treated similarly
using vector-mediated co-transfer
G. Pharmaceuticals and Methods of Treating Cancer
[0266] In a particular aspect, the present invention provides
methods for the treatment of various malignancies. Treatment
methods will involve treating an individual with an effective
amount of a viral particle, as described above, containing a
therapeutic gene of interest. An effective amount is described,
generally, as that amount sufficient to detectably and repeatedly
to ameliorate, reduce, minimize or limit the extent of a disease or
its symptoms. More rigorous definitions may apply, including
elimination, eradication or cure of disease.
[0267] To kill cells, inhibit cell growth, inhibit metastasis,
decrease tumor size and otherwise reverse or reduce the malignant
phenotype of tumor cells, using the methods and compositions of the
present invention, one would generally contact a "target" cell with
the therapeutic expression construct. This may be combined with
compositions comprising other agents effective in the treatment of
cancer. These compositions would be provided in a combined amount
effective to kill or inhibit proliferation of the cell. This
process may involve contacting the cells with the expression
construct and the agent(s) or factor(s) at the same time. This may
be achieved by contacting the cell with a single composition or
pharmacological formulation that includes both agents, or by
contacting the cell with two distinct compositions or formulations,
at the same time, wherein one composition includes the expression
construct and the other includes the second agent.
[0268] Alternatively, the gene therapy may precede or follow the
other agent treatment by intervals ranging from minutes to weeks.
In embodiments where the other agent and expression construct are
applied separately to the cell, one would generally ensure that a
significant period of time did not expire between the time of each
delivery, such that the agent and expression construct would still
be able to exert an advantageously combined effect on the cell. In
such instances, it is contemplated that one would contact the cell
with both modalities within about 12-24 h of each other and, more
preferably, within about 6-12 h of each other, with a delay time of
only about 12 h being most preferred. In some situations, it may be
desirable to extend the time period for treatment significantly,
however, where several d (2, 3, 4, 5, 6 or 7) to several wk (1, 2,
3, 4, 5, 6, 7 or 8) lapse between the respective
administrations.
[0269] Administration of the therapeutic expression constructs of
the present invention to a patient will follow general protocols
for the administration of chemotherapeutics, taking into account
the toxicity, if any, of the vector. It is expected that the
treatment cycles would be repeated as necessary. It also is
contemplated that various standard therapies, as well as surgical
intervention, may be applied in combination with the described gene
therapy.
[0270] Where clinical application of a gene therapy is
contemplated, it will be necessary to prepare the complex as a
pharmaceutical composition appropriate for the intended
application. Generally this will entail preparing a pharmaceutical
composition that is essentially free of pyrogens, as well as any
other impurities that could be harmful to humans or animals. One
also will generally desire to employ appropriate salts and buffers
to render the complex stable and allow for complex uptake by target
cells.
[0271] Aqueous compositions of the present invention comprise an
effective amount of the compound, dissolved or dispersed in a
pharmaceutically acceptable carrier or aqueous medium. Such
compositions can also be referred to as inocula. The phrases
"pharmaceutically or pharmacologically acceptable" refer to
molecular entities and compositions that do not produce an adverse,
allergic or other untoward reaction when administered to an animal,
or a human, as appropriate. As used herein, "pharmaceutically
acceptable carrier" includes any and all solvents, dispersion
media, coatings, antibacterial and antifungal agents, isotonic and
absorption delaying agents and the like. The use of such media and
agents for pharmaceutical active substances is well known in the
art. Except insofar as any conventional media or agent is
incompatible with the active ingredient, its use in the therapeutic
compositions is contemplated. Supplementary active ingredients also
can be incorporated into the compositions.
[0272] The compositions of the present invention may include
classic pharmaceutical preparations. Dispersions also can be
prepared in glycerol, liquid polyethylene glycols, and mixtures
thereof and in oils. Under ordinary conditions of storage and use,
these preparations contain a preservative to prevent the growth of
microorganisms.
[0273] Depending on the particular cancer to be, administration of
therapeutic compositions according to the present invention will be
via any common route so long as the target tissue is available via
that route. This includes oral, nasal, buccal, rectal, vaginal or
topical. Topical administration would be particularly advantageous
for treatment of skin cancers. Alternatively, administration will
be by orthotopic, intradermal, subcutaneous, intramuscular,
intraperitoneal or intravenous injection. Such compositions would
normally be administered as pharmaceutically acceptable
compositions that include physiologically acceptable carriers,
buffers or other excipients.
[0274] In certain embodiments, ex vivo therapies also are
contemplated. Ex vivo therapies involve the removal, from a
patient, of target cells. The cells are treated outside the
patient's body and then returned. One example of ex vivo therapy
would involve a variation of autologous bone marrow transplant.
Many times, ABMT fails because some cancer cells are present in the
withdrawn bone marrow, and return of the bone marrow to the treated
patient results in repopulation of the patient with cancer cells.
In one embodiment, however, the withdrawn bone marrow cells could
be treated while outside the patient with an viral particle that
targets and kills the cancer cell. Once the bone marrow cells are
"purged," they can be reintroduced into the patient.
[0275] The treatments may include various "unit doses." Unit dose
is defined as containing a predetermined-quantity of the
therapeutic composition calculated to produce the desired responses
in association with its administration, i.e., the appropriate route
and treatment regimen. The quantity to be administered, and the
particular route and formulation, are within the skill of those in
the clinical arts. Also of import is the subject to be treated, in
particular, the state of the subject and the protection desired. A
unit dose need not be administered as a single injection but may
comprise continuous infusion over a set period of time. Unit dose
of the present invention may conveniently may be described in terms
of plaque forming units (pfu) of the viral construct. Unit doses
range from 10.sup.3, 10.sup.4, 10.sup.5, 10.sup.6, 10.sup.7,
10.sup.8, 10.sup.9, 10.sup.10, 10.sup.11, 10.sup.12, 10.sup.13 pfu
and higher.
[0276] Preferably, patients will have adequate bone marrow function
(defined as a peripheral absolute granulocyte count of
>2,000/mm.sup.3 and a platelet count of 100,000/mm.sup.3),
adequate liver function (bilirubin <1.5 mg/dl) and adequate
renal function (creatinine <1.5 mg/dl).
[0277] i) Cancer Therapy
[0278] One of the preferred embodiments of the present invention
involves the use of viral vectors to deliver therapeutic genes to
cancer cells. Target cancer cells include cancers of the lung,
brain, prostate, kidney, liver, ovary, breast, skin, stomach,
esophagus, head and neck, testicles, colon, cervix, lymphatic
system and blood. Of particular interest are non-small cell lung
carcinomas including squamous cell carcinomas, adenocarcinomas and
large cell undifferentiated carcinomas.
[0279] According to the present invention, one may treat the cancer
by directly injection a tumor with the viral vector. Alternatively,
the tumor may be infused or perfused with the vector using any
suitable delivery vehicle. Local or regional administration, with
respect to the tumor, also is contemplated. Finally, systemic
administration may be performed. Continuous administration also may
be applied where appropriate, for example, where a tumor is excised
and the tumor bed is treated to eliminate residual, microscopic
disease. Delivery via syringe or catherization is preferred. Such
continuous perfusion may take place for a period from about 1-2
hours, to about 2-6 hours, to about 6-12 hours, to about 12-24
hours, to about 1-2 days, to about 1-2 wk or longer following the
initiation of treatment. Generally, the dose of the therapeutic
composition via continuous perfusion will be equivalent to that
given by a single or multiple injections, adjusted over a period of
time during which the perfusion occurs.
[0280] For tumors of >4 cm, the volume to be administered will
be about 4-10 ml (preferably 10 ml), while for tumors of <4 cm,
a volume of about 1-3 ml will be used (preferably 3 ml). Multiple
injections delivered as single dose comprise about 0.1 to about 0.5
ml volumes. The viral particles may advantageously be contacted by
administering multiple injections to the tumor, spaced at
approximately 1 cm intervals.
[0281] In certain embodiments, the tumor being treated may not, at
least initially, be resectable. Treatments with therapeutic viral
constructs may increase the resectability of the tumor due to
shrinkage at the margins or by elimination of certain particularly
invasive portions. Following treatments, resection may be possible.
Additional viral treatments subsequent to resection will serve to
eliminate microscopic residual disease at the tumor site.
[0282] A typical course of treatment, for a primary tumor or a
post-excision tumor bed, will involve multiple doses. Typical
primary tumor treatment involves a 6 dose application over a
two-week period. The two-week regimen may be repeated one, two,
three, four, five, six or more times. During a course of treatment,
the need to complete the planned dosings may be re-evaluated.
[0283] Cancer therapies also include a variety of combination
therapies with both chemical and radiation based treatments.
Combination chemotherapies include, for example, cisplatin (CDDP),
carboplatin, procarbazine, mechlorethamine, cyclophosphamide,
camptothecin, ifosfamide, melphalan, chlorambucil, bisulfan,
nitrosurea, dactinomycin, daunorubicin, doxorubicin, bleomycin,
plicomycin, mitomycin, etoposide (VP16), tamoxifen, taxol,
transplatinum, 5-fluorouracil, vincristin, vinblastin and
methotrexate or any analog or derivative variant thereof.
[0284] Other factors that cause DNA damage and have been used
extensively include what are commonly known as .gamma.-rays,
X-rays, and/or the directed delivery of radioisotopes to tumor
cells. Other forms of DNA damaging factors are also contemplated
such as microwaves and UV-irradiation. It is most likely that all
of these factors effect a broad range of damage on DNA, on the
precursors of DNA, on the replication and repair of DNA, and on the
assembly and maintenance of chromosomes. Dosage ranges for X-rays
range from daily doses of 50 to 200 roentgens for prolonged periods
of time (3 to 4 wk), to single doses of 2000 to 6000 roentgens.
Dosage ranges for radioisotopes vary widely, and depend on the
half-life of the isotope, the strength and type of radiation
emitted, and the uptake by the neoplastic cells.
[0285] Various combinations may be employed, gene therapy is "A"
and the radio- or chemotherapeutic agent is "B": TABLE-US-00005
A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B B/B/B/A B/B/A/B
A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B A/A/A/B B/A/A/A
A/B/A/A A/A/B/A
[0286] The terms "contacted" and "exposed," when applied to a cell,
are used herein to describe the process by which a therapeutic
construct and a chemotherapeutic or radiotherapeutic agent are
delivered to a target cell or are placed in direct juxtaposition
with the target cell. To achieve cell killing or stasis, both
agents are delivered to a cell in a combined amount effective to
kill the cell or prevent it from dividing.
[0287] The therapeutic compositions of the present invention are
advantageously administered in the form of injectable compositions
either as liquid solutions or suspensions; solid forms suitable for
solution in, or suspension in, liquid prior to injection may also
be prepared. These preparations also may be emulsified. A typical
composition for such purpose comprises a pharmaceutically
acceptable carrier. For instance, the composition may contain 10
mg, 25 mg, 50 mg or up to about 100 mg of human serum albumin per
milliliter of phosphate buffered saline. Other pharmaceutically
acceptable carriers include aqueous solutions, non-toxic
excipients, including salts, preservatives, buffers and the like.
Examples of non-aqueous solvents are propylene glycol, polyethylene
glycol, vegetable oil and injectable organic esters such as
ethyloleate. Aqueous carriers include water, alcoholic/aqueous
solutions, saline solutions, parenteral vehicles such as sodium
chloride, Ringer's dextrose, etc. Intravenous vehicles include
fluid and nutrient replenishers. Preservatives include
antimicrobial agents, anti-oxidants, chelating agents and inert
gases. The pH and exact concentration of the various components the
pharmaceutical composition are adjusted according to well known
parameters.
[0288] Additional formulations are suitable for oral
administration. Oral formulations include such typical excipients
as, for example, pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate and the like. The compositions take the form of
solutions, suspensions, tablets, pills, capsules, sustained release
formulations or powders. When the route is topical, the form may be
a cream, ointment, salve or spray.
H. EXAMPLES
[0289] The following examples are included to demonstrate preferred
embodiments of the invention. It should be appreciated by those of
skill in the art that the techniques disclosed in the examples
which follow represent techniques discovered by the inventor to
function well in the practice of the invention, and thus can be
considered to constitute preferred modes for its practice. However,
those of skill in the art should, in light of the present
disclosure, appreciate that many changes can be made in the
specific embodiments which are disclosed and still obtain a like or
similar result without departing from the spirit and scope of the
invention.
Example 1
Apoptotic Mechanisms Following Adenovirus-Mediated
p53 Replacement Gene Therapy
[0290] Induction of program cell death pathway is a critical step
in most anticancer therapies including adenovirus mediated
wild-type p53 gene therapy. The transient expression of the
adenovirus vector requires either induction of apoptosis, terminal
differentiation, or cellular senescence in order to result in
effective therapy. As the a further understanding of the mechanisms
involved in this process is gained, this will enable us to design
more effective therapeutic approaches to anticancer treatment.
Materials and Methods
[0291] Cell Culture. H358 and H1299 are non-small cell lung cancer
cell lines with both copies of the p53 deleted and were obtained
from A. Gazdar and J. Minna. H322j is a non-small cell lung cancer
cell line with a p53 mutation. Cells were maintained in RPMI-1640
medium supplemented with 10% fetal calf serum, 10 mM glutamine, 100
units/ml of penicillin, 100 .mu.g/ml of streptomycin, and 0.25
.mu.g/ml of amphotericin B (Gibco-BRL, Life Technologies, Inc.,
Grand Island, N.Y.) and incubated at 37.degree. C. in a 5% CO.sub.2
incubator.
[0292] Adenovirus production. The construction and properties of
the Adp53 have been reported elsewhere (Fujiwara et al., 1994;
Zhang et al., 1993). The Ad5/CMV/.beta.-gal virus was obtained from
F. Graham, McMaster University, Hamilton, Ontario. The E1 deleted
vector dl312 (obtained from T. Shenk, Princeton, N.J.) was utilized
as a control vector. Adenovirus was prepared as previously
described (Graham and Prevec, 1991) and purified by two rounds of
cesium chloride ultracentrifugation. Purified virus was mixed with
10% glycerol and dialyzed twice against 1000 ml of a buffer
containing 10 mM Tris HCl (pH 7.5), 1/.mu.M MgCl.sub.2, and 10%
glycerol at 4.degree. C. for 6 h. Purified virus was aliquoted and
stored at -80.degree. C. until used. Viral titer was determined by
UV-spectrophotometric analysis (viral particles/ml) and by plaque
assay (pfu/ml) (Zhang et al., 1995). Final viral concentrations for
in vitro and in vivo infections were made by dilution of stock
virus in PBS. Adenovirus preparations were free of
replication-competent adenovirus as determined by previously
described techniques (Zhang et al., 1995).
[0293] Gene delivery. In vitro transfection studies for all cell
lines were performed by plating 5.times.10.sup.5 cells in 100 mm
plates (Falcon Plastics, Lincoln Park, N.J.). Forty-eight h after
plating, cells were incubated for 2 h with purified virus in 2 mls
of RPMI-1640 medium supplemented with 2% fetal calf serum. The
multiplicity of infection (MOI) was based on cell counts of
untreated plates. The MOI used for each cell line was chosen to
result in an approximately 70-80% transduction based on preliminary
studies using the Ad5/CMV/.beta.-gal vector. These were an MOI of 5
pfu for the H1299 cell line, 70 pfu for the H358 cell line and 50
pfu for the H322j cell line. After 2 h, fresh RPMI-1640 medium
supplemented with 10% fetal calf serum was added to the plates.
Cells and cell lysate were collected at 6 h intervals up to 36 h
following infection for western blot, cell cycle, and TUNEL assay
analysis. This time course was chosen based on preliminary data
indicating a large fraction of apoptotic cells were evident at
these times and later time points resulted in the observance of
degraded cellular proteins.
[0294] Western blot analysis. Total cell lysates were prepared by
lysing monolayered cells in dishes with sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer
after rinsing cells with phosphate buffered saline (PBS). Each lane
was loaded with 50 .mu.g of cell lysate protein as determined by
BCA protein assay (Pierce, Rockford, Ill.). After SDS PAGE at 100
volts for two h, the proteins in the gels were transferred to
hybond-ECL membrane (Amersham International PLC, Little Chalfont,
Buckingham Shire, England). Membranes were blocked with 3% milk and
0.1% Tween 20 (Sigma Chemical Company) in PBS and incubated with
antibody against the specified protein overnight at 4.degree. C.
The mouse anti-human p53 (D0-7) (Pharmigen, San Diego, Calif.),
mouse anti-human Bcl-2 (124) (Dako Corp., Carpintenia, Calif.),
mouse anti-human Bak (Oncogene Science), mouse anti-human Bax
(Pharmigen, San Diego, Calif.), mouse anti-human Bcl-x.sub.L
(Pharmigen, San Diego, Calif.), and mouse anti-human (.beta.-actin
monoclonal antibody (N350) (Amersham International PLC, Buckingham
Shire, England) were used. The membranes were developed according
to Amershams ECL western blotting protocol.
[0295] Flow cytometry analysis for cell cycle. To measure the DNA
histogram, cells were fixed in 70% ethanol at 4.degree. C. for
greater than 24 h. The cells were incubated in propidium iodide (20
.mu.g/ml) and ribonucleases (20 .mu.g/ml) for 30 min at 37.degree.
C. All measures were made with an Epics Profile II (Coulter Corp.,
Hialeath, Fla.) equipped with an air-cooled argon ion laser
admitting 488 NM at 15 MW. A minimum of 10,000 events per sample
were analyzed and FITC fluorescence was collected using a 525 BP
filter. Coulters cytologic program was used for data analysis. Mean
peak fluorescence was determined for each histogram.
[0296] Terminal deoxynucleotidyl transferase-mediated dUTP-biotin
nick end labeling (TUNEL) Assay. The TUNEL assay was performed
utilizing the procedure described by Gorczyca et al. (1992).
Briefly, fixed cells were washed in PBS and resuspended in 50 .mu.l
of TdT buffer with 5 units of TdT enzyme (Sigma Chemical Co.) and
0.5 nmol biotin-16-dUTP (Boehringer Mannheim Co.). Controls were
prepared without TdT enzyme. Cells were incubated at 37.degree. C.
for 1 hour, rinsed in PBS, and resuspended in 100 ml of
avidin-FITC, 2.5 mg/ml, (Boehringer Mannheim Co.) in saline-citrate
buffer containing 0.1% Triton X-100, 0.1% BSA, 0.5 M NaCl, and 0.06
M Na citrate. Specimens were incubated in the dark for 30 min,
washed in PBS with 0.1% Triton X-100, resuspended in propidium
iodine (5 .mu.g/ml) and 0.1% RNAse A. After incubation for 30 min
the specimens were analyzed with the use of an EPICS Profile II
flow cytometer (Coulter Corp., Hialeah, Fla.). An analysis region
was set based on the negative controls and the percent of labeled
cells was calculated from this region.
[0297] Evaluation of apoptosis. For evaluation of apoptosis induced
by the Ad-Bax vector, the breast carcinoma cell lines MDA-MB-468,
MCF-7, and SKBr3 were used. The cells were plated at
0.5.times.10.sup.6 and then treated with Ad-Bax or viral control at
an MOI of 100 viral particles per cell. Media alone was used for
mock infection. At 2 and 4 days post transfection, the cells were
harvested and fixed in 80% ETOH. After 24 hours, propidium iodide
was added to each sample and the cells were analyzed by flow
cytometry. The subdiploid cell population was assessed and the
percent recorded as apoptotic cells.
[0298] Further analysis of apoptosis was determined by a cell death
ELISA kit from Boehringer Mannheim. This is a photometric "sandwich
enzyme immunoassay" which allows quantitative in vitro
determination of histone-associated DNA fragments which are
specific for apoptotic cell death. Briefly, MDA-MB-468 cells and
MCF-7 cells were transfected at an MOI of 100 (Ad-Bax, viral
control or media alone) and cells collected at 72 hours. Samples
were incubated with anti-histone biotin and anti-DNA peroxidase in
streptavidin coated plates. After removal of unbound antibodies,
the amount of peroxidase retained was determined photometrically.
The results are recorded as an enrichment factor which is a
photometric quantitation above the control samples.
Results
[0299] Adp53 Infection Results in Overexpression of p53 Protein and
Induction of p21.
[0300] Expression of p53 protein in the H1299 cells was measured at
6 h intervals following Adp53 infection by western blot analysis.
The control cells (mock infected) and dl312 (control vector)
infected cells expressed no measurable p53 protein. p53 protein was
observed at the 6 h time point following infection with Adp53. High
expression at multiple phosphorylation states was observed at 24 h
and continued to the 36 h time point. Induction of p21 was observed
following infection with Adp53. Control cells and dl312 infected
cells expressed low levels of p21 by western blotting analysis.
However, induction of p21 was observed early following infection
with Adp53. High levels of p21 were observed at the 18 h time point
and continued to with high expression observed at 36 h. Similar
results were observed at the 24 h time point for the H358 and H322J
cell lines.
[0301] Adp53 Infection Results in a G.sub.1 Cell Cycle Arrest and
Induction of Apoptosis.
[0302] Cell cycle analysis of the H1299 cell line demonstrated an
increase in the G.sub.1 population of cells following infection
with Adp53 compared to the control and dl312 infected cells (FIG.
2A. This increase in G.sub.1 population of cells was observed as
early as 12 h following Adp53 infection and was clearly evident at
the 18 h time point (percent G.sub.1: control=38%, Adp53=59%) and
continued to 36 h. Interestingly, accumulation of the sub 2N
population of cells was observed at a time point slightly delayed
from the time of accumulation of cells in G.sub.1 cell cycle
arrest. The sub 2N population of cells were observed at 24 h
following infection with Adp53 and continued to accumulate up to 36
h following infection. This increase sub 2N population of cells
corresponded to an increase labeling by TUNEL assay (FIG. 2B. These
data are consistent with increases in apoptotic cell death.
[0303] Adp53 Infection Result in Decreased Levels of CPP32 and Parp
Cleavage.
[0304] Levels of the inactive zymogen of CPP32 were observed in
control and dl312 infected cells. Adp53 infection resulted in
decreased levels of the inactive zymogen form of CPP32. These
diminished levels of the CPP32 zymogen were observed at the 24 h
time point and continued through the 36 h time point (FIG. 3A).
This reduction in CPP32 levels was accompanied by concomitant
evidence of cleavage of its early target Parp by western blot
analysis. Similar results were observed at the 24 h time point for
the H358 and H322J cell lines (FIG. 3B). The above data is again
consistent with induction of apoptotic cell death, activation of
the ICE-like protease CPP32, and cleavage of the CPP32 target
Parp.
[0305] Adp53 Infection Did not Effect the Bcl-2 or Bcl-x.sub.L
Expression.
[0306] No significant changes in the levels Bcl-x, and Bcl-2
proteins were observed by western blotting following infection with
Adp53 as compared to control or dl312 infected cells. Similar
results were observed at the 24 h time point for the H358 and H322J
cell lines.
[0307] Overexpression of p53 Results in Induction of Proapoptotic
Bax and Bak Proteins.
[0308] Bax protein levels were detectable in control and dl312
infected cells. Infection with Adp53 resulted in increased levels
of Bax protein. This was especially evident at the 24 h time point
and continued to 36 h. Bak protein expression was detectable by
western blot analysis in control and dl312 infected cells.
Following infection with Adp53, a significant increase in Bak
protein levels were observed compared to controls. Again, peak
levels were present at the 24 h time point and continued to the 36
h time point. Similar results were observed at the 24 h time point
for the H358 and H322J cell lines.
Example 2
The Adenoviral Bax Vector
[0309] Using the insights gained herein above, the inventors
reasoned that the overexpression of p53 gene induces apoptosis by
upregulating Bax. Thus if a vector could be designed that in itself
mediated the upregulation of over-expression of Bax, there may be
enough of an induction of Bax to induce apoptosis. In order to
investigate this further the inventors constructed a new and novel
adenoviral Bax vector as described herein below.
[0310] Cloning of the Human Bax cDNA.
[0311] Total RNA was isolated from SRB I squamous cell carcinoma
cell line using Ultraspec RNA isolation reagent (Biotecx). First
strand cDNA was synthesized using 5 .mu.g of RNA, 500 ng oligo
(dT), 5.times. strand buffer, 0.1 M DTT, 10 .mu.M dNTP mix 1 .mu.l
of superscripapt II.TM. in a RT-PCR.TM. reaction. Polymerase chain
reaction was then performed to amplify Bax cDNA using forward oligo
primer 5'-GGAATTCGCGGTGATGGAC GGGTCCGG-3' (SEQ ID NO:5) and reverse
oligo primer 5'-GGGAATTCTCAGCCCATCTTCTTCCA GA-3' (SEQ ID NO:6). The
reaction was incubated at 95.degree. C. for 1 min, 56.degree. C.
for 2 min and 72.degree. C. for 3 min for a total of 35 cycles. The
PCR.TM. reaction was then resolved on 1.5% agarose gel. The Bax
cDNA sequence was assessed with the M13 and T7 primers and was
found to differ from the wildtype Bax sequence in the amino
terminus. The highly conserved BH3 region which appears necessary
for apoptosis was intact but a frameshift mutation existed which
eliminated the BH1 and BH2 regions.
[0312] Construction of Adenoviral Bax Vector
[0313] The TA PCR.TM.II cloning vector (Invitrogen) containing the
truncated Bax cDNA (SEQ ID NO:1 cDNA encodes protein of SEQ ID
NO:2) was amplified and purified using Qiagen kit. The truncated
Bax gene DNA fragment was isolated by digestion with restriction
enzymes EcoRI (for the 5' side) and Not I (for the 3' side) and
electroeluted on a 1.5% agarose gel. The truncated Bax gene was
recovered from the gel with Qiagen DNA recovery kit and inserted
into a polylinker between the Xba I and Cla I sites in the pXCJL.1
shuttle vector. The shuttle vector contains the left end of the
adenovirus type 5 genome with the E1 region deleted. The resulting
plasmid, p12 Bax, was cotransfected with the recombinant plasmid
pJM17 into 293 kidney carcinoma cells which provided the deleted E1
region in ttrqns. pJM17 carries the bulk of the right side of the
adenovirus type 5 genome.
[0314] Calcium phosphate mediated cotransfection of the two
plasmids (p12Bax and pJM17) was performed with homologous
recombination producing the adenoviral truncated Bax vector
(AdBax). Successful adenoviral recombinants were identified by
cytopathic changes in the transfected 293 cells. The adenoviral
recombinants were amplified on 293 cells and harvested when a
complete cytopathic effect was evident. The virus was isolated by
free-thawing the cell pellets three times in dry ice ethanol bath
and a 37.degree. C. water bath.
[0315] Purification of the virus was performed with two cesium
chloride gradient ultracentrifugations. The isolated virus was then
dialyzed against a buffer (10 mM Tris-HCL, 1 mM MgCl.sub.2 and 10%
glycerol) to remove contaminating cesium chloride. Quantification
of the virus was then performed with O.D. readings ad plaque assay
on 293 cells. The purified virus was then analyzed for the presence
of the truncated Bax gene by dideoxy DNA sequencing with PCR.TM.
and two primers. The internal forward oligo primer
5'-GGGACGAACTGGACAGTAA-3' (SEQ ID NO:7) and reverse oligo primer
5'-GCACCAGTTTGCTGGCAAA-3' (SEQ ID NO:8) were used to sequence both
strands of the adenoviral Bax gene. Additional confirmations was
obtained with PCR.TM. primers located just upstream and downstream
of the Bax insert in the adenovirus genome. These primers included
the forward oligo 5'-ACGCAAATGGGCGGTAG-3' (SEQ ID NO:9) and reverse
5'-CAACTAGAAGGCACAGT-3' (SEQ ID NO:10). Sequencing confirmed that
the truncated Bax gene was correctly inserted in the adenoviral
recombinant AdBax.
Example 3
Induction of Apoptosis in Human Breast Cancer by Adenoviral
Mediated Overexpression of Bax
[0316] Apoptosis is controlled, at least in part, by the balance
between the proapoptotic (Bax, Bak, Bcl-xs) and antiapoptotic
(Bcl-2, Bcl-x.sub.L) members of the Bcl-2 family. Altering the
balance of these mediators can result in the suppression or
induction of apoptosis. The present example describes the use of
the novel adenoviral vector, Ad-Bax, to determine whether
overexpression of Bax could induce apoptosis in human breast
cancer.
[0317] The human Bax cDNA was isolated, sequenced and used to
construct the Type 5, E1 deleted adenoviral vector as described
herein above. The Ad-Bax vector contained a truncated Bax with an
intact death (BH3) domain. Human breast cancer cell lines
MDA-MB-468, SKBr3 and MCF-7 were transduced with Ad-Bax, E1 deleted
viral control (AdV) or media alone (Cont.) at multiplicity of
infection (MOI) of 100 to achieve an 85% transduction
efficiency.
[0318] Apoptosis was evaluated by changes in cellular morphology,
evidence of DNA-Histone complexes by ELISA and by FACS (FIG. 4A,
FIG. 4B and FIG. 4C) analysis of subdiploid cells with propidium
iodide staining.
[0319] Western blot analysis confirmed overexpression of the Bax
protein in the transduced cells. Apoptosis, by morphology, occurred
four days after transduction with Ad-Bax in 468 and SKBr3 cells but
not in MCF-7 cells (Table 5). FACS revealed a two-fold increase in
apoptosis (FIG. 4A, FIG. 4B, and FIG. 4C). DNA-Histone complexes
increased 40% in 468 cells with no increase in MCF-7 cells. Further
Western analysis revealed similar levels of Bcl-xL in all cell
lines. However, there were high levels of Bcl-2 only in the
apoptosis-resistant MCF-2 cells (Table 5; FIG. 4A, FIG. 4B, and
FIG. 4C). TABLE-US-00006 TABLE 5 Adenovirally-mediated Bax induced
Apoptosis and the Bcl-2 levels in MDA-MB-468, SKBr3 and MCF-7 cell
lines % Apoptosis Cell lines Cont AdV. Ad-Bax BCL-2 Level
MDA-MB-468 24 26 49 Low SKBr3 13 10 24 Low MCF-7 17 12 14 High
[0320] The present example demonstrates that adenoviral mediated
gene transfer of Bax induces apoptosis in human breast cancer cell
lines. Resistance to Ad-Bax induced apoptosis in MCF-7 cells may be
due to the high cellular levels of Bcl-2. These results suggest
that overexpression of the proapoptotic mediator Bax will be a
novel and useful gene therapy strategy. Further, such gene therapy
may be combined with inhibition of endogenous Bcl-2 to shift the
proapoptotic/antiapoptotic equilibrium in favor of death promotion
in cancer cells.
Example 4
[0321] Construction of Wild-Type AdBax and AdBak Using a Cosmid
System
[0322] Traditional methods of producing recombinant adenoviral
vectors involve co-transfection of a plasmid encoding the transgene
of interest and a shuttle vector carrying adenoviral genome
sequences into a cell line such as 293 cells that express the E1A
gene product. This allows for transactivation of adenoviral gene
transcription and homologous recombination to produce a recombinant
adenovirus that is replication deficient. Some drawbacks of this
system are a low efficiency of homologous recombination, tedious
cloning and plaque screening to identify the desired end product,
and the production of a relatively high level of non-recombinant
viruses in the viral preparation.
[0323] A relatively new method of producing recombinant adenoviral
particles is the use of a cosmid adenoviral vector cloning system
(Chartier et al., 1996, Fu and Deisseroth, 1997). The advantages to
such a system high recombination efficiency in recA+ E. coli
bacteria, high capacity for heterologous DNA, a stable genome, easy
isolation of recombinant virus, and the ability to construct
recombinant adenoviruses that carry cytotoxic gene. In the present
invention, pro-apoptotic genes such as bax and bak are capable of
being introduced into the adenoviral genome and produced by this
system while not killing the producer cell.
[0324] The inventors used the Supercos vector (Stratagene, La
Jolla, Calif.) as the base vector for this system (FIG. 5).
Initially the SV40 origin of replication and the Neo gene were
removed by restriction digestion to generate pCOS/LJ07 (FIG. 6).
The cloning of the adenovirus genome in to the cosmid was attained
by cotransfection of pCOS/LJ07 and pAdv-dlE1-dlE3-Gal4 (U.S.
application No. 60/030,675, herein incorporated by reference) into
NM522 E. coli cells to allow homologous recombination to occur. The
resultant vector, pCOS/Ad/LJ17 (FIG. 7) was purified and the
recombinant adenovirus then constructed by co-transfection of
pCOS/Ad/LJ17 and a shuttle plasmid pCMV/Bak (FIG. 8) into NM522 E.
coli cells. The resultant vector pCOS/Ad-Bak (FIG. 9) contains the
Bak gene under the control of the CMV IE promoter. Verification of
the Ad-CMV-Bak construct by PCR.TM. confirmed that the proper
insert was incorporated into the recombinant virus, and sequencing
of the bak gene confirmed the sequence to be wild-type. Similar
procedures were used to generate the wild-type bax gene adenovirus
recombinant. Linearization of the vector containing the adenoviral
genome, and then transfection into 293/GV16 cells results in the
generation of recombinant vectors. FIG. 10, FIG. 11, and FIG. 12
outline these procedures.
[0325] Thus it is evident that the use of a system such as this is
useful for the construction of adenoviral vectors, and that a wide
variety of transgenes may be incorporated into the adenoviral
genome using this or similar techniques. It will be appreciated
that those of skill in the art may modify or improve such a system
to produce better results or achieve greater efficiency.
Example 5
Expression of the Bax Gene by Adenovirus Mediated Gene
Co-Transfer
Materials and Methods
[0326] Cell lines. Human non-small cell lung cancer cell lines
H1299 and A549 were cultured in RPMI 1640 and HAM/F12 medium,
respectively, supplemented with 10% FBS and antibiotics. Human
embryonic kidney 293 cells were maintained in Dulbecco's modified
Eagle's medium (DMEM) containing 4.5 g/l of glucose with 10% FBS
and antibiotics and used in the construction and amplification of
adenovirus vectors.
[0327] Construction of recombinant adenovirus vectors. The
construction of Ad/PGK-GV16, Ad/GT-Luc, and Ad/GT-LacZ as described
by Fang et al. (1998). Ad/CMV-GFP was obtained from Fueyo et al
(1998). Mutations found in the bax cDNA were corrected by combining
two PCR products of the gene. The authenticity of the bax-.alpha.
cDNA sequence was then confirmed by automatic DNA sequencing
performed at M. D. Anderson Cancer Center's Core DNA Sequencing
Facility. For construction of Ad/GT-Bax, the bax gene was first
cloned downstream of the GT promoter to generate the shuttle
plasmid pAd/GT-Bax. Then, the vector was constructed by
cotransfecting 293 cells with a 35-kb cal fragment from Ad/p53 and
pAd/GT-Bax (Zhang et al., 1993). The virus titers cited in this
study were determined by optical absorbency at A.sub.260 (one
A.sub.260 unit=10.sup.12 viral particle/ml). Particle/plaque ratios
usually fell between 30:1 and 100:1. All viral preparations were
tested for E1.sup.+ adenovirus contamination by PCR (Fang et al.,
1996) and for endotoxin contamination by assays with a
third-generation pyrogen testing kit from BioWhittaker
(Walkersville, Md.).
[0328] PCR analysis. Viral DNA was isolated from the supernatant of
viruses expanded in 293 cells. A primer located in the bax gene was
then used with a second primer located in the adenoviral backbone
in PCR to identify recombinants via PCR. The plasmid pAd/GT-Bax was
used as a positive control for Ad/GT-Bax. Primers used for
detecting E1.sup.+ adenovirus were the same as in Fang et al.
(1996).
[0329] Transduction of target cells with adenoviral vectors. All
cells were seeded on 100-mm dishes at a density of
2.times.10.sup.6/dish 1 day prior to infection. H1299 and A549
cells were infected at a total MOI of 900 and 1500, respectively.
For coadministration of two vectors, the ratio of the first vector
to second vector was 2:1. A preliminary study showed that such a
ratio resulted in optimal transduction of H1299 cells. Cells were
either harvested at 24 h and 48 h after infection for western blot
analysis or morphological observation by Hoechst staining.
[0330] Western blot analysis. Cell samples were lysed or liver
samples from the in vivo study were homogenized in a buffer
consisting of 62.5 mM Tris, pH 6.8, 6 M urea, 10% glycerol, 2%
sodium dodecyl sulfate (SDS), and 0.003% bromophenol blue. All
samples were sonicated for 30 sec on ice before the subsequent
analysis. Protein concentration was determined using BCA Protein
Assay Reagent (Pierce, Rockford, Ill.). Fifty micrograms of protein
was mixed with 5% 2-mercaptoethanol, boiled for 5 min, and then
loaded onto a SDS-polyacrylamide gel. After electrophoresis, the
proteins were transferred onto PROTRAN nitrocellulose membranes
(Schleicher & Schuell, Keene, N.H.), which were then blocked
for 1 h in PBS containing 10% milk. To detect various proteins, the
membranes were probed overnight with primary antibodies against bax
(N-20; Santa Cruz Biotechnology, Santa Cruz, Calif.), PARP (C2-10;
PharMingen, San Diego, Calif.), caspase-3 (PharMingen), and
.beta.-actin (Amersham, Arlington Heights, Ill.) at concentrations
recommended by the manufacturers. The membranes were washed 3 times
and probed with horseradish peroxidase-conjugated, species-specific
secondary antibodies (Amersham). Finally, bands were visualized
using the ECL system (Amersham) according to the manufacturer's
instructions and the density of each band was quantified using
Optimas software (Media Cybernetics, Silver Spring, Md.).
[0331] Hoechst staining. Cells were seeded on 4-chamber slides at a
density of 5.times.10.sup.4/chamber 1 day prior to infection.
Forty-eight hours after infection at the MOI described above, cells
were fixed with 4% glutaraldehyde and stained with 100 .mu.g/ml
Hoechst 33342 (Sigma, St. Louis, Mo.) for 15 min, followed by a
gentle washing with PBS. Photographs were taken under a fluorescent
microscope.
[0332] Animal experiments. Balb/c mice 6-8 weeks old were purchased
from the National Cancer Institute (Frederick, Md.). Prior to
injection, Ad/GT-Bax (or Ad/GT-LacZ) was mixed with Ad/PGK-GV16 (or
Ad/CMV-GFP) at a ratio of 1:2. A total of 6.times.10.sup.10
particles/mouse were injected into the tail vein in a volume of 100
.mu.l. Mice were killed 1 day after injection. Their livers were
then harvested and frozen at -80.degree. C. for later western blot
analysis or fixed in 10% buffered formalin for later histochemical
analysis. Sectioning and staining was with hematoxylin and
eosin.
Results
[0333] Construction of Adenoviruses Expressing Bax.
[0334] Shuttle plasmids were constructed in which bax cDNA was
driven by GT. Recombinant viral vectors were obtained after a
single transfection of 293 cells with pAd/GT-Bax plus a 35-kb ClaI
fragment from Ad/p53 and identified by polymerase chain reaction
(PCR) analysis with viral DNA. The functionality of Ad/GT-Bax was
documented by the coadministration of Ad/GT-Bax and Ad/PGK-GV16 to
the cultured human lung carcinoma cell line H1299 (FIG. 13). Virus
from a single plaque was expanded in 293 cells and twice purified
by ultracentrifugation on a cesium chloride gradient. The vector
titer determined by optical absorbency at A.sub.260 was
3.3.times.10.sup.12 viral particles/ml, equivalent to that of the
other E1-deleted vectors, such as Ad/CMV-GFP and Ad/CMV-LacZ. The
total yield for Ad/GT-Bax also was the same as for the other
E1-deleted vectors produced in our laboratory, about
1.5.times.10.sup.4 particles/cell. The vector preparation was free
of E1.sup.+ adenovirus and endotoxin.
[0335] Induction of Bax Expression After Adenovirus-Mediated Gene
Codelivery.
[0336] To demonstrate induction of the bax gene in cultured
mammalian cells by adenovirus-mediated gene co-transfer, human lung
carcinoma cell lines H1299 and A549 were infected with Ad/GT-Bax
and Ad/PGK-GV16 at a vector ratio of 2:1 and at a total
multiplicity of infection (MOI) of 900 and 1500, respectively. A
preliminary experiment showed that this ratio gave optimal
transduction efficiency in H1299 cells treated at a fixed total
MOI. Cells treated with PBS or infected either with Ad/GT-Bax plus
Ad/CMV-GFP or with Ad/GT-LacZ plus Ad/PGK-GV16 at the same vector
ratio and MOIs were used as controls. Cells were harvested 24 h
after the treatment and their lysates subjected to western blot
analysis. Levels of .beta.-actin in the same western blots were
also analyzed and used to ensure equal protein loading in all
lanes. Though background levels of the bax protein expression
differed between H1299 and A549 cells and though the treatment with
control vectors did not increase those background levels, a strong
induction of bax expression was detected in both cell lines when
they were treated with Ad/GT-Bax plus Ad/PGK-GV16. The induction
was seen to be 67.2- and 8.7-fold in H1299 and A549 cells,
respectively, when the densities of the bax-specific bands were
quantified and normalized to the density of .beta.-actin bands.
[0337] Triggering Apoptosis by Induction of the Bax Expression.
[0338] Overexpression of the bax gene has been demonstrated to
induce the release of Cyt c from mitochondria (Jurgensmeier et al.,
1998; Pastorino et al., 1998; Rosse et al., 1998) which leads to
cleavage first of caspase-3/CPP32 followed by cleavage of poly(ADP
ribose) polymerase (PARP) (Tewari et al., 1995). To demonstrate the
induction of bax expression and apoptosis by adenovirus-mediated
gene codelivery in H1299 and A549 cells, samples of the same cell
lysate from the above-mentioned experiments were subjected to
western blot analysis of the cleavage of caspase-3 and PARP. The
cleavage of caspase-3 into a 17-kD fragment and PARP into a 85-kD
fragment was detected in cells treated with Ad/GT-Bax plus
Ad/PGK-GV16 but not in cells from any other experimental groups. To
further document the apoptosis in these cells, H1299 and A549 cells
were treated with various vectors as mentioned above and observed
for cytopathology and morphology changes at 48 h after treatment.
Over 80% of the cells treated with Ad/GT-Bax plus Ad/PGK-GV16
showed signs of cytopatholgy, and became rounded and detached,
whereas the cells in all other treated groups remained in
monolayers with normal morphology. Nuclear fragmentation, a
hallmark of cell apoptosis, was detected only in cells treated with
Ad/GT-Bax plus Ad/PGK-GV16 (FIG. 14), indicating that bax
expression by this system did activate not only the caspase
cascade, but ultimately extensive apoptosis in these human lung
cancer cell lines.
[0339] Induction of Bax Gene Expression In Vivo.
[0340] To demonstrate bax gene expression by adenovirus-mediated
gene codelivery in vivo, adult Balb/c mice were infused via their
tail veins with PBS, Ad/GT-Bax plus Ad/CMV-GFP, Ad/GT-Bax plus
Ad/PGK-GV16, or Ad/GT-LacZ plus Ad/PGK-GV16 at a total vector dose
of 6.times.10.sup.10 particles/mouse and a vector ratio of 2:1.
Mice were then killed at 24 h after treatment, after which liver
samples were harvested for western blot analysis and
histopathological examination. Western blot analysis showed a
14-fold increase in bax protein levels in animals treated with
Ad/GT-Bax plus Ad/PGK-GV16, but only background level in all other
treatment groups. These results clearly demonstrated that the
Ad/GT-Bax plus Ad/PGK-GV16 strictly regulated bax expression by
expressing GAL4/VP16 protein even in vivo. Expression of the bax
gene also induced typical apoptosis in normal liver cells, as
revealed by nuclear fragmentation and condensation in hematoxylin-
and eosin-stained liver sections (FIG. 15). Together, these results
demonstrate that adenovirus-mediated gene co-transfer can produce
sufficient bax expression and induce apoptosis in vivo.
[0341] All of the compositions and/or methods disclosed and claimed
herein can be made and executed without undue experimentation in
light of the present disclosure. While the compositions and methods
of this invention have been described in terms of preferred
embodiments, it will be apparent to those of skill in the art that
variations may be applied to the compositions and/or methods and in
the steps or in the sequence of steps of the method described
herein without departing from the concept, spirit and scope of the
invention. More specifically, it will be apparent that certain
agents which are both chemically and physiologically related may be
substituted for the agents described herein while the same or
similar results would be achieved. All such similar substitutes and
modifications apparent to those skilled in the art are deemed to be
within the spirit, scope and concept of the invention as defined by
the appended claims.
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Sequence CWU 1
1
10 1 578 DNA Human 1 atggacgggt ccggggagca gcccagaggc gggggtccca
ccagctctga gcagatcatg 60 aagacagggg cccttttgct tcagggtttc
atccaggatc gagcagggcg aatggggggg 120 gaggcacccg agctggccct
ggacccggtg cctcaggatg cgtccaccaa gaagctgagc 180 gagtgtctca
agcgcatcgg ggacgaactg gacagtaaca tggagctgca gaggatgatt 240
gccgccgtgg acacagactc cccccgagag gtctttttcc gagttgcagc tgacatgttt
300 tctgacggca acttcaactg ggccgggttg tcgccctttt ctactttgcc
agcaaactgg 360 tgctcaaggc cctgtgcacc aaggtgccgg aactgatcag
aaccatcatg ggctggacat 420 tggacttcct ccgggagcgg ctgttgggct
ggatccaaga ccagggtggt tgggacggcc 480 tcctctccta ctttgggacg
cccacgtggc agaccgtgac catctttgtg gcgggagtgc 540 tcaccgcctc
gctcaccatc tggaagaaga tgggctga 578 2 131 PRT Human 2 Met Asp Gly
Ser Gly Glu Gln Pro Arg Gly Gly Gly Pro Thr Ser Ser 1 5 10 15 Glu
Gln Ile Met Lys Thr Gly Ala Leu Leu Leu Gln Gly Phe Ile Gln 20 25
30 Asp Arg Ala Gly Arg Met Gly Gly Glu Ala Pro Glu Leu Ala Leu Asp
35 40 45 Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Leu Ser Glu Cys
Leu Lys 50 55 60 Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Glu Leu
Gln Arg Met Ile 65 70 75 80 Ala Ala Val Asp Thr Asp Ser Pro Arg Glu
Val Phe Phe Arg Val Ala 85 90 95 Ala Asp Met Phe Ser Asp Gly Asn
Phe Asn Trp Ala Gly Leu Ser Pro 100 105 110 Phe Ser Thr Leu Pro Ala
Asn Trp Cys Ser Arg Pro Cys Ala Pro Arg 115 120 125 Cys Arg Asn 130
3 579 DNA Human 3 atggacgggt ccggggagca gcccagaggc ggggggccca
ccagctctga gcagatcatg 60 aagacagggg cccttttgct tcagggtttc
atccaggatc gagcagggcg aatggggggg 120 gaggcacccg agctggccct
ggacccggtg cctcaggatg cgtccaccaa gaagctgagc 180 gagtgtctca
agcgcatcgg ggacgaactg gacagtaaca tggagctgca gaggatgatt 240
gccgccgtgg acacagactc cccccgagag gtctttttcc gagtggcagc tgacatgttt
300 tctgacggca acttcaactg gggccgggtt gtcgcccttt tctactttgc
cagcaaactg 360 gtgctcaagg ccctgtgcac caaggtgccg gaactgatca
gaaccatcat gggctggaca 420 ttggacttcc tccgggagcg gctgttgggc
tggatccaag accagggtgg ttgggacggc 480 ctcctctcct actttgggac
gcccacgtgg cagaccgtga ccatctttgt ggcgggagtg 540 ctcaccgcct
cgctcaccat ctggaagaag atgggctga 579 4 192 PRT Human 4 Met Asp Gly
Ser Gly Glu Gln Pro Arg Gly Gly Gly Pro Thr Ser Ser 1 5 10 15 Glu
Gln Ile Met Lys Thr Gly Ala Leu Leu Leu Gln Gly Phe Ile Gln 20 25
30 Asp Arg Ala Gly Arg Met Gly Gly Glu Ala Pro Glu Leu Ala Leu Asp
35 40 45 Pro Val Pro Gln Asp Ala Ser Thr Lys Lys Leu Ser Glu Cys
Leu Lys 50 55 60 Arg Ile Gly Asp Glu Leu Asp Ser Asn Met Glu Leu
Gln Arg Met Ile 65 70 75 80 Ala Ala Val Asp Thr Asp Ser Pro Arg Glu
Val Phe Phe Arg Val Ala 85 90 95 Ala Asp Met Phe Ser Asp Gly Asn
Phe Asn Trp Gly Arg Val Val Ala 100 105 110 Leu Phe Tyr Phe Ala Ser
Lys Leu Val Leu Lys Ala Leu Cys Thr Lys 115 120 125 Val Pro Glu Leu
Ile Arg Thr Ile Met Gly Trp Thr Leu Asp Phe Leu 130 135 140 Arg Glu
Arg Leu Leu Gly Trp Ile Gln Asp Gln Gly Gly Trp Asp Gly 145 150 155
160 Leu Leu Ser Tyr Phe Gly Thr Pro Thr Trp Gln Thr Val Thr Ile Phe
165 170 175 Val Ala Gly Val Leu Thr Ala Ser Leu Thr Ile Trp Lys Lys
Met Gly 180 185 190 5 27 DNA Artificial Sequence Description of
Artificial Sequence Synthetic 5 ggaattcgcg gtgatggacg ggtccgg 27 6
28 DNA Artificial Sequence Description of Artificial Sequence
Synthetic 6 gggaattctc agcccatctt cttccaga 28 7 19 DNA Artificial
Sequence Description of Artificial Sequence Synthetic 7 gggacgaact
ggacagtaa 19 8 19 DNA Artificial Sequence Description of Artificial
Sequence Synthetic 8 gcaccagttt gctggcaaa 19 9 17 DNA Artificial
Sequence Description of Artificial Sequence Synthetic 9 acgcaaatgg
gcggtag 17 10 17 DNA Artificial Sequence Description of Artificial
Sequence Synthetic 10 caactagaag gcacagt 17
* * * * *